Citation
Cloning of xenopus laevis phospholipase D

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Title:
Cloning of xenopus laevis phospholipase D
Creator:
Batbayar, Khulan
Publication Date:
Language:
English
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50 leaves : ; 28 cm

Thesis/Dissertation Information

Degree:
Master's ( Master of arts)
Degree Grantor:
University of Colorado Denver
Degree Divisions:
Department of Integrative Biology, CU Denver
Degree Disciplines:
Biology

Subjects

Subjects / Keywords:
Xenopus laevis ( lcsh )
Cloning ( lcsh )
Phospholipases ( lcsh )
Cloning ( fast )
Phospholipases ( fast )
Xenopus laevis ( fast )
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bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

Notes

Bibliography:
Includes bibliographical references (leaves 47-50).
General Note:
Department of Integrative Biology
Statement of Responsibility:
by Khulan Batbayar.

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|University of Colorado Denver
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Auraria Library
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All applicable rights reserved by the source institution and holding location.
Resource Identifier:
47826392 ( OCLC )
ocm47826392
Classification:
LD1190.L45 2001m .B37 ( lcc )

Full Text
CLONING OF XENOPUS LAEVIS
PHOSPHOLIPASE D
B. S., Masaryk University at Brno, 1995
M. S., Masaryk University at Brno, 1997
A thesis submitted to the
University of Colorado at Denver
in partial fulfillment
of the requirements for the degree of
Master of Arts
Biology
by
Khulan Batbayar
2001


This thesis for the Master of Arts
degree by
Khulan Batbayar
has been approved
by
Htz 7/0!
Date
\


Batbayar, Khulan (M. A., Biology)
Cloning of Xenopus laevis Phospholipase D
Thesis directed by Associate Professor Bradley J. Stith
ABSTRACT
Phospholipase D (PLD) catalyzes the hydrolysis of
phosphatidylcholine to phosphatidic acid (PA) and choline. PA may
increase intracellular [Ca2+] levels to induce most events of fertilization.
PLD sequences from many species have been reported. There are
three mammalian isoforms of PLD: PLDla, PLDlb, and PLD2. PLDlb is a
splice form of PLDla and has a 38 amino acid deletion in the loop region.
Sequence homology between PLD1 isoforms and PLD2 is about 50%. PLD
has four conserved regions (I-IV). Two of these regions (II and IV) are
"HKD" motifs that are involved in catalysis.
Due to our belief that PLD plays a role in fertilization and in order to
study PLD, we sought to clone PLD from Xenopus laevis. RT-PCR
(Reverse Transcriptase-Polymerase chain Reaction) using degenerate
primers amplified 3 fragments (220 base pair, 615 base pair, 820 base pair).
All three fragments were cloned and sequenced. Multiple sequence


alignment revealed that the 615 bp fragment is from PLDlb. This is the first
sequence data from a non-mammalian vertebrate. These data should tell us
much about the evolution of this enzyme. The 220 bp and 820 bp fragments
did not show any significant homology to PLD and were artifacts (e.g.
sequence of ribosomal proteins that play no role in fertilization).
This abstract accurately represents the content of the candidate's thesis. I
recommend its publication.
IV


ACKNOWLEDGEMENTS
I would like to thank Dr. Bradley Stith for allowing me work in his
laboratory. I have learned so much from him. Thank you.
I would like to thank Dr. Douglas Petcoff for the hours he spent with
me and teaching me the intricate skills of the lab. He has been a great
teacher and friend. Thank you.
I would like to thank Dr. Linda Dixon for her support,
encouragement and assistance in all academic matters.
Next, I would like to thank Dr. Todd Bergren for allowing me to
perform part of my thesis experiments in his lab.
I would like to thank William Holland, Erin Stauter and Kai Savi for
their support and friendship.


CONTENTS
Figures..........................................................viii
Chapter
1 Introduction...................................................1
1.1 Phospholipase D............................................. 1
1.2 Enzymology of PLD...........................................4
1.3 Isoforms of PLD.............................................5
1.4 Structure of PLD............................................6
1.4.1 HKD motif................................................ 7
1.4.2 IYIENQFF motif........................................... 8
1.4.3 PH domain.................................................. 8
1.4.4 PX domain.................................................. 10
1.4.5 Amino-and carboxy-termini.................................. 10
1.4.6 Loop sequences............................................. 11
1.5 Regulation of PLD activity................................... 12
1.5.1 PIP2 is a cofactor for PLD................................. 13
1.5.2 The ARF family of G-proteins stimulates PLD................ 14
1.5.3 Modulatory effect of Rho proteins.......................... 15
1.5.4 Regulation of PLD by PKC................................... 15
1.5.5 Regulation of PLD by calcium ions..........................16
1.5.6 PLD inhibitors.............................................16
VI


1.6 Xenopus laevis PLD............................................17
1.7 Cloning of Xenopus laevis PLD.................................18
2 Materials and methods............................................20
2.1 Xenopus eggs...................................................20
2.2 Isolation of total RNA from Xenopus eggs......................21
2.3 Synthesis of degenerate primers for RT-PCR...................22
2.4 Cleavage and deprotection of primers.........................23
2.5 First-strand cDNA synthesis...................................24
2.6 Purification of first-strand cDNA.............................25
2.7 Polymerase chain reaction with degenerate primers............26
2.8 Polymerase chain reaction with ODC primer.....................27
2.9 Electrophoresis of PCR products.............................. 27
2.10 Extraction of DNA fragments from the agarose gel.............28
2.11 Cloning and transformation of PCR product....................29
2.12 Isolation of plasmid DNA.....................................30
2.13 Seqeuncing of plasmids.......................................31
3. Results........................................................ 35
3.1 RT-PCR........................................................ 35
3.2 Cloning of PCR products.......................................40
3.3 Sequence analysis............................................ 41
4 Discussion...................................................... 44
References........................................................ 47
vii


FIGURES
Figure
1.1 Phospholipase D mediated breakdown of phosphatidylcholine....2
1.2 Reactions catalyzed by phospholipases C and D................ 3
1.3 Structural domains of human phospholipase D1................. 7
1.4 Model of PLD activity........................................ 9
1.5 Regulation o f PLD........................................... 13
2.1 Experimental steps necessary to clone the PCR product........ 32
2.2 The map of pCR4-TOPO vector................................. 33
3.1 PCR with ODC primers..........................................36
3.2 PCR with degenerate primers using FailSafe Premixes......... 37
3.3 PCR with degenerate primers using FailSafe Premixes......... 38
3.4 PCR with degenerate primers using FailSafe Premixes..........39
3.5 Nucleotide sequence oiXenopus PLD fragment...................42
3.6 Multiple amino acid sequence alignment.......................43
viii


1. Introduction
1.1 Phospholipase D
Phosphatidylcholine-specific phospholipase D (PLD) is a ubiquitous enzyme
that has been found in a variety of plants, mammals and microorganisms. It has
been implicated in a wide range of physiological responses including metabolic
regulation, cell proliferation, mitogenesis, oncogenesis, inflammation, secretion and
diabetes (Frohman et al., 1999; Exton, 2000). This enzyme catalyzes the hydrolysis
of phosphatidylcholine to generate phosphatidic acid (PA) and choline (Figure 1.1).
PA can be converted to the second messenger diacylglycerol (DAG) or
lysophosphatidic acid; DAG and lysophosphatidic acid are "second messengers"
that activate various cell pathways. Consistent with a role in signaling, PLD1 can
be activated by small G-proteins, elevated intracellular [Ca2+], protein kinase C
(PKC), and protein tyrosine kinases (Liscovtch and Cantley, 1995)(Figure 1. 2). In
mammalian cells, at least two different isoforms of PLD can be differentiated by
their susceptibility to regulation by G-proteins, or requirements for
phosphatidylinositol 4,5-bisphosphate (PIP2) or fatty acids (Massenburg et al.,
1


0
Phosphatidic Acid
Figure 1. 1
Phospholipase D mediated breakdown of phosphatidylcholine (from Savi, 2000,
with permission)
2


Cell Membrane
Figure 1. 2
Reactions catalyzed by phospholipases C and D (from Savi, 2000, with permission)
3


1994). For example, ARF (ADP-ribosylation factor), Rho, and PKC stimulation of
PLD1 activity requires PIP2 as an essential cofactor. PLD2 may not require any
cofactor.
1. 2 Enzvmology of PLD
In mammalian tissues, the main substrate of PLD is phosphatidylcholine
(PC), whereas in other systems, it can hydrolyze different phospholipids
(phosphatidylethanol and phosphatidylinositol). PLD la, PLD lb and PLD2
isoforms have been cloned recently from mammalian tissues (Hammond et al.,
1995). These mammalian isoforms share highly conserved sequences that are also
found in PLDs from plant, yeast, and bacteria (Exton, 1998).
Regulation of PLD 1 in vivo is through mechanisms such as phosphorylation
or translocation (Sung et al., 1999). Mammalian PLD2 is similarly constitutively
active in vitro and how PLD2 is regulated in vivo is not fully understood.
4


1. 3 Isoforms of PLD
There are three iso forms of mammalian PLD: PLD la, PLD lb (-120 kDa)
and PLD2 (-105 kDa). Only one form of PLD has been reported in worms, flies or
yeast (Frohman et al., 1999). The crystal structure of a 54 kDa PLD from
Streptomvces strain PMF has been established (Leiros et al., 2000).
MammalianPLDl has two splice variants: a and b. Variant b has a 38 amino acid
deletion between conserved sequences, but it does not make any difference in
catalytic activity and the response to PIP2 (Hammond et al., 1997; Katayama et al.,
1998; Nakashima et al., 1997). The deletion occurs in the unique loop region,
which is 505-620 amino acid long (Sung et al., 1999). Mammalian PLD2 lacks the
116 amino acids of this region; yeast PLD lacks an additional 30 amino acids.
Caenorhabditis elegans has an additional 300 amino acids in the loop region (Sung
et al., 1999).
The sequence identity of PLD 1 and PLD2 is about 50%. PLD2 also has
alternatively spliced isoforms. PLD2 is different in many w'ays from PLD1. It is
the product of a different gene, which is located on a different chromosome. PLD2
appears to be localized to the plasma membrane, whereas PLD1 is located at the
perinuclear region (endoplasmic reticulum, Golgi apparatus, and endosomes). Also,
5


its regulatory properties are very different from those of PLD1 (Peng and Rhodes,
2000). Northern blot and RNA protection analysis indicate that both PLD1 and
PLD2 are expressed in many different tissues and cell lines, although the message
levels vary significantly (Exton, 1998).
1. 4 Structure of PLD
All eukaryotic PC-specific PLD genes cloned to date share a relatively
conserved catalytic core flanked with much less conserved N- and C-terminal
regions (Liscovitch et al., 2000). The catalytic core of all eukaryotic PLDs consists
of domains I-IV (Figure 1. 3). These domains are also found in bacterial PLD.
There is significant internal similarity in four short sequence motifs; between
domains I and II, and between domains III and IV (CRII and IV are HKD motifs).
This suggests that the eukaryotic genes arose through duplication of an ancestral
gene after divergence occurred between lower eukaryotes and animals, raising the
possibility that PLDs are bilobed enzymes (Sung, et al., 1998).
6


Catalysis
Catalysis
PKC Interaction
\
PLD1 N "
Nmbr*n Association?
SubcsHiilsr Targeting?
WPj / mombniw
interaction
I HKD
hdohkeee
ill
Interaction with
vesicular trafficking
murhinery?
(Negative regulation*!
targeting signal
tor caveolae ?
Interaction with
choline ?
t
HKD
Membrane
Association?
t
-------1
Rho Interaction
Figure 1. 3
Structural domains of human phospholipase D1
1.4.1 HKD motif
Two of these conserved sequences (CRII and CRJV) are essential for
catalytic activity and are called HKD motifs (HxxxxKxDxDE) (Hammond et al.,
1995). It is required that Both HKD motifs must be intact for PLD to be active.
This suggests that the protein folds such that the domains are brought into close
proximity (Frohman et al., 1999). Because of their conservation and amino acid
content (such as those amino acids which mediate enzymatic activity), it is thought
that the HKD motifs are directly involved in catalysis. Mutagenesis analysis of
PLD from several species has shown that these amino acids are critical for catalysis
in Vitro and PLD function in vivo, and has led to a hypothetical model for the
7


catalytic cycle via a covalent phosphatidyl-enzyme intermediate (ping-pong
mechanism; see Figure 1. 4) (Sung et al., 1997).
1.4.2 IYIENQFF motif
The IYIENQFF region (CRIII) is as critically important as the HKD motifs,
but its role is not known (Sung et al., 1997). Since this region is rich in aromatic
amino acids, it is possible that CRIII interacts with the choline headgroup of PC to
increase the rate of catalysis, or, more likely, to determine the specificity of PLD
only to phospholipids having choline headgroup (Frohman et al., 1999).
1. 4. 3 PH domain
Surprisingly, PLD isoforms lack identifiable motifs such as SH2, SH3, PTB
etc, in their sequences. However, there is a weakly defined pleckstrin homology
(PH) domain (CRi), as suggested by the binding of PIP2, which activates almost all
mammalian isoforms. Deletion analysis of the PH domain revealed that the PH
domain is not essential for enzymatic activity and dependence of enzymatic activity
is not affected by deletion of the PH domain. This suggests that there are other PIP2
binding sites (Frohman et al., 1999).
8


Figure 1.4
Model of PLD activity
9
PLD-PA Irvternedlate


1. 4. 4 PX domain
PX motifs are proposed to mediate a variety of protein-protein interactions.
A significantly conserved PX domain is present in yeast and animal PLDs and its
function is critical for mammalian PLD (Sung et al., 1999). However, its role is still
unknown. It might play a role in regulatory interactions with factors that promote
translocation or activation. In mammalian PLD1 it is more likely involved in
interactions with the vesicular membrane trafficking machinery (Frohman et al.,
1999). Candidate roles for yeast PLD (SP014) include interaction with unknown
kinases that regulate its phosphorylation (Rudge et al., 1998).
1. 4. 5 Amino- and carboxv-termini
The amino-termini of PLDs from different species are relatively poorly
conserved. Recent studies have shown that the N-terminus of PLD 1 interacts with
protein kinase Ca (PKCa) (Sung et al., 1999). PLD membrane association is not
mediated by the N-terminus (Frohman et al., 1999).
In contrast the C-terminus is well conserved, especially in higher eukaryotes.
More recent studies have demonstrated that Rho interacts with the C-terminus (Sung
10


et al., 1997). Experiments that used chimeric PLD1 and PLD2 proteins generated
evidence that the ARF binding site may be in the C-terminus.
Studies on rat PLD1 by Zhi et al. (2000) demonstrated that the N-terminal
fragment containing one HKD motif can associate with the C-terminal fragment
containing the other HKD motif to form the functional complex. Mutation to
alanine of conserved hydrophobic amino acids within or close to the conserved
HKD domains decreased the association between the N- and C-terminal domains
indicating that the association of the N- and C-terminal domains is important for the
catalytic activity of rat PLD. Also N- and C-terminal domains are required for
Ser/Thr phosphorylation of rat PLD1 at the N-terminal half (Zhi et al., 2000).
1.4.6 Loop sequences
PLD1 has a region of 100-150 amino acids at the center of the protein that is
not present in PLD2 or in PLDs from most lower organisms (Hammond et al.,
1995). Since the loop region is highly variable among PLDs from closely related
species and undergoes alternative splicing, it does not significantly affect PLD1
activity, regulation, or subcellular localization (Frohman et al., 1999). It can be
deleted from PLD1 or inserted into PLD2, and it does not change the activity
significantly (Sung et al., 1999).
11


1.5 Regulation of PLD activity
Many regulators of PLD were identified in studies of permeabilized cells
and cell lysates. Some of these regulatory factors interact directly with PLD in
vitro, e.g. PKC, ADP-ribosylation factor and Rho family proteins. The mechanisms
of action for other factors, such as ceramide and calmodulin remain unclear. The
Gm2 activator and [i-hexaminidase A have been demonstrated to activate PLD, but
the physiological significance of this is not known (Exton, 1999). Physiological
inhibitors that have been characterized include fodrin, the clathrin assembly protein
AP3 and synaptojanin (Frohman et al., 1999). Synaptojanin acts indirectly by
hydrolyzing PIP2 (Singer et al., 1997).
The majority of agonists that stimulate PLD also activate the classic PI-
specific PLC pathway, and activation of PLD appears to occur downstream from
activation of PLC and consequent stimulation of PKC. In some systems, PLD is
activated by ligands, which do not stimulate PLC, mobilize Ca2+ or activate PKC
(Singer et al., 1997)(Figure 1. 5).
12


Figure 1. 5
Regulation of PLD
1. 5. 1 PIP2 is a cofactor for PLD
PIP2 is an essential cofactor for ARF-activated PLD. Both PLD1 and PLD2
activity are strongly dependent on PIP2 (Frohman et al., 1999). An exogenous
substrate assay to measure guanine nucleotide-dependent PLD activity
associated with membranes demonstrated the dependence on the inclusion of PIP2
in phospholipid vesicles containing radiolabeled PC as a substrate (Morris et al.,
1996) . A different study has shown the similar effect of PIP3 (Hammond et al.,
1997) . Permeabilization of the cells in the presence of antibodies directed against
PI4-kinase resulted in a remarkable reduction in the level of PIP2 that correlated
with inhibition of PLD activity (Singer et al., 1997).
13


Phosphoinositides seem to play important roles in membrane anchoring of
many proteins. The binding phosphoinositides anchor the PLDs to the phospholipid
surface, placing them closer to their substrates (Frohman et al., 1999). PIP2 binds to
a highly conserved region containing basic and hydrophobic amino acids (Exton,
2000).
1. 5. 2 The ARF family of G-proteins stimulates PLD
One of the small G-proteins that stimulate PLD activity is ADP-ribosylation
factor (ARE), which is an approximately 20 kDa GTP-binding protein that is a
member of the Ras superfamily. ARF was originally identified as a protein cofactor
required for efficient ADP-ribosylation of the a-subunit of Gs (the heteromeric G
protein that stimulates adenylate cyclase) by cholera toxin (Exton, 1999). This
activity is characteristic for all six known mammalian ARF proteins.
Studies with purified or homogenous PLD have shown that ARF proteins
can directly activate the enzyme (Exton, 2000). Stimulatory effects of ARFs on
PLD activity in cytosolic and membrane fractions have been observed. ARF-
responsive PLD activity also has been found in the Golgi, nuclei, and plasma
membranes. Chen et al. (1997) proposed that activation of PLD in the Golgi is
important for vesicle trafficking in this organelle.
14


1. 5. 3 Modulatory effects of Rho proteins
The Rho family of monomeric G-proteins comprises a second class of
cytosolic GTP-dependent activators of PLD. Members of this family play various
roles in cellular processes involving cytoskeletal rearrangements, membrane
movement, and cell growth. The Rho proteins stimulate Pl-5 kinase, the enzyme
that synthesizes P1P2 (Singer et al., 1997).
Rho proteins interact with the domain of PLD located in the C-terminus
(Exton, 2000). These proteins seem to mediate changes in gene transcription by
activation of protein kinase cascades and also control changes in cell morphology
and motility (Frohman et al., 1999).
1. 5. 4 Regulation of PLD by PKC
Phorbol esters are very potent stimuli of PLD activity in intact cells (Exton,
1997). There are two modes of regulation of PLD by PKC. The first has been
suggested to involve phosphorylation of PLD itself in vitro (Exton, 2000). The
second is a phosphorylation-independent mechanism by protein-protein interaction,
which takes place in vivo (Singer et al., 1997).
15


It appears that PKC is a major physiological regulator of PLD. Mutagenesis
binding studies have demonstrated that PKC isozymes interact with N-terminal
domain of PLD 1 (Zhi et al., 2000).
1. 5. 5 Regulation of PLD by calcium ions
Studies with homogenous, expressed PLD1 have shown that it is activated
by micromolar concentrations of Ca2+ and Mg2+. However, in the presence of a
physiological concentration of Mg2+ in cytosol, Ca2+ effects are not observed. Thus,
control of PLD by changing cytosolic [Ca ] seems unlikely. However, changes of
2+ 2+
[Ca ] in cytosol can alter activity of Ca -dependent proteins that regulate PLD.
1. 5. 6 PLD inhibitors
The polyphosphoinositide 5-phosphatase synaptojanin, fodrin, clathrin
assembly protein-3 (AP-3), synucleins are reported to inhibit PLD. Synaptojanin
and fodrin inhibit PLD indirectly through inhibition of PIP2. AP-3 interacts with
PLD and inhibits it. Synucleins are potent inhibitors of PLD2 in vitro (Frohman et
al., 1999).
16


PLD is able to use either water or a primary alcohol in its enzymatic activity.
Transphosphatidylation is the transfer of phosphatidate from phosphocholine to
water or a primary alcohol (Meier et al., 1999). The treatment of eggs or of the
zygote after fertilization with 1-butanol inhibits the PA increase at fertilization.
However, 2-butanol does not have this effect (Stith et al., unpublished data).
1. 6 Xenopus laevis PLD
Xenopus laevis, the South African clawed frog is a widely used model
organism for the areas of developmental, cell and molecular biology, because of its
relatively long life, short life cycle, easy handling and maintenance, and the large
size of its oocytes and eggs. Xenopus laevis is a tetraploid organism (Gurdon,
1992).
Previous work in our lab demonstrated that PLD plays an important role in
fertilization of Xenopus. PA, a product of hydrolysis of PC by PLD, is thought to
cause an increase in intracellular Ca2+ levels after fertilization by binding PKC and
Activating the PLC pathway (Stith, 1997)(Figure 1. 1 and 1. 2). PA also can be
converted to DAG, which can activate PKC to induce fertilization events. Two
products of PLD catalysis increase about the same amount after fertilization (Stith et
al., 1997).
17


Western blot analysis of PLD using different anti-PLD antibodies (raised
against mammalian PLD1 and against mammalian PLD2) has generated two bands;
53kDa and 100 kDa (Savi, 2000). Human membrane-associated PLD1 is 124 kDa,
another form of human PLD1 was found to be 95 kDa (Gibbs and Meier, 2000).
This indicates that a band at 100 kDa could be PLD1. Also densitometry readings
of bands suggested that PLD1 levels in Xenopus oocytes and eggs are much higher
than PLD2 levels. There was a difference of PLD 1 bands between eggs and
oocytes. Eggs appeared to have higher levels of PLD1.
1. 7 Cloning of Xenopus laevis PLD
Recently, many laboratories have been putting their efforts into cloning
different isoforms of PLD from different mammalian species. Sequences for PLD1
from human (Hammond et al., 1995), rat (Park et al., 1997; Katayama et al., 1998),
and mouse (Colley et ah, 1997b), and for PLD2 from human (Lopez et ah, 1998),
rat (Kodaki and Yamashita, 1997), and mouse (Colley et ah, 1997a) have been
published. From lower vertebrates partial sequence of puffer fish PLD has been
reported (GenBank).
We sought to clone Xenopus PLD from eggs via RT-PCR using degenerate
primers. Degenerate primers are designed from conserved amino acid sequences
18


(WAHHEK and PRMPWHD) of PLD of human and yeast (Nakashima et al., 1997).
Degenerate primers for these amino acid sequences were previously used
successfully to clone the PLD la, PLD lb and PLD2 from rat C6 glioma cells
(Yoshimura et al., 1996), rat pheochromocytoma cells (Nakashima et al., 1997), and
rat brain (Kodaki and Yamashita, 1997).
19 .


2. Materials and methods
2. 1 Xenopus eggs
Selected female frogs were primed 4 days in advance of the experiment with
100 I.U. of PMSG (Calbiochem, La Jolla, CA). Twelve to sixteen hours before the
experiment, they were injected with 850 I.U. of HCG (human chorionic
gonadotropin) (Sigma, St. Louis, MO). On the day of the experiment the eggs are
squeezed out of the female frogs into OR2 solution (83 mM NaCl, 0.5 mM Ca CI2,
ImM MgCh, lOmM HEPES, pH adjusted to 7.9). Expelled eggs were incubated in
2% cysteine solution (pH 8.0) for 10-15 minutes in order to remove the jelly coat.
Dejellied eggs were rinsed three times in rinse solution (0.1 M NaCl, 0.05 M Tris,
pH 7.0) and twice in 100% MBS solution (440 mM NaCl, 5mM KC1, 50 mM
HEPES, 4.1 mM Magnesium Sulfate Hydrate, 1.65 mM Calcium Nitrate
tetrahydrate, 2.05 mM Calcium Chloride dihydrate, 0.202 g/1 NaHCCL).
20


2. 2 Isolation of total RNA from Xenopus eggs
To reduce RNA degradation, 40 eggs (-100 mg) were flash-frozen in liquid
nitrogen as soon as possible after harvesting. Frozen eggs were placed into a tube
containing homogenization/denaturation solution and homogenized using a 2-ml
teflon pestle tissue grinder. For each sample, homogenization/denaturation solution
was prepared by adding 3.2 pi of {3-mercaptoethanol to 500 pi of denaturing
solution containing guanidine isothiocyanate (Micro RNA Isolation Kit, Stratagene,
La Jolla, CA). After the homogenate was transferred to a microcentrifuge tube, 50
pi of 2 M sodium acetate (pH 4.0), 500 pi of phenol and 100 pi of chloroform:
isoamyl alcohol were added. The sample was centrifuged for 5 minutes at 13,000 x
g. After centrifugation the upper phase was transferred to a new RNase-free
microcentrifuge tube. The conditions for extraction enable the partitioning of
proteins and DNA into the organic layer of the biphasic solution interface, while
retaining RNA in the upper aqueous phase. The RNA was precipitated by adding
500 pi of isopropanol followed by centrifugation for 5 minutes at 13,000 x g. The
pellet was washed twice with 75% ethanol and dried under vacuum. The RNA was
21


resuspended in 250 pi of DEPC-treated water1 by heating the sample at 65C for 10
minutes with intermittent vortexing. The RNA amount was determined by UV
absorbance at 260 nm (Sambrook et al., 1988).
2. 3 Synthesis of degenerate primers for RT-PCR
The mixed oligonucleotide primers were designed from amino acid
sequences (WAHHEK and PRMPWHD) within phospholipase D (PLD) that are
conserved between yeast and human (Nakashima et al., 1997); 5-
TGGGCICAYCAYGARAA -3(sense) and 5- TCRTGCCAIGGCATICKIGG -3
(antisense). Y represents C or T, R represents A or G, K represents G or T, and I
represents inosine. This degeneracy allowed synthesizing eight sense and four
antisense primers. Primers were synthesized on a Beckman Oligo 1000
oligonucleotide synthesizer (Fullerton, CA).
Sense:
5-TGGGCICACCACGAAAA-3
1 Diethylthiocarbonate-treated water is prepared by adding DEPC to a known volume of
deionized water, yielding a final concentration of 0.1 % (v/v). Following vigorous mixing this
solution is incubated overnight at 37C. To remove the DEPC which is highly toxic the lid
of the bottle is loosened and the solution is autoclaved.
22


5 -TGGGCIC ACC ACGAGAA-3
5-TGGGCICACCATGAAAA-3
5-TGGGCIC ACC ATG AG AA-3
5 -T GGGCIC ATC ACG AAAA-3
5 -TGGGCICATCACGAGAA-3
5-TGGGCICATCATG AAAA-3
5 -T GGGCIC AT CAT GAG AA-3
Antisense:
5 -TC ATGCC AIGGC ATICGIGG-3
5-TCATGCCAIGGCATICTIGG-3
5 -TCGTGCCAIGGCATICGIGG-3
5 -TCGTGCCAIGGCATICTIGG-3
2. 4 Cleavage and deprotection of primers
When the synthesis was complete, the synthesis columns were removed
from the Oligo 1000 and were attached to the supplied syringes (Ultrafast Cleavage
and Deprotection Kit, Beckman, Fullerton, CA). A vial containing 0.5 ml of AMA
reagent was also attached to the synthesis column. The vials supplied with this kit
contain a special fluorocarbon O-ring. Common O-ring materials such as EPR,
23


Viton, silicone, etc. are not acceptable and will leach material into the AM A
reagent.
The vial/column/syringe assembly was inverted so that the syringe was at
the bottom and the syringe was pumped several times to make sure all air is
displaced from the column. Then the assembly was propped so that the AMA
reagent remains within the column. After a 5-minute incubation at room
temperature the vial/column/syringe assembly was put in a position so that the vial
is at the bottom. The syringe was pumped several times to push all the AMA
reagent into the vial and the vial was removed from the assembly and capped
tightly. Then the vial was heated for 5 minutes at 65C and cooled to room
temperature. The sample was dried on a SpeedVac to remove the AMA reagent and
was then redissolved in 500 pi of sterile water. The concentration of
oligonucleotides was determined by UV absorbance at 260 nm (Sambrook et al.,
1988).
2. 5 First-strand cDNA synthesis
Total RNA (25 pi solution containing 5 pg RNA) was heated at 65C for 10
minutes then chilled on ice for 2 minutes. This sample was placed in the First-
strand Reaction Mix tube (Ready to Go Kit, Amersham Pharmacia Biotech,
24


Piscataway, NJ) containing 2 beads and 0.2 pg of random hexamer primers
(Promega, Madison, WI) was added. Ready to Go You Prime First-strand Beads
utilize Moloney Murine Leukemia Virus (M-MuLV) reverse transcriptase to
generate first-strand cDNA. The sample was brought to 33 pi with DEPC-treated
water. After a 1 -minute incubation at room temperature the sample was vortexed
and incubated at 37C for 60 minutes.
2. 6 Purification of first strand cDNA
First strand cDNA was purified using a High Pure PCR Purification Kit
(Boehringer Mannheim, Indianapolis, IN). A High Pure filter tube was combined
with a collection tube. Binding buffer (3 M guanidine-thiocyanate, 10 mM Tris-
HC1, 5% ethanol, pH 6.6-165 pi: 100 pi per each 20 pi) was added to the first-strand
cDNA reaction and mixed well. The sample was added to the upper reservoir and
centrifuged (1 minute, 13,000 x g). Nucleic acids bind specifically to the surface of
glass fibers in the High Pure tubes. The synergistic process requires a minimum
length of DNA fragment (ca. 100 bp) thus small oligonucleotides and dimerized
primer from PCR reactions are selectively removed. The flowthrough was
discarded and the filter tube was combined with the same collection tube again. The
filter was washed with 500 pi of wash buffer (20 mM NaCl, 2 mM Tris-HCl, pH
25


7.5, 80% ethanol). This step was repeated (200 pi of wash buffer) and after that the
collection tube was discarded. Then the filter tube was combined with the sterile
microcentrifuge tube. Purified cDNA was eluted with 50 pi of 10 mM Tris-HCl,
pH 8.0 and centrifugation (1 minute, 13,000 x g).
2. 7 Polymerase chain reaction with degenerate primers
The FailSafe PCR PreMix Selection Kit (Epicentre Technologies, Madison,
WI) contains a unique mix of thermostable DNA polymerases and a set of 12
reaction premixes. The FailSafe PCR Enzyme Mix is an enzyme blend containing a
3>5' proofreading DNA polymerase for high fidelity. The twelve PreMixes
contain buffer, all deoxyribonucleotides, various concentrations of MgCl 2 and
proprietary FailSafe PCR Enhancer (betaine).
The final reaction mix contained 25 pi of a premix, 1.25 U of enzyme, 500
ng of cDNA (product of the first strand cDNA synthesis purified with High Pure
PCR product purification kit), 4 pi of antisense primer cocktail (each pi contains 40
pMole of a primer), and 8 pi of sense primer cocktail. PCR amplification was
performed for 30 cycles at 94C for 30 seconds, 45C for 2 minutes and 72C for 3
minutes with a final hold at 72C for 7 minutes on a Gene Amp PCR System 2400,
26


(Perkin Elmer, Boston, MA) or Mastercycler (Eppendorf Scientific, Germany) with
hot start.
2. 8 Polymerase chain reaction with ODC primers
PCR mix contained 33 pi of first strand cDNA purified with High Pure PCR
Product Purification Kit, sense and antisense primers (34 pmol of each) and 0.5 U of
Taq polymerase (AmpliTaq Gold, Perkin Elmer, Branchburg, NJ). The mix was
brought to 100 pi with nuclease-free water. PCR amplification was performed for
50 cycles at 94C for 1 minute, at 55C for 1 minute and at 72C for 1 minute with
final hold at 72C for 7 minutes on Gene Amp PCR System 2400 (Perkin Elmer,
Boston, MA).
2. 9 Electrophoresis of PCR products
E-Gels (Lnvitrogen, Carlsbad, CA) are bufferless, agarose gels (1.2%) that
contain electrodes embedded in the agarose matrix. They also contain ethidium
bromide. The E-Gel cassette is 8 cm by 10 cm and 0.6 cm thick. The thickness of
the E-Gel is 3 mm and the volume of the E-gel is 20 ml. The gel (with the comb in
place) was inserted into the E-Gel base unit and run for 1 minute at 60 V. Then the
27


power was turned off and the comb was gently removed from the gel by lifting
straight up from both sides. Twenty pi of each sample was loaded into a sample
well and the gel was run at 60 V for 25 minutes. At the end of the run the gel was
removed from the base and placed on a uv transilluminator for analysis.
2.10 Extraction of DNA fragments from the agarose gel
The product of the PCR reaction was separated by agarose (1%) gel
electrophoresis. For extraction of DNA fragments an 8 cm by 9 cm and 0.8 cm
thick gel was used. A band of interest was excised from the agarose gel with a
clean, sharp scalpel and weighed in a colorless tube. Three volumes of Buffer QG
(QLAquick Gel Extraction Kit, QIAGEN, Germany) to one volume of gel (for 100
mg of gel add 300 pi of Buffer QG) were added and the sample was incubated at
50C for 10 minutes (until the gel slice completely dissolved). Then one gel volume
of isopropanol was added to the sample.
A QIAquick spin column was placed in a provided 2 ml collection tube. The
silica-gel membrane in a spin column is uniquely adapted to isolate DNA from both
aqueous solutions and agarose gels, and up to 10 mg DNA can bind to each column.
The binding buffers provide the correct salt concentration and pH for adsorption of
DNA to the membrane. The sample was added to the QIAquick column and
28


centrifuged for 1 minute to introduce the cDNA into the silica-gel membrane. Then
the column was washed with 0.75 ml of Buffer PE and the cDNA fragment was
eluted by adding 30 (j.1 of Buffer EB (lOmM Tris-HCl, pH 8.5) to the center of the
QIAquick membrane, letting the column stand for 1 minute and centrifuging for 1
minute. The DNA amount was determined on a spectrophotometer at 260 nm
(Sambrook et al., 1988).
2.11 Cloning and Transformation of PCR Product
The PCR product was inserted into the TOPO TA cloning vector by
combining 4 pi of the PCR product, 1 pi of Salt Solution, and 1 pi of pCR4-TOPO
vector (TOPO TA Cloning Kit, Invitrogen, Carlsbad, CA). This 6 pi solution was
incubated for 5 minutes at room temperature (Figure 2.1). The plasmid vector is
supplied linearized with single 3 thymidine overhangs for TA cloning. Then 2 pi
of the TOPO Cloning reaction was added to a vial of TOP 10 one shot chemically
competent E. coli and mixed well. After 30 minutes of incubation (4C), the
reaction mix with the cells was heat-shocked for 30 seconds at 42 C without
shaking followed by transfer to ice. SOS medium (250 pi at room temperature) was
added and the mix was incubated at 37C (1 hour) with shaking. Different volumes
29


of each transformation were spread on pre-warmed (to 37C) LB- agar plates
containing 50 pg/ml ampicillin. The plates were incubated overnight at 37C
(Figure 2.2).
The plasmid pCR4-TOPO allows direct selection of recombinants via
disruption of the lethal E. coli gene, ccdB (Bernard and Couturier, 1992). The
vector contains the ccdB gene fused to the C-terminus of the LacZa fragment.
Ligation of a PCR product disrupts expression of the lacZa-ccdB gene fusion
permitting growth of only positive recombinants upon transformation in TOP 10
cells. Cells that contain non-recombinant vector are killed upon plating. Single
colonies were picked and cultured overnight in LB medium containing 50 pg/ml
ampicillin at 37C. The overnight broth cultures were used for isolation of plasmid
DNA.
2. 12 Isolation of plasmid DNA
Overnight culture (between one and five ml) was harvested by centrifugation
at 10,000 x g for 5 minutes. The supernatant was poured off and the tube was
inverted on a paper towel to remove excess media. The cell pellet was resuspended
(250 pi of cell resuspension solution, Wizard Plus SV Minipreps DNA Purification
30


Kit, Promega, Madison, WI) with vortexing. Then another 250 (0.1 of Cell Lysis
Solution, 10 pi of alkaline protease solution and 350 pi of Wizard Plus
Neutralization Solution were added. The solution was mixed by inverting the tube
after each addition. The bacterial lysate was centrifuged (14,000 x g for 10 minutes)
and the supernatant was transferred to a plasmid DNA purification unit (the Spin
Column was inserted into two ml Collection Tube) and centrifuged at 14,000 x g for
1 minute. The flowthrough was discarded. The Spin Column was washed twice
with 750 pi and twice 250 pi of column wash solution. After the washes, the Spin
column was transferred to a new sterile microcentrifuge tube. The plasmid DNA
was eluted by adding 100 pi of nuclease-free water (supplied with the kit) to the
Spin Column and centrifugation (14,000 x g, 1 minute). The amount of plasmid
DNA was determined on a spectrophotometer at 260 nm (Sambrook et al., 1988).
2,13 Sequencing of plasmids
Forward and reverse Ml3 primers (TOPO TA Cloning Kit, Invitrogen,
Carlsbad, CA) were used for amplification of the insert (Figure 2. 2). The PCR mix
contained 500 ng plasmid DNA, 3.2 pmole of primer (either forward or reverse), 4
pi of Ready Reaction Premix, and 2 pi of 5x Sequencing Buffer brought up to a 20
31


c
Incubate 5 minutes
at room temperature
Transform into TOP 10 E. coli cells
1 '
Select and analyze colonies

Isolate plasmid DNA and sequence
Figure 2. 1
Experimental steps necessary to clone the PCR product
32


LacZa initiation codon
M13 Reverse pnmmq site I T3 pnming site
i I i i
201 CACACAGGAA ACAGCTATGA CCATGATTAC GCCAAGCTCA GAATTAACCC TCACTAAAGG
GTGTGTCCTT TGTCGATACT GGTACTAATG CGGTTCGAGT CTTAATTGGG AGTGATTTCC
261
. Spe I Sse8387 l (Pst I) Pme I EcoR I
T" III
GACTAGTCCT GCAGGTTTAA ACGAATTCGC CCTT|
CTGATCAGGA CGTCCAAATT TGCTTAAGCG GG£
fc'coR I Not I
2agggc gaattcgcgg
TTCCCG CTTAAGCGCC
311
________T7 priming site___^ M13 Forward (-20) priming site
CCGCTAAATT CAATTCGCCC TATAGTGAGT CGTATTACAA TTCACTGGCC GTCGTTTTAC
GGCGATTTAA GTTAAGCGGG ATATCACTCA GCATAATGTT AAGTGACCGG CAGCAAAATG
RNA polymerase binding site: bases 133-178
Lac repressor binding site: bases 179-199
Start of transcription: base 179
M13 Reverse priming site: bases 205-221
LacZa-ccdB gene fusion: bases 217-810
LacZa portion of fusion: bases 217-497
ccdB portion of fusion: bases 508-810
T3 priming site: bases 243-262
Polylinker bases 262-312
TOPO Cloning site, bases 294-295
T7 priming site: bases 328-347
M13 Forward (-20) priming site: bases 355-370
neo (kanamycin) promoter region: bases 1021-1070
neo (kanamycin) resistance gene (ORF): bases 1159-1953
bla promoter region: bases 2062-2156
RNA polymerase binding site: bases 2062-2143
-35 region: bases 2087-2093
bla promoter (P3): bases 2116-2122
Start of transcription: base 2122
Ribosome binding site: bases 2145-2149
bla (ampicillin) resistance gene (ORF): bases 2157-3017
pUC origin: bases 3162-3835
Figure 2. 2
The map of pCR 4-TOPO vector (3957 bp)
33


pi final volume with sterile water. The asymmetric PCR amplification was
performed for 25 cycles at 96C for 10 seconds, 50C for 5 seconds and at 60C for
4 minutes.
The PCR products were purified before sequencing using the Centrifix Gel
Filtration Cartridge (Eagle). The cartridge was placed in a microcentrifuge tube,
which is supplied with the kit and centrifuged (2,800 x g, 2 minutes). Then the
cartridge was transferred to a clean microcentrifuge tube and the plasmid DNA
sample was added to the packed tube. The cartridge and microcentrifuge tube unit
was centrifuged (2800 x g, 2 minutes) and the eluate was dried (SpeedVac). After
resuspending, the dried pellet in 12-25 |il of Template Suppression Reagent (TSR)
the sample was heated at 95C for 2 minutes to denature DNA and chilled on ice.
The nucleotide sequence was determined by the dideoxy nucleotide
termination method (Sanger et al., 1977), using sequenase (ABI Prism 310 Genetic
Analyzer). A homology search of obtained sequence was performed using the
BLAST algorithm at the National Center for Biotechnology Information
(http://www.ncbi.nlm.nih.gov). Sequence comparisons were performed using
CLUSTAL W (www.ebi.ac.uk).
34


3. Results
3.1. RT-PCR
Five pg of total RNA (total yield 20.8 jag) isolated from Xenopus eggs was
used for first-strand cDNA synthesis with random hexamers. The quality of cDNA
was checked via PCR using ODC primers (ornithine decarboxylase gene).
Ornithine decarboxylase is known for its abundant expression and is used as a
positive control. In Xenopus the expression of ornithine decarboxylase remains
constant throughout development. A 385 bp fragment was successfully amplified as
expected (Figure 3.1). The cDNA sample was used for PCR with degenerate
primers. Since 12 different PreMixes were used each PreMix result was somehow
different (Figure 3. 2 and 3. 3). Another band about 600 bp long was amplified in
every PreMix except PreMix C. There were also two bands about 800 bp and 250
bp amplified in each PreMix except PreMixes C and L. PreMix L had only one 600
bp long band.
Total RNA samples from human brain and lung were used for comparison.
The result is also shown in Figure 3. 3.
35


1
1
3
Figure 3. 1
PCR with ODC primers
DNA ladder (Stratagene, La Jolla, CA), (lane 1)
PCR product with ODC primers, (lane 2)
PCR product with ODC primers purified with High Pure PCR
Purification Kit (Boehringer Mannheim, Indianapolis IN), (lane 3)
36


1 2 3
6
7 8
9
10
4 5
1 1
800 bp
600 bp
250 bp
Figure 3. 2
PCR with degenerate primers using FailSafe Premixes
-Premixes A-K (lanes 1-11)
37


1
1
4
J
Figure 3. 3
PCR with degenerate primers using FailSafe Premixes
DNA ladder (lane 1)
Premix L (lane 2)
Human HC (hippocampal) cDNA (lane 3)
Human lung cDNA (lane 4)
38


1
2
3 4 5 6 7
600 bp
Figure 3. 4
PCR with degenerate primers using FailSafe Premixes
Repeat PCR with Premix D (annealing temperatures: 45C, 47C
53.5C, and 58.5C) (lane 1-4)
Human HC (annealing temperature: 53.5C) (lane 5)
Repeat PCR with Premix C (lane 6)
Product of re-PCR with Premix L (lane 7)
800 bp
~ 250 bp
39


In order to reduce effects of mispriming PCR with PreMix D, which had the
strongest signal, the amplification was repeated at four different annealing
temperatures (45C, 47C, 53.8C, and 58.5C)(Figure 3. 4). The results of
reactions at 45C and 47C were identical to previous generating three fragments
(-250 bp, -600 bp, and -800 bp). The reaction at 53.8C generated two smaller
fragments and the reaction at 58.5C amplified a weak band about 250 bp indicating
that this temperature is too high for annealing these primers.
PCR with cDNA generated from human brain RNA was also repeated at
53.8C (Figure 3. 4). This reaction generated three major fragments (-250 bp, -600
bp, and -800 bp).
Also, the PCR product of PreMix L was used for re-PCR at the same
conditions. The results are shown in Figure 3. 4. Surprisingly, there were three
bands seen (-250 bp, -600 bp, and -800 bp). These PCR products were used for
cloning.
3. 2 Cloning of PCR products
The fragments were cut and extracted from the gel. Each fragment was
directly cloned into pCR 4-TOPO plasmid vector (Invitrogen, Carlsbad, CA),
transformed in E.coli TOP 10 competent cells and plated on LB plates containing
40


100p.g/ml ampicillin. Incubation at 37C generated 2-14 well-isolated colonies for
each fragment. These colonies were picked and plasmid DNA was isolated and
sequenced.
3. 3 Sequence analysis
Three fragments amplified via PCR using degenerate primers were
sequenced on an ABI 310 sequencer. Actual sizes of fragments were 850 bp, 615
bp, and 220 bp. A homology search of obtained sequences was performed using the
BLAST algorithm at the National Center for Biotechnology Information
(www.nbci.nlm.nih.aov). Only the 615 bp fragment has shown homology to PLD
(65% identical to rat PLD lb; with conservative substitutions, 80%
conservation)(Figure 3. 5). The other two fragments did not yield significant
matches.
Multiple sequence alignment performed using CLUSTAL W (version 1.81)
has shown that the cloned Xenopus PLD fragment has the same 38 amino acid
deletion as mammalian PLDlb. (Figure 3. 6). This indicates that the fragment
cloned from Xenopus is a fragment of PLDlb. The cloned fragment begins a few
amino acids upstream of the first HKD, continues through the loop domain and ends
in the middle of CRII domain.
41


TGG GCG CAT CAT GAA AAG ATT GTT GTG ATA GAT CAG TCT GTG GCT
TTT GTC GGA GGA ATT GAC CTG GCC TAT GGA AGA TGG GAT GAT GAT
GAA CAC AGA CTA ACT GAT GTT GGC AGT GTA AAG CGG ATT GTA ACA
ACA CAG TCA ACA ACT GCA ATA AAT AAG ATC CCA TCT GAT CCT ACT
CTG TCT TTG AAT GGT GAT AAT AGG GCA GGG AAA AGA AGC TTT CAA
GTT CCA GGC AAG AAA GAT GAG GAT GCA GTG GAT TCA TCG AGA ATC
AGA GGA CTG GGG AAA TCT AAG AAG TTT GCA AAA TTT AGC CTT TAC
AAG CAG CTC CAC AAA CAT AAC CTC CAG CAT GCA GAC AGT GTG AGC
AGC ATA GAC AGC GAA TCT CAT AGA GGA TCA GTT CGC AGT TTG CAG
ACA GGC GTG GGA GAA CTT TTG GGT GAA ACC CGC TTT TGG CAT GGA
AAG GAC TAT TGC AAC TTT GTT TTT AAG GAC TGG GTA CAG CTG GAC
AAA CCA TTT GCT GAT TTC ATT GAT CGA TAC CAG ACT CCC CGC ATG
CCC TGG CAC GAA
Figure 3. 5
Nucleotide sequence o/~Xenopus PLD fragment
Primer binding sequences are underlined
42


Rat. PLDla
Rat PLDlb
Human PLDla
Xenopus PLDlb
Rat PLD2
WAHHEKLVIIDQSVAFVGGI DLA YGRWDDNEHRLTDVGS VKR VTSGQSLGSLTAAS VESM 60
WAHHE KLV11 DQSVAFVGG I DLA YGRWDDNEHRLTDVGS VKR VTSGQS LGS LTAAS VESM 6 0
WAHHEKLVI IDQSVAFVGGIDLAYGRWDDNEHRLTDVGSVKRVTSGPSLGSLPPAAMESM 6 0
WAHHEKIVVI DQSVAFVGG I DLA YGRWDDDEHRLTDVGS VKR IVTTQSTTAINKI PSDPT 60
WAHHEKLLWDQAVAFLGGLDLAYGRWDDVQYRLTDLG----------------------- 3 8
Rat PLDla ESLSLKDKH-QSHKNEPVLKSVDDTD-MKLKGIGKSRKFSKFSLYRQLHRRNLHNSDSIS 118
Rat PLDlb ESLSLKDKH-QSHKNEPVLKSVDDTD-MKLKGIGKSRKFSKFSLYRQLHRRNLHNSDSIS 118
Human PLDla ESLRLKDKN- EPVQNLPIQKSIDDVD-SKLKGIGKPRKFSKFSLYKQLHRHHLHDADSIS 118
Xenopus PLDlb LSVNGDNRAGKRSFQVPGKKDEDAVDSSRIRGLGKSKFFAKFSLYKQLHKHNLQHADSVS 120
Rat PLD2 --------------------------------------------------------------DPSE 4 2
Rat PLDla
Rat PLDlb
Human PLDla
Xenopus PLDlb
Rat PLD2
SVDSASSYFNHYRSHQNLIHGIKPHLKLFRPSSESEQGLTRHSADTGSIRSVQTGVGELH 178
SVDSAS--------------------------------------NTGS IRSVQTGVGELH 140
SIDSTSSYFNHYRSHHNLIHGLKPHFKLFHPSSESEQGLTRPHADTGSIRSLQTGVGELH 178
SIDSES--------------------------------------HRGSVRSLQTGVGELL 14 2
SADSQT----------------------------------------PTPGSDPAAT PDLS 62
* * . *
Rat PLDla
Rat PLDlb
Human PLDla
Xenopus PLDlb
Rat PLD2
GETRFWHGKDYCNFVFKDWVQLDKPFADFIDRYSTPRMPWHD 220
GETRFWHGKDYCNFVFKDWVQLDKPFADFIDRYSTPRMPKHD 182
GETRFWHGKDYCHFVFKDWVQLDKPFADFIDRYSTPRKPWHD 220
GETRFWHGKDYQJFVFKDWVQLDKPFADFIDRYQTPRMPWHE 184
HNHFFWLGKDYSNLITKDWVQLDRPFEDFIDRETTPRMPWHD 104
** *** *.. *******.** ***** ******
Figure 3. 6
Multiple amino acid sequence alignment ofXenopus PLD fragment to human
PLDla, rat PLDla, rat PLDlb, and rat PLD2
* indicates identical amino acids in all sequences, : indicates
conservative substitutions,. indicates semi-conservative substitutions.
38 amino acid splice deletion of PLDla
43


4. Discussion
PCR with degenerate primers amplified three fragments from rat
pheochromocytoma PC 12 cells (Nakashima et al., 1997), and rat C6 glioma cells
(Yoshimura et al., 1996). Sequence analysis of these fragments has shown that all
three fragments are indeed fragments of different PLD iso forms (PLDla, PLDlb,
and PLD2). Using the same degenerate primers we were also able to amplify three
fragments, but sequence analysis has shown that only the 616 bp fragment has
significant homology to PLD, especially to PLDlb. We strongly believe that the
cloned fragment is a part of PLDlb because it has the same 38 amino acid splice
deletion as mammalian PLDlb. Splice variants of PLD 1 are well conserved among
mammalian species.
Katayama et al. (1998) reported that rat PLDla and PLDlb are both
responsive to PIP2 and ARF. However, PLDlb is less responsive than PLDla to
RhoA. Sung et al., (1997) reported that the Rho interaction site is located in the C-
terminus. The deleted part of the loop region might be involved in the C-terminus
and RhoA interaction. The investigators also reported there is a difference in tissue
distribution of PLDla and PLDlb transcripts; PLDlb is expressed in brain, liver,
kidney, small intestines, and colon. PLDla is expressed in heart and spleen. They
are both expressed in the lung.
44


The other two cloned fragments did not show significant homology to PLD.
These bands could be a product of mispriming, which is a common occurrence
when degenerate primers are used. With degenerate primers a relatively low
annealing temperature is used to increase the annealing of primers to DNA.
The results of RT-PCR left a question as to whether PLD la and PLD2 are
expressed in Xenopus eggs. Western blot analysis, performed previously in our lab,
using anti-PLDl and anti-PLD2 antibodies indicated that PLD1 is expressed in
Xenopus eggs and oocytes much more than PLD2. PLD2 antibodies produced very
little or no signal. Also there was a difference of PLD 1 level between eggs and
oocytes. PLD1 level in eggs is higher than PLD1 level in oocytes.
Liscovitch et al. (2000) reported that PLD2 is expressed in the prostate,
placenta, thymus, heart, pancreas and kidney of humans. In rat brains PLD2 level is
very low after birth, but increases during the development and reaches the
maximum in the adult brain indicating that rat brain PLD2 is regulated
developmentally (Peng and Rhodes, 2000). This might be the reason why we were
not able to amplify PLD2 from Xenopus eggs.
In order to confirm these results RT-PCR with degenerate primers should be
performed using RNA isolated from different tissues and cells. That would tell us
how many isoforms of PLD are present in Xenopus and their tissue distribution.
45


Repeating RT-PCR with oocytes could generate an interesting result showing if
there is any difference in PLD mRNA expression between eggs and oocytes.
Since we have a partial sequence of the Xenopus PLD gene we are able to
design homologous primers to it and repeat the RT-PCR at higher annealing
temperature. That would increase the efficiency amplification eliminating the
mispriming.
Since RT-PCR generated only a fragment of PLDlb 5/3-RACE technique
should be used to get the complete sequence of the gene. Then the complete
sequence can be used as a homologous probe for screening of the cDNA or genomic
library.
46


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