Identification oftbe Sea Urcbin Egg Myosin Binding Protein Gene
by
Laura R. Shea, B.S.
Submitted in Partial Fulfillment ofthe Requirements
for the Degree of
Master ofScience
in the-
Biology
Program
SCHOOL OF GRADUATE STUDIES
YOUNGSTOWN STATE UNIVERSITY
AUGUST 1999
Identification ofthe Sea Urchin Egg Myosin Binding Protein Gene
by
Laura R. Shea, B.S.
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:
Student I 1
Approvals:
n J~A)()/V~~~
Committee Member
6jf3/ )"? ?
Date
Date
iii
Abstract
A novel myosin binding protein designated 53K (p53EMBP) has been identified
in unfertilized sea urchin eggs. (Yabkowitz and Burgess, 1987). Antibody against
p53EMBP was used to select recombinant cDNAs from a sea urchin oocyte library. An
approximately 1,000 base pair sequence was obtained, a very small fragment ofa much
larger gene. Based on Southern blot analysis it was found that this gene codes for a very
large rnRNA. This indicated that the gene was much larger than the approximately 1,000
base pair sequence that had been obtained thus far. In addition, some data suggest that
p53EMBP is a fragment ofa much larger protein (Yabkowitz and Burgess, 1987). The
goal ofthis study is to obtain the entire EMBP gene from sea urchin genomic DNA. Sea
urchin genomic DNA was isolated from sperm ofsea urchin species Stongylocentrotus
purpuratus. Following the isolation ofgenomic DNA, a restriction digest was done to
create appropriate ends in the DNA obtained. DNA agarose gel electrophoresis was
performed and the proper size fragments were obtained by e1ectroelution ofthe gel.
Vector DNA (A DNA) also prepared by a restriction digest was ligated to the insert
genomic DNA. Next, the ligated DNA were packaged into phage particles in vitro and
then were used to infect a E.Coli culture. The bacteria containing the genomic library
were grown on bacterial lawns in the hopes that plaques would be formed. In continuing
work on this project p53EMBP cDNA probes will be used to screen the library. This will
enabled us to obtain the gene for the sea urchin myosin binding protein designated
p53EMBP.
IV
Acknowledgments
I would fIrst like to thank with utmost appreciation my wonderful
thesis advisor Dr. Gary Walker. Without all ofDr. Walker's time, patience,
understanding, and help, the work included here in this thesis would not have
been possible. I would like to thank Dr. David K. Asch who has really been
like a co-advisor on this project for all ofhis time, advise, and help on this
project. I would like to thank Dr. Paul C. Peterson for taking time out ofhis
busy schedule as a department chair to serve on my thesis committee. I would
like to thank Ms. Renee' A. Pitts for all ofthe valuable information she
gained for this project by searching protein databases on the World Wide
Web. I would like to thank my fellow graduate students in the Walker lab,
Mr. Nader Atway, Ms. Staci Raab, and Mr. Thomas Watkins for all oftheir
support and advise. My parents, William T. Shea, and Inez P. Haddock, and
my sister, Kathleen A. Shea I would like to thank for all oftheir love and
support. For always supporting my academic pursuits I would like to thank
my uncle, Maurice J. Shea And lastly, I would like to thank Mr. Bryan E.
Mosora for all ofhis unconditional love and support.
Table ofContents
I. Abstract
Acknowledgments
List ofFigures
List ofTables
II. Introduction
The Sea Urchin Egg Myosin Binding protein
Strongylocentrotus Myosin Binding Protein
III. Materials and Methods
Experimental Design
Construction ofthe Sea Urchin Genomic Library
DNA Isolation
Restriction Digest
Density Gradient Centrifugation
Gel Purification
Ligation, Packaging, and Plating
IV. Results
V. Discussion
111
IV
Vll
IX
I
16
17
22
22
25
25
28
30
34
35
40
53
v
Future Work
VI. Literature Cited
VII. Appendix A
60
63
64
vi
List ofFigures
Figure 1. The muscle sarcomere 1
Figure 2. Myosin I and myosin II 4
Figure 3. Actin and myosin in non-muscle cells 6
Figure 4. Similarities in myosin binding proteins 13
Figure 5. Block diagram ofMyBP-C and MyBP-H 15
Figure 6. DNA Sequence ofp53EMBP 18
Figure 7. Comparison ofp53EMBP and VAP-l 20
Figure 8. Overview ofgenomic library construction 23
Figure 9. Selection ofthe gene for p53EMBP 25
Figure 10. Serial dilution ofpackaging reaction 39
Figure 11. Purified sea urchin sperm DNA 41
Figure 12. Attempted DNA isolation 43
Figure 13. DNA isolation containing impurities 44
Figure 14. Restriction digest with diluted enzyme Mho I
in a 1: 10 concentration. 45
Figure 15. Restriction digest with diluted enzyme Mho I
in a 1:50 concentration 46
vii
Figure 16. Glycerol Gradient Gel 48
Figure 17. Gel purification gel 50
Figure 18. DNA ligation gel 51
Figure 19 Bam HI digested DNA 52
Figure 20. Test ligation plate with plaques 57
Figure 21. DNA gel showing inserts 60
Vl1I
Table I
List ofTables
Selected DNA using UV absorption 42
IX
II. Introduction
The work included in this study shows how the gene for the sea urchin
egg myosin binding protein (p53EMBP)is to be cloned from genomic DNA.
In 1987 a novel sea urchin myosin binding protein was discovered in the
laboratory ofDavid Burgess (Yabkowitz and Burgess, 1987). This protein
was given the name 53kDa protein. Furthennore, 53kDa protein was shown
to effect the solubility ofmyosin at low ionic strengths. It was found by
Northern blot analysis that this protein fIrst designated as 53kDa protein
codes for a very large mRNA (unpublished data). This indicated that this
protein was much larger than the approximately 1,000 base pairs that had
been isolated.
In order to understand the background for this study it is necessary to
frrst include the structure and function ofmyosin in the muscle sarcomere
and the contractile ring ofnon-muscle cells.
n~bulin
Figure 1. The muscle sarcomere
This figure is redrawn from Alberts fig. 16-89 and fig.16~95 pg. 851 and 855.
2
Myosin is a motor protein that moves along actin filaments and is
plentiful in skeletal muscle. In skeletal muscle, it forms a major part ofthe
contractile apparatus (Alberts et al., 1994). Muscle contraction is produced
by the interaction ofmyosin filaments with actin filaments. This occurs by
the sliding ofactin filaments against myosin filaments. In the presence of
ATP, the myosin head regions (which are found at the end ofthe myosin
filaments) become attached to actin filaments. Next, a conformational change
occurs in the myosin head region. This conformational change allows the
myosin filament to encounter the actin filament and then become unattached.
Accessory proteins aid the actin and myosin in accomplishing this task.
These proteins hold the actin and the myosin in a parallel overlapping
manner. In many non-muscle cells and in smooth muscle cells, the
contraction is performed with actin and myosin in much the same way as it is
above with skeletal and cardiac muscle. However, in smooth muscle and in
non-muscle cells the contractile units are smaller. Furthermore, Ca2+
regulated phosphorylation ofa myosin light chain control the activity and
assembly in non-muscle cells (Alberts et al., 1994).
Because there are more than one type ofmyosin molecule, it is
necessary to distinguish between them. Myosin molecules comprise all ofthe
3
actin motor proteins that have been identified so far. When myosin molecules
bind to actin filaments they hydrolyze ATP into ADP and Pi. This is how
myosin was fITst identified in skeletal muscle. The myosin found in muscle is
called myosin II. The myosin II molecule has two heads and a rod like tail.
The head region contains the motor activity as well as the ATPase activity.
Myosin II has two heavy chains each ofwhich becomes a pair oflight chains
at the head region. The function ofthe myosin II molecule is to move actin
filaments past each other. The function ofthe tail region ofthe myosin II
molecule is to permit polymerization ofthe molecules into bipolar filaments.
Myosin II is also found in the cell cortex as well as the contractile ring ofcell
division. Furthermore, myosin II is thought to be responsible for the tension
in stress fibers and the tension in which to keep the cell surface frrm (Alberts
et aI., 1994). It should also be mentioned that Ca2+ dependent
phosphorylation ofthe myosin II molecule increases its interaction with actin
as well as enables its assembly into short bipolar filaments (Alberts et al.,
1994).
Also found in non-muscle cells is a type ofmyosin known as myosin I.
This type ofmyosin is smaller than myosin II. It is thought that myosin I is
the predecessor from which myosin II originated. Myosin I like myosin II
also has a motor head region as well as the ability to hydrolyze ATP (Alberts
4
et al., 1994). The structure ofthese two molecules can better be seen in figure
two.
N terminus
Jight chains
~1YOSI N 11
MYOSI N I
Figure 2. Myosin I and myosin II. This fig. is redrawn from Alberts et al., fig. 16-69 and fig. 16 
70 (This figure is not drawn to scale)
Itwas first thought that the muscle thick filament was bare. However,
in the 1970's a entire class ofproteins were found to associate with the
myosin. This was the beginning ofthe discovery ofmyosin-binding proteins
(Gautel, 1996). At fITst these proteins were thought to be contaminants of
myosin preparations. Later it was found that these proteins make up almost
four percent ofthe mass ofthe myofibril (Seiler et al., 1996). Most ofthe
myosin binding proteins identified have been in skeletal and cardiac muscles.
The fITst myosin binding proteins found were C-protein, H-protein, and X-
protein. C-protein and H-protein are now known as MyBP-C and MyBP-H
respectively. X-protein is now thought to be the slow isoform ofC-protein
5
and not considered a separate myosin binding protein (Gautel, 1996). In
conjunction with the thick filaments ofmuscle sarcomeres, the myosin
binding proteins: titi~ nebulin, myomesin, 86-kDa protein (MyBP-H), and
MyBP-C have been identified (Yabkowitz and Burgess, 1987).
However, myosin also plays a large role in non-muscle cells (Alberts et
aI., 1994). Myosin binding proteins also exist in non-muscle cells. This is
because myosin undergoes changes in distribution and polymerization in cells
and it is believed that the myosin binding proteins regulate the "super
molecular organization" ofmyosin in the cytoplasm (Yabkowitz and
Burgess, 1987). In the cytoplasm ofeukaryotic cells, actin is known to be the
most abundant protein. It comprises five to ten percent ofthe total protein in
eukaryotic cells. In addition, myosin makes up about one-fourth ofthe
amount ofactin (Voet, 1995). Studies have shown that in some eukaryotic
cells actin and myosin II come together for very short periods, accomplish a
task, and then disassemble. This has been shown to occur in cell division. In
this case, actin and myosin II filaments form a structure called a contractile
ring. This contractile ring aids in cytokinesis by appearing during M phase
ofcell division. The contractile ring pulls on the plasma membrane and helps
to contract the middle ofthe cells thereby forming two daughter cells (Alberts
et al., 1994). After cell division is complete the myosin II molecules separate
(Alberts et al.,1994).
Stress fibers in fibroblasts are another example ofthe existence of
actin and myosin in non-muscle cells. Here they are smaller and not as
organized as in muscle cells. Stress fibers attach to the plasma membrane at
focal contacts and at intermediate filaments that surround the cell nucleus.
The stress fibers permit the cells to apply tension to collagen molecules
around them (Alberts et al., 1994).
In epithelial cells, actin and myosin also playa role. Actin filaments
stretch across the cytoplasm from cell to cell junction. Tension is formed
across a "multicellular sheet". Also, in epithelial cells actin and myosin are
found in adhesion belts. In adhesion belts they fold epithelial cell sheets in
the developing embryo (Alberts et al., 1994). See figure 3 below.
6
Dividing Cell Epithelial Cell Fibroblast Cell
_ contractIle nng ~
(ca" teJ adheSion~ , 1'\, ./.~ belt ~ '[
~ /~ stress fiher
Figure 3. Actin and myosin in non-muscle cells. This figure is redrawn from Alberts et aI., fig.
16-72.
7
General Description ofMyosin Binding Proteins
Titin (which was fIrst known as Connectin) is the largest protein
known to date with its molecular weight of3000 kiloDaltons. Titin is the
third most abundant myofibrillar protein. It should be noted that it is third
only to actin and myosin (Wang, 1996). In skeletal muscle titin molecules
extend from the thick filaments M line to the Z line (see fig.l)(Alberts et aI.,
1994). This means that titin is over IlJ,m in length (Higgins et aI., 1994). It
is believed that titin molecules keep the myosin thick filaments centered
(Alberts et at, 1994) and provides the resting tension in the muscle
sarcomere (Ayme-Southgate et aI., 1991).
Due to the extremely large molecular weight oftitin, it took a longer
time to discover it. It wasn't until researchers reduced the acrylamide
concentration ofprotein gels from between 6% and 15% to only 2% that titin
was discovered. The titin molecule has a rod with a beaded substructure. This
is due to the Ig and Fn III domains ofthe titin molecule (Labeit et aI., 1997).
It has been shown that between six and twelve titin molecules associate with
each muscle thick filament. At the point where titin molecules attach to the
thick filament, it is believed to be the place offilament assembly (Eilertsen et
aI., 1994).
8
Titin is also believed to exist in non-muscle cells. It is thought to play
a role in the organization ofthe cytoskeletal myosin II filaments (Keller,
1995). It was first discovered in non-muscle cells as a protein called T 
protein. T-protein had the same molecular weight, molecular morphology,
and immunocrossreactivity as titin. Therefore, it is believed to be the non 
muscle cell isoform oftitin (Eilertsen et al., 1994).
The brush border ofisolated intestinal epithelial cells is an excellent
place to characterize cytoskeletal components and this is where the non 
muscle cell isoform oftitin was discovered. Here, the non-muscle cell
isoform oftitin is found in a region called the terminal web region.
Moreover, it is here at the terminal web region that titin could playa role in
the association ofmyosin II with the cytoskeleton (Eilertsen et al., 1994). In
studies by Eilertsen et al., it was found that only one non-muscle cell titin
molecule associates with one bipolar filament. This is in contrast to the
already noted six to twelve titin molecules that associate per muscle thick
filament (Eilertsen et al., 1994).
It is also worthy to note that Eilertsen et al. found that titin-myosin
interactions are isoform specific. He noted that non-muscle cell titin would
not bind to muscle myosin and muscle titin would not bind to non-muscle cell
myosin. This supports the theory that non-muscle cell titin is a factor in
9
cytoskeletal organization ofmyosin II bipolar filaments (Eilertsen et aI.;
1994).
Titin is not only found in vertebrate muscles and the cytoskeleton but
also mini-titins are found in invertebrate muscle. These mini-titins include the
C. elegans protein, twitchin, and the Drosophila protein, projectin. Myosin II
filaments are found to associate with all ofthe known titins. However, the
role that each ofthe titins play in muscle structure and function may differ
(Keller, 1995).
Twitchin
As already mentioned one ofthe mini-titins is known as twitchin. Both
titin and twitchin contain large numbers oftwo-conserved amino acid motifs
that occur in "regular arrays" (Ayme-Southgate et al., 1991). One ofthe
motifs is similar to the fibronectin type III domains and the other is similar to
immunoglobin C2 domains (Ayme-Southgate et al., 1991). The sequences of
these two proteins show marked similarities. Not to mention the fact that
they both have a protein kinase domain near the C-terminus. Even though
there has been remarkable similarity between titin and twitchin sequences,
their roles in function appear to be different (Higgins et aI., 1994). Animals
that lack the gene for twitchin have a constant twitching ofthe body wall
10
muscles. This shows that twitchin may be involved in the
contraction/relaxation cycle ofmuscle (Ayme-Southgate et aI., 1991).
New members ofthe Ig superfamily
Because ofits Ig domains, twitchin was the fITst intracellular protein
that was considered a member ofthe Ig superfamily. Now, due to the
reoccurring Ig and Fn III domains a whole "family" ofintracellular and
muscle proteins have added a new branch to the Ig superfamily (Benian et al.,
1996). This "family" can be broken down into groups based on their location
in the sarcomere and their function. The fITst ofthese groups is the already
mentioned titin family and consists oftwitchin, titin/connectin, and projectin
(Benian et al., 1996). The second group includes telokin ofsmooth muscle,
and MLCKs (myosin light chain kinases) ofsmooth muscle and non-muscle
cells. It should be noted that the phosphorylation by MLCKs is necessary in
order to initiate contraction in smooth muscle and in non-muscle cells.
Moreover, telokin has only one Ig domain. Furthermore, the third group in the
new branch ofthe Ig superfamily consists ofC-protein and 86-kDa protein
(MyBP-H)(Benian et aI., 1996). The proteins ofthis group can be found at
the A-band ofvertebrate striated muscle. Specifically, to the inner third of
the A-band at transverse stripes of43 11m spacing. A fourth group is a single
11
protein called kettin (Benian et aI., 1996). Kettin is not associated with
myosin or the thick filament. In addition, the last group includes M-protein,
skelemin, and myomesin. All the proteins ofthe last group are located at the
M-line in vertebrate striated muscle (Benian et al., 1996).
Nebulin
Another well-known giant myosin binding protein is nebulin. Nebulin
has a molecular weight of600-900 kDa. There are 150-200 repeating
structural domains in a nebulin molecule. Each one ofthese domains
interacts with actin molecules in the thin filament. Nonetheless, nebulin is
known to associate with the thin filament only in vertebrate skeletal muscle
(Keller, 1995). The nebulin molecule extends from one end ofthe actin thin
filament to the other and during muscle development regulates the assembly
ofactin and length ofthe actin thin filament (Alberts et al., 1994). Nebulin is
not known to exist in non-muscle cells. Nevertheless, because ofthe
regulation ofactin filament length there may be a possibility that nebulin
could exist in non-muscle cells. A protein called N-protein was discovered in
non-muscle cells that had a comparable molecular weight to that ofnebulin.
However, unlike the cellular isoform oftitin it was later found that it had
properties that were different from nebulin (Keller, 1995).
12
Projectin
Another ofthe mini-titins is a protein similar in size to twitchin. It is
the Drosphila protein known as projectin. It has been identified in the
connecting filaments ofinsect flight muscle (Ayme-Southgate et al., 1991).
Partial sequences ofprojectin shows the same repeat patterns as sequences of
twitchin. Therefore, it is thought that projectin is the insect homologue of
twitchin. The partial sequences that show the same repeat patterns can be
precisely aligned with sequences oftwitchin (see fig.4) (Higgins et al., 1994).
When analyzed in honeybees, a single molecule ofprojectin extends from Z
line to the thick filament (Benian et aI., 1996). In Drosophila, in the A band,
the myosin binding protein projectin is located. Moreover, in the Drosophila I
band a smaller projectin molecule is located. So, these two isoforms for this
protein are both derived from one gene (Benian et aI., 1996) by differential
RNA splicing.
13
t1~m super re;:>e3t
-Ii[anv:o:m:u] x b IT: -
;'-I",i
1;'3 >If .. ,,. 't1"fIIiHj"'I'r~njil~.sI/.?J':':J.J~ J~.a .. .J"
Titin =D:"rrr]ILTl[IJ]!r.tLlm[I"JiN_~ -
ml?;n
l\vitcbin
Projectin
o class 1 domain (fit,ronectin I! I motif)
II class 11 domain (Ig C2 motif )
~ protem kiro~..e domain
Figure 4. Similarities in myosin-binding proteins. This figure is redrawn from Higgins et al. fig
1. Layout ofFn II, Ig domains, and kinase domains. The first titin sequence is a series ofeleven
domain super repeats from the center ofthe protein. The second lies towards the C-terminus. The
titin domains from the super repeat are numbered one through sixty-nine. The class I and class II in
the second titin sequence are numbered m1 to m27.
Myomesin
Myomesin anchors titin in the region ofthe M-band (van der Yen and
Furst, 1997). Myomesin has a molecular weight of 185-kDa (Grove et al.,
1985). It is thought to playa role in maintenance and assembly ofthe
myofibril (Eppenberger et al., 1981) and in myosin thick filament
organization and alignment (Yabkowitz and Burgess, 1987).
14
Myosin-binding protein-M
Myosin-binding protein M anchors titin in the region ofthe M-band
(van der Ven and Furst, 1997). MyBP-M has a molecular weight of 165-kDa
(Grove et al., 1985).
Myosin-binding protein-C
MyBP-C is localized to the A-band ofstriated muscle. The region in
the A-band that it is localized is called the C zone. It is called the C zone due
to the fact that MyBP-C is localized there (Vaughan et al., 1993). The C zone
is the cross-bridge bearing zone. MyBP-C and MyBP-H have remarkable
similarities including structural similarities. These two proteins are highly
charged. Ultraviolet spectra and amino-acid composition are very similar in
MyBP-C and MyBP-H. Both MyBP-C and MyBP-H are rich in proline
residues (Vaughan et al., 1993). However, these two proteins show different
distribution in the A-bands.
Depending on the muscle which it is isolated MyBP-C has different
numbers ofphosphorylation sites. In the isoform found in cardiac muscle
there are four phosphorylation sites. Furthermore, these sites are kept under
control with help from cAMP and calmodulin regulated kinases. Whereas, in
skeletal muscle there is only one phosphorylation site, this could indicate a
regulatory function for the phosphorylation sites in cardiac muscle (Weisberg
15
and Winegrad, 1996). Fast and slow isoforms ofMyBP-C have been
identified. No isoforms ofMyBP-C have been identified in non-muscle or in
smooth muscle (Seiler et aI., 1996). MyBP-C could playa role in myosin
thick filament organization and alignment (Yabkowitz and Burgess, 1987).
Myosin-binding protein-H
Myosin-binding protein-H is the protein formally known as 86 kDa
protein. MyBP-H is also localized to the C zone ofthe A-band ofstriated
muscle. Both fast and slow isoforms have been identified in MyBP-H (Seiler
et al., 1996). Not unlike MyBP-C and myomesin, MyBP-H is thought to play
a role in myosin thick filament alignment and organization (Yabkowitz and
Burgess, 1987).
'",11 ':rr::
Figure 5. Block diagram representing motifstructure ofMyBP-H and MyBP-C. This fig. is
redrawn from Fig. 5 in Vaughan et aI. Note the conserved motifstructure ofe-terminal ofeach
molecule.
16
The sea urchin egg myosin binding protein
The 53 kDa myosin binding protein found in sea urchin eggs
(Yabkowitz and Burgess, 1987) is different from the skeletaI myosin binding
proteins in a number ofways. The 53 kDa protein (p53EMBP) binds to
myosin in a nucleotide dependent manner. Furthermore, it effects the
solubility ofmyosin at low ionic strengths (Yabkowitz and Burgess, 1987;
Walker et aI., 1991). This is believed to be because ofthe area in which the
p53EMBP binds to the myosin molecule. The head-rod junction ofthe
myosin molecule is important in myosin structure and function. Hence, any
proteins that bind at this region are likely to effect myosin's function (Walker
et aI., 1991). It was found that the p53EMBP binds to myosin at the head-rod
junction region (Walker et aI., 1991). Therefore, showing participation in
myosin regulation because this is an important area in the regulation of
myosin (Walker et aI., 1991). This is consistent with p53EMBP effects on
the solubility ofmyosin.
Antibody against p53EMBP was used to select recombinant cDNAs
from a sea urchin oocyte cDNA library. The major recombinant 53 kDa
protein (p56rEMBP) studied so far contains a number ofglycine rich
domains (unpublished data). Furthermore, the 53 kDa cDNAs contain a
17
Casein kinase phosphorylation site (Ck2) and ATP binding motifs
(unpublished data). The p56rEMBP also shows protein kinase activity similar
to that demonstrated for p53EMBP (unpublished data).
It is believed that the 53kDa obtained thus far is only a fragment ofa
very high molecular weight protein. Only a fragmentary sequence ofthis
gene has been obtained thus far. This 1,000 base pair sequence is only an
extremely small piece ofa much larger gene. Northern blot analysis indicates
that this gene codes for a very large mRNA (unpublished data). This
indicates that the gene is much larger than the approximately 1,000 base pair
sequence that has already been obtained. In addition, some data suggest that
the 53kDa protein is a fragment ofa much larger protein (Yabkowitz and
Burgess, 1987). This study is interested in obtaining and preliminary
characterization ofthe entire EMBP gene.
SPMBP: Strongylocentrotus Myosin Binding Protein
Amino acid sequence analysis was done on the world wide web using
the DNA sequence obtained for a portion ofthe myosin binding protein
(figure 6). This allowed for the comparison of the EMBP sequence to other
known proteins. As shown in figure 7 this sequence is both rich in the amino
acid glycine and has repeated sequences (unpublished data).
18
gaattccgat attgagagac ctgaccttga tgtcagtggg gatgcagacc
ttccatcagg aggagttggc ctggatgttg gaggagggat cggaggcgga
ctcggtggag gactagacat tgatgccaat ggtcctgatg ttgacatcaa
ggggccaaaa gttggaggtg acatctcagg .cccagacct tgatgtgagt
ggacccgatc tggatatcga tgtagatgga aagaaaaagg gaaaaggtgg
atttggattt ggaatgaaaatgcccaaa????.....???????ttcggat
ttggaggcca tggcaaaggt gatattgacg tagatgcaga cgttgatatt
gagagacctg accttgatgt cagtggggat gcagaccttc catcaggagg
agttggcctg gatatcggtg gtggagctgg aggtaatatt ggaatt..?..
Figure 6. The cDNA sequence from a p53EMBP clone. This was used as the starting
point for searching protein database SWISS-PROT
Following the search ofthe SWISS-PROT database punitive homologs
ofp53EMBP were identified. A vesicle associated protein (VAP-I) was one
ofthe homologs identified. The VAP-I protein was cloned from the sea
urchin species Strongylocentrotus Purpuratus the same species that
p53EMBP was been identified in. The characteristics ofthe protein known as
19
VAP-I include: tissue specificity in the egg cortex, contains four RNA
recognition motifs (RNP), the subcellular location is initially a peripheral
membrane protein that is associated with the microsomal membrane fractions
and that it may be targeted to the nucleus later in development, and it may
function as a multidomain RNA-binding protein and playa role in nuclear
RNA processing and in early development (Barton et al., 1992).
Furthermore, a protein database was used to obtain the protein
sequence ofthe DNA sequence shown in figure 6. Shown in figure 7 part A
is the most likely amino acid sequence ofSPMBP. This is further compared
to the amino acid sequence for VAP-l shown in part B offigure 7.
20
Part A.
NSDIERPDLDVSGDADLPSGGVGLDVGGGIGGGLGGGLDIDANGPDVDIKGP
KVGGDISGPDLDVSGPDLDIDVDGKKKGKGGFGFGMetKMetPKFGFGGHGK
GDIDVDADVDIERPDLDVSGDADLPSGGVGLDIGGGAGGNIGI
Part B.
TDIGGGLDVG GGLRGGLDID AKGPDVDIKG PKVGGDISGP DLDVSGPDLD
IDGGGKKGKG GFGFGLKMPK FGFGGHGKGD IDVDADVDIE
RPDLDVSGDA DLPSGGVGLD VGGGIGGGLG GGLDIDANGP DVDIKGPKVG
GDISGPDLDV SGPDLDIDVD GKKKGKGGFG FGMKMPKFGF
GGHGKGDIDV DADVDIERPD LDVSGDADLP SGGVGLDVGG GIGGGLGGGL
DIDANGPDVD IKGPKVGGDI SGPDLDVSGP DLDIDVDGKK KGKGGFGFGL
KIPKFMDPTF GFGGHGKGDI DVDADGGVVI PEGDIKVKTG KPDIGGDVDL
PSGGVDLDVG GGIGGGLGGG LDIDAKGPDV DIKGPRVGGD ISGPDLDVSG
PDLDIDGDGK KKGKGGFGFG LKMPKFGFGG HGKGDIDVDA
DVDIERPDLN VSG
Figure 7. Comparison ofEMBP and VAP-l. Comparison ofthe amino acid sequence thought to be the
most likely representation ofEMBP (Shown in Part A) and the known sequence for the protein known as
VAP-l (Shown in Part B). The sequences that are the same in each ofthe protein sequences are underlined.
Specific Aim ofthis study
Once the entire gene is obtained for the EMBP then it can be
confmned that p53EMBP is homologous to the protein known as VAP-I.
However, this is unable to be known for certain until the entire gene for
p53EMBP is obtained. This study has two primary aims. The ftrst one is to
construct a genomic DNA library from sea urchin sperm DNA. The second
one is to identify and isolate the entire gene for p53EMBP.
21
22
III. Materials and Methods
Experimental Design
The first specific aim ofthis project was to construct a genomic
DNA library from sea urchin sperm DNA (this can be seen in figure
8). This was to be done by first isolating genomic DNA from sea
urchin sperm. A restriction digest was then performed on the genomic
DNA using a enzyme in order to cut the DNA into certain size pieces.
The isolated genomic DNA fragments that are cut by the restriction
digest needs to be incorporated into a vector. In this project a
bacteriophage lambda vector was used. The bacteriophage lambda
vector also has to be cut with the same restriction enzyme as the
isolated DNA in order to enable the sticky ends to be ligated together.
This creates left and right arms ofthe lambda vector. The central
portion ofthe lambda vector is not needed and can be discarded. The
arms and restriction digested DNA are then ligated together. Once the
ends are ligated together the ligated DNA can then be packaged into
phage heads. This creates a library ofgenomic DNA within the
phages. The phage heads are then used to infect a E.Coli culture. The
E. Coli is then plated out on agar plates. The E. Coli will then grow a
23
confluent bacterial lawn on the agar~ Ifthe ligated DNA has been
incorporated into the E.Coli, plaques will be obtained. The presence of
a phage plaque on a bacterial lawn indicates a recombinant phage
bearing a insert. Once plaques are obtained the library can be screened
using aDNA probe ofthe gene ofinterest. In this case the gene of
interest is p53EMBP (this can be better seen in figure nine).
right arm
(
Digest with
enzyme
Bactcriophagl; Avector
/.,.--I--+-~\-
right arm
~leI>;
leli arm
Ligate
nRestr.ictionV
Digest----
-------
Package into
phages
Library ofgenomic DNA
D.Infect E. Coli
Plaques.. ~-::::7
Screen Libnu)'.. -
~ c:::::-==t> .With probe
Figure 8. Overview ofgenomic library construction
Once plaques have been obtained, the absorption ofthe phage DNA
to nitrocellulose will occur by placing the nitrocellulose on the surface
24
ofthe plates containing the plaques. The phage protein is then
dissolved with NaOH leaving the recombinant DNA denatured so that
it can stick to the filter. The filter is then incubated with a 32p labeled
probe. In this case the probe is from two EMBP cDNAs. They will be
allowed to hybridize to complimentary sequences on the nitrocellulose
and the position ofthe clone having the DNA we are looking for can
be revealed by autoradiography. Now the desired clone can be selected
and manufactured in a bacterial host.
Nitrocellulosl: tiltet"
Autoradiograph to
locate the desired clone
Desired done-_-_-J
/--------~---...
incubate filter with / .. .~
probe \/._ .)L_=-=C> .. ,I
'" . . /" I
_.._-~~
tiller
(
film
Figure 9. Selection ofthe gene for p53EMBP.
25
Construction ofthe sea urchin genomic library
DNA Isolation
In order to begin the creation ofthe sea urchin genomic library, the
first step was to isolate the DNA. The source ofthe sea urchin genomic
DNA was sperm from the sea urchin species Strongylocentrotus
purpuratus. This procedure has been performed according to the protocol
9.16 from Molecular Cloning, Analysis and Cloning ofEukaryotic
Genomic DNA (Maniatis et al., 1989). The sea urchin sperm used was
collected and frozen. The sperm was taken out ofthe freezer and thawed
by placing the conical tube containing the sperm in a beaker ofwarm
water. The amount ofsperm to be used for the isolation was weighed to
calculate its weight in grams. The amount ofsperm used in the isolation
was approximately 3 grams. The sperm was placed into a Erlenmeyer
flask and 40 milliliters ofextraction buffer was added. Next, the sperm
and extraction buffer (see Appendix A) solution was pippetted up and
down using an electric pippetter. This was to break up the mass ofsperm
which was clumped together. The sperm and extraction buffer solution
was then placed into a 37?C incubator for a period ofone hour. After
removing from the incubator the proteinase K was added to a
26
concentration of 10mg/ml. Therefore, I ml ofproteinase K was added.
The Erlenmeyer flask containing the solution was then placed into a
incubator/shaker for three hours at 50?C (Maniatis et al., 1989).
Proteinase K was then added and placed into the incubator/shaker at 50?C
for three hours (Maniatis et al., 1989). The mixture as agitated every few
minutes to resuspend. Following the three hour incubation the solution
was placed into two 50 ml conical tubes. An equal volume ofphenol was
added to each ofthe solutions in the iJJcubator (Maniatis et al., 1989). The
conical tubes were then turned end over end for ten minutes to mix the
two phases and then centrifuged for thirty minutes at 3600g. The aqueous
phase was extracted offofeach ofthe conical tubes and placed into a new
conical tube. The phenol extraction was repeated to a total ofthree times.
Then 0.27 volumes of7.5M ammonium acetate were added (5.4 mls to
each conical tube because each contained approximately 20 mls). The
solutions in the two conical tubes were then added back together into a
Erlenmeyer flask. In addition, two volumes of100% ethanol at room
temperature were added and swirled (Maniatis et al., 1989). The DNA
precipitated immediately. The amount ofthe isolated DNA filled the flask
and was then removed by a glass hook which was made by melting a glass
27
pipette (Maniatis et al., 1989). The DNA was placed into a new conical
tube and 70% ethanol was added to 50mls. The DNA was centrifuged for
eight minutes at 3600g. The ethanol was decanted and more 70% ethanol
was added to a volume of50mIs. This was centrifuged at 3600g for eight
minutes. After the ethanol was decanted the pellet was allowed to dry
(but not completion) by leaving the tube open (Maniatis et aI., 1989). Five
mIs ofTE (pH 8.0)were added to the conical tube and the DNA was
allowed to dissolve. The DNA in TE was allowed to rock on the moving
platform for twenty-four hours. An aliquot ofisolated DNA was then run
out on both a 0.3% agarose gel (see Appendix A) and a 1% agarose gel
(see Appendix A) assess the size and state ofthe DNA (Maniatis et al.,
1989). Due to the high concentration ofthe isolated DNA, one MI ofDNA
was placed into ten MI ofTAB. And then one MI ofthat was loaded into
three separate wells ofeach ofthe two gels (This can better be seen in
figure 11 ofthe results section).
In separate DNA isolations another method was also followed in order
to attempt to obtain DNA with the greatest purity and largest size.
Following the third phenol extraction, the aqueous phase was allowed to
dialyze against four liters ofTE (pH 8.0) (see Appendix A). This was
28
done by taking the aqueous phase and pippetting it into a dialysis bag
which was then clamped at both ends. Enough room was left in the
dialysis bag to allow the solution to increase by 1.5 - 2.0 fold. The dialysis
bag was placed into one liter ofTE in a beaker containing a stir bar. The
solution was allowed to dialyze for approximately twelve hours while
stirring. This procedure was followed until the four liters ofTE had been
used to dialyze the solution containing the DNA (Maniatis et al., 1989).
Restriction Digest
The next step in the preparation ofthe DNA library was to digest the
DNA using restriction enzymes. Two different methods were used to digest
the sea urchin genomic DNA. At fIrst a partial digest ofthe sea urchin
genomic DNA was completed. Lastly, a complete digest ofthe sea urchin
genomic DNA was accomplished.
Partial restriction digest ofthe isolated genomic DNA
Partial digestion ofsea urchin DNA using the restriction enzyme Mho
I was done (Mho I cuts at GATe) (Maniatis et al., 1982). This procedure
was at fIrst performed using only a very small amount ofDNA. Once the
optimum partial digest conditions were established, the digestion was
necessarily scaled up. The enzyme was diluted for the small scale partial
digest using a dilution buffer (see Appendix A). At fIrst the partial digest
29
was performed diluting the enzyme Mho I in a 1: 10 concentration. This
was done by taking one III ofthe enzyme and diluting it in nine Ills of
dilution buffer (see Appendix A).This proved not to be dilute enough.
Therefore, enzyme Mbo I was then diluted in a 1:50 concentration by
taking 49111 ofthe dilution buffer and adding 1III ofthe concentrated
enzyme Mho I. Next an eppendor~ tube ofthe following was made: 1 J..lI
ofthe DNA isolated earlier, 1 J..lI ofthe 1:50 dilution ofenzyme Mbo I, 2
III of lOx Rxn buffer (obtained from Promega), and 16 J..lI ddH20.
The eppendo~ tube containing the above was then placed in the
incubator at 37?C for 5 minutes. After precisely 5 minutes the eppendor~
tube was placed into a hot water bath set at 65?C. Next, four J..lI ofstop buffer
was added to the eppendor~ tubes and the DNA digested by Mbo I was run
out on a 0.3% agarose gel. After the optimal time point and dilution ofthe
enzyme was determined the conditions were scaled up and a large scale
digestion ofthe DNA was completed.
The digest was then performed again by scaling up the smaller scale
digestion. However, at fITst when these conditions were employed and the
DNA was viewed on a long DNA gel after being fractionated on a glycerol
gradient (see below) the amount ofDNA used was too little to view. So,
30
when scaling up some alterations were made. The amount of dH20 used
was replaced by DNA. The conditions used were as follows: 340 JlI ofthe
isolated DNA, 20 JlI ofthe 1:50 concentration ofenzyme Mbo I, and 40 JlI of
Rxn buffer C. The above ingredients were added to a eppendorf!!' tube and
immediately placed into an incubator at 37?C. After exactly 5 minutes the
eppendorf!!' tube containing the reaction was placed into a -80?C freezer. This
step was necessary to stop the reaction.
Complete restriction digest ofthe isolated genomic DNA
A complete digest ofthe isolated DNA was also attempted. A small scale
restriction digest was done using the enzyme Bam HI. Once optimal
conditions were determined then the reaction was scaled up. Added together
were: 50 JlI isolated genomic DNA, 75 Jll dH20, 15 JlIIOx buffer, and 2 Jll
enzyme Bam HI. Once added together the digest was allowed three hours for
completion by incubation at 37?C.
Density Gradient Centrifugation
This was done by fIrst making 500 mIs ofNET buffer according to the
Maniatis protocol for sucrose gradients(2.85)(See Appendix A). Then a 40%
glycerol solution and a 10% glycerol solution were made with the NET buffer
(see Appendix A). The denser glycerol (40%) solution into a centrifuge tube
31
that is placed straight in a test tube rack. Carefully, the lighter (10%) glycerol
solution was slowly pippetted on top ofthe denser glycerol solution. The
interface is visible at this point. The test tube was then closed with a rubber
stopper. Next, the test tube rack was slowly laid down on its side and the
glycerol solutions were allowed to diffuse with one another for three hours.
When the three hours were up the test tube rack was very slowly straightened
back up (Abe and Davis, 1986). The restriction digested DNA was then
added to the top ofthe gradient. A blank gradient was then weighed and
balanced with the gradient containing the restriction digested genomic DNA.
The gradients were then loaded into a swinging bucket rotor and centrifuged
for 18 hours at 26K.
Sucrose gradients were also made. A 600/0 sucrose solution and a 15%
sucrose solution were made (see Appendix A). The gradients were then
made the same as above with the glycerol gradients. The restriction digested
DNA was added to the top ofthe gradient. A blank gradient was then
weighed and balanced with the gradient containing the restriction digested
genomic DNA. The gradients were then loaded into a swinging bucket rotor
and centrifuged for 18 hours at 26K.
The next step was to fractionate the gradients. Forty eppendor~ tubes
were numbered from 1-40 for each ofthe gradients. The eppendor~ tubes
32
were then placed in order in a long row so that they would be handy for the
fractionation. The gradient to be fractionated was then clamped onto a stand
and a rubber stopper was placed onto the top ofthe test tube. An 18 gauge
needle was placed into the bottom ofthe test tube and approximately 0.5ml
fractions were collected in the numbered eppendor~ tubes.
For the fractions collected from the glycerol gradient, every other fraction
collected was run out on a 0.3% long agarose gel (see Appendix A)
overnight. Three Jlls ofstop buffer was added to the fractions to be run on the
gel. The voltage on the gel was run at 45 volts for approximately 20 minutes
until the dye ran out ofthe wells and then the voltage was turned down to 16
volts for 16.5 hours. The gel was dyed with ethidium bromide and viewed on
?
a large lightbox.
For the fractions collected from the sucrose gradient, every other fraction
was also run out on a 0.3% long agarose gel however, with the sucrose
gradient the fractions to be run on the gel had to be precipitated fIrst.
Therefore, 300JlI ofeach ofthe fractionated samples ofthe sucrose gradient
had two volumes of 1000/0 ethanol added to them in order to precipitate the
DNA. The fractions were then microfuged at 7000g. The supernatant was
then discarded. Thirty Jlls ofTE were placed onto each ofthe pellets and
33
placed on the shaker for ten minutes. Three J.lls ofstop buffer was added to
each ofthe samples. Lastly, twenty J.lls ofthe fractionated, precipitated DNA
was loaded onto the 0.3% long agarose gel and electrophoresed overnight for
16.5 hours. The gel was run at 45 volts for approximately 20 minutes until
the dye front was out ofthe wells and then the voltage was turned down to 16
volts. The next day the gel was stained with ethidium bromide and viewed on
a large lightbox.
Gel Purification
Once the restriction digest is completed the next step is to make sure that
the fragments to be ligated are ofa certain size range. Once the partial
restriction digest was performed, a long 0.3% agarose gel was made. The
comb which is used to make wells in a agarose gel were taped together in this
case in order to create one large trough. One hundred J.lls ofthe partially
digested genomic DNA and five J.lls ofStop buffer were loaded into the
trough. The gel was then electrophoresed overnight for sixteen and a half
hours. Starting with forty-five volts until the dye was out ofthe trough and
then turned down to sixteen volts for the remainder ofthe time. The next day
the gel was stained with ethidium bromide and viewed on a ultraviolet
lightbox. Using a razor blade a portion ofthe gel containing the high
34
molecular weight DNA was cut out. The gel containing the band ofhigh
molecular weight DNA was then placed into a dialysis bag and the ends were
clamped. The dialysis bag was then placed into a electrophoresis chamber
containing 0.5X TAE and electrophoresed at 60 volts for approximately an
hour. This step allows the DNA to be removed from the gel and into the
surrounding fluid contained in the dialysis bag. In the next step a column was
then primed with three mls ofhigh salt buffer by pushing the buffer through
the column with a syringe. Then column was then subjected to a second wash
with three mls oflow salt buffer. The DNA which was collected from the
dialysis bag was then pushed through the column with even pressure. The
DNA was then eluted from the column with four hundred fJ.ls ofhigh salt
buffer and into a eppendor~ tube. The DNA was then extracted with four
hundred fJ.ls ofphenol, microfuged at 14,000 for five minutes, and the top
layer was collected into a fresh eppendor~ tube. The DNA is next extracted
with 400 fJ.l ofchloroform, microfuged at 14,000 for five minutes, and the
top layer was then collected into a fresh eppendor:f? tube. Next, five hundred
fJ.ls ofisopropanol was added and spun for fifteen minutes, the top layer is
drawn off. The top layer was then washed one time with 80% EtOH, spun
for five minutes at 14,000, the liquid was then removed from the pellet and
35
dried. The DNA was then resuspended in twenty f..tls ofTE (Tris-EDTA).
Ligation, Packaging, and Plating
DNA Ligation
The ligation was then done by adding together the genomic DNA
fragments, the pre-digested lambda vector arms, lOx ligation buffer, and
ligase. The following concentrations were used: 2 f..tls lambda vector arms, 6
f..tls genomic DNA fragments, 2 f..tls lOx ligation buffer, I f..tls ligase, and 10
f..tls sterile H20.
Once added together the ligation mixture was then run overnight in the
PCR machine on the ligation setting at 14?C. The next day the packaging
reaction was performed.
Cultures ofE.Coli were grown in order to get ready for plating. An
overnight culture ofE.Coli strain KW392 in 2 mIs ofLB broth (see
Appendix A) was grown. Added to a sterile tube containing LB was a
toothpick which was used to scrape the top ofthe frozen glycerol stock of
E.Coli strain KW392. The tube was then placed in a incubator/shaker
overnight at 37?C. From the overnight culture the next day a two hour
culture ofE.Coli strain KW392 was grown. Five hundred f..tls ofthe
overnight culture of KW392 was placed into fifty mIs ofLB and placed into
36
the incubator/shaker at 37?C for a couple ofhours. Once the culture was
grown up the cells were placed into a centrifuge tube and pelleted by
centrifugation. The cells were then resuspended in 25 mls ofMgS04 to
prepare them for plating.
Packaging
Next, the ligation reaction is packaged by adding together the ligation
mixture and the packaging extracts. The vector DNA (A phage) was
purchased prepared by enzyme digestion and removal ofunnecessary
intermediate fragments was already completed. A Stratagene gigapak?
packaging extract tube was removed from the -80?C freezer and placed on
ice. The packaging extract tube is held between fmgers until it just begins to
thaw. Ligation ofsea urchin Mbo I digestion DNA to Mho I digested ADNA
was then carried out. After ligation, the phage DNA were packaged into
phage particles in vitro. The packaged phage, then, were used to infect E.Coli
culture KW392 (Maniatis et al., 1982). In doing so, immediately eleven Jlls
ofthe thawed ligation mixture was added to the tube with the packaging
extracts. It was then placed on ice. Carefully the tube was stirred with a
pippette tip being careful not to introduce bubbles. The tube was then
microfuged briefly in order to ensure that all the contents were at the bottom
37
ofthe tube. The tube was then incubated at room temperature for exactly one
hour and forty-five minutes. After the incubation period was over, 500 mls of
SM buffer was added to the tube. Next, a serial dilution using SM buffer was
done to prepare for plating (this can be better seen in figure ten below). This
allows for better distinction ofplaques. The phages and cells are then
incubated at 37?C for fifteen minutes. Following the incubation three mls of
soft agar was added to each tube. The contents ofeach ofthe four tubes are
then dumped onto four agar plates consis!ing ofNZCYM media. The plates
were then incubated overnight at 37?C. The bacteria containing this Alibrary
will then be grown on bacterial lawns. Plaques should form overnight. The
plaques are then harvested. This last step results in the amplification ofthe
library (Maniatis et al., 1982).
lOuI
38
S06uI
SM Buffer +
p8ckae;o& no.
Figure 10. Serial dilutions of the packaging reactions
39
IV. Results
The gene coding for EMBP is expected to be large based on Northern
blot analysis. The mRNA is larger than the 28s RNA which corresponds to
4,718 nucleotides (unpublished data).
The isolation ofthe DNA from sea urchin spenn yielded high
molecular weight DNA. This procedure had been repeated several times with
varying results. The DNA showing the highest level ofpurity and largest size
was selected to continue on to the next step in genomic library creation which
was the restriction digest. In the DNA from the selected isolation, the DNA
banded at about 23kbp on 0.30/0 agarose gels (Figure 9). It indicated a more
or less unifonn size, with smaller amounts ofintennediate fragments. The
small size indicated by a smeared appearance. High molecular weight DNA
is what is desired in order to exclude randomly broken DNA that will not
give good restriction digested ends. The selected DNA has been quantified
using UV absorption. Purity was assessed by a A26o:A28o ratio, and this was
found to be 1.92. This indicates highly purified DNA. The amount ofDNA
purified was 73.5J..lg/ml total of73J..lg (table 1).
A26011m = 1.4570
A28011m = 0.759
concentration = A260 11m = 1 = 50j.lg/ml
so, 1.4570 x 50j.lg/ml
73.5 j.lg/ml
Table 1. Selected DNA using UV absorption
40
A 3 5 7
Figure 11. Shown here is isolated sea urchin sperm
DNA with the highest degree ofpurity and largest
size. Lane 1 is the Hind m digested lambda DNA
ladder used as a size marker, Lane 2 is blank, Lane 3
isolated sperm DNA, 15/.11 load, Lane 4 is blank, Lane 5
isolated sperm DNA, 24~ load. Lane 6 is blank. Lane 7
isolated sperm DNA, 8Jllload. The isolated DNA shown
here was chosen to create the genomic library.
41
The procedure ofisolating the DNA was performed several times
under varying conditions. In the end the DNA shown in figure eleven was
chosen to continue on with the restriction digest. However, isolation ofsea
urchin DNA was attempted from a different source other than sea urchin
sperm. This source was sea urchin eggs. This was done following the same
protocol from Maniatis et al. as used with the sea urchin sperm. After the
isolation was done the purity was assessed by U. V. absorption and the A260 to
A280 ratio was found to be : 2.012/1.936 =: 1.039. This ratio suggests
impurities in the DNA prep. The ratio expected should be greater than 1.75.
A 0.3% agarose gel was run and the results showed a large amount ofRNA
(figure 12). The highest molecular weight ofthe RNA was at the 2.0kbp
marker and increasingly became lower molecular weight.
42
Lanes
3 5
Figure 12. Attempted DNA isolation. In this attempted
DNA isolation large amounts ofRNA were obtained, this
is indicated slightly in lane 3 and by the large illuminated
appearance in lane 5.
In a different sea urchin spenn DNA isolation, results turned out fairly
well and it was thought that this would be the DNA used to create the sea
urchin genomic library. However, it was later found out that this DNA
contained a high level ofimpurities. The DNA shown in figure 11 was then
used to attempt library construction. This DNA in this isolation attempt can
43
be seen in figure 13 below. Thespenn used in this isolation were freshly
collected.
Lanes
3 5 7
Figure 13. DNA isolation containing impurities.
At first it was thought that the DNA from this isolation would be the one used to create the genomic library.
It was later found out that this DNA contained a high level ofimpurities. Lanes 3, 5, and 7 are different
amounts ofDNA from the same isolation.
Isolated genomic DNA was subjected to limited restriction enzyme
digestion using Mho I. In the gel shown below in figure 14, the restriction
digest was perfonned using different time points ofdiluted enzyme (Mbo I),
in a I: 10 concentration (see Appendix A). The goal was to obtain fragments
44
ofapproximately 20kbp. In the four time points indicated here the fifteen
minute and the twenty minute time points were entirely too long. Entirely too
much digestion ofthe genomic DNA occurred. Therefore, the digest was
redone using diluted enzyme in a 1:50 concentration.
Minutes
Figure 14. Restriction digest with diluted enzyme Mbo I in a 1:10 concentration.
Lane 1 shows EcoRI digested lambda DNA used as a standard, Lane 2 is blank, Lane 3 has a five minute
time point, Lane 4 is blank, Lane 5 has a ten minute time point, Lane 6 is blank, Lane 7 shows a fifteen
minute time point, and Lane 8 is showing a twenty minute time point.
45
Minutes
o 2.5 5 10
Figure 15. Restriction digest with enzyme Mbo I diluted 1:50. Lane 1 shows the EcoRI digested lambda
DNA ladder, Lane 2 is blank (however, some ofthe ladder spilled over), Lane 3 is a zero time point of
undigested DNA, Lane 4 is a 2.5 minute time point, Lane 5 is a 5 minute time point, Lane 6 shows a ten
minute time point.
Nevertheless, the restriction digested genomic DNA with the enzyme
in a 1: 10 dilution failed to produce the high molecular weight DNA needed.
As shown in figure 15 three more time points were run. This time the
concentration ofthe enzyme Mho I was in a 1:50 dilution. The fragments at
the 2.5 minute time point appear barely to be cut at all when compared to the
46
zero time point. The five minute time point was selected as the optimal time
point and the digest was necessarily scaled up.
In order to isolate restriction DNA fragments, the digests were
subjected to density gradient centrifugation. Glycerol or sucrose gradients
were made, centrifuged, and fractionated. Every other fraction was
electrophoresed overnight on a 0.3% agarose large format gel. Density
gradient centrifugation was repeated various times, all with unsuccessful
results. The gradients did not separate the DNA according to size as had been
planned (see figure 16 below). Both glycerol and sucrose gradients were tried
(results ofsucrose gradient not shown) as an effort to produce uniform
gradients. A gradient maker was also tested in establishing gradients.
However, due to mechanical problems with the gradient maker this method
was abandoned.
47
Fractions
A 2 3 5 6 8 9 11 12 14
Figure 16. Glycerol Gradient Gel. This gel shows fractions that were electrophoresed on a 0.3% agarose
gel. As shown the lanes do not slope downward as one would expect a properly separated gradient to look.
Lane 1 shows the Hind III Digested DNA ladder. Lane 2 shows fraction #2. Lane 3 shows fraction #3. Lane
4 shows fraction #5. Lane 5 shows fraction #6. Lane 6 shows fraction #8. Lane 7 shows fraction #9. Lane 8
shows fraction #11. Lane 9 shows fraction #12. Lane 10 shows fraction #14. Lane 2 signifies the bottom of
the gradient and Lane 14 signifies the top.
In an effort to determine the problem in the density gradient
separation, test gradients were run using lambda DNA. This would
allow optimalization ofconditions without using the isolated genomic
DNA. This method showed that gradients were unable to be
established. Ifa gradient was established, when analyzed by gel
48
electrophoresis and the lanes ran in order, the lanes should slope in a
downward fashion.
Due to the inability to establish a gradient with density gradient
DNA separation on sucrose or glycerol gradients, a different method to
obtain the optimal size DNA fragments was done. This method
involves gel purification (gel purification was only necessary for the
genomic DNA that was digested under partial digest conditions).
The results ofthe gel purification turned out to be very good on
the first try. The excess small fragments were gotten rid ofupon
purification from the 0.3% large gel and the approximately 20 kbp
fragments necessary for inserting into the lambda vector were obtained
(this can be seen in figure 17).
49
Figure 17. Gel purification gel. Lane 1 is the pre-digested lambda DNA ladder. Lanes 2 and 3 are blank.
Lane 4 contains the gel purified DNA ofapproximately 20 kbp. Lanes 5and 6 are blank. The pre-digested
ladder fragments are as follows: (starting from the top) 23kbp, 9.4 kbp, 6.5 kbp, 4.3 kbp, 2.3 kbp, and 2
kbp.
Ligation, packaging, and plating were done. The result was that there
were no plaques. This could possibly be due to the fact that ligation may not
have occurred. The leftover ligation mixture was run out on a 0.3% agarose
gel. As indicated below in figure 16, there are three bands in the lane where
the ligation mix was loaded. This may indicate that two ofthese bands are
the lambda DNA arms and the third is the fragments that were to be inserted.
50
So ligation probably did not take place. The ligation was redone several
times with the same results.
lanes
A 234 5 67 8
Figure 18. DNA ligation gel. Lane 1 shows the pre digested lambda ladder. Lanes 2,3, and 4 are blank.
Lane 5 shows what was left over from the ligation mixture. Lanes 6, 7, and 8 are blank. Three bands are
shown in lane 5 indicating that ligation may not have taken place.
Due to the problems occurring with sea urchin genomic library
construction under partial digest conditions, it was decided that the direction
taken in creating the library should be changed. Now, a complete restriction
digest would be performed. A small scale digest was performed with enzyme
Bam HI. Several times the digest was performed and no digestion occurred.
This was found to be because the enzyme was old. New enzyme was
51
purchased and once optimal results were established, the digest was
necessarily scaled up. The results can be seen in figure 19 below.
Iv 2 3 4 5 6
Figure 19. Bam HI digested DNA. This DNA was used for the complete digest ofthe sea urchin genomic
library. Lane 1 shows the lambda DNA digested ladder as a standard. Lane 2 is blank. Lane 3 shows the
uncut sea urchin genomic DNA Lane 4 is blank. Lane 5 shows the complete restriction digested DNA by
Bam HI. Lane 6 is blank.
Again, the ligation, packaging, and plating was carried out as before.
Once again, plaques were not formed. A test ligation was performed to check
the protocol used. The test ligation produced plaques on the agar plates
indicating that the procedure used could indeed produce positive results.
52
v. Discussion
In the initial stage ofthis project problems were encountered in
restriction digestion and the subsequent ligation steps resulting in a failure to
construct a DNA library. Thereby hindering accomplishment ofthe second
specific aim, which was to isolate the gene for the sea urchin 53kDa myosin
binding protein. A possible conclusion to the difficulties that have occurred
while trying to create this sea urchin genomic library is the high quantity of
methylated DNA in the sea urchin genom..e and/or in sperm DNA alone. In
previous research it has been found that about forty percent ofthe sea urchin
genome is methylated (Bird et aI., 1979) Methylation ofthis DNA could
possibly be the reason that this genomic library was so difficult to create.
Furthermore, methylation could have been the reason for many or all ofthe
problems that have arisen in creation ofthis library.
Although, the DNA isolation was performed various times with
various results. In the end the DNA from the very first isolation proved to be
ofthe highest molecular weight and the greatest purity. The isolation was
performed several times while changing variables with the hopes ofobtaining
better results. However, the fIrst isolation was chosen to continue on with rest
ofthe project. Upon isolation ofthe DNA from sea urchin eggs and gonads it
53
was found that this tissue contained too much RNA. The best luck in
acquiring the large amount ofDNA needed was isolated from sea urchin
sperm. However, this DNA proved to be contaminated with a large quantity
ofimpurities. Alternate sources ofsea urchin DNA should be investigated.
However, due to lack ofdemand a commercial sea urchin genomic library
was unable to be located.
The state ofsperm DNA could possibly make it difficult to cleave by
restriction enzymes (Bird et al., 1979). It is also possible that methylation
occurring in the sperm is causing the difficulties and sea urchin DNA from
sources other than sperm and eggs should be researched.
The restriction digest had to be performed both as a partial digest and
then a complete digest since creating a genomic library with the former
proved to be unsuccessful. The ligation performed with the partial digest
appeared not to ligate according to the results seen in figure 18. However,
this conclusion was based on results from agarose gel electrophoresis which
is well-known for uncertainty for results such as these. Although it is hard to
confmn from the agarose gel, the results ofthe gel run from the leftover
ligation mixture indicate that no ligation took place. Had recombinant
molecules transpired, plaques on the agarose plates would have been
indicative ofthe ligation occurring. It could be that methylation is not
S4
allowing all sequences to be restriction digested. The enzyme that was
chosen for the partial restriction digest (Mho I) is not sensitive to all types of
methylation.
Other problems in this project have to do with the inability to establish
uniform gradients. When the fractionated gradients were analyzed by gel
agarose electrophoresis it was found that the lanes were the same all the way
across instead ofsloping downward. Ifthe lanes were sloping downward it
would indicate that the DNA was in fact separated by molecular weight. In
all ofthe methods chosen to create the gradients this turned out to be the
problem. When the gradients were created with both sucrose and glycerol
this same dilemma occurred, so the substance used to create the gradients
was not the problem. This could have happened because the centrifuge
stopped too abruptly or perhaps other problems with the swinging bucket
rotor. A gradient maker was also tried in creating these gradients but, due to
problems with the tubing this method also had to be abandoned. These
gradients were fractionated and weighed before running to see ifthe gradient
was uniform and it proved not to be. Ifthe gradient was uniform weighing
out the same quantity ofeach ofthe fractions should have showed a gradual
difference in weight.
55
Due to the problems occurring with density gradient centrifugation, gel
purification was used to obtain the needed size DNA fragments. This proved
to be the method in the project with the least uncertainty. Upon the first try
the approximately 20kbp DNA fragments needed were purified. The
fragments were then used to attempt the DNA ligation with the Avector arms
that were pre-digested with the same enzyme.
A complete restriction digest was performed when ligation problems
occurred in the partial digested sea urchiI] sperm DNA. This was digested
with enzyme Bam HI. Along with the partial digested DNA problems also
transpired in the ligation step with sperm DNA that had been digested to
completion. Restriction enzymes other than Mho I and Bam HI that are less
sensitive to DNA methylation should be tried as an effort to create
recombinant molecules.
The problems occurring in the ligation are not from the procedure
chosen. A test ligation was run in order to ensure that the mechanics ofthe
procedure were being done properly. Plaques were formed on the test plates.
56
This indicates that the problems occurring were indeed from the DNA.
Figure 20. Plaques on agar plate from test ligation. Although hard
to see, this plate contained 102 plaques from the test ligation. This plate
was the 10-2 test plate.
Ifligation had occurred, perhaps another strain ofE. Coli could be tried
in order to package the recombinant molecules. Perhaps ifrecombinant
molecule were formed and ifthey contain methylated portions they may not
be being recognized by E.Coli's genome. Trying different strains ofE.Coli
that might be able to recognize fragments that are methylated should be
explored. Ifindeed the problem with ligation is the sea urchin genome
containing large quantities ofmethylated DNA that are not being recognized
other methods ofovercoming this will have to be explored.
57
In the event that recombinant molecules were able to be made and
plaques had formed, the second specific aim ofthis project was to selected
for the entire gene for p53EMBP. EMBP cDNA probes (discussed below)
were to be used to screen the sea urchin genomic phage DNA library,
described above. Once the phage were able to be plated out on bacterial
lawns at a density giving distinct plaques, nitrocellulose filter disks would
then have been placed over the bacterial lawns. Absorption ofphage DNA to
nitrocellulose would then occur, and nitrocellulose would then be vacuum
dried at 80?C. This fixes the DNA in place. Next, the nitrocellulose would
be moistened with a solution containing 32p labeled probe DNA (Maniotis,
1982). The 32p labeled probe DNA would be allowed to hybridize to
complimentary sequences present on the nitrocellulose. The nitrocellulose
would then be washed extensively under conditions ofhigh stringency, dried,
and autoradiographed. The positions ofthe labeled plaques could then be
cross-referenced back to the original cultures. The phage would be recovered
from these positive plaques. Each plaque would be subjected to a second
round ofplaque selection as described above. This would insure clonality.
58
Once the library is established, it must be probed in hopes ofobtaining
several clones for p53EMBP. This would be done using two plasmids
available as a source ofcDNA. These plasmids are pGEX rEMBP 400 and
pGEX rEMBP 1600 (pGEX is the plasmid, rEMBP (recombinant egg
myosin-binding protein) is the insert, and 400 or 1600 is the number ofbase
pairs) (figure 21). These two sea urchin egg myosin binding protein cDNAs
were prepared to be utilized as probes for the screening. The cDNA in these
plasmids are oftwo sizes, 400 bp (rEMBP400) and 1600 bp (rEMBPI600).
They would have both be used in order to maximize the number ofclones
detected and to increase the amount ofthe gene that would be probed for.
The cDNAs would be gel purified and 32p labeled (please see figure 21).
These two plasmids code for different portions of p53EMBP. These
plasmids are easily purified using the PERFECT prep TMplasmid DNA kit.
The inserted cDNAs can be cut out with EcoRl (Figure 21) and used as the
probe.
59
Hind III)., digest
rEMBP 1600
~
rEMBP400
~ PGr3X
- 23 kbp
_ 9.4 kbp
- 6.5 kbp
- 4.3 kbp
_ 2.3 kbp
- 2 kbp
- 500 bp
1% agarose ge I of Eco Ridigested
Plasmids. Stained with ethidium bromide.
A, Iinearized plasmid DNA
B, 1600 bp cDNA coding forEMBP
C, 400 bp cDNA coding for EMBP
Figure 21. This is a DNA gel showing the inserts.
Future work
In order to establish the genomic library alternate sources ofDNA
other than sea urchin sperm and eggs should be tried. A library could be
established from a closely related species such as the star fish in hopes that
another species would be similar enough to probe for the gene and the
60
problems encountered in creation ofthe sea urchin genomic library could be
avoided. Ifthis does not work, perhaps ifthere are any methods available to
demethylate the sea urchin DNA these could be tried in order to establish the
sea urchin genomic library. Furthermore, alternate methods ofobtaining
DNA from the sea urchin eggs in order to avoid the large quantities ofRNA
could be tried.
Once the problems ofcreating this genomic library are worked out,
and the gene for p53EMBP is obtained further characterization ofthe gene
will be accomplished by analysis ofits genetic sequence. This analysis will
identify any functional domains that are known. Furthermore, comparative
studies can be done between this gene and other known myosin binding
protein sequences using recombinant EMBP. Transformation experiments
then could be done to understand the role ofEMBP in cells. Introduction of
altered EMBP genes into sea urchin eggs can be used to address the questions
ofthe developmental role ofEMBP. Transformation ofother cell lines may
suggest the fundamental role EMBP plays in cellular physiology.
Further investigation into the sequence analysis on the world wide web that
p53EMBP could indeed be VAP-I, a vesicle transport protein should be
attempted. Until the entire gene is isolated for p53EMBP it won't be known
for certain whether these two proteins are homologous or not.
61
Once the gene is isolated characterization ofhe gene can take place.
The restriction mapping ofthe isolated clones will be useful in characterizing
the gene for further manipulations. The specific enzymes to be used should
be determined at that time.
62
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Appendix A
l.Extraction buffer
10mM Tris-CI
O.IMEDTA
0.5% SDS
2. TE (for dialysate 4L)
50mM Tris-CI (pH 8.0)
10mM EDTA (pH 8.0)
3. Dilution buffer
10111 BSA (Bovine Serum Albumin)
10111 Buffer B (made earlier according to Promega protocol)
80111 ofdH20
4. NET buffer (according to the Maniotis protocol for sucrose
gradients[2.85])
1M NaCI
20mM TrisCI
5mMEDTA
66
67
brought to pH 8.0.
5. Glycerol solutions
For the 40% solution 40mIs ofglycerol and 60 mIs ofNET buffer were
added together. For the 10% solution 10mIs ofglycerol and 90 mls of
NET buffer were added together.
6. Sucrose solutions
For the 60% solution approximately 77g ofsucrose and 5lg ofdH20
were added together. For the 15% sucrose solution approximately 16g
ofsucrose and 90g ofdH20 were added together.
7.0.30/0 agarose gel
0.175g ofagarose and 50mIs ofTAE
8. 1% agarose gel
0.5g ofagarose and 50 mIs ofTAE
9. 0.3% Long agarose gel
0.350g ofagarose and 100 milliliters ofTAE.
10. 8M (Phage storage and dilution): IL
5.8gNaCI
2.0gMgS04
50.0ml 1M Tris, pH 7.5
5.0mI 2% Gelatin
11. NZCYM (liquid) : 500ml
5g NZamine (Casein, enzymatic lysate)
2.5g NaCI
O.5g Casamino acids
2.5g Yeast Extract
IgMgS04
NaOH to pH 7.5
12. NZCYM Plates
Bottom agar: 15g Bacto agar/L
Soft agar: 7g Bacto agar/L
68
13. LB Broth (IL)
109 Bacto-trypton
5g Bacto-Yeast extract
109 NaCI
to 1000 ml dH20
pH to 7.5, autoclave
69