The Localization of the qa -1S - qa -1F Intergenic Region of Neurospora africana by Scott Michael Raidel Submitted In Partial Fulfillment of the Requirements for the Degree of Master of Science in the Department of Biology Program YOUNGSTOWN STATE UNIVERSITY September, 1997 The Localization of the qa -1S - qa -1F Intergenic Region of Neurospora africana by Scott Michael Raidel Submitted In Partial Fulfillment of the Requirements for the Degree of Master of Science in the Department of Biology Program YOUNGSTOWN STATE UNIVERSITY September, 1997 The Localization of the qa -1 S - qa -1 F Intergenic Region of Neurospora africana Scott Michael Raidel I hereby release this thesis to the public. I understand this thesis will be housed at the Circulation Desk ofthe University library and will be available for public access. I also authorize the University or other individuals to make copies ofthis thesis as needed for scholarly research. Signature: Approvals: ~.. J .) CA;L!Ll~ Tesis Advisor Committee Member Cj11qa-4 -->qa-3 (Schweizer et aI., 1981a; 1981b) (Figure 4). It was also determined using transformation experiments involving stable qa-1Sand qa-1F mutants as recipients that the regulatory genes were located to the right of the gene qa-3. These experiments, along with genetic mapping, also determined the order of these regulatory genes (Figure 4) (Schweizer et aI., 1981a; 1981b). This cloning of genes allowed for the identification of all the structural genes mRNAs. DNA RNA hybridization studies revealed the existence of five, rather then three structural genes. Each structural gene was transcribed as a separate mRNA (Giles et aI., 1985). The two additional structural genes which were identified were termed qa-x and qa-y. The qa-y gene was originally identified as a qUlmc acid inducible transcript of unknown function (Patel et aI., 1981). The qa-y gene was found to be located between qa-3 and qa 1S (Giles et aI., 1985) (Figure 4). The qa-y gene showed 61 % 23 respectively. Based on these results it was now necessary to revise the original hypothesis that the qa-1 gene encoded a single regulatory protein with separate functional domains (Case and Giles, 1975). The new hypothesis proposed that the two qa genes (q a-1 Sand qa-1 F) encoded a repressor protein (qa-1 S) and an activator protein (qa-1 F), whose interactions controlled qa gene expression (Huiet, 1984). Using subclones of a 42 kb region of cloned N. crassa DNA, which was centered around the gene qa-2, it was possible to localize and determine the order of the three structural genes as qa-2 -->qa-4 -->qa-3 (Schweizer et aI., 1981a; 1981b) (Figure 4). It was also determined using transformation experiments involving stable qa-1Sand qa-1F mutants as recipients that the regulatory genes were located to the right of the gene qa-3. These experiments, along with genetic mapping, also determined the order of these regulatory genes (Figure 4) (Schweizer et aI., 1981a; 1981b). This cloning of genes allowed for the identification of all the structural genes mRNAs. DNA RNA hybridization studies revealed the existence of five, rather then three structural genes. Each structural gene was transcribed as a separate mRNA (Giles et aI., 1985). The two additional structural genes which were identified were termed qa-x and qa-y. The qa-y gene was originally identified as a qUlmc acid inducible transcript of unknown function (Patel et aI., 1981). The qa-y gene was found to be located between qa-3 and qa 1S (Giles et aI., 1985) (Figure 4). The qa-y gene showed 61 % 23 amino acid identity with the qutD gene of Aspergillus nidulans, which was predicted to encode a quinate permease (Whittington et aI., 1987). Experiments also showed amino acid structural similarities to a family of human hepatoma cells (Mueckler et aI., 1985) and bacterial transporters for arabinose (AraE), xylose (XylE) and citrate (cit+) (Maiden et aI., 1987; Geever et aI., 1989). In addition to these results, it was found that when the qa-y gene of N. crassa was mutated these strains had a reduced ability to absorb quinic acid and grow on quinic acid as a sole carbon source. These strains also had very low levels of quinate pathway enzymes (qa-2, qa-3, and qa-4) when compared to wild-type strains (Case et aI., 1992). With this comparative and experimental evidence it has been established that the qa-y gene encodes a quinic acid permease. The qa-x gene was also originally identified as a qUlll1C acid inducible transcript of unknown function (Patel et aI., 1981). It was found that the qa-x gene was located to the left of qa-2 (Giles et aI., 1985) (Figure 4). Using null mutations of the qa-x gene it was found that these strains could still grow on quinic acid as a sole carbon source, and over time they accumulated a brown pigment (Asch, unpublished data). It was hypothesized that the qa-x gene encoded an enzyme capable of hydrolyzing chlorogenic acid (Giles et aI., 1985). However, these mutants still grew on chlorogenic acid seemingly to disprove this hypothesis. A possible role for the qa-x gene was seen by evidence obtained with the galactose (GAL) system in S. cerevlszae. Both the GAL and qa systems 24 amino acid identity with the qutD gene of Aspergillus nidulans, which was predicted to encode a quinate permease (Whittington et aI., 1987). Experiments also showed amino acid structural similarities to a family of human hepatoma cells (Mueckler et aI., 1985) and bacterial transporters for arabinose (AraE), xylose (XylE) and citrate (cit+) (Maiden et aI., 1987; Geever et aI., 1989). In addition to these results, it was found that when the qa-y gene of N. crassa was mutated these strains had a reduced ability to absorb quinic acid and grow on quinic acid as a sole carbon source. These strains also had very low levels of quinate pathway enzymes (qa-2, qa-3, and qa-4) when compared to wild-type strains (Case et aI., 1992). With this comparative and experimental evidence it has been established that the qa-y gene encodes a quinic acid permease. The qa-x gene was also originally identified as a qUlll1C acid inducible transcript of unknown function (Patel et aI., 1981). It was found that the qa-x gene was located to the left of qa-2 (Giles et aI., 1985) (Figure 4). Using null mutations of the qa-x gene it was found that these strains could still grow on quinic acid as a sole carbon source, and over time they accumulated a brown pigment (Asch, unpublished data). It was hypothesized that the qa-x gene encoded an enzyme capable of hydrolyzing chlorogenic acid (Giles et aI., 1985). However, these mutants still grew on chlorogenic acid seemingly to disprove this hypothesis. A possible role for the qa-x gene was seen by evidence obtained with the galactose (GAL) system in S. cerevlszae. Both the GAL and qa systems 24 I j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j J I I I I I I I I I I I I I I I I I I I I I I I I I I I I I STRUCTURAL GENES REGULATORY GENES I I qa-x qa-2 qa-4 qa-3 qa-y qa-1S qa-1F STRUCTURAL GENES REGULATORY GENES I I qa-x qa-2 qa-4 qa-3 qa-y qa-1S qa-1F are subject to carbon catabolite repression. It was found that expression of the qa-x gene was strongly affected by catabolite regression, more so than the other qa genes. This was shown by a 20-fold increase in qa-x mRNA when a culture was shifted to a carbon limiting growth condition (Tyler and Geever, unpublished data). This suggested that a preferred carbon source may directly effect repression of qa-x transcription (Giles et aI., 1991). However, it was also suggested that the product of qa-x is itself involved directly in affecting catabolite repression. This is supported by the comparison of qa-x to a gene (GAM1) implicated in carbon-regulated dephosphorylation of the GAL4 activator. The gene qa-x was found to have 31 % amino acid identity to the GAM1 gene, suggesting homology (Giles et aI., 1991). If qa-x plays a similar role to GAM1 it has yet to be determined. Therefore, the function of the gene qa-x still remams unknown. Finally, by using transformation experiments, Northern blot analysis, 51 nuclease mapping and nucleotide sequencing the structure of the qa gene cluster has been determined (Giles et aI., 1985; Geever et aI., 1989). The seven qa genes were found to cover approximately 17.3 kb of DNA on chromosome VII (Figure 5). The locations of each gene and lengths of their mRNAs was also established (Geever et aI., 1989) (Figure 5). The direction of transcription for each gene has also been established and the genes were found to be divergently transcribed in pairs (qa-x/qa-2, qa-4/qa-3, and qa-lS/qa-lF) 27 are subject to carbon catabolite repression. It was found that expression of the qa-x gene was strongly affected by catabolite regression, more so than the other qa genes. This was shown by a 20-fold increase in qa-x mRNA when a culture was shifted to a carbon limiting growth condition (Tyler and Geever, unpublished data). This suggested that a preferred carbon source may directly effect repression of qa-x transcription (Giles et aI., 1991). However, it was also suggested that the product of qa-x is itself involved directly in affecting catabolite repression. This is supported by the comparison of qa-x to a gene (GAM1) implicated in carbon-regulated dephosphorylation of the GAL4 activator. The gene qa-x was found to have 31 % amino acid identity to the GAM1 gene, suggesting homology (Giles et aI., 1991). If qa-x plays a similar role to GAM1 it has yet to be determined. Therefore, the function of the gene qa-x still remams unknown. Finally, by using transformation experiments, Northern blot analysis, 51 nuclease mapping and nucleotide sequencing the structure of the qa gene cluster has been determined (Giles et aI., 1985; Geever et aI., 1989). The seven qa genes were found to cover approximately 17.3 kb of DNA on chromosome VII (Figure 5). The locations of each gene and lengths of their mRNAs was also established (Geever et aI., 1989) (Figure 5). The direction of transcription for each gene has also been established and the genes were found to be divergently transcribed in pairs (qa-x/qa-2, qa-4/qa-3, and qa-lS/qa-lF) 27 with the unpaired qa-y gene separating the structural pairs from the regulatory pair (Figure 5). XII. Mechanisms of the qa-1S Repressor Protein It was found USIng DNA sequence analysis that the qa-1S gene encodes a protein of 918 amino acids (Huiet, 1983; Huiet and Giles, 1986; Geever et al., 1989). Evidence to support the thought that the qa-1S gene encodes a repressor protein was seen when a deletion of the gene caused constitutive transcription of all the qa genes at high levels (Case et al., 1992). Studies using the two classes of qa-1S pleiotropic mutations (noninducible and constitutive) showed the possible location of two functional domains within the repressor protein (Huiet and Giles, 1986). The semidominant noninducible (qa- 1S- ) class of mutants all contained a missense mutation which caused these mutants to encode a functional repressor that acted as a superepressor. The location of these mutations (between codons 627 and 743) showed that this domain of the repressor protein interacted with the inducer quinic acid (Huiet and Giles, 1986). Here these mutant superepressor proteins failed to bind the inducer quinic acid and caused them to remain bound to its target. However, an alternative thought believes that these qa-1S- mutations affect the affinity of the repressor for the activator protein, as in the comparable GAL80s mutations in yeast (Salmeron et al., 1990). 28 with the unpaired qa-y gene separating the structural pairs from the regulatory pair (Figure 5). XII. Mechanisms of the qa-1S Repressor Protein It was found USIng DNA sequence analysis that the qa-1S gene encodes a protein of 918 amino acids (Huiet, 1983; Huiet and Giles, 1986; Geever et al., 1989). Evidence to support the thought that the qa-1S gene encodes a repressor protein was seen when a deletion of the gene caused constitutive transcription of all the qa genes at high levels (Case et al., 1992). Studies using the two classes of qa-1S pleiotropic mutations (noninducible and constitutive) showed the possible location of two functional domains within the repressor protein (Huiet and Giles, 1986). The semidominant noninducible (qa- 1S- ) class of mutants all contained a missense mutation which caused these mutants to encode a functional repressor that acted as a superepressor. The location of these mutations (between codons 627 and 743) showed that this domain of the repressor protein interacted with the inducer quinic acid (Huiet and Giles, 1986). Here these mutant superepressor proteins failed to bind the inducer quinic acid and caused them to remain bound to its target. However, an alternative thought believes that these qa-1S- mutations affect the affinity of the repressor for the activator protein, as in the comparable GAL80s mutations in yeast (Salmeron et al., 1990). 28 j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j J qa-x qa-2 qa-4 qa-3 qa-y qa-1 S qa-1 F...... ~.... ~.... ~ ~ 1.3 kb mRNAs 1.5 kb 1.0 kb 1.2 kb 1.5 kb 1.7 kb 1.4 kb 1.6 kb 1.25 kb 2.3 kb 4.1 kb 3.4 kb 2.9 kb 3.0 kb REGULATORY GENESQA INDUCIBLE STRUCTURAL GENES " QA GENE CLUSTER , 17.3 KB CHROMOSOME VII qa-x qa-2 qa-4 qa-3 qa-y qa-1S qa-1F .....- ~ ~ ....- ~ 1.3 kb 1.0 kb 1.5 kb 1.4 kb 2.3 kb 4.1 kb 2.9 kb mRNAs 1.5 kb 1.2 kb 1.7 kb 1.6 kb 3.4 kb 3.0 kb 1.25 kb REGULATORY GENESQA INDUCIBLE STRUCTURAL GENES '--- QA GENE CLUSTER _ 17.3 KB CHROMOSOME VII The constitutive (qa-l SC) mutants were the result of a frameshift or nonsense mutation, which caused these mutants to encode inactive repressors. These mutations appeared to show that the carboxy terminus of the repressor protein interacts with the target. Experiments using overexpressed repressor protein produced m baculovirus showed that the qa 1SC repressor protein failed to bind to the DNA itself (Geever and Baum, unpublished data). This along with other evidence (Giles et aI., 1985; 1987) suggested that the target for the repressor protein is the activator protein. XIII. Mechanisms of the qa-lF Activator Protein It was found usmg DNA sequence analysis that the qa-lF gene encodes a protein of 816 amino acids (Huiet, 1983; Geever et aI., 1989). Experiments have shown that qa-lF mRNAs of Neurospora wild-types are produced at basal levels in the absence of quinic acid. However, a 50-fold increase was observed upon quinic acid induction (Giles et aI., 1985). Additional experiments using noninducible qa-lF- mutations resulted in noninduced transcription of all the qa genes at low basal levels, like uninduced wild-type (Avalos, Geever, and Case, unpublished data). Genetic and molecular studies (Patel and Giles, 1985) have also shown that the qa-lF activator protein plays a positive role in transcription of all the qa genes, including itself (autoregulation). 3 1 The constitutive (qa-l SC) mutants were the result of a frameshift or nonsense mutation, which caused these mutants to encode inactive repressors. These mutations appeared to show that the carboxy terminus of the repressor protein interacts with the target. Experiments using overexpressed repressor protein produced m baculovirus showed that the qa 1SC repressor protein failed to bind to the DNA itself (Geever and Baum, unpublished data). This along with other evidence (Giles et aI., 1985; 1987) suggested that the target for the repressor protein is the activator protein. XIII. Mechanisms of the qa-lF Activator Protein It was found usmg DNA sequence analysis that the qa-lF gene encodes a protein of 816 amino acids (Huiet, 1983; Geever et aI., 1989). Experiments have shown that qa-lF mRNAs of Neurospora wild-types are produced at basal levels in the absence of quinic acid. However, a 50-fold increase was observed upon quinic acid induction (Giles et aI., 1985). Additional experiments using noninducible qa-lF- mutations resulted in noninduced transcription of all the qa genes at low basal levels, like uninduced wild-type (Avalos, Geever, and Case, unpublished data). Genetic and molecular studies (Patel and Giles, 1985) have also shown that the qa-lF activator protein plays a positive role in transcription of all the qa genes, including itself (autoregulation). 3 1 On the basis of certain studies (Geever et aI., 1987; 1989; Beri et aI., 1987; Salmeron and Johnston, 1986; Avalos, Geever, and Case, unpublished data) at least four functional domains within the activator protein have been identified, when compared to the A. nidulans activator. These are: a DNA binding domain, a dimerization domain, a transcriptional activation domain, and a domain for interaction with the q a repressor. The DNA-binding domain was localized to the first 183 amino acids (Baum et aI., 1987). Within this region a 28 ammo acid sequence containing a six cysteine motif shows conservation with several lower eukaryotic activator proteins (Baum et aI., 1987; Pfeifer et aI., 1989). Direct evidence was obtained which implicated this conserved segment in DNA binding (Geever et aI., 1987; 1989, and unpublished data). The second domain occurs over a broad central segment. Studies using qa-lF noninducible and inducible mutants indicated that alterations in this regIOn affected DNA binding. However, within this domain is a yet identified segment between codons 296 and 562 which did not bind to DNA. This segment is believed to contain residues needed for protein dimerization (Giles et aI., 1991). The third domain is located at the carboxy terminus of the activator and contains mainly acidic residues. This region is believed to be implicated in interactions with transcriptional factors by comparison to a similar region in the GAL4 activator (Ma and Ptashine, 1987a). 32 On the basis of certain studies (Geever et aI., 1987; 1989; Beri et aI., 1987; Salmeron and Johnston, 1986; Avalos, Geever, and Case, unpublished data) at least four functional domains within the activator protein have been identified, when compared to the A. nidulans activator. These are: a DNA binding domain, a dimerization domain, a transcriptional activation domain, and a domain for interaction with the q a repressor. The DNA-binding domain was localized to the first 183 amino acids (Baum et aI., 1987). Within this region a 28 ammo acid sequence containing a six cysteine motif shows conservation with several lower eukaryotic activator proteins (Baum et aI., 1987; Pfeifer et aI., 1989). Direct evidence was obtained which implicated this conserved segment in DNA binding (Geever et aI., 1987; 1989, and unpublished data). The second domain occurs over a broad central segment. Studies using qa-lF noninducible and inducible mutants indicated that alterations in this regIOn affected DNA binding. However, within this domain is a yet identified segment between codons 296 and 562 which did not bind to DNA. This segment is believed to contain residues needed for protein dimerization (Giles et aI., 1991). The third domain is located at the carboxy terminus of the activator and contains mainly acidic residues. This region is believed to be implicated in interactions with transcriptional factors by comparison to a similar region in the GAL4 activator (Ma and Ptashine, 1987a). 32 The final fourth domain is a regIOn that overlaps with the acidic region in the carboxy terminus. It is proposed that this region interacts with the qa repressor protein. Experiments which exchanged the C-terminus of the N. crassa activator with that of A. nidulans found that when this chimeric activator was transformed into N. crassa modest levels of transcription was seen. These transformants grew slowly on quinic acid, which said that the chimeric was capable of activating transcription. However, transcription was found to be constitutive and not inducible (Avalos, Geever, and Case, unpublished data). These results suggested that the carboxy terminus of the activator was involved in interactions with the repressor protein. This was supported by the finding that a deletion of this carboxy terminus produced a mutant with constitutive activity greater than (20%) that of an induced wild-type (Giles et aI., 1991). Evidence supporting the mechanisms of the activators function came from studies using genetic analysis of mRNA transcription and chromatin structure in the qa gene cluster. These studies provided evidence that the activator protein interacts at specific sites in the 5' flanking region of the qa genes (Baum and Giles, 1985; 1986; Geever et aI., 1983; 1986). From these studies, a 16 base-pair (bp) sequence element, found one or more times 5' to each of the qa genes, was identified as a potential binding site for the activator protein. Evidence for the activator binding DNA was found in overexpressing the qa 1F gene in a baculovirus expression vector (Miller et aI., 1986). This overexpressed qa-lF activator protein was used in DNA 33 The final fourth domain is a regIOn that overlaps with the acidic region in the carboxy terminus. It is proposed that this region interacts with the qa repressor protein. Experiments which exchanged the C-terminus of the N. crassa activator with that of A. nidulans found that when this chimeric activator was transformed into N. crassa modest levels of transcription was seen. These transformants grew slowly on quinic acid, which said that the chimeric was capable of activating transcription. However, transcription was found to be constitutive and not inducible (Avalos, Geever, and Case, unpublished data). These results suggested that the carboxy terminus of the activator was involved in interactions with the repressor protein. This was supported by the finding that a deletion of this carboxy terminus produced a mutant with constitutive activity greater than (20%) that of an induced wild-type (Giles et aI., 1991). Evidence supporting the mechanisms of the activators function came from studies using genetic analysis of mRNA transcription and chromatin structure in the qa gene cluster. These studies provided evidence that the activator protein interacts at specific sites in the 5' flanking region of the qa genes (Baum and Giles, 1985; 1986; Geever et aI., 1983; 1986). From these studies, a 16 base-pair (bp) sequence element, found one or more times 5' to each of the qa genes, was identified as a potential binding site for the activator protein. Evidence for the activator binding DNA was found in overexpressing the qa 1F gene in a baculovirus expression vector (Miller et aI., 1986). This overexpressed qa-lF activator protein was used in DNA 33 binding and DNase I footprinting experiments. These identified the precise location of 14 sites in the cluster, each characterized by a conserved, symmetrical 16 bp sequence (GGRTAARYRYTTAYCC) to which the activator bound (Buam et aI., 1987; Geever et aI., 1989). Of particular interest was the finding of a single binding site in the common 5' region of the two regulatory genes. This suggested bidirectional control and supported the findings for activator autoregulation and transcriptional control of repressor gene expression by the activator. XIV. Comparisons Between the Quinic Acid (qa) Gene Cluster of Neurospora crassa and the Quinic Acid Utilization (qut) Gene Cluster of Aspergillus nidulans Comparative studies of the quinic acid pathways in A. nidulans and N. crassa revealed many similarities and differences between the two. First, the regulation of the pathway in A. nidulans, which is controlled by the q utA activator protein and the qutR repressor protein, seem to be analogous to the regulation caused by the qa-lF activator protein and the qa-lS repressor protein in the N. crassa pathway. However, when the sequence of the qut regulatory proteins (Beri et aI., 1987; Geever, unpublished data) was compared to the qa regulatory proteins they were found to diverge substantially. The amino acid identity of the two activators was only 25%, and 50% between the two repressors. 34 binding and DNase I footprinting experiments. These identified the precise location of 14 sites in the cluster, each characterized by a conserved, symmetrical 16 bp sequence (GGRTAARYRYTTAYCC) to which the activator bound (Buam et aI., 1987; Geever et aI., 1989). Of particular interest was the finding of a single binding site in the common 5' region of the two regulatory genes. This suggested bidirectional control and supported the findings for activator autoregulation and transcriptional control of repressor gene expression by the activator. XIV. Comparisons Between the Quinic Acid (qa) Gene Cluster of Neurospora crassa and the Quinic Acid Utilization (qut) Gene Cluster of Aspergillus nidulans Comparative studies of the quinic acid pathways in A. nidulans and N. crassa revealed many similarities and differences between the two. First, the regulation of the pathway in A. nidulans, which is controlled by the q utA activator protein and the qutR repressor protein, seem to be analogous to the regulation caused by the qa-lF activator protein and the qa-lS repressor protein in the N. crassa pathway. However, when the sequence of the qut regulatory proteins (Beri et aI., 1987; Geever, unpublished data) was compared to the qa regulatory proteins they were found to diverge substantially. The amino acid identity of the two activators was only 25%, and 50% between the two repressors. 34 Despite this divergence, the functional domains in both regulatory pairs appear to be conserved. Next, when the qutD gene product of A. nidulans was compared to the qa-y gene product of N. crassa 61 % amino acid identity was seen, which suggested that qutD also encoded a quinic acid permease. Also, the qutG gene product of A. nidulans showed 68.5% amino acid identity to the qa-x gene product of N. crassa, and both were found to encode quinate-inducible messages of unknown function. Two organizational features of both clusters stand out: (l) their genes are arranged as divergently transcribed pairs; and (2) structural and regulatory genes occupy separate regions of the cluster. Eventhough the genes of both remained clustered, their gene orders are different (Grant et aI., 1988; Hawkins et aI., 1988) (Figure 6). Not all the same pairs of genes are divergently transcribed in the two, and the order of the regulatory genes in A. nidulans is inverted (Figure 4). Finally, the gene of unknown function between the two q u t regulatory genes (Figure 6) appears not to be under quinic acid regulation and N. crassa does not posses this gene. xv. Comparisons Between the Quinic Acid (qa) Gene Clusters of Neurospora Species With the detection of qa catabolic enzymes in other fungi (Ahmed and Giles, 1969; Berlyn and Giles, 1972) comparative studies of the qa gene cluster were initiated. Now with all of the information gathered on the qa gene cluster of N. crassa 35 Despite this divergence, the functional domains in both regulatory pairs appear to be conserved. Next, when the qutD gene product of A. nidulans was compared to the qa-y gene product of N. crassa 61 % amino acid identity was seen, which suggested that qutD also encoded a quinic acid permease. Also, the qutG gene product of A. nidulans showed 68.5% amino acid identity to the qa-x gene product of N. crassa, and both were found to encode quinate-inducible messages of unknown function. Two organizational features of both clusters stand out: (l) their genes are arranged as divergently transcribed pairs; and (2) structural and regulatory genes occupy separate regions of the cluster. Eventhough the genes of both remained clustered, their gene orders are different (Grant et aI., 1988; Hawkins et aI., 1988) (Figure 6). Not all the same pairs of genes are divergently transcribed in the two, and the order of the regulatory genes in A. nidulans is inverted (Figure 4). Finally, the gene of unknown function between the two q u t regulatory genes (Figure 6) appears not to be under quinic acid regulation and N. crassa does not posses this gene. xv. Comparisons Between the Quinic Acid (qa) Gene Clusters of Neurospora Species With the detection of qa catabolic enzymes in other fungi (Ahmed and Giles, 1969; Berlyn and Giles, 1972) comparative studies of the qa gene cluster were initiated. Now with all of the information gathered on the qa gene cluster of N. crassa 35 Neurospora QA cluster.. .... ..... . qa-x qa-2 qa-4 qa-3 qa-y qa-1S qa-1F ---~...... ..~ .. ~ qutC qutD qutB qutG qutE qutA ? qutR Aspergillus OUT cluster qa-x qutC Neurospora QA cluster ......~--~~~-----1_~~-- .....~---~~ qa-2 qa-4 qa-3 qa-y qa-1S qa-1 F ---.....~---- ......~----- .......~--- ..~-~~ qutD qutB qutG qutE qutA ? qutR Aspergillus OUT cluster more detailed comparative studies could be done on different Neurospora species. One such study, (Asch et aI., 1991) compared the qa genes of various heterothallic (different nuclei) and homothallic (same nuclei) Neurospora species. It was found that the qa gene cluster of N. crassa (heterothallic) was highly conserved in various species of Neurospora. However, there were many restriction fragment length poly morphisms that distinguished N. crassa from the homothallic species. Despite this difference, the gene organization of the cluster remained highly conserved (qa-x, qa-2, qa-4, qa-3, qa y, qa-lS, and qa-lF). With the amount of conservation observed in the various species focused then turned to examme if the mechanisms controlling expression of the qa genes In both heterothallic and homothallic species were similar. To determine if the same control circuits operate in homothallic and heterthallic species, Asch et aI. (1991) measured qa-2 gene expression in N. africana (homothallic) under non-inducing conditions (no carbon source), inducing conditions (quinic acid alone), and catabolite repression conditions (quinic acid and dextrose). Results showed at least a 1,500-fold induction of the qa-2 gene in the presence of quinic acid over basal levels compared to a 2,OOO-fold in N. crassa. Under catabolite repression, N. africana showed a 3-fold reduction in expression of qa-2 verse a 65-fold reduction In N. crassa. It was thought that this difference might be due to specific sequence differences between the homothallic species and N. crassa. 38 more detailed comparative studies could be done on different Neurospora species. One such study, (Asch et aI., 1991) compared the qa genes of various heterothallic (different nuclei) and homothallic (same nuclei) Neurospora species. It was found that the qa gene cluster of N. crassa (heterothallic) was highly conserved in various species of Neurospora. However, there were many restriction fragment length poly morphisms that distinguished N. crassa from the homothallic species. Despite this difference, the gene organization of the cluster remained highly conserved (qa-x, qa-2, qa-4, qa-3, qa y, qa-lS, and qa-lF). With the amount of conservation observed in the various species focused then turned to examme if the mechanisms controlling expression of the qa genes In both heterothallic and homothallic species were similar. To determine if the same control circuits operate in homothallic and heterthallic species, Asch et aI. (1991) measured qa-2 gene expression in N. africana (homothallic) under non-inducing conditions (no carbon source), inducing conditions (quinic acid alone), and catabolite repression conditions (quinic acid and dextrose). Results showed at least a 1,500-fold induction of the qa-2 gene in the presence of quinic acid over basal levels compared to a 2,OOO-fold in N. crassa. Under catabolite repression, N. africana showed a 3-fold reduction in expression of qa-2 verse a 65-fold reduction In N. crassa. It was thought that this difference might be due to specific sequence differences between the homothallic species and N. crassa. 38 To examine this the intergenic region between qa-x and qa-2 of N. afrieana (homothallic) was sequenced and compared to its counterpart in N. erassa (Asch et aI., 1991). An earlier study (Asch and Case, unpublished data), found that the N. afrieana qa-lF binding domain was identical to the N. erassa domain (GGRTAARYRYTTATCC). This seemed to state that N. afrieana employs the same binding sites as N. erassa for activation. Indeed, it was found that all four binding sites for the activator protein located in the qa-x-qa-2 intergenic regIOn of N. erassa could be aligned with those of N. afrieana, eventhough the N. afrieana sequence (1088 bp) was smaller than the N. erassa region (1194 bp) (Asch et aI., 1991). Since sequence analysis provided no conclusive evidence to the differences of the qa expreSSIOn between species under carbon catabolite repressing conditions, Asch et aI. (1991) examined whether the control circuits expressing the qa genes in N. erassa would operate in the presence of N. afrieana sequences. To do this the qa-x-qa-2 intergenic region of N. erassa was replaced with the qa-x-qa-2 intergenic region of N. afrieana. Using this transformant, qa-2 gene expression was measured under non-inducing conditions (no carbon source), inducing conditions (quinic acid alone), and catabolite repres sion conditions (quinic acid and dextrose). Results showed increased expression of the qa-2 gene, which were approximately 70% of those seen in wild-type N. erassa, under inducing conditions. Furthermore, under catabolite repression conditions, the transformed strains showed over 100-fold 39 To examine this the intergenic region between qa-x and qa-2 of N. afrieana (homothallic) was sequenced and compared to its counterpart in N. erassa (Asch et aI., 1991). An earlier study (Asch and Case, unpublished data), found that the N. afrieana qa-lF binding domain was identical to the N. erassa domain (GGRTAARYRYTTATCC). This seemed to state that N. afrieana employs the same binding sites as N. erassa for activation. Indeed, it was found that all four binding sites for the activator protein located in the qa-x-qa-2 intergenic regIOn of N. erassa could be aligned with those of N. afrieana, eventhough the N. afrieana sequence (1088 bp) was smaller than the N. erassa region (1194 bp) (Asch et aI., 1991). Since sequence analysis provided no conclusive evidence to the differences of the qa expreSSIOn between species under carbon catabolite repressing conditions, Asch et aI. (1991) examined whether the control circuits expressing the qa genes in N. erassa would operate in the presence of N. afrieana sequences. To do this the qa-x-qa-2 intergenic region of N. erassa was replaced with the qa-x-qa-2 intergenic region of N. afrieana. Using this transformant, qa-2 gene expression was measured under non-inducing conditions (no carbon source), inducing conditions (quinic acid alone), and catabolite repres sion conditions (quinic acid and dextrose). Results showed increased expression of the qa-2 gene, which were approximately 70% of those seen in wild-type N. erassa, under inducing conditions. Furthermore, under catabolite repression conditions, the transformed strains showed over 100-fold 39 repression of the qa-2 gene, which were highly comparable to wild-type N. erassa. The latter result suggested that any sequence differences between the N. erassa and N. afrieana qa x-qa-2 intergenic region had no impact on catabolite repression of the qa genes (Asch et aI., 1991). XVI. Two Regulatory Circuits Regulate the Quinic Acid (qa) Gene Cluster of Neurospora erassa The expressIOn of the qa genes appear to be controlled by two levels of genetic regulation. The first regulatory circuit controlling transcription of the qa genes is mediated by the interactions of the qa-lSand qa-lF proteins in response to quinic acid. This is supported by the findings that uninduced wild-type and mutants (qa-l S- and qa-lF-) which were grown in the presence or absence of quinic acid both contained only small amounts of qa mRNAs. However, constitutive qa-lse mutants grown in the absence of quinic acid contained elevated levels of qa mRNAs (Giles et aI., 1985). These findings together, showed that qa gene expression is controlled at the transcriptional level by the qa-lSand qa-lF gene products, and is regulated by the presence or absence of the inducer, quinic acid (Patel et aI., 1981; Huiet, 1984). It has been shown that in the presence of the inducer, quinic acid, transcription of all the qa genes is increased 50- to 1,000-fold by the action of the activator. This is also seen in the galactose (GAL) system in yeast. Here the inducer, galactose, induces transcription of the 40 repression of the qa-2 gene, which were highly comparable to wild-type N. erassa. The latter result suggested that any sequence differences between the N. erassa and N. afrieana qa x-qa-2 intergenic region had no impact on catabolite repression of the qa genes (Asch et aI., 1991). XVI. Two Regulatory Circuits Regulate the Quinic Acid (qa) Gene Cluster of Neurospora erassa The expressIOn of the qa genes appear to be controlled by two levels of genetic regulation. The first regulatory circuit controlling transcription of the qa genes is mediated by the interactions of the qa-lSand qa-lF proteins in response to quinic acid. This is supported by the findings that uninduced wild-type and mutants (qa-l S- and qa-lF-) which were grown in the presence or absence of quinic acid both contained only small amounts of qa mRNAs. However, constitutive qa-lse mutants grown in the absence of quinic acid contained elevated levels of qa mRNAs (Giles et aI., 1985). These findings together, showed that qa gene expression is controlled at the transcriptional level by the qa-lSand qa-lF gene products, and is regulated by the presence or absence of the inducer, quinic acid (Patel et aI., 1981; Huiet, 1984). It has been shown that in the presence of the inducer, quinic acid, transcription of all the qa genes is increased 50- to 1,000-fold by the action of the activator. This is also seen in the galactose (GAL) system in yeast. Here the inducer, galactose, induces transcription of the 40 GAL4 activator gene, which in turn induces transcription of the GAL genes (GAL1,-7, and-10). In the absence of the inducer all of the qa genes are transcribed at low basal levels. This is attributed to the interaction of the qa-1 S repressor protein with the q a-1 F activator protein, which in turn inhibits activator function (Geever et aI., 1989; Giles et aI., 1991). Again, this is also seen in the GAL system. Here in the absence of the inducer, the GAL80 repressor product interacts with the GAL4 activator product to inhibit activator function. The second regulatory circuit, which IS superimposed on the first, acts to repress qa gene transcription in the presence of a preferred carbon source, such as glucose or dextrose. It has been shown that wild-type N. crassa when grown in the presence of quinic acid and a preferred carbon source, such as glucose, have a reduced level of qa gene transcription compared to wild-type N. crassa grown on quinic acid alone. The mechanism by which this apparent catabolite repression IS acting to repress qa transcription is unknown. However, evidence from the GAL system of S. cerevlszae (Flick and Johnston, 1990; Johnston et aI., 1994) provides three possible explanations to this repression. The first is that the qa -1 F activator protein may not be able to bind to the activator sites when in the presence of a preferred carbon source, which represses transcription of the qa genes. This is believed to be due to protein modification, proteolysis, or direct repression of the qa-1F gene expressIOn. The second is the possible 41 GAL4 activator gene, which in turn induces transcription of the GAL genes (GAL1,-7, and-10). In the absence of the inducer all of the qa genes are transcribed at low basal levels. This is attributed to the interaction of the qa-1 S repressor protein with the q a-1 F activator protein, which in turn inhibits activator function (Geever et aI., 1989; Giles et aI., 1991). Again, this is also seen in the GAL system. Here in the absence of the inducer, the GAL80 repressor product interacts with the GAL4 activator product to inhibit activator function. The second regulatory circuit, which IS superimposed on the first, acts to repress qa gene transcription in the presence of a preferred carbon source, such as glucose or dextrose. It has been shown that wild-type N. crassa when grown in the presence of quinic acid and a preferred carbon source, such as glucose, have a reduced level of qa gene transcription compared to wild-type N. crassa grown on quinic acid alone. The mechanism by which this apparent catabolite repression IS acting to repress qa transcription is unknown. However, evidence from the GAL system of S. cerevlszae (Flick and Johnston, 1990; Johnston et aI., 1994) provides three possible explanations to this repression. The first is that the qa -1 F activator protein may not be able to bind to the activator sites when in the presence of a preferred carbon source, which represses transcription of the qa genes. This is believed to be due to protein modification, proteolysis, or direct repression of the qa-1F gene expressIOn. The second is the possible 41 interactions of carbon repressors with sequences 5' to the vanous qa genes which act to block transcription while in the presence of a preferred carbon source. However, the presence of such sequences within the qa gene cluster have not yet been identified. The third possible mechanism might be the direct repression of the qa-1F gene. This repression would in turn repress the other qa genes by limiting the amount of the qa-1 F activator protein. In order to isolate the mechanism of carbon repressIOn, N. crassa strains carrying a complete deletion of the qa-1S gene were examined. These strains displayed increased levels of q a expression while in the absence of quinic acid (Case et aI., 1992). These mutants also showed slightly repressed qa-x, qa 2 and qa-4 gene expression and highly repressed qa-3, qa-y, and qa-1F gene expression while in the presence of glucose (Asch and Case, unpublished data). This suggests that each gene of the qa gene cluster may be regulated by different mechanisms, or that carbon catabolite repression acts on the qa-1F gene. Evidence for this type of repression was not seen when the qa-x-qa-2 intergenic region of N. africana was compared to its counterpart in N. crassa (Asch et aI., 1991). However, qa-2 was shown to not be dramatically affected by carbon repression directly. Therefore, since qa-1F seems to be directly affected by a preferred carbon source, the qa-1 S-qa-1 F intergenic region of N. africana will be examined and compared to its counterpart in N. crassa to see if it contains sequences which may play a role in carbon catabolite repression of the q a 42 interactions of carbon repressors with sequences 5' to the vanous qa genes which act to block transcription while in the presence of a preferred carbon source. However, the presence of such sequences within the qa gene cluster have not yet been identified. The third possible mechanism might be the direct repression of the qa-1F gene. This repression would in turn repress the other qa genes by limiting the amount of the qa-1 F activator protein. In order to isolate the mechanism of carbon repressIOn, N. crassa strains carrying a complete deletion of the qa-1S gene were examined. These strains displayed increased levels of q a expression while in the absence of quinic acid (Case et aI., 1992). These mutants also showed slightly repressed qa-x, qa 2 and qa-4 gene expression and highly repressed qa-3, qa-y, and qa-1F gene expression while in the presence of glucose (Asch and Case, unpublished data). This suggests that each gene of the qa gene cluster may be regulated by different mechanisms, or that carbon catabolite repression acts on the qa-1F gene. Evidence for this type of repression was not seen when the qa-x-qa-2 intergenic region of N. africana was compared to its counterpart in N. crassa (Asch et aI., 1991). However, qa-2 was shown to not be dramatically affected by carbon repression directly. Therefore, since qa-1F seems to be directly affected by a preferred carbon source, the qa-1 S-qa-1 F intergenic region of N. africana will be examined and compared to its counterpart in N. crassa to see if it contains sequences which may play a role in carbon catabolite repression of the q a 42 genes, III the presence of a preferred carbon source. 43 genes, III the presence of a preferred carbon source. 43 MATERIALS AND METHODS Materials I. Ethanol was purchased from Aaper Alcohol and Chemical Company, Shelbyville, KY; isopropanol was purchased from Baxter Healthcare Corporation, MCGraw Park, IL; restriction endonucleases [EcoRI (10 D/ul), BamHI (10 D/ul), KpnI (10 D/ul), Sad (10 D/ul), SmaI (10 D/ul)], T4 DNA ligase (1 D/ul), DIG Taq DNA Sequencing Kit for Standard and Cycle Sequencing, DIG DNA Labeling and Detection Kit, Blocking reagent, disodium 3-(4-methoxyspiro {I,2-dioxetane 3,2'(5'chloro)tricyclo[3.3.1.1.3,7]decan}-4-yl) phenyl phosphate [CSPD], anti-digoxigenin-AP fab fragments, acrylamide, bis acrylamide, and positively charged nylon membranes were purchased from Boehringer Mannheim, Indpls, IN; bacto trypton, bacto-agar and yeast extract were purchased from Difco Laboratories, Detroit MI; Polaroid film, developer and replenisher, fixer and replenisher and maleic acid were purchased from Eastman Kodak Co., Rochester, NY; agarose was purchased from EM Science, Cherry Hill, NJ; ethidium bromide [EtOH], 85% phosphoric acid [H3P04], sodium citrate and acetic acid [HOAc] were purchased from Fisher Scientific, Fair Lawn, NJ; PERFECTprep Plasmid DNA Kit, and PCR SELECT-II Spin Columns were purchased from 5 Prime---> 3 Prime, Inc., Boulder, Co; restriction endonucleases [PstI (15 D/ul), HindIII (15 D/ul), XhoI (15 D/ul)], isopropyl-B-D-thiogalactoside [IPTG], 44 MATERIALS AND METHODS Materials I. Ethanol was purchased from Aaper Alcohol and Chemical Company, Shelbyville, KY; isopropanol was purchased from Baxter Healthcare Corporation, MCGraw Park, IL; restriction endonucleases [EcoRI (10 D/ul), BamHI (10 D/ul), KpnI (10 D/ul), Sad (10 D/ul), SmaI (10 D/ul)], T4 DNA ligase (1 D/ul), DIG Taq DNA Sequencing Kit for Standard and Cycle Sequencing, DIG DNA Labeling and Detection Kit, Blocking reagent, disodium 3-(4-methoxyspiro {I,2-dioxetane 3,2'(5'chloro)tricyclo[3.3.1.1.3,7]decan}-4-yl) phenyl phosphate [CSPD], anti-digoxigenin-AP fab fragments, acrylamide, bis acrylamide, and positively charged nylon membranes were purchased from Boehringer Mannheim, Indpls, IN; bacto trypton, bacto-agar and yeast extract were purchased from Difco Laboratories, Detroit MI; Polaroid film, developer and replenisher, fixer and replenisher and maleic acid were purchased from Eastman Kodak Co., Rochester, NY; agarose was purchased from EM Science, Cherry Hill, NJ; ethidium bromide [EtOH], 85% phosphoric acid [H3P04], sodium citrate and acetic acid [HOAc] were purchased from Fisher Scientific, Fair Lawn, NJ; PERFECTprep Plasmid DNA Kit, and PCR SELECT-II Spin Columns were purchased from 5 Prime---> 3 Prime, Inc., Boulder, Co; restriction endonucleases [PstI (15 D/ul), HindIII (15 D/ul), XhoI (15 D/ul)], isopropyl-B-D-thiogalactoside [IPTG], 44 5'bromo-4-chloro-3-indoyl-B-D-galactopyranoside [X-gal], ethylenediaminetetraacetic acid-disodium salt [EDTA], sodium dodecyl sulfate [SDS] and ammonium persulfate [AMPS] were purchased from International Biotechnologies, Inc., New Haven, CT; chloroform and phenol were purchased from J.T. Baker Chemical Co., Phillipsburg, NJ; calcium chloride [CaCI2] and magnesium chloride [MgCI2] were purchased from Mallinckrodt, Inc., Paris, KY; QIAGEN 100 Tips were purchased from QIAGEN Inc., Chatsworth, CA; ELUTIP-D columns were purchased from Schleicher & Schuell, Keene, NH; ampicillin, sodium chloride [NaCl], potassium acetate [KOAc], 3-N morpholino-propanesulfonic acid [MOPS], Trizma base, RNase A, octyl phenoxy polyethoxyethanol [Triton X-IOO], urea, polyoxythlene-sorbitan monolaurate [Tween 20], N,N,N',N' Tetramethylethylenediamine [TEMED], sigmacote, lithium chloride [LiCI], and N-Iauroylsarcosine were purchased from Sigma Chemical Co., St. Louis, MO;sodium hydroxide [NaOH] was purchased from VMR, Media, PA. Methods II. Strains and Media Recombinant plasmids were transformed into Escherichia coli strain JMIOI. E. coli JMIOI was cultured in Luria Broth [LB] (l% bacto-tryptone; 0.5% yeast extract; 1% NaCI). Transformants were selected on Luria Agar [LA100] (1 % bacto- 45 5'bromo-4-chloro-3-indoyl-B-D-galactopyranoside [X-gal], ethylenediaminetetraacetic acid-disodium salt [EDTA], sodium dodecyl sulfate [SDS] and ammonium persulfate [AMPS] were purchased from International Biotechnologies, Inc., New Haven, CT; chloroform and phenol were purchased from J.T. Baker Chemical Co., Phillipsburg, NJ; calcium chloride [CaCI2] and magnesium chloride [MgCI2] were purchased from Mallinckrodt, Inc., Paris, KY; QIAGEN 100 Tips were purchased from QIAGEN Inc., Chatsworth, CA; ELUTIP-D columns were purchased from Schleicher & Schuell, Keene, NH; ampicillin, sodium chloride [NaCl], potassium acetate [KOAc], 3-N morpholino-propanesulfonic acid [MOPS], Trizma base, RNase A, octyl phenoxy polyethoxyethanol [Triton X-IOO], urea, polyoxythlene-sorbitan monolaurate [Tween 20], N,N,N',N' Tetramethylethylenediamine [TEMED], sigmacote, lithium chloride [LiCI], and N-Iauroylsarcosine were purchased from Sigma Chemical Co., St. Louis, MO;sodium hydroxide [NaOH] was purchased from VMR, Media, PA. Methods II. Strains and Media Recombinant plasmids were transformed into Escherichia coli strain JMIOI. E. coli JMIOI was cultured in Luria Broth [LB] (l% bacto-tryptone; 0.5% yeast extract; 1% NaCI). Transformants were selected on Luria Agar [LA100] (1 % bacto- 45 tryptone; 0.5% yeast extract; 1% NaCI; 1.5% bacto-agar) usmg ampicillin (100 ug/mL), 100 uL IPTG (200 mM) and 50 uL X gal (2%). Transformants were picked to LB 100 [LB; ampicillin (100 ug/mL)]. Single stranded phages were infected into E. coli strain JM101. E. coli JM101 was cultured in LB. Transformants were selected on YT plates (0.8% bacto-tryptone; 0.5% yeast extract; 0.5% NaCI; 1.5% bacto-agar) using 100 uL IPTG (200 mM) and 50 uL X-gal (2%). Transformants were picked to 2xYT (1.6% bacto-tryptone; 1% yeast extract; 0.5% NaCI) containing 200 uL of E. coli JM101 cells. III. pBluescript II KS (+/-) Phagemid This 2,961 basepair (bp) phagemid, which was derived from pUC19, was purchased from Stratagene, La Jolla, CA. Located within this phagemid is a portion of the lacZ gene, which confers blue/white color selection of recombinants m the presence of IPTG and X-gal. It also contained a multiple cloning site (MCS) which was oriented in such a way that cloning into this region resulted in the disruption of lacZ translation producing white recombinants. Finally it contained an ampicillin resistance gene which was utilized in antibiotic selection of recombinants. 46 tryptone; 0.5% yeast extract; 1% NaCI; 1.5% bacto-agar) usmg ampicillin (100 ug/mL), 100 uL IPTG (200 mM) and 50 uL X gal (2%). Transformants were picked to LB 100 [LB; ampicillin (100 ug/mL)]. Single stranded phages were infected into E. coli strain JM101. E. coli JM101 was cultured in LB. Transformants were selected on YT plates (0.8% bacto-tryptone; 0.5% yeast extract; 0.5% NaCI; 1.5% bacto-agar) using 100 uL IPTG (200 mM) and 50 uL X-gal (2%). Transformants were picked to 2xYT (1.6% bacto-tryptone; 1% yeast extract; 0.5% NaCI) containing 200 uL of E. coli JM101 cells. III. pBluescript II KS (+/-) Phagemid This 2,961 basepair (bp) phagemid, which was derived from pUC19, was purchased from Stratagene, La Jolla, CA. Located within this phagemid is a portion of the lacZ gene, which confers blue/white color selection of recombinants m the presence of IPTG and X-gal. It also contained a multiple cloning site (MCS) which was oriented in such a way that cloning into this region resulted in the disruption of lacZ translation producing white recombinants. Finally it contained an ampicillin resistance gene which was utilized in antibiotic selection of recombinants. 46 IV. Single-Stranded M13mp18 This phage IS described by Messing and Vieira (1982). V. Restriction Digest of the Vector Approximately five mIcrograms of either vector was placed in the presence of sterile water, enzyme of choice, and that enzymes lOX reaction buffer and incubated at 37?C overnight. Next a small sample was run on a 1% agarose gel. If the DNA sample was properly digested, then 200 uL of neutralized phenol was added to the eppendorf tube. The sample was then centrifuged at 12,000-16,000 xg for 10 minutes. The top layer was then removed and placed III a clean eppendorf tube. Next, 200 uL of chloroform was added and then this was also centrifuged at 12,000-16,000 xg for 5 minutes. The top layer was removed again and placed into a clean eppendorf tube and 200 uL of isopropanol was added. This was then centrifuged at 12,000-16,000 xg for 15 minutes. After this centrifugation the liquid was decanted, the DNA pellet was washed two times in 80% ethanol and then allowed to dry for 10-20 minutes. The pellet was then resuspended in 20 uL of IX TE buffer (0.001 M Trizma base; 0.001 M EDTA, pH 8.0) and stored at -20?C for later use. 47 IV. Single-Stranded M13mp18 This phage IS described by Messing and Vieira (1982). V. Restriction Digest of the Vector Approximately five mIcrograms of either vector was placed in the presence of sterile water, enzyme of choice, and that enzymes lOX reaction buffer and incubated at 37?C overnight. Next a small sample was run on a 1% agarose gel. If the DNA sample was properly digested, then 200 uL of neutralized phenol was added to the eppendorf tube. The sample was then centrifuged at 12,000-16,000 xg for 10 minutes. The top layer was then removed and placed III a clean eppendorf tube. Next, 200 uL of chloroform was added and then this was also centrifuged at 12,000-16,000 xg for 5 minutes. The top layer was removed again and placed into a clean eppendorf tube and 200 uL of isopropanol was added. This was then centrifuged at 12,000-16,000 xg for 15 minutes. After this centrifugation the liquid was decanted, the DNA pellet was washed two times in 80% ethanol and then allowed to dry for 10-20 minutes. The pellet was then resuspended in 20 uL of IX TE buffer (0.001 M Trizma base; 0.001 M EDTA, pH 8.0) and stored at -20?C for later use. 47 VI. Agarose Gel Electrophoresis The condition of the DNA used in all of the experiments was analyzed by electrophoresis. Here, DNA fragments were resolved by a 1% agarose gel electrophoresis in IX tris phosphate (TPE) buffer [0.08 M Trizma base; 0.005 M EDTA; 85% H3P04 (1.679 mg/mL)]. The gel was stained with EtBr (50 mg/mL) and the DNA was visualized on a transilluminator VII. Preparation of Fragments Fragments were prepared by digesting approximately 10 ug of the plasmid pRI (supplied by Kim Rutledge) with the restriction enzyme(s) of choice. The fragments were then resolved by a 1% agarose gel electrophoresis. The fragment of interest was then cut from the gel and placed into a dialysis bag filled with 0.5X tris-acetate [TAE] buffer (0.04 M Trizma base; 0.2 M NaOAc; 0.002 M Na2EDTA, pH 7.9). The bag containing the fragment was then placed into an electrophoresis chamber and electrophoresised for 45 minutes. After this time period the polarity was switched and the sample was electrophoresied for 1 minute to release any DNA bound to the inside of the bag. The liquid, containing the DNA, was then drawn out of the bag and placed into an eppendorf tube. Next, an Elutip column was primed by passing 3 mL of high salt buffer (l M NaCL; 0.02 M Trizma base; 0.001 M EDTA; pH 7.4) and then 3 mL of low salt buffer (0.02 M NaCL; 0.02 M 48 VI. Agarose Gel Electrophoresis The condition of the DNA used in all of the experiments was analyzed by electrophoresis. Here, DNA fragments were resolved by a 1% agarose gel electrophoresis in IX tris phosphate (TPE) buffer [0.08 M Trizma base; 0.005 M EDTA; 85% H3P04 (1.679 mg/mL)]. The gel was stained with EtBr (50 mg/mL) and the DNA was visualized on a transilluminator VII. Preparation of Fragments Fragments were prepared by digesting approximately 10 ug of the plasmid pRI (supplied by Kim Rutledge) with the restriction enzyme(s) of choice. The fragments were then resolved by a 1% agarose gel electrophoresis. The fragment of interest was then cut from the gel and placed into a dialysis bag filled with 0.5X tris-acetate [TAE] buffer (0.04 M Trizma base; 0.2 M NaOAc; 0.002 M Na2EDTA, pH 7.9). The bag containing the fragment was then placed into an electrophoresis chamber and electrophoresised for 45 minutes. After this time period the polarity was switched and the sample was electrophoresied for 1 minute to release any DNA bound to the inside of the bag. The liquid, containing the DNA, was then drawn out of the bag and placed into an eppendorf tube. Next, an Elutip column was primed by passing 3 mL of high salt buffer (l M NaCL; 0.02 M Trizma base; 0.001 M EDTA; pH 7.4) and then 3 mL of low salt buffer (0.02 M NaCL; 0.02 M 48 Trizma base; 0.001 M EDTA; pH 7.4) through the column. Next, the DNA collected from the dialysis bag was then passed over the primed column as described by the manufacturer. Finally, the DNA was eluted from the column with 400 uL of high salt buffer and collected (Figure 7). The DNA was then washed and extracted as above (see section V). VIII. Construction of Recombinant Plasmids and Phages Recombinant DNA was constructed by ligating an isolated fragment from the plasmid pR1 with the vector (pBLUESCRIPT; M13) which was cut with the same enzyme(s). This was done by placing 5 uL of the fragment, 5 uL of the vector, 3 uL of lOX ligase buffer, 16 uL of sterile water, and 1 uL of T4 DNA ligase into a sterile eppendorf tube. This mixture was then incubated at 15?C overnight. IX. Transformation of E. coli JM101 with pBluescript DNA First 2 mL of LB was inoculated with JM101 and incubated at 37?C overnight. The following day, 50 mL of LB was inoculated with 0.5 mL of the overnight growth and incubated at 37?C for 3 hours. These cells were then placed on ice for 10 minutes and then centrifuged (10K; 4?C) for 10 minutes. The resulting pellet was resuspended in 15 mL of 0.1 49 Trizma base; 0.001 M EDTA; pH 7.4) through the column. Next, the DNA collected from the dialysis bag was then passed over the primed column as described by the manufacturer. Finally, the DNA was eluted from the column with 400 uL of high salt buffer and collected (Figure 7). The DNA was then washed and extracted as above (see section V). VIII. Construction of Recombinant Plasmids and Phages Recombinant DNA was constructed by ligating an isolated fragment from the plasmid pR1 with the vector (pBLUESCRIPT; M13) which was cut with the same enzyme(s). This was done by placing 5 uL of the fragment, 5 uL of the vector, 3 uL of lOX ligase buffer, 16 uL of sterile water, and 1 uL of T4 DNA ligase into a sterile eppendorf tube. This mixture was then incubated at 15?C overnight. IX. Transformation of E. coli JM101 with pBluescript DNA First 2 mL of LB was inoculated with JM101 and incubated at 37?C overnight. The following day, 50 mL of LB was inoculated with 0.5 mL of the overnight growth and incubated at 37?C for 3 hours. These cells were then placed on ice for 10 minutes and then centrifuged (10K; 4?C) for 10 minutes. The resulting pellet was resuspended in 15 mL of 0.1 49 I j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j J I I I I I I I I I I I I I I I I I I I I I I I I I I I I I + I -- -- - _I- - - - - - - -;.-1+- .....,~ 1 1- Electrophoresis sample I + I Cut out band of interest and place in dialysis bag Electrophoresis extracted band I I collect buffer from bag andpass through a collection collumn c:::::J c::=J- --- ? -- Q -... gel check sample + I -- -- - _I- - - - - - - -;.-1+- .....,~ 1 1- Electrophoresis sample I + I Cut out band of interest and place in dialysis bag Electrophoresis extracted band I I collect buffer from bag andpass through a collection collumn c:::::J c::=J- --- ? -- Q -... gel check sample M CaCl2 and placed on ice for 30 minutes. Next, the cells were centrifuged (10K; 4?C) for 10 minutes and the pellet was resuspended in 0.5 mL of CaCI2. Then, 100 uL of these competent cells were then dispensed into two eppendorf tubes, one being the control and the other the experimental. To the experimental tube 10 uL of the ligation mix was added and no DNA was added to the control tube. These tubes were then incubated on ice for 30 minutes and transferred to a 37?C heat block for 5 minutes. Next, 1 mL of LB was added to each tube and they were incubated at 37?C for 60 minutes. After this incubation 100 uL of the transformation mixes were spread onto selective media (LAI00; ampicillin; IPTG; X-gal). These plates were then incubated at 37?C overnight (Figure 8). X Transformation of E. coli JMI0l with M13 DNA First, 2 mL of LB was inoculated with JMI0l and incubated at 37?C overnight. The following day, 50 mL of LB was inoculated with 0.5 mL of the overnight growth and incubated at 37?C for 3 hours. Next,S mL of these cells were then collected and placed back at 37?C while the rest of the cells were placed on ice for 10 minutes. These cells were then centrifuged (10K; 4?C) for 10 minutes. The resulting pellet was then resuspended in 15 mL of CaCl2 and placed on ice for 30 minutes. After the incubation, the cells were centrifuged (10K; 52 M CaCl2 and placed on ice for 30 minutes. Next, the cells were centrifuged (10K; 4?C) for 10 minutes and the pellet was resuspended in 0.5 mL of CaCI2. Then, 100 uL of these competent cells were then dispensed into two eppendorf tubes, one being the control and the other the experimental. To the experimental tube 10 uL of the ligation mix was added and no DNA was added to the control tube. These tubes were then incubated on ice for 30 minutes and transferred to a 37?C heat block for 5 minutes. Next, 1 mL of LB was added to each tube and they were incubated at 37?C for 60 minutes. After this incubation 100 uL of the transformation mixes were spread onto selective media (LAI00; ampicillin; IPTG; X-gal). These plates were then incubated at 37?C overnight (Figure 8). X Transformation of E. coli JMI0l with M13 DNA First, 2 mL of LB was inoculated with JMI0l and incubated at 37?C overnight. The following day, 50 mL of LB was inoculated with 0.5 mL of the overnight growth and incubated at 37?C for 3 hours. Next,S mL of these cells were then collected and placed back at 37?C while the rest of the cells were placed on ice for 10 minutes. These cells were then centrifuged (10K; 4?C) for 10 minutes. The resulting pellet was then resuspended in 15 mL of CaCl2 and placed on ice for 30 minutes. After the incubation, the cells were centrifuged (10K; 52 I j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j J I I I I I I I I I I I I I I I I I I I I I I I I I I I I I ICE 200 uL? Overnight JM101 50 mL LB grow at 37 (@ 2hrs) 5-10 mins ~ ~ \ 7?ICE %%~~~ ~~~w spin 10 mins Resuspend in CaCL2 30 mins 100 uL ~ Add 1 uL of DNA Control (no DNA) spin 10 mins Resuspend in CaCL2 30 mins 5 mins Add 1 mL of LB to each tube and incubate at 37 for 1 hr Plate 200 uL of each and grow at 37 0 overnight ICE 200 uL? Overnight JM101 50 mL LB grow at 37 (@ 2hrs) 5-10 mins ~ ~ \ /.ICE %%~~~ ~~~w spin 10 mins Resuspend in CaCL2 30 mins 100 uL ~ Add 1 uL of DNA Control (no DNA) spin 10 mins Resuspend in CaCL2 30 mins 5 mins Add 1 mL of LB to each tube and incubate at 37 for 1 hr Plate 200 uL of each and grow at 37 0 overnight 4?C) for 10 minutes and the pellet was resuspended in 0.5 mL of CaCI2. Then, 100 uL of these competent cells were then dispensed into two sterile eppendorf tubes, one being the control and the other the experimental. To the experimental tube 10 uL of the ligation mix was added and no DNA was added to the control tube. These tubes were then incubated on ice for 30 minutes and then transferred to a 37?C heat block for 5 minutes. While the samples were on ice, YT soft agar was melted and into two falcon tubes 100 ul IPTG and 50 uL of X gal was dispensed, and labeled control and experimental. After the heat shock, 3 mL of YT soft agar, 35 uL of the ligation mix, and 200 uL of JM101 lawn cells was added to the experimental tube. While to the control tube only 3 mL of YT soft agar, and 200 uL of lawn cells were added. Each of these mixtures were then spread out on YT plates and incubated at 37?C overnight. XI. Direct Electrophoresis of M13 DNA White transformants were picked to 2 mL of 2XYT and incubated at 37?C overnight. While, one blue plaque was also picked to 2 mL of 2XYT broth and incubated at 37?C overnight. Next, 1.5 mL of the overnight cultures were placed into sterile eppendorf tubes and placed into a microcentrifuge (12,000 16,000 xg) for 5 minutes. The supernatants were then drawn off and placed into sterile eppendorf tubes while the pellets were discarded. Next, 50 uL of each supernatant were then placed into sterile eppendorf tubes along with 5 uL of 2% SDS 55 4?C) for 10 minutes and the pellet was resuspended in 0.5 mL of CaCI2. Then, 100 uL of these competent cells were then dispensed into two sterile eppendorf tubes, one being the control and the other the experimental. To the experimental tube 10 uL of the ligation mix was added and no DNA was added to the control tube. These tubes were then incubated on ice for 30 minutes and then transferred to a 37?C heat block for 5 minutes. While the samples were on ice, YT soft agar was melted and into two falcon tubes 100 ul IPTG and 50 uL of X gal was dispensed, and labeled control and experimental. After the heat shock, 3 mL of YT soft agar, 35 uL of the ligation mix, and 200 uL of JM101 lawn cells was added to the experimental tube. While to the control tube only 3 mL of YT soft agar, and 200 uL of lawn cells were added. Each of these mixtures were then spread out on YT plates and incubated at 37?C overnight. XI. Direct Electrophoresis of M13 DNA White transformants were picked to 2 mL of 2XYT and incubated at 37?C overnight. While, one blue plaque was also picked to 2 mL of 2XYT broth and incubated at 37?C overnight. Next, 1.5 mL of the overnight cultures were placed into sterile eppendorf tubes and placed into a microcentrifuge (12,000 16,000 xg) for 5 minutes. The supernatants were then drawn off and placed into sterile eppendorf tubes while the pellets were discarded. Next, 50 uL of each supernatant were then placed into sterile eppendorf tubes along with 5 uL of 2% SDS 55 and 5 uL of tracking dye. Each sample was then loaded in a 1% agarose gel and electrophoresied to view for shifts in the samples. XII. Isolation of Single-Stranded M13 Phages E. coli JMI0l was inoculated in 2 mL of 2XYT and grown at 37?C for 3 hours. One milliliter of this culture was then used to inoculate 50 mL of 2XYT along with 200 uL of the supernatant from section XI, that contained the shift, and incubated at 37?C overnight. This culture was then centrifuged (10K; 4?C) for 10 minutes and the supernatant was collected. The supernatant was then checked that it contained the phage (as in section XL). If the phage was present 2 mL of LB was inoculated with E. coli JMI0l and grown at 37?C overnight. The next day 200 uL of the overnight and 50 uL of the phage suspenSIOn was used to inoculate 2 mL of YT and grown overnight at 37?C. Next, 1.5 mL of this culture was transferred into a sterile eppendorf tube and placed into a micro-centrifuge (12,000 16,000 xg) for 5 minutes. Next, 1 mL of the supernatant was transferred into a sterile eppendorf tube along with 200 uL of 27% PEG 8000 and 200 uL of 3.3M NaCI and mixed. The phages were allowed to precipitate for a minimallS minutes at room temperature [RT]. This mixture was then placed into a microcentrifuge (12,000-16,000 xg) for 5 minutes. The supernatant was decanted and the sides of the eppendorf tube 56 and 5 uL of tracking dye. Each sample was then loaded in a 1% agarose gel and electrophoresied to view for shifts in the samples. XII. Isolation of Single-Stranded M13 Phages E. coli JMI0l was inoculated in 2 mL of 2XYT and grown at 37?C for 3 hours. One milliliter of this culture was then used to inoculate 50 mL of 2XYT along with 200 uL of the supernatant from section XI, that contained the shift, and incubated at 37?C overnight. This culture was then centrifuged (10K; 4?C) for 10 minutes and the supernatant was collected. The supernatant was then checked that it contained the phage (as in section XL). If the phage was present 2 mL of LB was inoculated with E. coli JMI0l and grown at 37?C overnight. The next day 200 uL of the overnight and 50 uL of the phage suspenSIOn was used to inoculate 2 mL of YT and grown overnight at 37?C. Next, 1.5 mL of this culture was transferred into a sterile eppendorf tube and placed into a micro-centrifuge (12,000 16,000 xg) for 5 minutes. Next, 1 mL of the supernatant was transferred into a sterile eppendorf tube along with 200 uL of 27% PEG 8000 and 200 uL of 3.3M NaCI and mixed. The phages were allowed to precipitate for a minimallS minutes at room temperature [RT]. This mixture was then placed into a microcentrifuge (12,000-16,000 xg) for 5 minutes. The supernatant was decanted and the sides of the eppendorf tube 56 were wiped down with a Kimwipe to remove residual liquid. The pellet was then resuspended in 90 uL TE, 10 uL lOX buffer (0.2% Sarkosyl; 0.1 M Trizma base, pH 7.8; 0.01 M EDTA), and 1 uL 5 mg/mL Proteinase K (50 ug/mL Proteinase K; 500 uL glycerol; 500 uL IX TE). This was incubated at 55?C for 20 minutes, allowed to cool to RT., and 8 uL of 5M NaCI was added. This sample was the extracted one time with phenol and two times with chloroform (as in V.) to remove the PEG. The phages were then precipitated with 2 volumes of isopropanol, washed one time with 80 % EtOH, and allowed to dry. The pellet was then resuspended in 20 uL of IX TE. Next, 5 uL of this sample was gel checked in a 1% agarose gel and the remainder of the sample was stored at 4?C until needed for sequencIng XIII. Isolation of Recombinant Plasmid DNA (Alkaline Plasmid Screen) White transformants were picked to 2 mL of LA100 broth and incubated at 37?C overnight. Then, 1.5 mL of the overnight culture was placed into a sterile eppendorf tube and placed in a microcentrifuge (12,000-16,000 xg) for 15 seconds. The supernatant was decanted and the pellet was resuspended in 200 uL of G buffer (0,05M dextose; 0.025M Trizma base, pH 8.0; O.OIM EDTA, pH 8.0). Next, 400 uL of Denaturing solution (0.2N NaOH; 1% SDS) was added and the tube was inverted several times to mix the solution. The mixture was then placed 57 were wiped down with a Kimwipe to remove residual liquid. The pellet was then resuspended in 90 uL TE, 10 uL lOX buffer (0.2% Sarkosyl; 0.1 M Trizma base, pH 7.8; 0.01 M EDTA), and 1 uL 5 mg/mL Proteinase K (50 ug/mL Proteinase K; 500 uL glycerol; 500 uL IX TE). This was incubated at 55?C for 20 minutes, allowed to cool to RT., and 8 uL of 5M NaCI was added. This sample was the extracted one time with phenol and two times with chloroform (as in V.) to remove the PEG. The phages were then precipitated with 2 volumes of isopropanol, washed one time with 80 % EtOH, and allowed to dry. The pellet was then resuspended in 20 uL of IX TE. Next, 5 uL of this sample was gel checked in a 1% agarose gel and the remainder of the sample was stored at 4?C until needed for sequencIng XIII. Isolation of Recombinant Plasmid DNA (Alkaline Plasmid Screen) White transformants were picked to 2 mL of LA100 broth and incubated at 37?C overnight. Then, 1.5 mL of the overnight culture was placed into a sterile eppendorf tube and placed in a microcentrifuge (12,000-16,000 xg) for 15 seconds. The supernatant was decanted and the pellet was resuspended in 200 uL of G buffer (0,05M dextose; 0.025M Trizma base, pH 8.0; O.OIM EDTA, pH 8.0). Next, 400 uL of Denaturing solution (0.2N NaOH; 1% SDS) was added and the tube was inverted several times to mix the solution. The mixture was then placed 57 on Ice for 5 minutes. Next, 300 uL of prechilled Neutralizing solution (3M KOAc; 2M HOAc) was added and the tube was again inverted several times to mix the solution. The mixture was then placed on ice for 15 minutes and then centrifuged (12,000-16,000 xg) for 5 minutes. The supernatant was then transferred to a sterile eppendorf tube, 540 uL of isopropanol was added, and the mixture was mixed well. The tube was then placed into a micro-centrifuge (12,000-16,000 xg) for 5 minutes. The pellet was then washed two times in 80% EtOH and allowed to dry. The pellet was then resuspended in 50 uL of IX TE. Next, 20 ul of this sample was then gel checked in a 1% agarose gel to observe shifts, while the remainder was stored at 4?C (Figure 9). XIV. Large Scale Isolation of Plasmid DNA (QIAGEN Preparation) Transformants (from section IX.) were picked to 2 mL of LA100 broth and incubated at 37?C overnight. The following day, 50 mL of LAI00 broth was inoculated with 500 uL of the overnight culture and incubated at 37?C overnight. The cells were then transferred to a sterile centrifuge tube and placed into an ultracentrifuge (10K; 4?C) for 15 minutes. The pellet was resuspended in 7.5 mL of buffer PI (100 ug/mL RNase A; 0.05M Trizma base; O.OIM EDTA, pH 8.0). Next 7.5 mL of buffer P2 (0.2M NaOH; 1% SDS) was added and the tube was 58 on Ice for 5 minutes. Next, 300 uL of prechilled Neutralizing solution (3M KOAc; 2M HOAc) was added and the tube was again inverted several times to mix the solution. The mixture was then placed on ice for 15 minutes and then centrifuged (12,000-16,000 xg) for 5 minutes. The supernatant was then transferred to a sterile eppendorf tube, 540 uL of isopropanol was added, and the mixture was mixed well. The tube was then placed into a micro-centrifuge (12,000-16,000 xg) for 5 minutes. The pellet was then washed two times in 80% EtOH and allowed to dry. The pellet was then resuspended in 50 uL of IX TE. Next, 20 ul of this sample was then gel checked in a 1% agarose gel to observe shifts, while the remainder was stored at 4?C (Figure 9). XIV. Large Scale Isolation of Plasmid DNA (QIAGEN Preparation) Transformants (from section IX.) were picked to 2 mL of LA100 broth and incubated at 37?C overnight. The following day, 50 mL of LAI00 broth was inoculated with 500 uL of the overnight culture and incubated at 37?C overnight. The cells were then transferred to a sterile centrifuge tube and placed into an ultracentrifuge (10K; 4?C) for 15 minutes. The pellet was resuspended in 7.5 mL of buffer PI (100 ug/mL RNase A; 0.05M Trizma base; O.OIM EDTA, pH 8.0). Next 7.5 mL of buffer P2 (0.2M NaOH; 1% SDS) was added and the tube was 58 o? o o? o Transformation plate spin cells down .. Pick white colonies and grow onernight in LB + Amp resuspend in G-buffer and add denaturing solution .. .. 5 mins 15 mins .. add neutralizing solution transfer supernatent.. spin down cells add isopropanol and spin down resuspend in 1x TE and gel check o? o o? o Transformation plate spin cells down .. Pick white colonies and grow onernight in LB + Amp resuspend in G-buffer and add denaturing solution .. .. 5 mins 15 mins .. add neutralizing solution transfer supernatent.. spin down cells add isopropanol and spin down resuspend in 1x TE and gel check inverted gently and incubated on ice for 5 minutes. Finally, 7.5 mL of buffer P3 (3M KOAc, pH 5.5) was added and mixed gently. The sample was then placed on ice for 20 minutes and centrifuged (15K; 4?C) for 30 minutes. The supernatant was then transferred to a sterile centrifuge tube and recentrifuged (l5K; 4?C) for 30 minutes. During this centrifugation, a Qiagen tip was equilibrated with 4 mL of buffer QBT (0.75M NaCI; 0.05M MOPS; 15% EtOH, pH 7.0; 0.15% Triton X-100) and allowing it to empty by gravity flow. The supernatant, containing the recombinant plasmid, was then applied to the tip allowing the plasmid DNA to bind to the primed resin. Next, the plasmid DNA was washed 2 times using 10 mL of buffer QC (lM NaCI; 0.05M MOPS; 15% EtOH, pH 7.0) each time. The DNA was then eluted with 5 mL of buffer QF (1.25M NaCI; 0.05M Trizma base; 15% EtOH, pH 8.5) and precipitated with 0.7 volumes of isopropanol and centrifuged (l3K; 4?C) for 30 minutes. The pellet was then washed in ice cold 70% EtOH and allowed to dry. The pellet was then resuspended in 3 mL of Ix TE and stored at 4?C. XV. Isolation of Plasmid DNA (PERFECT prep Preparation) Here, isolation was done as described by the manufacturer (PERFECT prep Plasmid DNA Kit). The protocol was as follows. First, 2 mL of LB was inoculated with JM101 containing the plasmid of interest and incubated at 37? overnight. Next, 1.5 mL of this bacterial culture was 61 inverted gently and incubated on ice for 5 minutes. Finally, 7.5 mL of buffer P3 (3M KOAc, pH 5.5) was added and mixed gently. The sample was then placed on ice for 20 minutes and centrifuged (15K; 4?C) for 30 minutes. The supernatant was then transferred to a sterile centrifuge tube and recentrifuged (l5K; 4?C) for 30 minutes. During this centrifugation, a Qiagen tip was equilibrated with 4 mL of buffer QBT (0.75M NaCI; 0.05M MOPS; 15% EtOH, pH 7.0; 0.15% Triton X-100) and allowing it to empty by gravity flow. The supernatant, containing the recombinant plasmid, was then applied to the tip allowing the plasmid DNA to bind to the primed resin. Next, the plasmid DNA was washed 2 times using 10 mL of buffer QC (lM NaCI; 0.05M MOPS; 15% EtOH, pH 7.0) each time. The DNA was then eluted with 5 mL of buffer QF (1.25M NaCI; 0.05M Trizma base; 15% EtOH, pH 8.5) and precipitated with 0.7 volumes of isopropanol and centrifuged (l3K; 4?C) for 30 minutes. The pellet was then washed in ice cold 70% EtOH and allowed to dry. The pellet was then resuspended in 3 mL of Ix TE and stored at 4?C. XV. Isolation of Plasmid DNA (PERFECT prep Preparation) Here, isolation was done as described by the manufacturer (PERFECT prep Plasmid DNA Kit). The protocol was as follows. First, 2 mL of LB was inoculated with JM101 containing the plasmid of interest and incubated at 37? overnight. Next, 1.5 mL of this bacterial culture was 61 transferred into a sterile eppendorf tube and centrifuged (12,000-16,000 xg) for 20 seconds. The supernatant was then removed and the pellet was resuspended in 100 uL of Solution I (0.05 M Tris-HCL, pH 7.6; 0.01 M EDTA, pH 8.0; 100 ug/mL RNase A). The cells were then lysed by adding 100 uL of Solution II (0.2 N NaOH; 1.0% SDS) and inverting the tube several times. The mixture was then neutralized by adding 100 uL of Solution III (1.32 M Potassium Acetate, pH 5.2) and inverting the tube vigorously. The mixture was then centrifuged (12,000-16,000 xg) for 30 seconds and the supernatant was then transferred to a PERFECT prep spin column in a collection tube. To this solution, 450 uL of PERFECT prep DNA Binding Matrix (PERFECT prep DNA Binding Matrix Suspension in Guanidine-HCL) was added and mixed by pipetting. The plasmid was then bound by centrifuging the PERFECT prep spin column/collection tube assembly (12,000 16,000 xg) for 30 seconds. Next, the filtrate was decanted, and 400 uL of diluted Purification Solution [Purification Solution Concentrate (Tris-CL; NaCL; EDTA) diluted 1: 1 in 95% ethanol] was added to the same PERFECT prep spin column. This was then centrifuged (12,000-16,000 xg) for 60 seconds. The PERFECT prep spin column was then transferred to a new collection tube and centrifuged (12,000-16,000 xg) again for 60 seconds to remove residual Purification Solution. The same PERFECT prep spin column was then transferred to another clean collection tube, and the purified plasmid DNA was eluted by adding 50 uL of TE and centrifuging (12,000-16,000 xg) for 62 transferred into a sterile eppendorf tube and centrifuged (12,000-16,000 xg) for 20 seconds. The supernatant was then removed and the pellet was resuspended in 100 uL of Solution I (0.05 M Tris-HCL, pH 7.6; 0.01 M EDTA, pH 8.0; 100 ug/mL RNase A). The cells were then lysed by adding 100 uL of Solution II (0.2 N NaOH; 1.0% SDS) and inverting the tube several times. The mixture was then neutralized by adding 100 uL of Solution III (1.32 M Potassium Acetate, pH 5.2) and inverting the tube vigorously. The mixture was then centrifuged (12,000-16,000 xg) for 30 seconds and the supernatant was then transferred to a PERFECT prep spin column in a collection tube. To this solution, 450 uL of PERFECT prep DNA Binding Matrix (PERFECT prep DNA Binding Matrix Suspension in Guanidine-HCL) was added and mixed by pipetting. The plasmid was then bound by centrifuging the PERFECT prep spin column/collection tube assembly (12,000 16,000 xg) for 30 seconds. Next, the filtrate was decanted, and 400 uL of diluted Purification Solution [Purification Solution Concentrate (Tris-CL; NaCL; EDTA) diluted 1: 1 in 95% ethanol] was added to the same PERFECT prep spin column. This was then centrifuged (12,000-16,000 xg) for 60 seconds. The PERFECT prep spin column was then transferred to a new collection tube and centrifuged (12,000-16,000 xg) again for 60 seconds to remove residual Purification Solution. The same PERFECT prep spin column was then transferred to another clean collection tube, and the purified plasmid DNA was eluted by adding 50 uL of TE and centrifuging (12,000-16,000 xg) for 62 60 seconds. Finally, 10 uL of this sample was gel checked to make sure the plasmid was isolated and, the remainder for the sample was stored at 4?. XVI. Restriction Digest of Recombinant DNA Recombinant DNA was digested with vanous enzymes (EcoR1; BamH1; KpnI; Sad; Pst!; HindIII; XhoI; and SmaI) as described by the manufacturer. Here, 10 uL of recombinant DNA was incubated at 37?C for 90 minutes in the presence of 16 uL of sterile water, 3 uL of the appropriate lOX reaction buffer, and 1 uL of enzyme. XVII. Sequencing Reactions for Single-Stranded DNA First, a pnmer annealing mixture was prepared as described by the manufacturer (DIG Taq DNA Sequencing Kit for Standard and Cycle Sequencing). This was done by adding 5-10 uL of single-stranded M13 (from section XII.), 2 uL of lOx reaction buffer, 2 uL of DIG-labeled M13/pUC19 forward sequencing primer, 10 uL of sterile water, and 1 uL of Taq DNA polymerase (3 U/uL) into a sterile eppendorf tube. Four 300 uL eppendorf tubes, labeled G, A, T, and C, were filled with 2 uL of the appropriate extension/termination mixture. Next, 4 uL of the primer annealing mixture was added to each PCR tube and overlaid with 10 uL of mineral oil. Each tube was then placed in the thermocycler. Here first, the mixture was 63 60 seconds. Finally, 10 uL of this sample was gel checked to make sure the plasmid was isolated and, the remainder for the sample was stored at 4?. XVI. Restriction Digest of Recombinant DNA Recombinant DNA was digested with vanous enzymes (EcoR1; BamH1; KpnI; Sad; Pst!; HindIII; XhoI; and SmaI) as described by the manufacturer. Here, 10 uL of recombinant DNA was incubated at 37?C for 90 minutes in the presence of 16 uL of sterile water, 3 uL of the appropriate lOX reaction buffer, and 1 uL of enzyme. XVII. Sequencing Reactions for Single-Stranded DNA First, a pnmer annealing mixture was prepared as described by the manufacturer (DIG Taq DNA Sequencing Kit for Standard and Cycle Sequencing). This was done by adding 5-10 uL of single-stranded M13 (from section XII.), 2 uL of lOx reaction buffer, 2 uL of DIG-labeled M13/pUC19 forward sequencing primer, 10 uL of sterile water, and 1 uL of Taq DNA polymerase (3 U/uL) into a sterile eppendorf tube. Four 300 uL eppendorf tubes, labeled G, A, T, and C, were filled with 2 uL of the appropriate extension/termination mixture. Next, 4 uL of the primer annealing mixture was added to each PCR tube and overlaid with 10 uL of mineral oil. Each tube was then placed in the thermocycler. Here first, the mixture was 63 denatured by heating at 95?C for 5 minutes. Following this step, the PCR reaction used for the forward primer was as follows, one cycle included; 95?C for 30 seconds, 60?C for 30 seconds, and 70?C for 60 seconds. The reaction cycled 29 times and after amplification the samples were stored at 4?C. To end the reaction 2 uL of formamide buffer was added to each tube. XVIII. Sequencing Reactions for Double-Stranded DNA First, a primer annealing mixture was prepared as described by the manufacturer (DIG Taq DNA Sequencing Kit for Standard and Cycle Sequencing). This was done by adding 5-10 uL of double stranded plasmid DNA (from section XV.), 2 uL of lOX reaction buffer, 2 uL of DIG-labeled M13/pUC19 forward or reverse sequencing primer, 10 uL of sterile water, and 1 uL of Taq DNA polymerase (3 U/uL) into a sterile eppendorf tube. Next, four 300 uL eppendorf tubes, labeled G, A, T, and C were filled with 2 uL of the appropriate extension/termination mixture. To these, 4 uL of the pnmer annealing mixture was added and, each was overlaid with 10 uL of mineral oil. Each tube was then placed into the thermocycler. Here first, the mixtures were denatured by heating at 95? for 5 minutes. Following this step, the PCR reaction varied depending on the primer used. For the forward primer, one cycle included; 95?C for 30 seconds, 60?C for 30 seconds, and 70?C for 60 seconds. The reverse primers cycle was, 95?C for 60 seconds, 56?C for 60 seconds, and 70?C for 60 64 denatured by heating at 95?C for 5 minutes. Following this step, the PCR reaction used for the forward primer was as follows, one cycle included; 95?C for 30 seconds, 60?C for 30 seconds, and 70?C for 60 seconds. The reaction cycled 29 times and after amplification the samples were stored at 4?C. To end the reaction 2 uL of formamide buffer was added to each tube. XVIII. Sequencing Reactions for Double-Stranded DNA First, a primer annealing mixture was prepared as described by the manufacturer (DIG Taq DNA Sequencing Kit for Standard and Cycle Sequencing). This was done by adding 5-10 uL of double stranded plasmid DNA (from section XV.), 2 uL of lOX reaction buffer, 2 uL of DIG-labeled M13/pUC19 forward or reverse sequencing primer, 10 uL of sterile water, and 1 uL of Taq DNA polymerase (3 U/uL) into a sterile eppendorf tube. Next, four 300 uL eppendorf tubes, labeled G, A, T, and C were filled with 2 uL of the appropriate extension/termination mixture. To these, 4 uL of the pnmer annealing mixture was added and, each was overlaid with 10 uL of mineral oil. Each tube was then placed into the thermocycler. Here first, the mixtures were denatured by heating at 95? for 5 minutes. Following this step, the PCR reaction varied depending on the primer used. For the forward primer, one cycle included; 95?C for 30 seconds, 60?C for 30 seconds, and 70?C for 60 seconds. The reverse primers cycle was, 95?C for 60 seconds, 56?C for 60 seconds, and 70?C for 60 64 seconds. Both reaction cycled 29 times and after amplification the samples were stored at 4?C. To end the reaction 2 uL of formamide buffer was added to each tube. XIX. Sequencing Gel Electrophoresis and Contact Blot An 8% polyacrylamide gel was cast In a mold between two sequencing plates, one of which was treated with siliconizing solution. The PCR products (from sections XVII. or XVIII.) were denatured at 95?C for 5 minutes and transferred to ice. Next, 3 uL of each of the four PCR reactions (G, A, T, and C) were loaded into the wells of the sequencing gel. After electrophoresis (2000V, 29 mAmps, 60 Watts~ short run/about 4 hours~ long run/about 8 hours) the plate treated with the siliconizing solution was removed. A positively charged nylon membrane, cut to match the size of the run, was then placed on the gel. The plate that was removed was then placed back on the gel along with approximately 20 kilograms of weight. After 20 minutes, the sandwich was disassembled. The DNA, now attached to the membrane was crossliked in a UV crosslinker under optimal conditions set by the manufacturer. The membrane then proceeded to the detection process. 65 seconds. Both reaction cycled 29 times and after amplification the samples were stored at 4?C. To end the reaction 2 uL of formamide buffer was added to each tube. XIX. Sequencing Gel Electrophoresis and Contact Blot An 8% polyacrylamide gel was cast In a mold between two sequencing plates, one of which was treated with siliconizing solution. The PCR products (from sections XVII. or XVIII.) were denatured at 95?C for 5 minutes and transferred to ice. Next, 3 uL of each of the four PCR reactions (G, A, T, and C) were loaded into the wells of the sequencing gel. After electrophoresis (2000V, 29 mAmps, 60 Watts~ short run/about 4 hours~ long run/about 8 hours) the plate treated with the siliconizing solution was removed. A positively charged nylon membrane, cut to match the size of the run, was then placed on the gel. The plate that was removed was then placed back on the gel along with approximately 20 kilograms of weight. After 20 minutes, the sandwich was disassembled. The DNA, now attached to the membrane was crossliked in a UV crosslinker under optimal conditions set by the manufacturer. The membrane then proceeded to the detection process. 65 xx. Detection All of the incubations here were performed at RT. First, the membrane was rinsed for 1 minute in 50 mL of washing buffer (Buffer 1: O.IM maleic acid; 0.15M NaCI, pH 7.5 plus 0.3% Tween 20). The washing buffer was removed and the membrane was incubated for 30 minutes in 50 mL of Buffer 2 (10% Blocking Stock Solution diluted 1:10 in Buffer 1). The Blocking Solution was decanted and 50 mL of antibody solution (anti-DIG-AP conjugate diluted 1:5000 in Buffer 2) was added and incubated for 30 minutes. Next, the antibody solution was removed and the membrane was washed 2 times, 15 minutes per wash, in 50 mL of Washing solution. The membrane was then equilibrated in 20 mL of Detection buffer, or Buffer 3 (O.IM Trizma base; O.IM NaCI; 0.05M MgCI2, pH 9.5). The membrane was then placed on plastic wrap and 2 mL of a CSPD solution (diluted 1:100 in Buffer 3) was placed on the membrane. The membrane was incubated for 5 minutes at RT. and then the CSPD solution was removed. The membrane was then sealed in a hybridization bag and incubated at 37?C for 15 minutes. The membrane was then exposed to X-ray film for about 3 hours. This film was then exposed to visualize the sequencing reactions. 66 xx. Detection All of the incubations here were performed at RT. First, the membrane was rinsed for 1 minute in 50 mL of washing buffer (Buffer 1: O.IM maleic acid; 0.15M NaCI, pH 7.5 plus 0.3% Tween 20). The washing buffer was removed and the membrane was incubated for 30 minutes in 50 mL of Buffer 2 (10% Blocking Stock Solution diluted 1:10 in Buffer 1). The Blocking Solution was decanted and 50 mL of antibody solution (anti-DIG-AP conjugate diluted 1:5000 in Buffer 2) was added and incubated for 30 minutes. Next, the antibody solution was removed and the membrane was washed 2 times, 15 minutes per wash, in 50 mL of Washing solution. The membrane was then equilibrated in 20 mL of Detection buffer, or Buffer 3 (O.IM Trizma base; O.IM NaCI; 0.05M MgCI2, pH 9.5). The membrane was then placed on plastic wrap and 2 mL of a CSPD solution (diluted 1:100 in Buffer 3) was placed on the membrane. The membrane was incubated for 5 minutes at RT. and then the CSPD solution was removed. The membrane was then sealed in a hybridization bag and incubated at 37?C for 15 minutes. The membrane was then exposed to X-ray film for about 3 hours. This film was then exposed to visualize the sequencing reactions. 66 XXI. Southern Transfer Recombinant DNA was digested with selected restriction enzymes, and run on a 1% agarose gel (1 7V for 8 hours) and stained in EtBr (50 mg/mL) to visualize the cut DNA. If the DNA was cut, the gel was incubated at RT. for 10 minutes III 0.5N HCI. The gel was then washed briefly in water, and incubated at RT. for 60 minutes in Denaturing solution (0.5N NaOH; 1.5M NaCI) with gentle shaking. The gel was then placed in Neutralization solution (0.5M Trizma base; 3M NaCI, pH 7.5) for 60 minutes at RT. with gentle shaking. The gel was then blotted overnight to remove the DNA by capillary transfer, and bind it to a positively charged nylon membrane using 20X SSC buffer (3M NaCI; 0.3M Sodium citrate, pH 7.0). See figure 10 for the setup of the transfer. XXII. DNA Fixation After the Southern Transfer the membrane was rinsed III 5X SSC (1:4 dilution of 20X SSC buffer) buffer at RT. for 60 seconds. The membrane was then placed on Whatman paper and baked for 60 minutes at 80aC. The membrane was place into a hybridization bag and now ready for hybridization of the probe (see section XXVI.) 67 XXI. Southern Transfer Recombinant DNA was digested with selected restriction enzymes, and run on a 1% agarose gel (1 7V for 8 hours) and stained in EtBr (50 mg/mL) to visualize the cut DNA. If the DNA was cut, the gel was incubated at RT. for 10 minutes III 0.5N HCI. The gel was then washed briefly in water, and incubated at RT. for 60 minutes in Denaturing solution (0.5N NaOH; 1.5M NaCI) with gentle shaking. The gel was then placed in Neutralization solution (0.5M Trizma base; 3M NaCI, pH 7.5) for 60 minutes at RT. with gentle shaking. The gel was then blotted overnight to remove the DNA by capillary transfer, and bind it to a positively charged nylon membrane using 20X SSC buffer (3M NaCI; 0.3M Sodium citrate, pH 7.0). See figure 10 for the setup of the transfer. XXII. DNA Fixation After the Southern Transfer the membrane was rinsed III 5X SSC (1:4 dilution of 20X SSC buffer) buffer at RT. for 60 seconds. The membrane was then placed on Whatman paper and baked for 60 minutes at 80aC. The membrane was place into a hybridization bag and now ready for hybridization of the probe (see section XXVI.) 67 a- (I) (I) t: C. C'lS ? C'lS>- a- Z C. J: .c Cc. E >-C'lS (I) C) J:a- E c. C) t: C'lSrn 0 t: a-1J - 1J C)(I) C'lS C'lS (I) C'lS 03: c. E C) -a- t: - 0 (I) a- 0 C'lS0 C'lS E- - C) a- C. J: (,)J: r:: J: C'lS (,) 0C) a- U C. a-.- (I) + (I) J:(I) C. Cl u3= C'lSa. (I)C) t:o c.rn C) t:-- o m a- (I) (I) t: C. C'lS ? C'lS>- a- Z C. J: .c Cc. E >-C'lS (I) C) J:a- E c. C) t: C'lSrn 0 t: a-1J - 1J C)(I) C'lS C'lS (I) C'lS 03: c. E C) -a- t: - 0 (I) a- 0 C'lS0 C'lS E- - C) a- C. J: (,)J: r:: J: C'lS (,) 0C) a- U C. a-.- (I) + (I) J:(I) C. Cl u3= C'lSa. (I)C) t:o c.rn C) t:-- o m XXIII. Probe Preparation First, 10 uL of DNA template (supplied by John Troutman) was placed into a PCR tube along with 2 uL of the primer Cl, 2 uL of the primer C2, # uL of lOx dNTP's, 3 uL of lOx reaction buffer, and 9 uL of sterile water. This mixture was heated at 95?C for 5 minutes, then 1 uL of Taq DNA polymerase was added, and the mixture was overlaid with 10 uL of mineral oil. The tube was then placed into the thermocycler and run under the program PCREX45 (92.5?C for 5 minutes, 45?C for 2 minutes, 72?C for 2 minutes, 92.5?C for 30 seconds; it then cycled 42 times at 45?C for 30 seconds, 72?C for 2 minutes, and 92.5?C for 30 seconds; the reaction was finalized by 45?C for 30 seconds, 72?C for 5 minutes, and 4?C for 1 minute; the mixture was then held at 4?C until needed). The following day, the product was collected and 5 uL was gel checked in a 1% agarose gel to see if the reaction occurred. If the reaction was successful the product was placed through a peR SELECT-II spin column according to the manufactures protocol in order to purify the PCR product. XXIV. Labeling of the Probe After the PCR sample was cleaned the DNA was labeled using the DIG DNA Labeling and Detection Kit as describe by the manufacturer. Here, 15 uL of the sample was place into a sterile eppendorf tube and denatured at 95?C for 5 minutes 70 XXIII. Probe Preparation First, 10 uL of DNA template (supplied by John Troutman) was placed into a PCR tube along with 2 uL of the primer Cl, 2 uL of the primer C2, # uL of lOx dNTP's, 3 uL of lOx reaction buffer, and 9 uL of sterile water. This mixture was heated at 95?C for 5 minutes, then 1 uL of Taq DNA polymerase was added, and the mixture was overlaid with 10 uL of mineral oil. The tube was then placed into the thermocycler and run under the program PCREX45 (92.5?C for 5 minutes, 45?C for 2 minutes, 72?C for 2 minutes, 92.5?C for 30 seconds; it then cycled 42 times at 45?C for 30 seconds, 72?C for 2 minutes, and 92.5?C for 30 seconds; the reaction was finalized by 45?C for 30 seconds, 72?C for 5 minutes, and 4?C for 1 minute; the mixture was then held at 4?C until needed). The following day, the product was collected and 5 uL was gel checked in a 1% agarose gel to see if the reaction occurred. If the reaction was successful the product was placed through a peR SELECT-II spin column according to the manufactures protocol in order to purify the PCR product. XXIV. Labeling of the Probe After the PCR sample was cleaned the DNA was labeled using the DIG DNA Labeling and Detection Kit as describe by the manufacturer. Here, 15 uL of the sample was place into a sterile eppendorf tube and denatured at 95?C for 5 minutes 70 and then transferred to ice. Next, 2 uL of lOX hexanucleotide mixture, 2 uL of lOX dNTP labeling mixture, and 1 uL of Klenow enzyme were added and the tube was incubated at 37?e overnight. The following day, 2 uL of 0.2M EDTA was added and the DIG-labeled nucleic acid was precipitated with 0.1 volume of 4M Liel and 2.5-3.0 volumes of cold 70% EtOH. The tube was incubated at -70oe for 30 minutes and then centrifuged (12,000-16,000 xg) for 15 minutes. The liquid was then decanted and the pellet was allowed to dry. The pellet was then resuspended in 50 uL of IX TE. XXV. Quantitation of the Probe Serial 10-fold dilution's of the DIG-labeled control DNA and the generated DIG-labeled experimental probe DNA were prepared as described by the manufacturer (DIG DNA Labeling and Detection Kit). Next, 1 uL of each dilution were spotted onto a positively charged nylon membrane and each corresponding dilution was marked near the appropriated spot. This membrane was then baked at 800 e for 30 minutes to fix the DNA for detection (all solutions used were the same as in section XX. unless noted). After baking, the membrane was washed for 1 minute III Washing buffer. The membrane was then incubated in Blocking solution for 5 minutes. The Blocking solution was then removed and the membrane was placed in antibody solution and incubated for 10 minutes. After the Blocking solution was 7 1 and then transferred to ice. Next, 2 uL of lOX hexanucleotide mixture, 2 uL of lOX dNTP labeling mixture, and 1 uL of Klenow enzyme were added and the tube was incubated at 37?e overnight. The following day, 2 uL of 0.2M EDTA was added and the DIG-labeled nucleic acid was precipitated with 0.1 volume of 4M Liel and 2.5-3.0 volumes of cold 70% EtOH. The tube was incubated at -70oe for 30 minutes and then centrifuged (12,000-16,000 xg) for 15 minutes. The liquid was then decanted and the pellet was allowed to dry. The pellet was then resuspended in 50 uL of IX TE. XXV. Quantitation of the Probe Serial 10-fold dilution's of the DIG-labeled control DNA and the generated DIG-labeled experimental probe DNA were prepared as described by the manufacturer (DIG DNA Labeling and Detection Kit). Next, 1 uL of each dilution were spotted onto a positively charged nylon membrane and each corresponding dilution was marked near the appropriated spot. This membrane was then baked at 800 e for 30 minutes to fix the DNA for detection (all solutions used were the same as in section XX. unless noted). After baking, the membrane was washed for 1 minute III Washing buffer. The membrane was then incubated in Blocking solution for 5 minutes. The Blocking solution was then removed and the membrane was placed in antibody solution and incubated for 10 minutes. After the Blocking solution was 7 1 removed, the membrane was then washed two times in Washing buffer, 5 minutes per wash. After then final wash the membrane was placed in Detection buffer and incubated for 2 minutes. The membrane was then removed from the Detection buffer and a Color Substrate Solution (45 uL of NBT; 35 uL of X phosphate solution in 10 mL of Detection buffer) was added. Color development was then allowed to occur in the dark for 45 minutes. This reaction was then terminated by washing the membrane in sterile water for 5 minutes. Finally, spot intensities of the control and experimental dilution's were compared to estimate the concentration of the experimental probe. XXVI. Prehybridization and Hybridization First, 50 mL of Prehybridization solution (5X SSC; 0.1 % N lauroylsarcosine; 0.02% SDS; 1% Blocking Reagent) was added to the bag containing the membrane (from section XXII). The membrane was then incubated at 65?C for 2-3 hours. After the incubation, the Prehybridization solution was collected and 20 mL of the Hybridization solution (contains the DIG-labeled probe from section XXIV.) was added to the bag. The probe was allowed to hybridize overnight at 65?C. The next day, the Hybridization solution was collected and the membrane was removed from the bag. The membrane was then wash two times, 5 minutes each wash, in 2X Wash solution (2X SSC; 0.1 % SDS) at RT.. The membrane was then washed two times, 15 72 removed, the membrane was then washed two times in Washing buffer, 5 minutes per wash. After then final wash the membrane was placed in Detection buffer and incubated for 2 minutes. The membrane was then removed from the Detection buffer and a Color Substrate Solution (45 uL of NBT; 35 uL of X phosphate solution in 10 mL of Detection buffer) was added. Color development was then allowed to occur in the dark for 45 minutes. This reaction was then terminated by washing the membrane in sterile water for 5 minutes. Finally, spot intensities of the control and experimental dilution's were compared to estimate the concentration of the experimental probe. XXVI. Prehybridization and Hybridization First, 50 mL of Prehybridization solution (5X SSC; 0.1 % N lauroylsarcosine; 0.02% SDS; 1% Blocking Reagent) was added to the bag containing the membrane (from section XXII). The membrane was then incubated at 65?C for 2-3 hours. After the incubation, the Prehybridization solution was collected and 20 mL of the Hybridization solution (contains the DIG-labeled probe from section XXIV.) was added to the bag. The probe was allowed to hybridize overnight at 65?C. The next day, the Hybridization solution was collected and the membrane was removed from the bag. The membrane was then wash two times, 5 minutes each wash, in 2X Wash solution (2X SSC; 0.1 % SDS) at RT.. The membrane was then washed two times, 15 72 minutes each wash, in 0.5X Wash solution (0.5X SSC; 0.1 % SDS) at 65?C. The membrane was now prepared for detection as in section XX. 73 minutes each wash, in 0.5X Wash solution (0.5X SSC; 0.1 % SDS) at 65?C. The membrane was now prepared for detection as in section XX. 73 RESULTS I. Construction of Plasmid pRI The qa-lS-qa-lF intergenic region of N. africana was chosen for study based on the role it may play in carbon catabolite repression of the qa genes. To start the characterization of the qa-lS-qa-lF intergenic region of N. africana the lambda clone NA3 was examined. This particular clone was known to contain most of the qa gene cluster (Asch, unpublished data) (Figure 11). It was found that when this clone was digested with the restriction endonuclease EcoRI, six fragments of various sizes were produced, four of which contained parts of the qa gene cluster (Figure 11) (Rutledge, unpublished data). In another study, it was found that the qa 1S-qa-lF intergenic region was contained within the 3.8 kb fragment of the NA3 clone, after digestion with EcoRl (Roys, unpublished data). Thus, this particular fragment was isolated, ligated into an EcoRl digested pBluescript vector, and transformed into E. coli JMI01. The resulting subclone was designated plasmid pRl (Rutledge, unpublished data) (Figure 11). II. Characterization of Plasmid pR1 To further characterize the qa-1S-qa-1F intergenic regIOn of N. africana, the subclone pR1 was examined. Here, a series 74 RESULTS I. Construction of Plasmid pRI The qa-lS-qa-lF intergenic region of N. africana was chosen for study based on the role it may play in carbon catabolite repression of the qa genes. To start the characterization of the qa-lS-qa-lF intergenic region of N. africana the lambda clone NA3 was examined. This particular clone was known to contain most of the qa gene cluster (Asch, unpublished data) (Figure 11). It was found that when this clone was digested with the restriction endonuclease EcoRI, six fragments of various sizes were produced, four of which contained parts of the qa gene cluster (Figure 11) (Rutledge, unpublished data). In another study, it was found that the qa 1S-qa-lF intergenic region was contained within the 3.8 kb fragment of the NA3 clone, after digestion with EcoRl (Roys, unpublished data). Thus, this particular fragment was isolated, ligated into an EcoRl digested pBluescript vector, and transformed into E. coli JMI01. The resulting subclone was designated plasmid pRl (Rutledge, unpublished data) (Figure 11). II. Characterization of Plasmid pR1 To further characterize the qa-1S-qa-1F intergenic regIOn of N. africana, the subclone pR1 was examined. Here, a series 74 I j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j J I I I I I I I I I I I I I I I I I I I I I I I I I I I I I NA3 Clone qa-2 qa-4- qa-y- qa-1S qa-1 F- -1 I I E E E ~- -;.~ -:b--- ---t3~1-kb- E ~ E pBluescript vector with insert rAmp Plasmid pR1 2.5 kb 1-----1 3.8 kb LacZ 3.8 kb insest within MCS (mutiple cloning site) Lacl NA3 Clone qa-2 qa-4- qa-y- qa-1S qa-1 F- -1 I I E E E ~- -;.~ -:b--- ---t3~1-kb- E ~ E pBluescript vector with insert rAmp Plasmid pR1 2.5 kb 1-----1 3.8 kb LacZ 3.8 kb insest within MCS (mutiple cloning site) Lacl of different restriction digests were performed to produce a restriction map of the plasmid pRI. The restriction enzymes used all contained a unique (or one) restriction site within the pBluescript vector. Therefore, if a restriction site for a particular enzyme also existed within the 3.8 kb insert, multiple fragments would be seen after digestion. The sizes of these fragments could then be estimated by comparison to a size standard (lambda DNA cleaved with HindIII) (Figure 12, lane 1). The restriction enzyme EcoRI produced two fragments of approximately 3.8 kb and 2.9 kb in size (Figure 12, lane 2). This result confirmed the presence of both the insert (3.8 kb) and the vector (2.9 kb) whose sizes were already known. The enzyme Xho 1 generated two fragments, one of 5.370 kb and the other 1.330 kb in length (Figure 12, lane 3). These two fragments suggested that a Xho 1 restriction site existed within the insert. The enzyme Pstl produced three fragments of 3.490 kb, 2.710 kb and 0.500 kb in size (Figure 12, lane 4). With the production of three fragments this suggested that the insert contained two Pstl restriction sites. Next, the enzyme Sacl, also generated three fragments of 3.360 kb, 2.800 kb and 0.540 kb in size (Figure 12, lane 5). This also suggested that the insert contained two restriction sites for the enzyme Sac1. The enzyme BamHI generated two fragments of the lengths, 4.570 kb and 2.130 kb (Figure 12, lane 6). This result suggested, like Xho 1, that only one restriction site for BamH 1 could be found within the insert. The restriction enzymes Kpn 1 (Figure 12, lane 7), HindIII (Figure 12, lane 8), and Sma 1 77 of different restriction digests were performed to produce a restriction map of the plasmid pRI. The restriction enzymes used all contained a unique (or one) restriction site within the pBluescript vector. Therefore, if a restriction site for a particular enzyme also existed within the 3.8 kb insert, multiple fragments would be seen after digestion. The sizes of these fragments could then be estimated by comparison to a size standard (lambda DNA cleaved with HindIII) (Figure 12, lane 1). The restriction enzyme EcoRI produced two fragments of approximately 3.8 kb and 2.9 kb in size (Figure 12, lane 2). This result confirmed the presence of both the insert (3.8 kb) and the vector (2.9 kb) whose sizes were already known. The enzyme Xho 1 generated two fragments, one of 5.370 kb and the other 1.330 kb in length (Figure 12, lane 3). These two fragments suggested that a Xho 1 restriction site existed within the insert. The enzyme Pstl produced three fragments of 3.490 kb, 2.710 kb and 0.500 kb in size (Figure 12, lane 4). With the production of three fragments this suggested that the insert contained two Pstl restriction sites. Next, the enzyme Sacl, also generated three fragments of 3.360 kb, 2.800 kb and 0.540 kb in size (Figure 12, lane 5). This also suggested that the insert contained two restriction sites for the enzyme Sac1. The enzyme BamHI generated two fragments of the lengths, 4.570 kb and 2.130 kb (Figure 12, lane 6). This result suggested, like Xho 1, that only one restriction site for BamH 1 could be found within the insert. The restriction enzymes Kpn 1 (Figure 12, lane 7), HindIII (Figure 12, lane 8), and Sma 1 77 I j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j J I I I I I I I I I I I I I I I I I I I I I I I I I I I I I 23.13 9.42 6.56 4.36- 2.32 2.03- 0.54- 1 2 345 6 789 23.13 9.42 6.56- 4.36- 2.32 2.03- 0.54- 1 2 345 6 789 (Figure 12, lane 9) each only generated one fragment. These results suggested that no restriction site for these enzymes existed within the insert. Now based on this information a preliminary restriction map was deduced (Figure 13). Localization of the qa-lS-qa-lF Intergenic Region III. Southern Blot Analysis of Plasmid pR1 To localize the qa-lS-qa-lF intergenic region contained within the 3.8 kb insert, a Southern blot analysis was performed on the plasmid pRl. Here the plasmid pR1 was subjected to a series of restriction digests. The restriction endonucleases chosen (EcoR1, Xho1, Pst1, BamH1, and Sac!) as the insert was known to contain these restriction sites (Figure 13). Next, an 800 bp DIG-labeled probe, which was a peR product, spanning a portion of the qa-lS-qa-lF intergenic region of N. crassa was generated from N. crassa genomic DNA (Roys, unpublished data). Based on an earlier study (Asch et al., 1991), it was thought that this N. crassa probe would hybridize to complementary N. africana qa-lS-qa-lF intergenic sequences, and was therefore used to confirm which fragments contained qa-lS-qa-lF intergenic sequences complementary to the probe. This probe covered from 14,300 to 15,000 on the qa gene sequence (Geever et al., 1989). and contained only sequences derived from the qa-1S-qa-1F intergenic region of N. crassa (Roys, unpublished data). As the blot shows, for the 80 (Figure 12, lane 9) each only generated one fragment. These results suggested that no restriction site for these enzymes existed within the insert. Now based on this information a preliminary restriction map was deduced (Figure 13). Localization of the qa-lS-qa-lF Intergenic Region III. Southern Blot Analysis of Plasmid pR1 To localize the qa-lS-qa-lF intergenic region contained within the 3.8 kb insert, a Southern blot analysis was performed on the plasmid pRl. Here the plasmid pR1 was subjected to a series of restriction digests. The restriction endonucleases chosen (EcoR1, Xho1, Pst1, BamH1, and Sac!) as the insert was known to contain these restriction sites (Figure 13). Next, an 800 bp DIG-labeled probe, which was a peR product, spanning a portion of the qa-lS-qa-lF intergenic region of N. crassa was generated from N. crassa genomic DNA (Roys, unpublished data). Based on an earlier study (Asch et al., 1991), it was thought that this N. crassa probe would hybridize to complementary N. africana qa-lS-qa-lF intergenic sequences, and was therefore used to confirm which fragments contained qa-lS-qa-lF intergenic sequences complementary to the probe. This probe covered from 14,300 to 15,000 on the qa gene sequence (Geever et al., 1989). and contained only sequences derived from the qa-1S-qa-1F intergenic region of N. crassa (Roys, unpublished data). As the blot shows, for the 80 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 r r 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 r r r r r r r 1 r r 1 1 r 1 1 . 1 . 1 I? I? I Plasmid pR1 K X H E P 8 P X B 8 E P 8m B 8 3.8 kb E I EI I 2.9 kb I I E E I 1.330 kb~ I 5.370 kb I X E II II EX 3.490 kb I I :.0.5':' I I I I I I I I I 2.710 kb E P I P P I I I4.570 kb .....2.130 kb II BI E B 2.80 kb ~_.3.360 kb -! 0.540 kb I 8 8 8 Plasmid pR1 K X H E P 8 P X B 8 E P 8m B 8 3.8 kb E I EI I 2.9 kb I I E E I 1.330 kb~ I 5.370 kb I X E II II EX 3.490 kb I I :.0.5':' I I I I I I I I I 2.710 kb E P I P P I I I4.570 kb .....2.130 kb II BI E B 2.80 kb ~_.3.360 kb -! 0.540 kb I 8 8 8 EcoRI digest (Figure 14A, lane 1) only the 3.8 kb fragment hybridized the probe (Figure 14B, lane 1). This result confirmed the previous data indicating that this fragment contained the qa-lS-qa-l F intergenic region of N. africana (Roys, unpublished data). The two fragments produced by the Xho 1 digest (Figure 14A, lane 2) both hybridized the probe (Figure 14B, lane 2). However the 5.370 kb fragment produced a greater intensity than the 1.330 kb fragment. This result suggested that only a small portion of the qa-lS-qa-lF intergenic region existed within the 1.330 kb fragment. The double digest of EcoRI and Xho 1 generated three fragments of 2.870 kb, 2.500 kb, and 1.300 kb (Figure 14A, lane 3). Of these three fragments only the 2.500 kb and the 1.300 kb hybridized the probe (Figure 14B, lane 3). However like the Xho 1 digest, the 2.500 kb fragment produced a greater intensity than the 1.300 kb fragment. This again suggested that the 1.300 kb fragment contained only a small portion of the qa-lS-qa-lF intergenic region. Of the three generated Pstl fragments (Figure 14A, lane 4), only the 2.710 kb fragment hybridized the probe (Figure 14B, lane 4). This result showed that this particular fragment contained the entire qa-lS-qa-lF intergenic region. Both of the BamHI generated fragments (Figure 14A, lane 5) hybridized the probe (Figure 14B, lane 5). This provided evidence that the BamHI restriction site existed within the qa-lS-qa-l F intergenic region. Finally, of the three fragments produced by Sac1 (Figure 14A, lane 6), only the 2.800 kb fragment hybridized the probe (Figure 14B, lane 6). 83 EcoRI digest (Figure 14A, lane 1) only the 3.8 kb fragment hybridized the probe (Figure 14B, lane 1). This result confirmed the previous data indicating that this fragment contained the qa-lS-qa-l F intergenic region of N. africana (Roys, unpublished data). The two fragments produced by the Xho 1 digest (Figure 14A, lane 2) both hybridized the probe (Figure 14B, lane 2). However the 5.370 kb fragment produced a greater intensity than the 1.330 kb fragment. This result suggested that only a small portion of the qa-lS-qa-lF intergenic region existed within the 1.330 kb fragment. The double digest of EcoRI and Xho 1 generated three fragments of 2.870 kb, 2.500 kb, and 1.300 kb (Figure 14A, lane 3). Of these three fragments only the 2.500 kb and the 1.300 kb hybridized the probe (Figure 14B, lane 3). However like the Xho 1 digest, the 2.500 kb fragment produced a greater intensity than the 1.300 kb fragment. This again suggested that the 1.300 kb fragment contained only a small portion of the qa-lS-qa-lF intergenic region. Of the three generated Pstl fragments (Figure 14A, lane 4), only the 2.710 kb fragment hybridized the probe (Figure 14B, lane 4). This result showed that this particular fragment contained the entire qa-lS-qa-lF intergenic region. Both of the BamHI generated fragments (Figure 14A, lane 5) hybridized the probe (Figure 14B, lane 5). This provided evidence that the BamHI restriction site existed within the qa-lS-qa-l F intergenic region. Finally, of the three fragments produced by Sac1 (Figure 14A, lane 6), only the 2.800 kb fragment hybridized the probe (Figure 14B, lane 6). 83 I j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j J I I I I I I I I I I I I I I I I I I I I I I I I I I I I I This suggested that this fragment also contained the entire qa lS-qa-1F intergenic region. This information was then used to construct subclones of plasmid pR1 in a hope to further localize the qa-lS-qa-lF intergenic region, and initiate DNA sequencing of this region. IV. Construction and Characterization of the Subclone Plasmid pRX1 To further localize the qa-lS-qa-lF intergenic regIOn, the 1.330 kb fragment produced by a Xho 1 digest of plasmid pR1 was isolated and ligated into a Xho 1 cleaved pBluescript vector (Figure 15). This particular fragment was chosen because the location of the Xho 1 restriction site essentially split the 3.8 kb insert into two halves and based on the Southern blot analysis of the plasmid pR1 (Figure 14B, lane 2) where this fragment showed apparent complementarity to the DIG-labeled probe. The resulting subclone, plasmid pRX1, was then subjected to a series of restriction enzymes to further identify the locations of there restriction sites within the insert. Again, as in section II, the sizes of the fragments produced by the restriction digests were compared to a size standard (Figure 16, lane 1). The restriction enzyme Xho 1 generated two fragments of 2.900 kb and 1.330 kb (Figure 16, lane 2). This result confirmed the presence of both the vector (2.900 kb) and the Xho 1 generated fragment (1.330 kb). The enzyme Sac1 produced two 86 This suggested that this fragment also contained the entire qa lS-qa-1F intergenic region. This information was then used to construct subclones of plasmid pR1 in a hope to further localize the qa-lS-qa-lF intergenic region, and initiate DNA sequencing of this region. IV. Construction and Characterization of the Subclone Plasmid pRX1 To further localize the qa-lS-qa-lF intergenic regIOn, the 1.330 kb fragment produced by a Xho 1 digest of plasmid pR1 was isolated and ligated into a Xho 1 cleaved pBluescript vector (Figure 15). This particular fragment was chosen because the location of the Xho 1 restriction site essentially split the 3.8 kb insert into two halves and based on the Southern blot analysis of the plasmid pR1 (Figure 14B, lane 2) where this fragment showed apparent complementarity to the DIG-labeled probe. The resulting subclone, plasmid pRX1, was then subjected to a series of restriction enzymes to further identify the locations of there restriction sites within the insert. Again, as in section II, the sizes of the fragments produced by the restriction digests were compared to a size standard (Figure 16, lane 1). The restriction enzyme Xho 1 generated two fragments of 2.900 kb and 1.330 kb (Figure 16, lane 2). This result confirmed the presence of both the vector (2.900 kb) and the Xho 1 generated fragment (1.330 kb). The enzyme Sac1 produced two 86 I j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j j J I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I t--t--+----ir(((??f((?f((((f?(?(1~-- ...---...--I--+---+---l I I I KXHE PSP x B S E PBS pR1 with Xho 1 I : Digest I? I I I I I RX1 ,---------------- I It---f1?1???t???I???t?I???;1 I II ? x x pBluescript Digest pBluescript with Xho 1 ... ... .... ... ... .......Lacl LacZ I RX1 I f--MOV$I.?.?.<$lS?S<,cStSltJ>o;}l{:l{:lt7:lHH~~~%':r}?'?{f}i"f/?{iR5S.5~~, -+--+----4 I I I I 1 I I I I I K X H E P S P X E P S X S I I I 1 I------I~???????}); 1.330 kb ~???????>}>>>~ I I I X ------5.370 kb-------------- 1 I I I ~??~0.800 kb ~??~'---------I I I I I kb f/?/f/ffC:&:ClJ>'1lJ:l]>~ I I S I I I I rrllll~0.500 kb ~!('~ I I I I I I I I : R????t~0.370 kb-l I I '; : : : P P [3.360 kb:~ _ I I ~0.500 I I I I I Ikb., E I I I I r>>>>>~0.500 kb~??~ I I I I kb~ : I I P : 0.35~ kb : ~?121 I I I P E A) I 1 I I I ~ll?l?lk&&~l(j~-- .....- .......--,--- I I 1 I I I I ~ I I I ,...... I Ie" P s P x "'--... B S E" ...... B) , ......... \ " I I I'""'1 1--+--~tS.5S.'i{;'l1>ltJ>o;}l{:l{:lt7:lHH~~~%':r}?'?{f}i"f/?{iR5S.5~~, -+--+----4 I I I I 1 I I I I I K X H E P S P X E P S X S I I I 1 I------I~???????}); 1.330 kb ~???????>}>>>~ I I I X ------5.370 kb-------------- 1 I I I ~??~0.800 kb ~??~'---------I I I I I kb f/?/f/ffC:&:ClJ>'1lJ:l]>~ I I S I I I I rrllll~0.500 kb ~!('~ I I I I I I I I : R????t~0.370 kb-l I I '; : : : P P [3.360 kb:~ _ I I ~0.500 I I I I I Ikb., E I I I I r>>>>>~0.500 kb~??~ I I I I kb~ : I I P : 0.35~ kb : ~?121 I I I P E Figure 18. Southern blot analysis of the subclone plasmid pRXl. Lane 1 is the positive control (pRI cleaved with EcoRl), showing the 3.800 kb fragment. Lane 2 is the Xho 1 digest. Lane 3 is the Sac1 digest. Lane 4 is the Pstl digest. Lane 5 is the double digest of EcoRI and Pstl. Lanes 2-5 each show no activity, suggesting no complementarity to the probe. 94 Figure 18. Southern blot analysis of the subclone plasmid pRXl. Lane 1 is the positive control (pRI cleaved with EcoRl), showing the 3.800 kb fragment. Lane 2 is the Xho 1 digest. Lane 3 is the Sac1 digest. Lane 4 is the Pstl digest. Lane 5 is the double digest of EcoRI and Pstl. Lanes 2-5 each show no activity, suggesting no complementarity to the probe. 94 1 2 3 4 5 Pstl (Figure 18, lane 4), or the double digest of EcoRI and Pstl (Figure 18, lane 5) generated fragments hybridized the probe. This suggested that no qa-lS-qa-lF intergenic sequences could be found in these portions of the original 3.8 kb insert. It IS believed that this particular portion of the insert contains most of the qa-lF gene of N. africana. This result contradicts the Southern blot analysis of the plasmid pRI (Figure 14B, lane 2), which showed this portion of the insert hybridizing the DIG labeled probe. However, it is believed that this hybridization was a false positive and is hoped to be confirmed with DNA sequencing of this region of the insert. VI. Construction and Characterization of the Subclone Plasmid pRX2 To further localize the qa-lS-qa-lF intergenic regIOn, the subclone plasmid pRX2 was generated (Figure 19). This plasmid was generated based on the location of the Xho 1 restriction site, which split the 3.8 kb insert into two halves and the Southern blot analysis of the plasmid pRI (Figure 14B, lane 2) which showed this fragment hybridized the DIG-labeled probe. This plasmid was then also subjected to a series of restriction enzymes to further reinforce there locations within the original 3.8 kb insert. The sizes of the fragments generated by these digests were then compared to a size standard to estimate their lengths (Figure 20A, lane 1). The double digest of Sad and Xho 1 generated three fragments of 2.830 kb, 2.000 96 Pstl (Figure 18, lane 4), or the double digest of EcoRI and Pstl (Figure 18, lane 5) generated fragments hybridized the probe. This suggested that no qa-lS-qa-lF intergenic sequences could be found in these portions of the original 3.8 kb insert. It IS believed that this particular portion of the insert contains most of the qa-lF gene of N. africana. This result contradicts the Southern blot analysis of the plasmid pRI (Figure 14B, lane 2), which showed this portion of the insert hybridizing the DIG labeled probe. However, it is believed that this hybridization was a false positive and is hoped to be confirmed with DNA sequencing of this region of the insert. VI. Construction and Characterization of the Subclone Plasmid pRX2 To further localize the qa-lS-qa-lF intergenic regIOn, the subclone plasmid pRX2 was generated (Figure 19). This plasmid was generated based on the location of the Xho 1 restriction site, which split the 3.8 kb insert into two halves and the Southern blot analysis of the plasmid pRI (Figure 14B, lane 2) which showed this fragment hybridized the DIG-labeled probe. This plasmid was then also subjected to a series of restriction enzymes to further reinforce there locations within the original 3.8 kb insert. The sizes of the fragments generated by these digests were then compared to a size standard to estimate their lengths (Figure 20A, lane 1). The double digest of Sad and Xho 1 generated three fragments of 2.830 kb, 2.000 96 Plasmid pR1 P I I I I I I I I 1--+---+-.-.........- ....~fG?1'????????~???~I---........---+----f I I I I X B S E PBS I I I Digest with Xho 1 I, E 1------1....1.330 kb--..I I 5.370 kb I I I ~???????????l???????15------.., I X E X X II I I DNA is phenol/chloroform extracted I+ I I : The 5.370 kb fragment is ligated I together with T4 DNA ligase resulting , in the plasmid pRX2 rAmp LacZ /IL.......... Lacl Plasmid pRX2 (5.370 kb) Plasmid pR1 P I I I I I I I I 1--+---+-.-.........- ....~fG?1'????????~???~I---........---+----f I I I I X B S E PBS I I I Digest with Xho 1 I, E 1------1....1.330 kb--..I I 5.370 kb I I I ~???????????l???????15------.., I X E X X II I I DNA is phenol/chloroform extracted I+ I I : The 5.370 kb fragment is ligated I together with T4 DNA ligase resulting , in the plasmid pRX2 rAmp LacZ /IL.......... Lacl Plasmid pRX2 (5.370 kb) A) B) 1 234 5 1 2 3 4 5 6 6.56 9.42 6.56 4.364.36 2.32 2.322.03 2.03 0.54 0.54 A) B) 1 234 5 1 2 3 4 5 6 23.13 6.56 9.42 6.56 4.36 4.36 2.32 2.03 2.322.03 0.54 0.54 kb, and 0.540 kb (Figure 20A, lane 3). The double digest of BamHl and Xhol produced three fragments of 2.840 kb, 2.130 kb, and 0.400 kb (Figure 20A, lane 4). Next, the double digest of BamHl and Sac1 also generated three fragments of 3.230 kb, 1.600 kb, and 0.540 kb (Figure 20A, lane 5). Finally the enzyme Sacl, when used alone generated two fragments of 4.830 kb and 0.54 kb (Figure 20A, lane 6). All of this information was then used to generate a restriction map the of the subclone plasmid pRX2 (Figure 21). VII. Southern Blot Analysis of the Subclone Plasmid pRX2 To isolate portions of the subclone plasmid pRX2 possibly containing qa-lS-qa-lF regions, a Southern blot analysis was performed on the subclone plasmid pRX2. Here, the subclone plasmid pRX2 was subjected to a series of restriction digests (Figure 20A, lanes 3-6). Next, the same DIG-labeled 800 bp probe used in sections III and V was used to confirm the presence of qa-lS-qa-lF intergenic sequences within these fragments. Here, as a positive control the plasmid pRl was digested with EcoRl to produce the entire 3.8 kb original insert (Figure 20A, lane 2). As the blot shows, the positive control hybridized to the probe (Figure 20B, lane 1). Of the three fragments generated by the double digest of Sac1 and Xho 1, only the 2.000 kb fragment hybridized the probe (Figure 20B, lane 2). With the three fragments generated by the double 101 kb, and 0.540 kb (Figure 20A, lane 3). The double digest of BamHl and Xhol produced three fragments of 2.840 kb, 2.130 kb, and 0.400 kb (Figure 20A, lane 4). Next, the double digest of BamHl and Sac1 also generated three fragments of 3.230 kb, 1.600 kb, and 0.540 kb (Figure 20A, lane 5). Finally the enzyme Sacl, when used alone generated two fragments of 4.830 kb and 0.54 kb (Figure 20A, lane 6). All of this information was then used to generate a restriction map the of the subclone plasmid pRX2 (Figure 21). VII. Southern Blot Analysis of the Subclone Plasmid pRX2 To isolate portions of the subclone plasmid pRX2 possibly containing qa-lS-qa-lF regions, a Southern blot analysis was performed on the subclone plasmid pRX2. Here, the subclone plasmid pRX2 was subjected to a series of restriction digests (Figure 20A, lanes 3-6). Next, the same DIG-labeled 800 bp probe used in sections III and V was used to confirm the presence of qa-lS-qa-lF intergenic sequences within these fragments. Here, as a positive control the plasmid pRl was digested with EcoRl to produce the entire 3.8 kb original insert (Figure 20A, lane 2). As the blot shows, the positive control hybridized to the probe (Figure 20B, lane 1). Of the three fragments generated by the double digest of Sac1 and Xho 1, only the 2.000 kb fragment hybridized the probe (Figure 20B, lane 2). With the three fragments generated by the double 101 A) I Ikb-----l Is I Ikb---l I I S x -----2.830 kb---------------- I I I I ~0.400 kb~4 I I I I I I : t<{<{<<<<<<<<<<<<2.130 kb~??????~I-------t I I I _~: B B x -----2.840 kb-------------- I I I I ~~~~1.600 kb~~~~~ I I II : ~?l&~0.400 I II I ? I I I S -----!~??????Wi I .B I Ikb-l I I B --------3.230 kb----------- A) I Ikb-----l Is I Ikb---l I I S x -----2.830 kb---------------- I I I I ~0.400 kb~4 I I I I I I : t<{<{<<<<<<<<<<<<2.130 kb~??????~I-------t I I I _~: B B x -----2.840 kb-------------- I I I I ~~~~1.600 kb~~~~~ I I II : ~?l&~0.400 I II I ? I I I S -----!~??????Wi I .B I Ikb-l I I B --------3.230 kb----------- digest of BamHI and Xhol, only the 2.130 kb and the 0.400 kb hybridized the probe (Figure 20B, lane 3). Here, the 0.400 kb fragment generated by this digest hybridized the probe very weakly suggesting only slight complementarity to the probe. The double digest of BamHI and Sacl generated three fragments, of which the 3.230 kb and the 1.600 kb hybridized the probe (Figure 20B, lane 4). Finally, of the two fragments generated by the enzyme Sacl, only the 4.830 kb fragment hybridized the probe (Figure 20B, lane 5). The 0.540 kb fragment generated by the Sac1 and Xhoi double digest and the BamHI and Sacl double digest was believed to contain a portion of the qa-lS gene of N. africana. With this information, along with the Southern blot analysis of subclone plasmid pRXI (section V) the qa-lS-qa-lF intergenic region of N. africana was believed to be narrowed down to a select region in the original 3.8 kb insert. VIn. Construction and Characterization of the Subclones Plasmid pRP1 and Plasmid pRB 1 With the thought that the qa-1S-qa-1F intergenic region had been narrowed down to a select region within the original 3.8 kb insert, subclones of plasmid pRI could now be constructed to initiate DNA sequencing. First, the plasmid pRPI was generated. This was done by digesting the plasmid pRI with the restriction enzyme Pstl, and then ligating the 3.490 kb fragment together (Figure 22). This particular plasmid was 104 digest of BamHI and Xhol, only the 2.130 kb and the 0.400 kb hybridized the probe (Figure 20B, lane 3). Here, the 0.400 kb fragment generated by this digest hybridized the probe very weakly suggesting only slight complementarity to the probe. The double digest of BamHI and Sacl generated three fragments, of which the 3.230 kb and the 1.600 kb hybridized the probe (Figure 20B, lane 4). Finally, of the two fragments generated by the enzyme Sacl, only the 4.830 kb fragment hybridized the probe (Figure 20B, lane 5). The 0.540 kb fragment generated by the Sac1 and Xhoi double digest and the BamHI and Sacl double digest was believed to contain a portion of the qa-lS gene of N. africana. With this information, along with the Southern blot analysis of subclone plasmid pRXI (section V) the qa-lS-qa-lF intergenic region of N. africana was believed to be narrowed down to a select region in the original 3.8 kb insert. VIn. Construction and Characterization of the Subclones Plasmid pRP1 and Plasmid pRB 1 With the thought that the qa-1S-qa-1F intergenic region had been narrowed down to a select region within the original 3.8 kb insert, subclones of plasmid pRI could now be constructed to initiate DNA sequencing. First, the plasmid pRPI was generated. This was done by digesting the plasmid pRI with the restriction enzyme Pstl, and then ligating the 3.490 kb fragment together (Figure 22). This particular plasmid was 104 P Plasmid pR1 1 I I I 1 I I I I--+--+-~~???(~~""'I ---....---------.. -+--1: I I I I I I KXHE PS P X B S EP BS 1 1 Digest with Pst 11, 1 I +-0.5 kb., I I 1 r-------1~W;StSt~ : 2.710 kb --------1 1 1 IP P ------------3.490 kb--------1 1 : DNA is phenol/chloroform extracted 1, I 1 1 The 3.490 kb fragment is ligated 1 1 together with T4 DNA ligase resulting , in the plasmid pRP1 rAmp LacZ Lacl Plasmid pRP1 (3.490 kb) P Plasmid pR1 1 I I I 1 I I I I--+--+-~~???(~~""'I ---....---------.. -+--1: I I I I I I KXHE PS P X B S EP BS 1 1 Digest with Pst 11, 1 I +-0.5 kb., I I 1 r-------1~W;StSt~ : 2.710 kb --------1 1 1 IP P ------------3.490 kb--------1 1 : DNA is phenol/chloroform extracted 1, I 1 1 The 3.490 kb fragment is ligated 1 1 together with T4 DNA ligase resulting , in the plasmid pRP1 rAmp LacZ Lacl Plasmid pRP1 (3.490 kb) made with the hope that DNA sequencing would reveal that indeed this portion of the 3.8 kb insert contained a section of the qa-1F gene. This plasmid, like the others was also subjected to a series of restriction digests to generate a restriction map of the subclone (Figure 23). Next, the plasmid pRB I was generated. This was done by digesting the plasmid pRI with the restriction enzyme BamH1, and then ligating the 4.570 kb fragment together (Figure 24). Again, a restriction map was deduced for this subclone, to reinforce the restriction sites contained within the original 3.8 kb insert (Figure 25). This plasmid was made based on the Southern blot analysis of plasmid pRI (Figure 14B) and the subclone plasmid pRX2 (Figure 20B). Since both the fragments produced by the BamHI digest (Figure 14A, lane 6) hybridized the DIG-labeled probe (Figure 14B, lane 5), the Southern blot of pRI showed that the BamHI site was located in the qa-1S-qa- 1F intergenic region. While the double digest of BamHI and Xho 1 performed on the subclone plasmid pRX2 generated three fragments (Figure 20A, lane 4). Two of these (2.130 kb and 0.400 kb) three hybridized the probe (Figure 20B, lane 3), but the 0.400 kb fragment hybridized the probe very weakly. This suggested that it contained only a small portion of the qa -1 S qa-1F intergenic region. It is hoped that DNA sequencing will reveal this section of the qa-1S-qa-1 F intergenic region contained within the subclone plasmid pRB 1. 107 made with the hope that DNA sequencing would reveal that indeed this portion of the 3.8 kb insert contained a section of the qa-1F gene. This plasmid, like the others was also subjected to a series of restriction digests to generate a restriction map of the subclone (Figure 23). Next, the plasmid pRB I was generated. This was done by digesting the plasmid pRI with the restriction enzyme BamH1, and then ligating the 4.570 kb fragment together (Figure 24). Again, a restriction map was deduced for this subclone, to reinforce the restriction sites contained within the original 3.8 kb insert (Figure 25). This plasmid was made based on the Southern blot analysis of plasmid pRI (Figure 14B) and the subclone plasmid pRX2 (Figure 20B). Since both the fragments produced by the BamHI digest (Figure 14A, lane 6) hybridized the DIG-labeled probe (Figure 14B, lane 5), the Southern blot of pRI showed that the BamHI site was located in the qa-1S-qa- 1F intergenic region. While the double digest of BamHI and Xho 1 performed on the subclone plasmid pRX2 generated three fragments (Figure 20A, lane 4). Two of these (2.130 kb and 0.400 kb) three hybridized the probe (Figure 20B, lane 3), but the 0.400 kb fragment hybridized the probe very weakly. This suggested that it contained only a small portion of the qa -1 S qa-1F intergenic region. It is hoped that DNA sequencing will reveal this section of the qa-1S-qa-1 F intergenic region contained within the subclone plasmid pRB 1. 107 A) 1 1 ~?~1 1 1 I 1 I ~, L I I I IP x B 8 E"- -... B) , -, -...... -..., - ...,I -I k?????<<<<<<<<<<<<<<<<<@I I I I K X H E P 8m B 8 1 1 r---------II 1 ~l)}?)?)~ I B '--------4.570 kb--------------- I : DNA is phenol/chloroformed extracted+ I I The 4.570 kb fragment is ligated : together with T4 DNA ligase resulting+ in the plasmid pRB1 rAmp LacZ Plasmid pRB1 (4.570 kb) Plasmid pR1 E PBS I I I I I I I I I I I I 1--+--+-~~S$S$S$S?$S$i$S$S$SW1$S$S?$S$S$~~---------If---+---+---I ? I I ? KXHE PS P X B I I I Digest with Bam H1I, B I I........2.130 kb--I--------; I I I I I I .-------~$)$???)$?)$)$)?}?>)}?)?)~ I B '--------4.570 kb--------------- I : DNA is phenol/chloroformed extracted+ I I The 4.570 kb fragment is ligated : together with T4 DNA ligase resulting+ in the plasmid pRB1 rAmp LacZ Plasmid pRB1 (4.570 kb) IX. Sequencing the Subclone Plasmid pRPI To establish that a portion of the qa-lF gene was located to the left of the Xho 1 restriction site within the original 3.8 kb insert, DNA sequence analysis was performed on the subclone plasmid pRPl. This subclone was sequenced using an M13/pUC forward sequencing primer. This primer recognizes a sequence ,in the 3 end of the multiple cloning site of the pBluescript vector and allows DNA sequencing to proceed towards the 5' end of the multiple cloning site. Therefore, sequencing of the insert within the subclone plasmid pRPl started at the Pst1 site and moved towards the EcoRl site (Figure 23). The DNA sequence which was generated by this reaction was then analyzed on DNA Strider 1.0. This software locates any restriction site which is located within the sequence entered and produces a restriction map of that sequence. Figure 26 shows the sequence generated with the subclone plasmid pRPl and its restriction sites. Next, it had to be determined if any sequence homology existed between the portions of the 3.8 kb insert of N. africana, contained within this subclone, and the qa gene cluster of N. crassa. To do this, the sequence generated by this subclone was entered into a database on the World Wide Web. The resource (URL) used was Bioscan Online (http://genome.cs.unc.edu/bin/nucl-match).This web site allows the user to enter sequences and it will compare and match the users sequence to know sequences contained within 114 IX. Sequencing the Subclone Plasmid pRPI To establish that a portion of the qa-lF gene was located to the left of the Xho 1 restriction site within the original 3.8 kb insert, DNA sequence analysis was performed on the subclone plasmid pRPl. This subclone was sequenced using an M13/pUC forward sequencing primer. This primer recognizes a sequence ,in the 3 end of the multiple cloning site of the pBluescript vector and allows DNA sequencing to proceed towards the 5' end of the multiple cloning site. Therefore, sequencing of the insert within the subclone plasmid pRPl started at the Pst1 site and moved towards the EcoRl site (Figure 23). The DNA sequence which was generated by this reaction was then analyzed on DNA Strider 1.0. This software locates any restriction site which is located within the sequence entered and produces a restriction map of that sequence. Figure 26 shows the sequence generated with the subclone plasmid pRPl and its restriction sites. Next, it had to be determined if any sequence homology existed between the portions of the 3.8 kb insert of N. africana, contained within this subclone, and the qa gene cluster of N. crassa. To do this, the sequence generated by this subclone was entered into a database on the World Wide Web. The resource (URL) used was Bioscan Online (http://genome.cs.unc.edu/bin/nucl-match).This web site allows the user to enter sequences and it will compare and match the users sequence to know sequences contained within 114 DNA sequence 59 b.p. GACTAGTTGCCT .. , GTCTATGGAACA !-.:"near MSp I ~ Hpa II Hga - Hga I ~ Mnl I Cf='0 I 35m:: 3s,U T II iii '! i GACTAGTTGCCTCTTGATGATGACCGGCTACGAC_~GAP~GCGTCGC~ACC~GTC GCGTCTATGGAACA 69 C~GATCAACGGAGAACTAC~ACTGGCCGATGCTGTTCTTACGCAGCGATGGACAGC GCAGATACCTTGT I! I II -I 2:0 23 37 'SS 3 24 41 56 24 DNA sequence 59 b.p. GACTAGTTGCCT .. , GTCTATGGAACA !-.:"near MSp I ~ Hpa II Hga - Hga I ~ Mnl I Cf='0 I 35m:: 3s,U T II iii '! i GACTAGTTGCCTCTTGATGATGACCGGCTACGAC_~GAP~GCGTCGC~ACC~GTC GCGTCTATGGAACA 69 C~GATCAACGGAGAACTAC~ACTGGCCGATGCTGTTCTTACGCAGCGATGGACAGC GCAGATACCTTGT I! I II -I 2:0 23 37 'SS 3 24 41 56 24 several databases. The sequence data from the subclone plasmid pRPl provided evidence that this portion of the insert contained part of the qa-lF gene of N. africana. This was seen with nucleotides 3 to 39 of the N. africana generated sequence showing homology with nucleotides 16547 to 16583 of the N. crassa qa gene cluster, which is a conserved qa-lF coding region (Figure 27). Based on this information, Southern blot analysis of the subclone pRXl (Figure 18), and physical analysis of the location of these complementary sequences with the q a gene cluster of N. crassa (Geever et aI., 1989) a conclusion can be drawn. It can be stated, with some certainty that the portion of the 3.8 kb insert located to the left of the Xho 1 restriction site contains no qa-lS-qa-lF intergenic sequences, but a large portion of the qa-lF gene of N. africana. X Sequencing the Subclone Plasmid pRB1 DNA sequencmg analysis of the subclone plasmid pRB1 was performed based on the Southern blot analysis of the subclone plasmid pRX2. Here, the 400 bp fragment, produced by the BamHl/Xho I digest, hybridized the DIG-labeled probe very weakly (Figure 20B, lane 3) suggesting that it contained only a small portion of the qa-1S-qa-1F intergenic region. Therefore, DNA sequencing was performed on this fragment, using the subclone plasmid pRBl, in a hope to identify if qa-lS qa-lF intergenic sequences existed within it. Sequencing of the 1 17 several databases. The sequence data from the subclone plasmid pRPl provided evidence that this portion of the insert contained part of the qa-lF gene of N. africana. This was seen with nucleotides 3 to 39 of the N. africana generated sequence showing homology with nucleotides 16547 to 16583 of the N. crassa qa gene cluster, which is a conserved qa-lF coding region (Figure 27). Based on this information, Southern blot analysis of the subclone pRXl (Figure 18), and physical analysis of the location of these complementary sequences with the q a gene cluster of N. crassa (Geever et aI., 1989) a conclusion can be drawn. It can be stated, with some certainty that the portion of the 3.8 kb insert located to the left of the Xho 1 restriction site contains no qa-lS-qa-lF intergenic sequences, but a large portion of the qa-lF gene of N. africana. X Sequencing the Subclone Plasmid pRB1 DNA sequencmg analysis of the subclone plasmid pRB1 was performed based on the Southern blot analysis of the subclone plasmid pRX2. Here, the 400 bp fragment, produced by the BamHl/Xho I digest, hybridized the DIG-labeled probe very weakly (Figure 20B, lane 3) suggesting that it contained only a small portion of the qa-1S-qa-1F intergenic region. Therefore, DNA sequencing was performed on this fragment, using the subclone plasmid pRBl, in a hope to identify if qa-lS qa-lF intergenic sequences existed within it. Sequencing of the 1 17 Best Sum Statistic for Each Similar Database Sequence Jb i .c..c::', Name ,-' '1 <:;,' -:=. r-----'---, Alignments Best Sum Scatlstic ?(~) = 6.9e-D3 LeilgIh: 18120 Date 30-0''L\Y-1996 ,Veurospora crassa qa gene clusrer. , Score = 169 L:ngrh = 37 Expec: = 6.ge-D3 P = 6.ge-D3 ,~ue':i: Best Sum Statistic for Each Similar Database Sequence Jb i .c..c::', Name ,-' '1 <:;,' -:=. r-----'---, Alignments Best Sum Scatlstic ?(~) = 6.9e-D3 LeilgIh: 18120 Date 30-0''L\Y-1996 ,Veurospora crassa qa gene clusrer. , Score = 169 L:ngrh = 37 Expec: = 6.ge-D3 P = 6.ge-D3 ,~ue':i: subclone plasmid pRBI was done usmg the same MI3/pUC forward primer used in section IX. Hence, sequencing of the insert within the subclone plasmid pRB 1 started at the BamH 1 site and moved towards the Xho 1 site (Figure 25). The sequence generated by this reaction was, as before, analyzed on DNA Strider 1.0. Figure 28 shows the sequence generated with the subclone plasmid pRB 1 and its restriction sites. Next, as in section IX, Bioscan Online was used to determine if any sequence homology existed between this portion of the 3.8 kb insert of N. africana and the qa gene cluster of N. crassa. The sequence generated by the subclone plasmid pRB1 (Figure 29) regretfully did not provide any homology to the qa gene cluster of N. crassa. However, this particular sequence did show complementary sequences to E. coli DNA, raising numerous questions. It was thought that since E. coli was the host used to propagate the subclone plasmid pRB 1, perhaps a section of E. coli DNA was inserted into the plasmid. To examine this, the plasmid pRI and the subclone plasmid pRBI were both digested with BamHI and Sacl, used in concert (Figure 30). Therefore, if the subclone plasmid pRB1 contained only its portion of the original 3.8 kb insert, it would be seen by producing two fragments the same size as two of the four produced by the digest of plasmid pR1. Indeed, this was seen in figure 30, where the digested subclone plasmid pRB1 (Figure 30, lane 2) showed the two expected fragments, of the same size, as two of the four produced by plasmid pR1 (Figure 30, lane 3). Hence, more examination of this subclone 120 subclone plasmid pRBI was done usmg the same MI3/pUC forward primer used in section IX. Hence, sequencing of the insert within the subclone plasmid pRB 1 started at the BamH 1 site and moved towards the Xho 1 site (Figure 25). The sequence generated by this reaction was, as before, analyzed on DNA Strider 1.0. Figure 28 shows the sequence generated with the subclone plasmid pRB 1 and its restriction sites. Next, as in section IX, Bioscan Online was used to determine if any sequence homology existed between this portion of the 3.8 kb insert of N. africana and the qa gene cluster of N. crassa. The sequence generated by the subclone plasmid pRB1 (Figure 29) regretfully did not provide any homology to the qa gene cluster of N. crassa. However, this particular sequence did show complementary sequences to E. coli DNA, raising numerous questions. It was thought that since E. coli was the host used to propagate the subclone plasmid pRB 1, perhaps a section of E. coli DNA was inserted into the plasmid. To examine this, the plasmid pRI and the subclone plasmid pRBI were both digested with BamHI and Sacl, used in concert (Figure 30). Therefore, if the subclone plasmid pRB1 contained only its portion of the original 3.8 kb insert, it would be seen by producing two fragments the same size as two of the four produced by the digest of plasmid pR1. Indeed, this was seen in figure 30, where the digested subclone plasmid pRB1 (Figure 30, lane 2) showed the two expected fragments, of the same size, as two of the four produced by plasmid pR1 (Figure 30, lane 3). Hence, more examination of this subclone 120 DNA sequence 324 b.p. AGCAGCACGCCG .,. CATATCGTGCGT linear MIll Hin;: I HinP I ?'nu4H I Hha 3bv I SC:;c- :: :,;cQR T'" 3se.;)" ~ ?'nu4H T Sbv I i AGCAGCACGCCGTTGCCGTCAGAAATCC~ACCTGGCACGC~GC~C~G~;TCCT~TGGTGTGAGCAG7TATC:AC~TGTTG 30 TCGTCGTGCGGCAACGGCAGTCTTTAGGATGGACCGTGCGACGCGAC~TAGGAGACCACACTCGTC~~TAGGTGAAC.~C ? ! i? 39 2 31 3:1. H 39 42 51 .J2 NspB ,.-, 3au3A ' HinP :: MIll I Mbo - ::lha - ~ Dpn - BspM I Mae -.,..,. Mse T Taq :: Xm!:L..I BspM .,. iii I I I CTTTTGTCCACC~GCGCCACCAGTT~GT.~CCGAGGCTT.~CTATAGGCTCGATCAGCGG~_;TGGTTTCCC.~CTGACC 160 GAAAACAGGTGGACGCGGTGGTC~~CATTGGCTCCGAATTTGATATCCGAGC~AGTCGCCTTACCA&~GGGTTGACTGG i I ?1 I ? I .! I 90 107 118 131 141 158 94 1 11 l33 94 113 ~J3 lJJ 136 Fnu4H I Bbv I 1 :1111 .,. Taq :: Mnl - ~~o TI ?nu4H - Tthlll r: TGCTGCTCGCTGGTTTCGCTGATGGTGCTGGTCAGGTTTCGAGGCTTTTCTCTT CCGCCTCCGCCGCCAGCGTGTTTGC~ 240 ACGACGAGCGAC2AAAGCGAC~ACCACGACCAGTC2.~GC~C:GAA~.GAGAAGGCGGAGGCGGCGGTCGCAC.~CGA i 233 I 223 ."1?1 199 201 I 162 162 218 Sau3A :: :1bo I Rsa J9n: 391 I ?"JU ~ Mae ?le:: Ksa I ?nu4H:: HinP I ~th111 ~aq'" SnaB I f:.Qk..J;. Hha I Mae II Hinf I S91 I Bbv I I I I 11'1II! i CATCCGTCCAGCGCAT~.CTC.~GCCAGCACGTTC.~~GC.~~CCGAGTCGATCGTACGTACGCAGCACGACATA~CGT 320 GTAGGCAGGTCGCGTATTTTGAGTTTCGGTCGTGC.~.GTTCGTTGGC~CAGCT AGCATGCATGCGTCGTGCTGTATAGCA 1?1 I III i III1 1 I 241 251 273 287 295 304 251 278 290 297 287 296 304 291 298 292 299 292 300 292 GCGT 324 CGCA DNA sequence 324 b.p. AGCAGCACGCCG .,. CATATCGTGCGT linear MIll Hin;: I HinP I ?'nu4H I Hha 3bv I SC:;c- :: :,;cQR T'" 3se.;)" ~ ?'nu4H T Sbv I i AGCAGCACGCCGTTGCCGTCAGAAATCC~ACCTGGCACGC~GC~C~G~;TCCT~TGGTGTGAGCAG7TATC:AC~TGTTG 30 TCGTCGTGCGGCAACGGCAGTCTTTAGGATGGACCGTGCGACGCGAC~TAGGAGACCACACTCGTC~~TAGGTGAAC.~C ? ! i? 39 2 31 3:1. H 39 42 51 .J2 NspB ,.-, 3au3A ' HinP :: MIll I Mbo - ::lha - ~ Dpn - BspM I Mae -.,..,. Mse T Taq :: Xm!:L..I BspM .,. iii I I I CTTTTGTCCACC~GCGCCACCAGTT~GT.~CCGAGGCTT.~CTATAGGCTCGATCAGCGG~_;TGGTTTCCC.~CTGACC 160 GAAAACAGGTGGACGCGGTGGTC~~CATTGGCTCCGAATTTGATATCCGAGC~AGTCGCCTTACCA&~GGGTTGACTGG i I ?1 I ? I .! I 90 107 118 131 141 158 94 1 11 l33 94 113 ~J3 lJJ 136 Fnu4H I Bbv I 1 :1111 .,. Taq :: Mnl - ~~o TI ?nu4H - Tthlll r: TGCTGCTCGCTGGTTTCGCTGATGGTGCTGGTCAGGTTTCGAGGCTTTTCTCTT CCGCCTCCGCCGCCAGCGTGTTTGC~ 240 ACGACGAGCGAC2AAAGCGAC~ACCACGACCAGTC2.~GC~C:GAA~.GAGAAGGCGGAGGCGGCGGTCGCAC.~CGA i 233 I 223 ."1?1 199 201 I 162 162 218 Sau3A :: :1bo I Rsa J9n: 391 I ?"JU ~ Mae ?le:: Ksa I ?nu4H:: HinP I ~th111 ~aq'" SnaB I f:.Qk..J;. Hha I Mae II Hinf I S91 I Bbv I I I I 11'1II! i CATCCGTCCAGCGCAT~.CTC.~GCCAGCACGTTC.~~GC.~~CCGAGTCGATCGTACGTACGCAGCACGACATA~CGT 320 GTAGGCAGGTCGCGTATTTTGAGTTTCGGTCGTGC.~.GTTCGTTGGC~CAGCT AGCATGCATGCGTCGTGCTGTATAGCA 1?1 I III i III1 1 I 241 251 273 287 295 304 251 278 290 297 287 296 304 291 298 292 299 292 300 292 GCGT 324 CGCA Best Sum Statistic for Each Similar Database Sequence :t: 1)? C::: , ~Tame ... ,....--_....... ,...~-?.-e::,-_ _ ~ ..... __..... =-::::CJ85a: ~'::1;]83S-=6 - __<'9"0,.... ....=-'-- ... "=:: '::5-=::-=:=:':'::':.=.. :~r;:.. . =.... -- Best Si.L.l1 StaIistic ?(J) = 3.3.:-29 ::"err?T.h: 11195 Dale: 19-DEC-1995 -, ". ''7 ~. -r, ~- ?? -7 'f V V~'O ""\ ... - c/'r 7\/{cscner:c.'1ia COil genes/or .ap;.. I_/l. feI/v, "C1.T,'1... 1'-'-/_, L;inj fcqL "C17.v, (afO and Y2,T? 3eSL Sll.rrl SC21:STIC ?(~~: = 1.3e-2i f....:ng-:h: 91~30 DaLe: lO-_-\..PR-1996 ::SC?1e;~C?!ZQ coii JtV-"'L Scm: =:401 ["e:1g7 = 91 ::::-;;e~: =3.-!.e-19 ? =3.4e-19 F'~l.-!., .y/on.-!. Din? ~ 2 S08 ~7~GGC~~~~~~~GC~G~~7~377~~::~~C~G~C~~~C~~C~~~C~~G~~~~~C~~~~~ .l..:'':;GGC:':::;... -:::. .. TGG:''':''':'C::::_~_:"C-:G_:"'C::-: ~ ::7~C'":' -:: -:.::'~.:; '":''':''':" -::: -::-'': .:._:,-: a~~ssc~=~ac=aqc~gaac;~~~~===aac~~ac=~~=~~=~=~==~~~::=;~~~a:~ :3~ 3~GC~GG7C~GG~~~~~~GGC~7~~:~~~~~~ ,-.~_..... .... .......... .-... -- .,., .........; .:.. '''';'- ~~ ..:.. -.1..= sa9S3 ~~~c~~~~=agc~====~ag~c~==~====== SCJre ...,.....,..., ::..~~gch -= .J f t.:'(De~: = ~.6e-06 ? -= 4.6e-06 S3980 ~7~~~~~GGC~~~~C~C~~~:~C:~::~C:~C:~GC~ G~~~C~~GGC77~7:7~77::~C:~C~~C:~C:~GC~ ~~~~~=as~c~~~:=~~~~~~=~~:~~~~~=~~~~c= Score Score 130 l~:1gr..h = 44 ::x'Pe~: ='Ue-1)3 Sum-Stac ?(2) = 2.7e-23 ~GC~GCAC~C:S7~~CC~7~~G~~~~~~~~C:~~GC~C~C~~C~ AGC~GC~CGc:~~GC~~7~GAAA~: ~c~c~c GC~ ~qc~~cac=c~~~:~c~=~=a~aaa~~~C=~~~==~2~=C==~= 171 l~:1gth = 39 Expe~: = 1.ge-D1 Sum-Scat 2(3) = 1.3e-27 :"22. 5390J Best Sum Statistic for Each Similar Database Sequence :t: 1)? C::: , ~Tame ... ,....--_....... ,...~-?.-e::,-_ _ ~ ..... __..... =-::::CJ85a: ~'::1;]83S-=6 - __<'9"0,.... ....=-'-- ... "=:: '::5-=::-=:=:':'::':.=.. :~r;:.. . =.... -- Best Si.L.l1 StaIistic ?(J) = 3.3.:-29 ::"err?T.h: 11195 Dale: 19-DEC-1995 -, ". ''7 ~. -r, ~- ?? -7 'f V V~'O ""\ ... - c/'r 7\/{cscner:c.'1ia COil genes/or .ap;.. I_/l. feI/v, "C1.T,'1... 1'-'-/_, L;inj fcqL "C17.v, (afO and Y2,T? 3eSL Sll.rrl SC21:STIC ?(~~: = 1.3e-2i f....:ng-:h: 91~30 DaLe: lO-_-\..PR-1996 ::SC?1e;~C?!ZQ coii JtV-"'L Scm: =:401 ["e:1g7 = 91 ::::-;;e~: =3.-!.e-19 ? =3.4e-19 F'~l.-!., .y/on.-!. Din? ~ 2 S08 ~7~GGC~~~~~~~GC~G~~7~377~~::~~C~G~C~~~C~~C~~~C~~G~~~~~C~~~~~ .l..:'':;GGC:':::;... -:::. .. TGG:''':''':'C::::_~_:"C-:G_:"'C::-: ~ ::7~C'":' -:: -:.::'~.:; '":''':''':" -::: -::-'': .:._:,-: a~~ssc~=~ac=aqc~gaac;~~~~===aac~~ac=~~=~~=~=~==~~~::=;~~~a:~ :3~ 3~GC~GG7C~GG~~~~~~GGC~7~~:~~~~~~ ,-.~_..... .... .......... .-... -- .,., .........; .:.. '''';'- ~~ ..:.. -.1..= sa9S3 ~~~c~~~~=agc~====~ag~c~==~====== SCJre ...,.....,..., ::..~~gch -= .J f t.:'(De~: = ~.6e-06 ? -= 4.6e-06 S3980 ~7~~~~~GGC~~~~C~C~~~:~C:~::~C:~C:~GC~ G~~~C~~GGC77~7:7~77::~C:~C~~C:~C:~GC~ ~~~~~=as~c~~~:=~~~~~~=~~:~~~~~=~~~~c= Score Score 130 l~:1gr..h = 44 ::x'Pe~: ='Ue-1)3 Sum-Stac ?(2) = 2.7e-23 ~GC~GCAC~C:S7~~CC~7~~G~~~~~~~~C:~~GC~C~C~~C~ AGC~GC~CGc:~~GC~~7~GAAA~: ~c~c~c GC~ ~qc~~cac=c~~~:~c~=~=a~aaa~~~C=~~~==~2~=C==~= 171 l~:1gth = 39 Expe~: = 1.ge-D1 Sum-Scat 2(3) = 1.3e-27 :"22. 5390J 23.13 9.42 6.56- 4.36 2.32 2.03 , 2 3 needs to be accomplished before a definitive explanation for this sequence can be reported. Finally, as an overview figure 31 shows the location of the qa-lS-qa-lF intergenic region of N. africana, while figure 32 shows all of the start sites of sequencing performed within the original 3.8 kb insert contained within the plasmid pR1. XI. Construction of the M13mp18 Subclone Plasmid pSB2 The Southern blot analysis of plasmid pRl (Figure 14) and the subclone plasmid pRX1 (Figure 18) and plasmid pRX2 (Figure 20), along with the sequence analysis of the subclone plasmid pRPl (Figure 27) provided evidence to the location of the qa-1S-qa-1F intergenic region of N. africana (Figure 31). The entire qa-lS-qa-lF intergenic region is believed to be contained within the 2.000 kb Xhol/Sacl fragment (Figure 20B, lane 2), and most within the 1.600 kb BamHlISacl fragment (Figure 20B, lane 4) produced by the subclone plasmid pRX2. With this information, the 1.600 kb BamHlISacl fragment was isolated and ligated into an M13mp18 vector (Figure 33). The resulting subcloned plasmid pSB2 was then transformed into E. coli JMI0l, and directly electrophoresised (Figure 34, lanes 2, 3, and 4) and compared to a control (Figure 34, lanes 1 and 5) to ensure that the 1.600 kb insert was successfully ligated into the vector. This was observed as the shift in size seen in figure 34. 127 needs to be accomplished before a definitive explanation for this sequence can be reported. Finally, as an overview figure 31 shows the location of the qa-lS-qa-lF intergenic region of N. africana, while figure 32 shows all of the start sites of sequencing performed within the original 3.8 kb insert contained within the plasmid pR1. XI. Construction of the M13mp18 Subclone Plasmid pSB2 The Southern blot analysis of plasmid pRl (Figure 14) and the subclone plasmid pRX1 (Figure 18) and plasmid pRX2 (Figure 20), along with the sequence analysis of the subclone plasmid pRPl (Figure 27) provided evidence to the location of the qa-1S-qa-1F intergenic region of N. africana (Figure 31). The entire qa-lS-qa-lF intergenic region is believed to be contained within the 2.000 kb Xhol/Sacl fragment (Figure 20B, lane 2), and most within the 1.600 kb BamHlISacl fragment (Figure 20B, lane 4) produced by the subclone plasmid pRX2. With this information, the 1.600 kb BamHlISacl fragment was isolated and ligated into an M13mp18 vector (Figure 33). The resulting subcloned plasmid pSB2 was then transformed into E. coli JMI0l, and directly electrophoresised (Figure 34, lanes 2, 3, and 4) and compared to a control (Figure 34, lanes 1 and 5) to ensure that the 1.600 kb insert was successfully ligated into the vector. This was observed as the shift in size seen in figure 34. 127 -- -- en ____ CD ____ 0- ____ W -- -- en -- -- en -- -- CD __ -- 0- _W -en c 0 0) Q) CD __ CD .... () c__ X -X Q)0).... Q)...... c-- __ 0- _0- l.L....- Icu 0-- - en -en I l.L.. U) U)..- ..- ..- I I I0- 0- cu cu cu 0- 0- 0- -- -- w -W ~ I ~ -- -- I -- -- I -- -- x -- -- x ..- ..-? -- ~ CD -- ~ -- -- en ____ CD ____ 0- ____ W -- -- en -- -- en -- -- CD __ -- 0- _W -en c 0 0) Q) CD __ CD .... () c__ X -X Q)0).... Q)...... c-- __ 0- _0- l.L....- Icu 0-- - en -en I l.L.. U) U)..- ..- ..- I I I0- 0- cu cu cu 0- 0- 0- -- -- w -W ~ I ~ -- -- I -- -- I -- -- x -- -- x ..- ..-? -- ~ CD -- ~ I 3.8 kb I I ....JI i I .... _.J I qa-1S-qa-1F intergenic region ~""""""""""""""""" I qa-IS I I qa-IF I I I !~ I I I !~ I I I I I I I I I I I I I I I I I I I F~~~~:::::::::: <.<.t.'".'.'".'.'.'.'.'.'.'.'.::.'.'.'.'.'.'.<.<.t.'.'.'_'".'.']. . I I I ! ! E S B x P S P E LpRP1 I pRX1------~1 I I pRB1 , ! I pRX2 --------il~--J, ! pSB2 ' r-----------------3.8kb----------------- qa-1S qa-1S-qa-1F intergenic region E s B I .... _.J I x L------+--pRX2 -+ ----J '-----pSB2-------I E PBS I I P S P I I i---+---+-------.....jl???(l(laaaaJ--It----i~~ I I I I K X H E SB2 : ~?}})})~?})>>~ I '!"; ----! I I I Digest pR1 with Bam H1 and Sac 1 I, M13mp18 Double-stranded Isolate the 1.600 kb Bam H 1/ Sac 1 fragment , I I I I f.'.D.DDD!,,~~~~ ~ ... -,'" S...' '- "",,""I'"+ Ligate fragment into cleaved vector with T4 DNA ligase ........ : Digest M13mp18 with : Bam H1 and Sac 1, ~---------------- Disrupt phages with 2% SDS and collect single-stranded plasmid [6 J G~;;-t~~~~~~~~d-~~Is-~ith--u_;,i~f;~t~.~ . E. coli JM101 lawn cells '---------' Growth produces phages containing single-stranded ,..' plasmid DNA...... ' " ... ........ " Centrifuge and save supernatant containing phages Transform into E. coli JM101 E PBS I I P S P I I i---+---+-------.....jl???(l(laaaaJ--It----i~~ I I I I K X H E SB2 : ~?}})})~?})>>~ I '!"; ----! I I I Digest pR1 with Bam H1 and Sac 1 I, M13mp18 Double-stranded Isolate the 1.600 kb Bam H 1/ Sac 1 fragment , I I I I f.'.D.DDD!,,~~~~ ~ ... -,'" S...' '- "",,""I'"+ Ligate fragment into cleaved vector with T4 DNA ligase ........ : Digest M13mp18 with : Bam H1 and Sac 1, ~---------------- Disrupt phages with 2% SDS and collect single-stranded plasmid [6 J G~;;-t~~~~~~~~d-~~Is-~ith--u_;,i~f;~t~.~ . E. coli JM101 lawn cells '---------' Growth produces phages containing single-stranded ,..' plasmid DNA...... ' " ... ........ " Centrifuge and save supernatant containing phages Transform into E. coli JM101 , 2 345 This M13mp18 vector was chosen over the standard pBluescript vector for sequencing purposes. DNA sequencing requires a single-stranded template to work correctly. Since pBluescript is double-stranded it requires denaturing to allow sequencmg. However, M13mp18 exists in a single-stranded state and eliminates this variable from the sequencing reaction, allowing for simpler sequencmg. It is hoped that sequencing of this subclone will reveal qa-lS-qa-lF intergenic sequences. However, this has yet to be accomplished. 136 This M13mp18 vector was chosen over the standard pBluescript vector for sequencing purposes. DNA sequencing requires a single-stranded template to work correctly. Since pBluescript is double-stranded it requires denaturing to allow sequencmg. However, M13mp18 exists in a single-stranded state and eliminates this variable from the sequencing reaction, allowing for simpler sequencmg. It is hoped that sequencing of this subclone will reveal qa-lS-qa-lF intergenic sequences. However, this has yet to be accomplished. 136 ? DISCUSSION Carbon catabolite repressIOn acts to regulate gene expressIOn III many microorganisms. Two examples of this are the regulation of the galactose (GAL) system of Saccharomyces cerevisiae and the quinic acid (qa) system of Neurospora crassa in the presence of a preferred carbon source. Wild-type N. crassa, grown in the presence of quinic acid and a preferred carbon source, displays a greatly reduced level of qa gene expression compared to wild-type N. crassa grown on quinic acid alone. The mechanisms which are acting to cause this repression remain unknown. However, the GAL system of S. cerevisiae may offer some explanations. Carbon catabolite repression of the GAL regulatory circuit appears to act on at least three separate levels. These include: (1) directly on the level of GAL4 activator protein, (2) on inducer levels, and (3) directly on the GAL gene promoters. The catabolite repressIOn seen in S. cerevisiae may be caused by the direct inhibition of the GAL4 activator protein. This inhibition may be an effect of the preferred carbon source: 1) directly repressing the expression of the GAL4 activator protein, 2) acting on the GAL80 repressor protein, 3) or recruiting unidentified gene products to prevent the GAL4 activator from binding to its activation sites. This same effect may be occurring with qa gene expression in Neurospora. Here, the qa -1 Factivator protein, in the presence of a preferred carbon source, may not be able to bind to its activation sites. 137 ? DISCUSSION Carbon catabolite repressIOn acts to regulate gene expressIOn III many microorganisms. Two examples of this are the regulation of the galactose (GAL) system of Saccharomyces cerevisiae and the quinic acid (qa) system of Neurospora crassa in the presence of a preferred carbon source. Wild-type N. crassa, grown in the presence of quinic acid and a preferred carbon source, displays a greatly reduced level of qa gene expression compared to wild-type N. crassa grown on quinic acid alone. The mechanisms which are acting to cause this repression remain unknown. However, the GAL system of S. cerevisiae may offer some explanations. Carbon catabolite repression of the GAL regulatory circuit appears to act on at least three separate levels. These include: (1) directly on the level of GAL4 activator protein, (2) on inducer levels, and (3) directly on the GAL gene promoters. The catabolite repressIOn seen in S. cerevisiae may be caused by the direct inhibition of the GAL4 activator protein. This inhibition may be an effect of the preferred carbon source: 1) directly repressing the expression of the GAL4 activator protein, 2) acting on the GAL80 repressor protein, 3) or recruiting unidentified gene products to prevent the GAL4 activator from binding to its activation sites. This same effect may be occurring with qa gene expression in Neurospora. Here, the qa -1 Factivator protein, in the presence of a preferred carbon source, may not be able to bind to its activation sites. 137 This may be due to the direct repression of the qa-1F activator gene or protein modifications and proteolysis of the activator by unidentified gene products. Both the GAL system and qa system encode a specific premease (GAL2 and qa-y) for their respective sugars. Within the GAL system the transport of galactose appears to be inhibited by a preferred carbon source at two levels. The first being that the GAL2 gene, which encodes the premease, IS subject to catabolite repression (Tschopp et aI., 1986) and the second IS that a preferred carbon source may interact with preexisting premeases inactivating them, a process called catabolite inactivation (Ma and Ptashne, 1987c). These same effects may occur within the qa gene cluster. Indeed this was seen, when a N. crassa strain containing a deletion of the qa-IS gene was created. This particular strain should have displayed constitutive expression of the qa genes. However, when grown in the presence of glucose alone the qa-3, qa-y, and qa-1F genes remained highly repressed (Asch and Case, unpublished data). This result suggested two things. First, that like GAL2, the quinic acid premease qa-y gene is affected by catabolite repression. Second, it seemingly disproved any thought that the qa-1S repressor protein acts on the qa-1F activator protein during carbon catabolite repressing conditions. These results when taken together suggest the possible role of yet identified gene products acting to cause repression. Finally, catabolite repression may act directly on the promoters of each system. This is the most compelling scheme 138 This may be due to the direct repression of the qa-1F activator gene or protein modifications and proteolysis of the activator by unidentified gene products. Both the GAL system and qa system encode a specific premease (GAL2 and qa-y) for their respective sugars. Within the GAL system the transport of galactose appears to be inhibited by a preferred carbon source at two levels. The first being that the GAL2 gene, which encodes the premease, IS subject to catabolite repression (Tschopp et aI., 1986) and the second IS that a preferred carbon source may interact with preexisting premeases inactivating them, a process called catabolite inactivation (Ma and Ptashne, 1987c). These same effects may occur within the qa gene cluster. Indeed this was seen, when a N. crassa strain containing a deletion of the qa -1 S gene was created. This particular strain should have displayed constitutive expression of the qa genes. However, when grown in the presence of glucose alone the qa-3, qa-y, and qa-1F genes remained highly repressed (Asch and Case, unpublished data). This result suggested two things. First, that like GAL2, the quinic acid premease qa-y gene is affected by catabolite repression. Second, it seemingly disproved any thought that the qa-1S repressor protein acts on the qa-1F activator protein during carbon catabolite repressing conditions. These results when taken together suggest the possible role of yet identified gene products acting to cause repression. Finally, catabolite repression may act directly on the promoters of each system. This is the most compelling scheme 138 for catabolite repreSSIOn. In the GAL system sequences termed upstream repression sequences (URSGAL) were found to exist between the upstream activating sequences (UASGAL) and the transcriptional initiation sites. These URSGAL sites are thought to act under catabolite repression conditions by binding unidentified repressor proteins (Erickson and Johnston, 1993). Recent experiments to find these unidentified proteins has yielded the MIG1, SSN6, and TUPI proteins. MIGI was found to bind to GAL promoters in the presence of glucose and may play a role in repression alone, or it may complex with SSN6 and TUPI (Keleher et aI., 1992). These possible interactions of carbon repressor proteins, with sequences 5' to the various GAL genes, which act to block transcription while in the presence of a preferred carbon source, may also act within the qa system of Neurospora. If similar sequences do exist within the qa gene cluster of Neurospora, they would most likely be found before the qa-3, qa-y, and the qa-lF genes. The reason for this, is that an N. crassa strain carrying a complete deletion of the qa-lS gene displayed highly repressed qa-3, qa-y, and qa-lF gene expression and slightly repressed qa-x, qa-2, and qa-lS gene expression when grown in the presence of a preferred carbon source. However, the existence of such sequences before these genes (qa-3, qa-y, and qa-lF) has yet to be determined. In an attempt to see if such sequences exist within the Neurospora qa gene cluster the qa-lS-qa-l F intergenic region of N. africana was chosen for study. This region was chosen 139 for catabolite repreSSIOn. In the GAL system sequences termed upstream repression sequences (URSGAL) were found to exist between the upstream activating sequences (UASGAL) and the transcriptional initiation sites. These URSGAL sites are thought to act under catabolite repression conditions by binding unidentified repressor proteins (Erickson and Johnston, 1993). Recent experiments to find these unidentified proteins has yielded the MIG1, SSN6, and TUPI proteins. MIGI was found to bind to GAL promoters in the presence of glucose and may play a role in repression alone, or it may complex with SSN6 and TUPI (Keleher et aI., 1992). These possible interactions of carbon repressor proteins, with sequences 5' to the various GAL genes, which act to block transcription while in the presence of a preferred carbon source, may also act within the qa system of Neurospora. If similar sequences do exist within the qa gene cluster of Neurospora, they would most likely be found before the qa-3, qa-y, and the qa-lF genes. The reason for this, is that an N. crassa strain carrying a complete deletion of the qa-lS gene displayed highly repressed qa-3, qa-y, and qa-lF gene expression and slightly repressed qa-x, qa-2, and qa-lS gene expression when grown in the presence of a preferred carbon source. However, the existence of such sequences before these genes (qa-3, qa-y, and qa-lF) has yet to be determined. In an attempt to see if such sequences exist within the Neurospora qa gene cluster the qa-lS-qa-l F intergenic region of N. africana was chosen for study. This region was chosen 139 agaIn based on the results that a N. crassa strain carrying a deletion of the qa-lS gene displayed slightly repressed qa-x, qa-2, and qa-4 gene expression and highly repressed qa-3, qa y, and qa-lF gene expression, when grown in the presence of glucose. Therefore, if sequences like the URSGAL existed within the cluster, they would most likely be found 5' to the genes which remained highly repressed when grown on glucose. Since the qa-lF gene remains highly repressed and since the sequence of this qa-1S-qa-1F intergenic region IS known in N. crassa (Geever et al., 1989) it allows comparisons to be made between the two species (N. crassa/heterothallic and N. africanal homothallic). To enable the isolation and characterization of the N. africana qa-lS-qa-lF intergenic region, a 3.8 kb fragment from the lambda clone NA3, known to contain the qa-1S-qa-1F intergenic region was isolated and ligated into a pBluescript vector, and termed pRI (Rutledge, unpublished data) (Figure 11). Plasmid pRI was then subjected to a senes of restriction enzymes to establish a preliminary restriction map of the 3.8 kb insert (Figure 13). Next, a Southern blot analysis was performed on the plasmid pR1 to localize those fragments which contained qa-lS-qa-l F intergenic sequences (Figure 14). The most interesting portion of this blot, for two reasons, was the two fragments generated by the restriction enzyme XhoI (Figure 14A, lane 3). First, the location of this restriction site essentially split the insert into two halves, and second, that both fragments hybridized the DIG-labeled probe (Figure 14B, 140 agaIn based on the results that a N. crassa strain carrying a deletion of the qa-lS gene displayed slightly repressed qa-x, qa-2, and qa-4 gene expression and highly repressed qa-3, qa y, and qa-lF gene expression, when grown in the presence of glucose. Therefore, if sequences like the URSGAL existed within the cluster, they would most likely be found 5' to the genes which remained highly repressed when grown on glucose. Since the qa-lF gene remains highly repressed and since the sequence of this qa-1S-qa-1F intergenic region IS known in N. crassa (Geever et al., 1989) it allows comparisons to be made between the two species (N. crassa/heterothallic and N. africana/ homothallic). To enable the isolation and characterization of the N. africana qa-lS-qa-lF intergenic region, a 3.8 kb fragment from the lambda clone NA3, known to contain the qa-1S-qa-1F intergenic region was isolated and ligated into a pBluescript vector, and termed pRI (Rutledge, unpublished data) (Figure 11). Plasmid pRI was then subjected to a senes of restriction enzymes to establish a preliminary restriction map of the 3.8 kb insert (Figure 13). Next, a Southern blot analysis was performed on the plasmid pR1 to localize those fragments which contained qa-lS-qa-l F intergenic sequences (Figure 14). The most interesting portion of this blot, for two reasons, was the two fragments generated by the restriction enzyme XhoI (Figure 14A, lane 3). First, the location of this restriction site essentially split the insert into two halves, and second, that both fragments hybridized the DIG-labeled probe (Figure 14B, 140 lane 2). However, the 1.330 kb fragment produced a weaker intensity than the 5.370 kb fragment (Figure 14B, lane 2). This result suggested that only a small portion of the qa -1 S-qa -1 F intergenic region existed within the 1.330 kb fragment. Based on these results, the subclones plasmid pRXI and plasmid pRX2 were produced (Figures 15 and 19, respectively). Like plasmid pRl, both subclones were then subjected to a series of restriction enzymes to generate restriction maps of their portions of the original 3.8 kb insert (Figures 17 and 21). Southern blot analysis was then performed on both subclones to establish if they both indeed contained portions of the qa- 1S-qa-1 F intergenic region of N. africana. The Southern blot analysis of the subclone plasmid pRXI revealed that none of the restriction fragments generated hybridized the DIG-labeled probe (Figure 18). This result contradicted that of the plasmid pRI (Figure 14B, lane 2) and suggested that none of the qa-1 S-qa-1 F intergenic region existed to the left of the Xho 1 site within the 3.8 kb insert. The construction (Figure 22) and subsequent sequencing (Figure 26) of the subclone plasmid pRPI provided evidence, based upon its location within the qa gene cluster of N. crassa (Figure 29), that this portion of the 3.8 kb insert contained a section of the qa-1F gene of N. africana (Figure 31). Thus the entire qa 1S-qa-1 F intergenic region had to be located in the subclone plasmid pRX2 (Figure 32). The Southern blot analysis of the subclone plasmid pRX2 (Figure 20) indeed provided evidence to support the conclusion 141 lane 2). However, the 1.330 kb fragment produced a weaker intensity than the 5.370 kb fragment (Figure 14B, lane 2). This result suggested that only a small portion of the qa -1 S-qa -1 F intergenic region existed within the 1.330 kb fragment. Based on these results, the subclones plasmid pRXI and plasmid pRX2 were produced (Figures 15 and 19, respectively). Like plasmid pRl, both subclones were then subjected to a series of restriction enzymes to generate restriction maps of their portions of the original 3.8 kb insert (Figures 17 and 21). Southern blot analysis was then performed on both subclones to establish if they both indeed contained portions of the qa- 1S-qa-1 F intergenic region of N. africana. The Southern blot analysis of the subclone plasmid pRXI revealed that none of the restriction fragments generated hybridized the DIG-labeled probe (Figure 18). This result contradicted that of the plasmid pRI (Figure 14B, lane 2) and suggested that none of the qa-1 S-qa-1 F intergenic region existed to the left of the Xho 1 site within the 3.8 kb insert. The construction (Figure 22) and subsequent sequencing (Figure 26) of the subclone plasmid pRPI provided evidence, based upon its location within the qa gene cluster of N. crassa (Figure 29), that this portion of the 3.8 kb insert contained a section of the qa-1F gene of N. africana (Figure 31). Thus the entire qa 1S-qa-1 F intergenic region had to be located in the subclone plasmid pRX2 (Figure 32). The Southern blot analysis of the subclone plasmid pRX2 (Figure 20) indeed provided evidence to support the conclusion 141 that it contained the entire qa-lS-qa-lF intergenic region. This was seen with the double digest of the plasmid with Xho 1 and Sac1. Here, the 2.000 kb fragment hybridized the DIG-labeled probe, while the 0.540 kb fragment did not (Figure 20B, lane 2). This suggested that this 2.00 kb fragment contained the entire qa- 1S-qa-lF intergenic region of N. africana. When this 2.000 kb Xhol/Sacl fragment was digested with the enzymes BamHI and Sacl, it produced a 0.400 kb fragment and a 1.600 kb fragment (Figure 21), both of which hybridized the DIG labeled probe. However, the 0.400 kb Xhol/BamHI fragment produced a weaker intensity than that of the 1.600 kb BamHI/Sacl fragment (Figure 20B, lanes 4 and 5, respectively). This result suggested that the 0.400 kb fragment contained only a small portion of the qa-1S-qa-1F intergenic regIOn. While, the 1.600 kb fragment contained most of the qa lS-qa-1F intergenic region. Based on this, the subclone plasmid pRB 1 was constructed (Figure 24). Next, sequencing of the subclone plasmid pRB 1 was conducted to try and identify the qa-1S-qa-1F intergenic sequences contained within this 0.400 kb Xho I/BamHI fragment. The sequence generated by this subclone (Figures 28) did not identify any homology to the qa gene cluster of N. crassa, in particular to the qa-lS-qa-lF intergenic region of N. crassa (Figure 29). However, a more detailed analysis of this sequence is needed before it is dismissed as not containing qa-lS-qa-l F intergenic sequences of N. africana. 142 that it contained the entire qa-lS-qa-lF intergenic region. This was seen with the double digest of the plasmid with Xho 1 and Sac1. Here, the 2.000 kb fragment hybridized the DIG-labeled probe, while the 0.540 kb fragment did not (Figure 20B, lane 2). This suggested that this 2.00 kb fragment contained the entire qa- 1S-qa-lF intergenic region of N. africana. When this 2.000 kb Xhol/Sacl fragment was digested with the enzymes BamHI and Sacl, it produced a 0.400 kb fragment and a 1.600 kb fragment (Figure 21), both of which hybridized the DIG labeled probe. However, the 0.400 kb Xhol/BamHI fragment produced a weaker intensity than that of the 1.600 kb BamHI/Sacl fragment (Figure 20B, lanes 4 and 5, respectively). This result suggested that the 0.400 kb fragment contained only a small portion of the qa-1S-qa-1F intergenic regIOn. While, the 1.600 kb fragment contained most of the qa lS-qa-1F intergenic region. Based on this, the subclone plasmid pRB 1 was constructed (Figure 24). Next, sequencing of the subclone plasmid pRB 1 was conducted to try and identify the qa-1S-qa-1F intergenic sequences contained within this 0.400 kb Xho I/BamHI fragment. The sequence generated by this subclone (Figures 28) did not identify any homology to the qa gene cluster of N. crassa, in particular to the qa-lS-qa-lF intergenic region of N. crassa (Figure 29). However, a more detailed analysis of this sequence is needed before it is dismissed as not containing qa-lS-qa-l F intergenic sequences of N. africana. 142 The Southern blot analysis of the subclone plasmid pRX2 also showed that the 0.540 kb fragment produced by the SacllXho1, BamHlISacl, and Sacl digests (Figure 20A, lanes 3,5, and 6, respectively) did not hybridize the DIG-labeled probe (Figure 20B, lanes 2, 4, and 5). Since, the Southern blot of the subclone plasmid pRXI (Figure 18), and the sequencing of the subclone plasmid pRPI (Figure 27) showed that the portion of the 3.8 kb fragment to the left of the Xho 1 site contained a section of the qa-lF gene of N. africana (Figure 31), it was thought that this 0.540 kb fragment contained a section of the qa-lS gene of N. africana. To verify this, the subclone plasmid pRX2 can be used to sequence this region of the insert. However, this has not been accomplished yet. Therefore, more analysis is needed before it can be definitively stated that this 0.540 kb fragment contains a section of the qa-lS gene of N. africana. The lack of sequence homology which was encountered with the sequencing performed was attributed to the use of the double-stranded pBluescript vector. Since, DNA sequencmg requires a single-stranded template to work correctly, the double-stranded pBluescript vector needs to be denatured to allow sequencing. In an attempt to eliminate this variable from the sequencing reaction, the single-stranded M13mp18 vector was chosen for the construction of any new subclones which were intended for DNA sequencing purposes. The first subclone to be constructed using this procedure was the subclone plasmid pSB2 (Figure 33). This plasmid was 143 The Southern blot analysis of the subclone plasmid pRX2 also showed that the 0.540 kb fragment produced by the SacllXho1, BamHlISacl, and Sacl digests (Figure 20A, lanes 3,5, and 6, respectively) did not hybridize the DIG-labeled probe (Figure 20B, lanes 2, 4, and 5). Since, the Southern blot of the subclone plasmid pRXI (Figure 18), and the sequencing of the subclone plasmid pRPI (Figure 27) showed that the portion of the 3.8 kb fragment to the left of the Xho 1 site contained a section of the qa-lF gene of N. africana (Figure 31), it was thought that this 0.540 kb fragment contained a section of the qa-lS gene of N. africana. To verify this, the subclone plasmid pRX2 can be used to sequence this region of the insert. However, this has not been accomplished yet. Therefore, more analysis is needed before it can be definitively stated that this 0.540 kb fragment contains a section of the qa-lS gene of N. africana. The lack of sequence homology which was encountered with the sequencing performed was attributed to the use of the double-stranded pBluescript vector. Since, DNA sequencmg requires a single-stranded template to work correctly, the double-stranded pBluescript vector needs to be denatured to allow sequencing. In an attempt to eliminate this variable from the sequencing reaction, the single-stranded M13mp18 vector was chosen for the construction of any new subclones which were intended for DNA sequencing purposes. The first subclone to be constructed using this procedure was the subclone plasmid pSB2 (Figure 33). This plasmid was 143 generated based on the Southern blot analysis of the subclone plasmid pRX2. As previously mentioned, the 1.600 kb BamHlISacl fragment (Figure 21), which hybridized the DIG labeled probe (Figure 20B, lane 5), is believed to contain most of the qa-lS-qa-lF intergenic region of N. africana. Therefore, it is thought that the sequencing of this subclone will reveal these qa-lS-qa-lF intergenic sequences. However, the sequencing of this subclone plasmid pRB2 has yet to be accomplished. In conclusion, the Southern blot analysis of plasmid pRI (Figure 14), subclone plasmid pRXI (Figure 18), and subclone plasmid pRX2 (Figure 20), along with the DNA sequencing performed on the original 3.8 kb insert, it is believed that the qa-lS-qa-lF intergenic region of N. africana has been isolated (Figure 32). In the future, the subclones plasmid pRB 1, plasmid pRX2, and plasmid pSB2 can be used to sequence the entire qa-lS-qa-l F intergenic region. Once this has been accomplished, the qa-lS-qa-lF intergenic region of N. africana can be compared to its N. crassa counterpart and examined for the existence of sequences 5' to the qa-lF gene acting under carbon catabolite repressing conditions. Ultimately, the qa-lS qa-lF intergenic region of N. africana will be used to replace its N. crassa counterpart to determine if the qa-lS-qa-lF intergenic sequences of N. africana can operate to cause carbon catabolite repression of the qa genes of N. crassa. 144 generated based on the Southern blot analysis of the subclone plasmid pRX2. As previously mentioned, the 1.600 kb BamHlISacl fragment (Figure 21), which hybridized the DIG labeled probe (Figure 20B, lane 5), is believed to contain most of the qa-lS-qa-lF intergenic region of N. africana. Therefore, it is thought that the sequencing of this subclone will reveal these qa-lS-qa-lF intergenic sequences. However, the sequencing of this subclone plasmid pRB2 has yet to be accomplished. In conclusion, the Southern blot analysis of plasmid pRI (Figure 14), subclone plasmid pRXI (Figure 18), and subclone plasmid pRX2 (Figure 20), along with the DNA sequencing performed on the original 3.8 kb insert, it is believed that the qa-lS-qa-lF intergenic region of N. africana has been isolated (Figure 32). In the future, the subclones plasmid pRB 1, plasmid pRX2, and plasmid pSB2 can be used to sequence the entire qa-lS-qa-l F intergenic region. Once this has been accomplished, the qa-lS-qa-lF intergenic region of N. africana can be compared to its N. crassa counterpart and examined for the existence of sequences 5' to the qa-lF gene acting under carbon catabolite repressing conditions. Ultimately, the qa-lS qa-lF intergenic region of N. africana will be used to replace its N. crassa counterpart to determine if the qa-lS-qa-lF intergenic sequences of N. africana can operate to cause carbon catabolite repression of the qa genes of N. crassa. 144 BIBLIOGRAPHY 1. Ahmed, S. I. and N. H. Giles. 1969. Organization of enzymes in the common aromatic synthetic pathway: evidence for aggregation in fungi. J. Bacteriol. 99: 231 237. 2. Alton, N. H., J. A. Havtala, N. H. Giles, S. R. Kushner, and D. Vapnek. 1978. Transcription and translation in E. coli of hybrid plasmids containing the catabolic dehydroquinase gene from Neurospora crassa. Gene. 4: 241-259. 3. Asch, D. K., M. Orejas, R. F. Geever, and M. E. Case. 1991. Comparative studies of the quinic acid (qa) cluster in several Neurospora species with special emphasis on the qa-x-qa-2 intergenic region. Mol. Gen. Genet. 230: 337 344. 4. Buam, J. A., R. F. Geever, and N. H. Giles. 1987. Expression of qa-lF activator protein: identification of upstream binding sites in the qa gene cluster and localization of the DNA-binding domain. Mol. Cell. BioI. 7: 1256-1266. 5. Buam, J. A. and N. H. Giles. 1985. Genetic control of chromatin structure 5' to the qa-x and qa-2 genes of Neurospora. J. Mol. BioI. 182: 79-89. 6. Buam, J. A. and N. H. Giles. 1986. DNase I hypersensitive sites in the inducible quinic acid (qa) gene cluster of Neurospora crassa. Proc. Natl. Acad. Sci. USA. 83: 6533 6537. 7. Beadle, G. W. and E. L. Tatum. 1945. Neurospora II. Methods of producing and detecting mutations concerned with nutritional requirements. Am. J. Botany. 32: 678 686. 8. Beri, R. K., H. Whittington, C. F. Roberts, and A. R. Hawkins. 1987. Isolation and characterization of the positively acting regulatory gene QUTA. Nucleic Acids Res. 15: 7991-8001. 145 BIBLIOGRAPHY 1. Ahmed, S. I. and N. H. Giles. 1969. Organization of enzymes in the common aromatic synthetic pathway: evidence for aggregation in fungi. J. Bacteriol. 99: 231 237. 2. Alton, N. H., J. A. Havtala, N. H. Giles, S. R. Kushner, and D. Vapnek. 1978. Transcription and translation in E. coli of hybrid plasmids containing the catabolic dehydroquinase gene from Neurospora crassa. Gene. 4: 241-259. 3. Asch, D. K., M. Orejas, R. F. Geever, and M. E. Case. 1991. Comparative studies of the quinic acid (qa) cluster in several Neurospora species with special emphasis on the qa-x-qa-2 intergenic region. Mol. Gen. Genet. 230: 337 344. 4. Buam, J. A., R. F. Geever, and N. H. Giles. 1987. Expression of qa-lF activator protein: identification of upstream binding sites in the qa gene cluster and localization of the DNA-binding domain. Mol. Cell. BioI. 7: 1256-1266. 5. Buam, J. A. and N. H. Giles. 1985. Genetic control of chromatin structure 5' to the qa-x and qa-2 genes of Neurospora. J. Mol. BioI. 182: 79-89. 6. Buam, J. A. and N. H. Giles. 1986. DNase I hypersensitive sites in the inducible quinic acid (qa) gene cluster of Neurospora crassa. Proc. Natl. Acad. Sci. USA. 83: 6533 6537. 7. Beadle, G. W. and E. L. Tatum. 1945. Neurospora II. Methods of producing and detecting mutations concerned with nutritional requirements. Am. J. Botany. 32: 678 686. 8. Beri, R. K., H. Whittington, C. F. Roberts, and A. R. Hawkins. 1987. Isolation and characterization of the positively acting regulatory gene QUTA. Nucleic Acids Res. 15: 7991-8001. 145 9. Berlyn, M. B. and N. H. Giles. 1972. Studies of aromatic biosynthetic and catabolic enzymes in Ustilago maydis and in mutants of U. violacea. Genet. Res. Camb. 19: 261-270. 10. Bevan, P. and H. C. Douglas. 1969. Genetic control of phosphoglucomutase variants in Saccharomyces cerevisiae. J. Bacteriol. 98: 532-535. 11. Case, M. E., R. F. Geever, and D. K. Asch. 1992. Use of gene replacement transformation to elucidate gene function in the qa gene cluster of Neurospora crassa. Genetics. 130: 729-736. 12. Case, M. E. and N. H. Giles. 1975. Genetic evidence on the organization and action of the qa-lF gene product: a protein regulating the induction of three enzymes in quinate catabolism in Neurospora crassa. Proc. Natl. Acad. Sci. USA. 72: 553-557. 13. Case, M. E., M. Schweizer, S. R. Kushner, and N. H. Giles. 1979. Efficient transformation of Neurospora crassa by utilizing hybrid plasmid DNA. Proc. Natl. Acad. Sci. USA. 76: 5259-5363. 14. Cha1eff, R. S. 1974. The inducible quinate-shikimate catabolic pathway in Neurospora crassa. Genetic organization. J. Gen. Microbiol. 81: 337-355. 15. Citron, B. A., and J. E. Donelson. 1984. Sequence of the Saccharomyces GAL region and its transcription in vivo. J. Bacteriol. 158: 269-278. 16. Douglas, H. C. and D. C. Hawthorne. 1964. Enzymatic expression and genetic linkage of genes controlling galactose utilization in Saccharomyces. Genetics. 49: 837-844. 17. Douglas, H. C. and D. C. Hawthorne. 1972. Uninducible mutants in the gall locus of Saccharomyces cerevlszae. J. Bacteriol. 109: 1139-1143. 146 9. Berlyn, M. B. and N. H. Giles. 1972. Studies of aromatic biosynthetic and catabolic enzymes in Ustilago maydis and in mutants of U. violacea. Genet. Res. Camb. 19: 261-270. 10. Bevan, P. and H. C. Douglas. 1969. Genetic control of phosphoglucomutase variants in Saccharomyces cerevisiae. J. Bacteriol. 98: 532-535. 11. Case, M. E., R. F. Geever, and D. K. Asch. 1992. Use of gene replacement transformation to elucidate gene function in the qa gene cluster of Neurospora crassa. Genetics. 130: 729-736. 12. Case, M. E. and N. H. Giles. 1975. Genetic evidence on the organization and action of the qa-lF gene product: a protein regulating the induction of three enzymes in quinate catabolism in Neurospora crassa. Proc. Natl. Acad. Sci. USA. 72: 553-557. 13. Case, M. E., M. Schweizer, S. R. Kushner, and N. H. Giles. 1979. Efficient transformation of Neurospora crassa by utilizing hybrid plasmid DNA. Proc. Natl. Acad. Sci. USA. 76: 5259-5363. 14. Cha1eff, R. S. 1974. The inducible quinate-shikimate catabolic pathway in Neurospora crassa. Genetic organization. J. Gen. Microbiol. 81: 337-355. 15. Citron, B. A., and J. E. Donelson. 1984. Sequence of the Saccharomyces GAL region and its transcription in vivo. J. Bacteriol. 158: 269-278. 16. Douglas, H. C. and D. C. Hawthorne. 1964. Enzymatic expression and genetic linkage of genes controlling galactose utilization in Saccharomyces. Genetics. 49: 837-844. 17. Douglas, H. C. and D. C. Hawthorne. 1972. Uninducible mutants in the gall locus of Saccharomyces cerevlszae. J. Bacteriol. 109: 1139-1143. 146 18. Erickson, J. R. and M. Johnston. 1993. Genetic and molecular characterization of GAL83: its interaction and similarities with other genes involved in glucose repression in Saccharomyces cerevisiae. Genetics. 135: 655-664. 19. Flick, J. S. and M. Johnston. 1991. Two systems of glucose repression of the GALl promoter in Saccharomyces cerevisiae. Mol. Cell. BioI. 10: 4757 4769. 20. Flick, J. and M. Johnston. 1992. Analysis of URSR mediated glucose repression of the GALl promoter of Saccharomyces cerevisiae. Genetics. 130: 295-304. 21. Geever, R. F., J. A. Baum, M. E. 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