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Analysis of Two Genes Encoding Prothrombin Activators in Australian
Common Brown Snake (Pseudonaja textilis)
Bachtiar, M. 1 , Kwong, S. 2 , and Kini, R. M. 3
Department of Biological Sciences, Faculty of Science, National University of Singapore
10 Kent Ridge Road, Singapore 117546
ABSTRACT
The presence of two parallel prothrombin activators for an extremely different
physiological role in the Pseudonaja textilis (P. textilis) snake is unique. One prothrombin
activator, pseutarin C, is a multi subunit protein found in the venom as a toxin, and can activate
blood clot in target preys. The pseutarin C enzymatic subunit (PCCS) and non-enzymatic
subunit (PCNS), is found to be structurally and functionally similar to endogenous blood
coagulation FXa and FVa, respectively. The project focuses on the analysis of PCNS and FVa
intron 2 gene structures in elucidating the different mechanism involved in their gene expression
regulations. This is because despite of the similarities, the two genes are expressed very
differently. The PCNS gene expression in the venom gland is inducible and in a great quantity,
in comparison to the constitutive FV gene expression in the liver. In explaining this intriguing
phenomenon, intron 2 DNA sequences of both genes were analysed in search of any similarities
and differences, as well as evolutionary relationship. Putative matrix attachment and
transcription factors sites search reveal no significant differences between the two. However,
DNA sequence alignment shows the presence of 283 bp insertion in the PCNS gene, suggesting
that the gene may have evolved from that of FV.
INTRODUCTION
The objective of the project is to determine the intron 2 region of P. textilis FV and PCNS
genes. The DNA sequence are analysed for identities, structures and other things such as the
presence of significant transcription regulatory sites. The result is used to improve our
understanding on the importance of FV and PCNS gene structures, in explaining their different
gene expression regulations and evolutionary relationship.
MATERIALS AND METHODS
Intron 2 of both FV and PCNS genes from P. textilis were specifically obtained thru
fragment amplification using the long range gDNA nested PCR approach. For each fragment
produced after the primary amplification, a secondary nested amplification was performed to
confirm for the accuracy of the primary PCR product (figure 1). Following agarose gel
electrophoresis, the attained FV and PCNS intron 2 fragments were purified by gel extraction
1
Student
Asst. Supervisor, Graduate Student
3
Supervisor, Professor
2
using Qiaquick Gel extraction kit. The purified DNA was then ligated into a plasmid vector for
cloning and sequencing.
1FN
Promoter
Exon 1
Intron 1
1F
Exon 2
1R
Intron 2
1RN
Exon 3
(A) Factor V
1P
Promoter
Exon 1
Intron 1
2P
Exon 2
3P
Intron 2
4P
Exon 3
(B) PCNS
Figure 1. gDNA PCR of FV (A) and PCNS (B) intron 2 regions. The FV PCR primer
pairs are FV Int2-2FN & 1RN and FV Int2-1F & 1R for the primary amplification
and secondary amplification, respectively. Similarly, the primary and secondary PCR
primers for PCNS are PCNS Int2-3FN & 1RN and PCNS Int2-2F & 3R, respectively.
BigDye® Terminators DNA sequencing protocol using the ABI 3100 Automated Capillary
DNA Sequencer was initiated from both ends of the insert within the pGEM®-T plasmid, which
contains T7 and SP6 promoter sequences. T7 and SP6 sequencing primer pairs were utilised for
the first sequencing step. Following the first sequencing result, sequence alignment was
performed to detect the presence of overlaps. A successive sequencing steps through the used of
internal primers was then follow suit, till the two sequencing fragments overlap.
RESULTS
Sequence analysis of P. textilis FV and PCNS intron 2
The DNA sequencing results showed that intron 2 of FV is 1,555 bp in length, whilst the
PCNS is 1,740 bp. Aside of the presence of two minor short insertions near the 3’ end of PCNS
intron 2, there is a 283 bp insertion near the 5’ end of the sequence.
There are two possible alternatives for which the optimal alignment can be arranged. The
difference between these alternatives is on the positioning of a “shadow-overlap-regions” (figure
2). The first alternative gives ~86% identity, whilst the second alternative gives ~70% identity.
In the second alternative, the ‘shadow-overlap-regions” is regarded as a sequence deletion in
PCNS intron 2. This is because of its low identity (~24%) after aligning them locally. After
performing a dotplot pairwise alignment, the second alternative is chosen as the best alignment
option. The dotplot optimal pairwise alignment result shows the presence of a gap at the position
where the PCNS deletion and insertion takes place.
S/MAR Analysis
S/MAR search result shows no significant difference on the potential probability of matrix
attachment region in both intron sequences. Highest S/MAR potential is about fifty percent,
which is located just after the mid point of the intron.
Transcriptional factors binding sites
The complete FV and PCNS intron 2 sequences, as well as the PCNS deletion and
insertion sequence were used as template for the transcription factors binding search. Among the
results that were found on both intron 2 sequences were sites of GATA-1 binding factor, HNF
(hepatic nuclear factor) proteins, CCAAT box, and SP1 transcription factor.
Alignment Alternative 1
109 bp overlap region within
FV intron 2
FV intron 2 (1,555 bp)
5 bp insertion
4 bp insertion
283 bp insertion
Overall Identity: 85.77%
109 bp “shadowoverlap-regions” within
PCNS intron 2 insertion
PCNS intron 2 (1,740 bp)
FV intron 2 (1,555 bp)
5 bp insertion
4 bp insertion
283 bp insertion
PCNS intron 2 (1,740 bp)
109 bp deletion
Overall Identity: 70.34%
Figure 2. Two alternatives for the optimal alignment of P. textilis FV and PCNS intron
2. The major difference is on the positioning of the so called “shadow-overlap-regions”
of 109 bp in the PCNS sequence, in respect to that of FV.
DISCUSSION
Complete DNA sequencing of the whole fragment of P. textillis FV and PCNS intron 2 is
a step forward for the study. It has given us another set of direction to which aspects of the gene
structure is worth investigating in order to elucidate the different mechanisms involved in the
regulating of transcription activities of the two genes. However no direct conclusion can be
made for the importance and significance of intron 2 in the mechanism for the differentiated
expressions of the two genes. The next step for observing the cis-regulatory activity of intron 2
can be assisted by the construction of the firefly luciferase expression assay, where intron 2 can
be used as the cis-element contributing to the regulation for reporter gene expression. Next, in
recognising for any important upstream promoter element, a 5’ deletion study can be performed
on the whole FV and PCNS promoter region before continuing the DNA sequencing of this
unfinished region (Chen, 2007).
The question on how far have the PCNS gene diverged from that of FV also awaits further
answers. Only intron 2 has so far been recognised to contain a significant amount of sequence
insertion and deletion, as none are found on the promoter and intron 1 regions of the two genes
(Chen, 2007). Pairwise alignment of intron 2 produced two alternatives, in which the second
alternative is suggested to be a more reasonable option following the dotplot alignment. More
answers can be derived from sequencing of the remaining introns of the two genes, so that the
complete DNA sequences are known for a global pairwise alignment to be performed.
Therefore, a lot of studies are needed in order to understand more on the structure-function
relationship of both FV and PCNS genes in their role as prothrombin activators.
REFERENCES
Chen Guanshen, M. (2007), "Putative Differences in the Regulation of Expression between
Factor V and Pseutarin C Non-enzymatic Subunit (PCNS) in Pseudonaja textilis Snake", a
Final Year Thesis in the Department of Biological Science, National University of
Singapore: Singapore.
Minh Le, T.N., Reza, M.A., Swarup, S. and Kini, R.M. (2005), "Gene duplication of coagulation
factor V and origin of venom prothrombin activator in Pseudonaja textilis snake",
Thrombosis and Haemostasis, Vol 93(3), 420-429.
Rao, V.S. and Kini, R.M. (2002), "Pseutarin C, a Prothrombin Activator from Pseudonaja
textilis Venom: Its Structural and Functional Similarity to Mammalian Coagulation Factor
Xa-Va Complex", Thrombosis and Haemostasis, Vol 4(88), 611-619.