|
Molecular
analyses of human and animal genome(s) |
| Principal Investigator : Sher Ali
Project
Associates/Assistants
Ph
D Students
Colloborators The
Protooncogene c-kit receptor having pleiotropic attributes is involved
in control of spermatogenesis, hematopoiesis, and melanogenesis. Pattern of c-kit
receptor protein expression in human and rodents show high level of
similarities suggesting that this gene operates under similar, if not
identical, conditions. Thus, study on c-kit receptor is envisaged to
enhance our understanding on its role in control and regulation of fertility
in the human system. The objectives of the project are (i) expression of c-kit
receptor in Brown Norway rat R.norvegicus testis during different
stages of postnatal development, analyzing each of the domains independently,
(ii) comparative analyses of rat c-kit cDNA sequences in related genomes using
sequences from the GenBank and (iii) assessing phylogenetic potential of the
organizational uniqueness of the sequences within the extracellular domain of
c-kit gene. Expression
of protooncogene c-kit receptor in rat testis and uniqueness of extracellular
domain across the species with potential in molecular phylogeny We
studied expression of protooncogene c-kit receptor in Brown Norway rat R.norvegicus
testis during different stages of postnatal development. Several regions from
within the c-kit gene encompassing different domains were amplified
employing reverse transcriptase polymerase chain reaction (Table-1) and the
resultant amplicons were cloned and characterized. Maximum expression of c-kit
was observed in the testis during the days 10 to 30 suggesting its involvement
in transition of primary spermatocytes towards formation of mature
spermatozoa. Multiple novel transcripts originating from the extracellular
domain were also identified, though their functions remained unknown. The
evolutionary divergence of c-kit cDNA of 11 other vertebrates was
studied downloading their sequences from the GenBank. Analyses of c-kit
cDNA and its protein sequences in rat and related genomes showed
organizational uniqueness across the species. Construction of phylogenetic
tree, based on c-kit cDNA and protein sequences delineated all the
species successfully and was found to be in accordance with the established
positioning of these animals. This may be exploited as a tool in delineating
phylogenetic relationship of different species. Extracellular
domain of c-kit in R.norvegicus reveals variant mRNA transcripts The
c-kit mRNA transcripts in testis from different stages of postnatal
development identified by RT-PCR were found to show size variation in the ECD.
Primers derived from the extracellular domain (nucleotides 17-867) including
5’UTR showed
several still uncharacterized transcripts of about 700, 600 and 550 bps in
addition to the major expected one of 850 bp. These transcripts were
relatively more prominent in the testis samples obtained during days 10-30.
Assessment of amplicons generated with primers specific to 3' untranslated
region revealed an alternate 3' UTR transcript of 600 bp in addition to the
1.25 kb expected one. Transcriptional analysis from the cytoplasmic domain and
the entire c-kit cDNA showed a single amplicon each of the expected
size of 971 bp and 3.7 kb, respectively. Differential expression of c-kit during
different stages of postnatal development was seen despite similar quantities
of template DNA used for reaction. This was evident from the b-actin
RT-PCR amplification used as control for quantification of cDNA showing
similar signal intensity. Table-1: Oligo primers encompassing all the domains of c-kit cDNA of rat Rattus norvegicus used for RT-PCR amplification
*
Primers derived from extracellular domain; #
cytoplasmic domain; #a
includes 5’UTR; Evolutionary
divergence of c-kit cDNA in two species of rats, R.rattus and R.norvegicus As
mentioned earlier, c-kit receptor gene is highly conserved with an
equally conserved biological function. However, in the present study,
tissue-specific organizational variation of the sequences within and between
the two species of rats (R.norvegicus and R.rattus) was observed
without an alteration in the overall charge of the protein. The nucleotide
changes within the ECD of the c-kit suggest that evolutionarily, these
are not unfavored and perhaps reflect fixation of allele. Sequences
from the RT-PCR generated amplicons of testis representing all the domains of
the c-kit receptor from R.norvegicus were compared to the known c-kit
cDNA of the R.rattus brain by blast similarity search using
NCBI default server (http://www.ncbi.nlm.nih.gov/blast). The result showed
80-90% homology at the cDNA level and 92-96% at the amino acid sequence level
involving alteration of the “aa” I®F
and L®F
(non-polar), DÛE
(acidic) and N®S
(polar uncharged) residues. Functionally, the 4-8% variation at “aa” level
was insignificant because it did not alter the overall charge of the protein.
However, this work led to an important observation that organizational
uniqueness of c-kit cDNA maintained across the species was reflected
even between the two closely related species of rats. The variations observed
at the nucleotide sequence level are greater than that at amino acids level
clearly distinguishing the two species of rats. Homology
of c-kit cDNA and protein sequences amongst different species Clustal
W based multiple alignment of c-kit cDNA from eleven different species
(Table-2) revealed primary structure of c-kit to be highly conserved,
although no consensus sequence could be deduced at the 5' and 3' UTRs. Protein
sequence alignment showed maximum variation within the extracellular domain
amongst different species indicating 36.2% completely conserved, 36% highly
conserved and 10.7% fairly conserved residues, amounting to a total of 82.9%
homology. In contrast, rest of the c-kit receptor showed 78.6%
completely conserved, 11.9% highly conserved and 4.8% fairly conserved
sequences giving rise to a total of 95.3% homology. The Cysteines (C) and
Tryptophans (W) in the extracellular domain were completely conserved. The GNN/SK
motif, a known splice site in the juxtamembrane domain was absent in mouse,
horse and chick. Multiple alignment of amino acid sequences showed high level
of conservation in closely related species such as cattle and goat, and rat
and mouse (data not shown), where L®
V; V®G;
F®
L; E®T
changes occurred only in mouse and rat. Similar changes (e.g. C®F)
were observed in the cattle and goat. Non-conserved changes in the amino acids
were observed at the same position in all the species conferring
organizational uniqueness of c-kit gene in these animals. Table-2:
Sequences of c-kit cDNA of eleven different speicies with their Gene
Bank accession numbers
Upregulation
of c-kit expression in rat R.norvegicus testis The
requirement of c-kit for the migration of gonocytes to produce type A
spermatogonia has been well established. In the present study, maximum signal
in testes during the days 10 to 30 and detection of transcripts in addition to
the expected ones suggested up-regulation of the c-kit gene and its
alternative splicing. Thus, up-regulation of c-kit expression
substantiates its role in meiosis and spermiogenesis. Evolutionary
conservation of c-kit protein The
c-kit receptor comprises 5 Ig-like domains in the extracellular domain
besides multiple intermolecular disulfide bonds. The conservation of disulfide
bonds guarantees that the overall topology and tertiary structure of the c-kit
receptor remains the same in all the species. This is corroborated by the fact
that interspecies chimeras of the c-kit molecule can be created without
significant loss of functional activity. In the present study, tryptophan
residue crucial for biological activity of the receptor was found to be
conserved. This is in accordance with the earlier observation where mutations
involving tryptophan residues in the extracellular domain of c-kit in
humans were found to downregulate receptor function. Phylogenetic
potentials of c-kit receptor Phylogenetic
trees based on c-kit cDNA alignment from different species showed a
close relationship between rat and mouse, goat and cattle and dog and cat. On
the other hand, Xenopus, chick and zebrafish were found to be the most
distant species. The bootstrap values of most of the nodes were greater than
90% in the neighbour joining trees. However, in the maximum parsimony tree,
the branches comprising human and horse were not supported by high bootstrap
value as it was only ~59%. Nonetheless, the overall positioning of the species
was found to be in accordance with the established phylogenetic trees based on
paleontological records. The
c-kit gene exemplifies evolution of ECD in a species-specific manner
while retaining its biological functions across the species. Thus, sequences
from the ECD may be exploited for the phylogenetic analysis of the species.
Publications Original peer-reviewed articles 1.
Prasanth SG, Chattopadhyay M, Bhat KV and Ali S (2004) Expression of
protooncogene c-kit receptor in rat testis and uniqueness of
extracellular domain across the species with potential in molecular phylogeny.
DNA Cell Biol 23:35-43. 2.
Prasanth SG and Ali S (2003) Expression of protooncogene c-kit
receptor in rats Rattus norvegicus and identification of a mutant mRNA
transcript implicated in spermatogenic failure. DNA Cell Biol 22:447-456
Reviews/Proceedings 1.
Prasanth SG, Giran HM and Ali S (2004)
Biology of protooncogene c-kit receptor and spermatogenesis. Curr
Pharmacogenomics 2:1-14. 2.
Ali S
and Hasnain SE (2003) Genomics of the human Y -chromosome: 1- association with
male infertility. Gene 321:25-37. 3.
Bashamboo A, Giran HM, Azfer MA and Ali S (2003) Genomics of the human
Y chromosome and molecular diagnosis. Proc Ind Nat Sci Acad B69:525-528. |
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