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correspond to a specific gene in the genome. The diversity is
generally thought to be attributable to such mechanisms as
gene mutation, gene conversion or alternative splicing of
pre-mRNA[4]. However, no definitive conclusion can yet be
drawn regarding the molecular mechanism by which their
structural diversity is achieved. Therefore, research effort in
this area may not only help to reveal the molecular evolution
of snake venom proteins, but also improve our
understanding of their structure-function relationships and provide
guidance for better exploitation of these substances, especially
with respect to recombinant production.
In the present study, some new TLE cDNAs from
Deinagkistrodon acutus were cloned. The cDNA sequences
were aligned and analyzed to determine possible rules for
sequence variations. A phylogenetic tree was constructed
and discussed.
Materials and methods
Materials Kits for RNA extraction, reverse transcription,
and plasmid rapid extraction were purchased from
BioDev-Tech Scientific and Technical Co, Beijing, China.
Taq DNA polymerase and T4 DNA ligase were from Beijing
Sino-American Company. The competent Escherichia
coli cell preparation kit was the product of Sangon, Shanghai, China.
Plasmid pGEM-T and E coli JM109 strain were from Promega.
Four adult Deinagkistrodon acutus snakes were collected
from Hunan Province, China.
Reverse transcription-polymerase chain reaction and
cDNA cloning Isolation of the total RNA from a snake venom
gland and the reverse transcription-polymerase chain
reactions (RT-PCR) were performed as described
previously[3]. The total RNA in the experiment was a preparation from a
single snake venom gland. For PCR, a pair of degenerate
primers was designed on the basis of the highly-conserved
N- and C-termini amino acid sequences of TLEs:
T1 5กฏ-GTC ATT GGA GGT GA(TC) GA(AG) T-3กฏ;
T2 5กฏ-A(CT)G GGG GGC AAG T(TC)G C(AG)-3กฏ. The 5กฏ dATP was designed as per
the first nucleotide in the stop codon.
The PCR products amplified with Taq DNA polymerase
were cloned into the pGEM-T vector and the inserted cDNAs
were sequenced with ABI Prism 377-96 by Sangon. Each
cDNA was sequenced in two directions.
Software cDNAs were translated and exported with
VISED, a freely distributed software (downloaded from
http://iubio.bio.indiana.edu). Sequence alignment was performed
with Omiga 2.0 (developed by Oxford Molecular
Office)[5] and exported with GenDoc (downloaded from
http://www.cris.com). Phylogenetic trees were constructed using
Megalign[6]. Similarity searches were carried out online
using the BLAST program at the website
http://www.ncbi.nlm.nih.gov.
Results
By using the RT-PCR and cDNA cloning strategy, we
obtained 7 complete cDNA sequences coding for mature
TLEs, that is, ac1, ac5, ctq, n1, R3, R5, and tler7. We
compared the cDNA sequences (Figure 1), and the statistical
reports show that the sequence identities range from 81% to
99%. Despite the differences, the TLE sequences have
certain common features. First, all the cloned cDNAs have
open reading frames that encode 234 amino acids. Second,
their deduced amino acid sequences were homologous with
known snake venom TLEs, and an NCBI Conserved Domain
Search[7] demonstrated that they were all homologous with
trypsin-like serine protease domains (CD accession
numbers cd00190, smart00020, pfam00089, and COG5640). Third,
no amino acid substitution of the catalytic triad residues
was found, which suggests that the proteins were probably
functionally active enzymes.
ctq By using the BLAST program we demonstrated that
the amino acid sequence deduced from ctq cDNA had 99%
identity with the thrombin-like enzyme precursor (GenPept
accession number AAK12273) and 83% identity with acubin
(GenPept accession number CAB46431) from
Deinagkistro-don acutus.
ac1, ac5 , R3, and n1 On the basis of BLAST analysis,
the amino acid sequence of ac1, ac5, R3, and n1 had the
highest degree of identity (99%) with the DAV-PA pre-cursor[8] (GenPept accession number AAF76378). Points of
difference from the DAV-PA precursor are that these
sequences have Asp-5 instead of Asn-5, which is a
conservative substitution; furthermore, Gln-191 is replaced by
His-191 in n1, and His-210 substitutes for Tyr-210 in ac1.
Comparison of the cDNAs of R3 and the DAV-PA precursor
indicates that there is a synonymous base substitution of CCT
with CCC, which both encode Ser-108.
ac1, ac5, R3, and n1 had the second highest degree of
identity (94%) with DFA1 (Deinagkistrodon
acutus thrombin-like defibrase1, GenPept accession number
AAD19350)[9]. DFA1 has only 233 amino acid residues because it lacks
Tyr-161 of ac1, ac5, R3 and n1.
R5 and tler7 R5 cDNA has only a single base difference
from the cDNA of the thrombin-like enzyme precursor of
Deinagkistrodon acutus (GenBank accession number
AF333768); that is, at site 612 the former has an A and the
latter has a G. But this change occurs at the third position of
the codon, and both CCA and CCG code for proline.
There-fore, the amino acid sequences remain unchanged.
tler7 also had the highest degree of identity (85%) with
thrombin-like enzyme precursor (AAK12273), and its degree
of identity with acubin was 83%. tler7 cDNA was accepted
by GenBank, and its accession number is AF362127.
Abnormal sequence T4 An abnormal sequence, T4, was
also cloned. Although T4 cDNA is similar to the other cDNAs,
it has 681 bp (shorter than the usual 702 bp), and it would
encode a truncated peptide, because a nonsense codon
appears in the middle of the reading frame. We compared the
sequence with those of other TLEs and with the batroxobin
gene (GenBank accession number X12747), a thrombin-like
enzyme from Bothrops atrox moojeni
venom[10], and found that a segment of T4,
GFPLNGFERQYFLFQAMRSA-PLVGDNGNYSSMHLGGKLZ was aberrant. It replaced the
segment normally encoded by exon 3, with the batroxobin
gene as reference, and BLAST analysis of its cDNA showed
that it has 91% identity with a region of intron 3 that
conforms to the Breathnach-Chambon
rule[11]. Intron 3 has a tandem repeat of TTGGTTGGAGACAATGGAAA (from 6712
to 6751) in the region (Figure 2). These results suggest three
things. First, the gene structures of TLEs might be similar in
Deinagkistrodon acutus and Bothrops
atrox. Second, a putative minisatellite site exists in intron
3. Third, T4 is a possible product of alternative splicing.
Discussion
Factors contributing to variations Snake venom TLEs
exhibit great variability, but their molecular scaffolding is
conserved[12]. From a genetic viewpoint, conservativeness
and variability are normally maintained in a delicate balance
that is probably ensured by a set of mechanisms or a
"super-visor". As for the TLEs, the balance seems to incline
towards variability. TLEs resemble antibodies in that they
also could be seen as armaments against all kinds of exterior
or environmental factors. A relaxed mechanism is used and
the possibility of variation is enhanced. Evolution has
selected the changeability itself.
Multiple alignments suggested that the variation could
be categorized into three types: type I, II, and III. Type I
relates to the differences involved in
relatively large seg-ments. The alignment of
the cDNAs of R3, R5 and tler7 illustrates variation of this type. Continuous sequence
identities between R3 and tler7, and between R5 and tler7,
suggest that the combination of R3 and R5 produced tler7 (Figure
3). Judging by the organization of the batroxobin gene, so
far the only known genomic structure of TLEs of snake
venom , it is obvious that the combination is neither the
result of alternative splicing of the pre-mRNAs nor that of
trans-splicing. A similar phenomenon was reported by Siigur
et al[13]. We propose that the variation is in fact the result of
post-transcriptional recombination, as we have previously
hypothesized for snake venom C-type lectin
proteins[14,15]. The putative crossover sites at around 20_40 and 260_280
were shared by the 3 cDNAs (Figure 4), and some other
dispersed sequence identities in R3, R5 and tler7 were also
observed. At the present time it is not clear whether these
regions are a prerequisite for or a consequence of
recombina-tion. If the former were true, the conserved crossover sites
could be explained as the feature of the gene family; whereas
if the latter were true we would infer that the recombination
was proceeding continually in the live venom gland.
Furthermore, we analyzed 3 cDNA sequences from
GenBank: Deinagkistrodon acutus thrombin-like protein 1
(AY861382), thrombin-like enzyme 2 (AY861138) and
thrombin-like protein 3 (AY861383). Multiple
alignment of the 3 sequences provided additional evidence for the
recombination hypothesis (Figure 5).
The characteristics of the recombination coincide with
those of some homologous or non-replicative homologous
RNA recombination models[16]. The mechanism and the
enzymatic basis of this proposed post-transcriptional
recombination remain to be elucidated, and its evolutionary origin is
also mysterious. Viral RNA genomes undergo rapid
evolution[17], and so far RNA recombination has been found only
in RNA molecules that have genomic functions, such as
genomes of RNA viruses, excluding RNA
processing[18,19]. We wonder if there exist evolutionary connections between
eukaryotic recombination and viral genomic recombination.
Type II variation in alignment relates to point mutations,
including deletions/insertions of one or several bases and
base substitutions (missense mutations or
synonymous/conservative mutations) as illustrated in Figures 6 and 7.
For base substitutions, all 6 possible shifts have been found,
but shifts between A and G seem to occur with the greatest
frequency, according to our preliminary statistics. In some
alignments, shifts between C and T were the second most
frequent. Our BLAST analyses and comparison data
suggest that type II variations were the most widely dispersed
in the present study. The origins of type II variations are not
yet understood, but RNA editing, gene conversion or point
mutation caused by RNA recombination might be the
primary cause[20,21]. Therefore, the relationships between type
I and II variations are worth studying in the future.
Type III variation in alignment comprises mistakes. T4 is
not the sole example of this type. We have also cloned
another partial cDNA sequence, O1, which had the same
abnormal splicing as T4 cDNA; that is, a segment of intron 3
took the place of exon 3. However, O1 is different from T4
cDNA because of some type II variations. These findings
again highlight the instability inherent in the flow of genetic
information in snake venom.
The preceding discussion is based on data gathered
using some traditional techniques, such as PCR. Therefore,
replication errors or template switching in DNA
amplification should be considered. To circumvent these potential
problems, high-fidelity Pfu DNA polymerase was also used
to validate the accuracy of PCR reactions catalyzed by
Taq DNA polymerase, and the errors proved to be negligible as
far as our experiments were concerned. In future
studies, cDNA libraries and genomic DNA libraries should be used.
However, an overall understanding of snake genomes is
necessary to answer questions regarding the diversity of TLEs.
Horizontal gene transfer Alignment of defirase 1, acubin
and ancrod (Figure 8) revealed a segment that is peculiar to
ancrod: PRTRWGE (81_87)[22]. Ancrod is purified from the
venom of Calloselasma rhodostoma, and is being clinically
used for the treatment of conditions such as acute ischemic
stroke. BLAST analysis (expect=10000) of the segment hit
only 2 unknown proteins from Arabidopsis
thaliana (aside from ancrod itself), and the degree of similarity between them
was 85%. That segment probably represents a novel motif,
and it possibly originated from other organisms through
horizontal gene transfer. A retrovirus might have played a
role in the horizontal transfer[14,23].
Phylogenetic analysis Phylogenetic trees (Figure 9)
constructed by using some TLE amino acid sequences are
consistent with conventional taxonomy; that is, the degree of
variation is greater between species than within species.
TLEs seem to have some value for relationship estimation.
However, TLEs of the species Deinagkistrodon
acutus do not possess geographic features according to our
phylogenetic analysis. Wang et al constructed TLE phylogenetic
trees to find functional groups[8]. Because functional
alterations may involve a very limited number of key amino acid
residues, direct experiments on structure-function
relationships at the DNA and protein level need to be conducted: an
important task for studying the structural biology of TLEs.
References
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