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Genes and proteins expressed specifically
in testis |
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Principal Investigator : Anil K Suri Project Associates/Assistants Collaborator Morphological and biochemical observations along
the molecular biological approaches suggest that germ cell differentiation is
accompanied by selective gene expression. However, the molecular mechanisms
regulating the cell type specific gene expression during spermatogenesis are
yet to be determined. Identification of testis specific genes and their
characterization would thus enhance our understanding about the functioning of
sperm at the molecular level. This would possibly highlight the mechanism of
sperm development, maturation and sperm-egg interaction. This project’s goal
is to investigate testis specific genes expressed in a restricted, temporal
and spatial manner during spermatogenesis. Cloning, expression and purification of hss
recombinant protein The human sperm surface recombinant protein hss
construct A (complete ORF having Leucine zipper LZ, antgenic determinant sites
A1-A3, Transmembrane domain T and cytosolic domain C), construct C (LZ, A1-A3,
DT,
DC), and construct D (A1-A3, DLZ, DT,
DC) were cloned in pET 28b (+) and successfully expressed in E.coli
BL 21 (DE3) pLyse. The recombinant protein was subjected to a purification
procedure. Since the recombinant protein was fused with 6 Histidine residue of
the expression vector at COOH terminus, the expressed protein was purified
using Ni-Histidine bind resin affinity chromatography. Initially small-scale
purification of recombinant protein was done by His- Bind resin using two
approaches: pH gradient and by eluting with imidazole. It was observed that
the recovery of recombinant protein was higher using imidazole as compared to
pH gradient. Therefore 1 M Imidazole was used for large-scale purification of
recombinant protein. Before initiating further studies with hss
recombinant protein, recombinant protein was subjected to mass spectrometry in
order to find whether our recombinant expressed protein had the same hss
deduced sequence which we have published (EMBL Acc. No. X91879). The
recombinant protein was run on SDS page gel and band of interest was
processed. The gel piece was transferred to a siliconized tube and washed and
destained in 200 ml 50% methanol overnight. The gel pieces were treated in 20 ng/ml
trypsin in 50 mM ammonium bicarbonate on ice for 10 min. Any excess trypsin
solution was removed and 20 ml 50 mM ammonium
bicarbonate added. The sample was digested over night at 37°C
and the peptides formed extracted from polyacrylamide in 30 ml
aliquots of 50% acetonitrle/5% formic acid. These extracts were combined and
evaporated to 25 ml for LC-MS analysis. The data thus
generated were analyzed by database searching using the Sequest search
algorithm. Results of the Mass Spectrometry revealed that micro-sequencing of
hss recombinant protein (approximately 45% of recombinant protein was analyzed
of various peptide lengths: in total 205 amino acids analyzed of deduced 766
amino acids) matched with our deduced protein which we have cloned. No other
protein was found to have homology with our protein, therefore it is a novel
protein. The hss recombinant
protein was used to immunize rabbits, rats and monkeys. Antibodies thus raised
against recombinant protein are being used to examine the properties of
antibodies to investigate the affect on sperm functioning using several
functional in vitro assays. Genomic structural organization and chromosomal
mapping of hss gene We have cloned a human sperm surface gene
designated as hss gene, which encodes for a protein having Leuzine
zipper (LZ), antigenic determinant sites (A1-A3), transmembrane domain (T) and
cytosolic domain (C). Tissue distribution of hss gene was studied using
different tissue mRNA that revealed that hss gene was exclusively
expressed in testis only. Expression of hss transcript was observed only in
round spermatids indicating post meiotic haploid gene expression. Human
genomic leucocytic library was screened using hss cDNA. Genomic clone was
successfully isolated and was sequenced. Attempts were made to do the
chromosomal mapping of hss gene. Fluorescent in situ hybridization to
normal human male metephase chromosome was carried out. Assessment of well
spread metaphase chromosomes indicated signals of hss gene on both
homologous chromosome 17, mapping long arm of chromosome 17 at band 17q 21.3-q
22. Studies on post-transcriptional regulation of
human hss gene during spermatogenesis The hss nucleotide sequence revealed the
presence of three unique palindromic motifs and hairpin loop in 5’ and 3’
sequence. It is observed that Pal I is present in 5’ UTR, Pal II is present
just before stop codon in ORF and Pal III is present in 3’ UTR. It was
earlier reported that using in vitro synthesized mRNA transcript of
respective Palindromic motifs, specific RNA protein complexes were formed
using testicular cytosolic extract. Further on Northwestern studies have been
carried out to find out the mass of those RNA binding proteins, which bind
specifically to these motifs. Attempts are also being made to screen the
testis cDNA expression library to fish out the clone of interest, which
expresses the RNA binding protein, specifically binding to these motifs. |