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Development of a universal probe for DNA fingerprinting
Dr Singh and his colleagues in the CCMB, at Hyderabad, developed a probe called Bkm-derived probe for DNA fingerprinting, as a fall out of their earlier internationally well known work on the mechanisms of determination of sex. Their probe is being extensively used for forensic investigation, paternity determination and seed stock verification. DNA fingerprinting evidence was presented in the court and for the first time in the annals of Indian history this result was accepted as an infallible evidence in the court of law. This verdict was upheld by the Kerala High Court. Since then, they have used this indigenous technique in 500 cases such as paternity disputes, identification of missing children, identification of mutilated bodies, exchange of babies in maternity wards and cases of rape and murder, etc. These include sensational cases of assassination of the late Prime Minister Shri Rajiv Gandhi, assassination of Punjab Chief Minister and the famous tandoor case of Naina Sahni.
A separate autonomous "Centre for DNA Fingerprinting and Diagnostics" has been set up by the Department of Biotechnology, Government of India, which is presently housed in CCMB to apply and further develop this technology for the benefit of the country. The Centre is already providing DNA diagnostic services for many genetic disorders such as Thalassemia, Sickle Cell Anemia, Mental Retardation, Fragile-X Syndrome, Duchenne Muscular Dystrophy, Huntington's Disease, Azoospermia, etc. The ultimate aim of the Centre is to develop, acquire and standardize the protocols for carrier detection, prenatal diagnosis and genetic counseling for all the genetic disorders prevalent in our country. This is an important achievement for CCMB and CSIR. In recognition of this contribution, Dr Singh and his group have recently been awarded the CSIR Technology Award for the year 1992.
DNA fingerprinting using indigenously developed Bkm-derived probe for phylogenetic analyses
DNA Fingerprinting has so far been used only in individual identification and establishment of biological relationships owing to its extreme power of resolving individual specific variation present in the genome.
At CCMB Dr Singh and his colleagues by using the indigenously developed Bkm-derived probe, which is successfully used for human DNA fingerprinting, have shown, for the first time, that DNA fingerprinting can effectively be used to infer the generic affinities among related group of animals like crocodilians. This was hitherto thought not to be feasible largely because the fingerprint profiles are believed to evolve too rapidly to be informative over large time intervals. Based on qualitative differences in the fingerprints and quantitative differences in the copy number of Bkm-related sequences in the genomes, they have been able to infer generic affinities among different species/genera of crocodilians, which are in agreement with the consensus phylogeny reconstructed using various other approaches together. This observation is of great importance as it establishes, for the first time, the potential utility of this molecular technique in the study of evolutionary relationships of plants and animals. This is presently being used by Dr Singh for wild life preservation and better management of endangered species in our zoos and also for identification and isolation of genes for useful characters in silkworm races. This technology is also being used for medical diagnosis.
Molecular basis of sex determination
Isolation of highly conserved sex chromosome-specific satellite DNA, 'Bkm', from the female Indian snake, the Banded Krait, by Dr Singh, made the beginning of our understanding of the molecular basis of sex-determination, which is one of the most important unsolved problems in modern biology. This discovery, published in Cell (1982), was highlighted at several occasions in Nature (News and views) and in New Scientist, and is considered to be a major breakthrough in the understanding of molecular basis of sex-determination. This has now become the basis of our understanding also of sex-reversal in humans.
Isolation and characterization of a testis organizing gene (TO)
Sex determination in mammals is regulated by gene/s present on the Y chromosome - the presence and activation of which makes the bipotential gonad to adopt the male differentiation pathway. Recently a new gene termed SRY was discovered (Sinclair et al., 1990) which is considered as the most likely candidate for testis determining factor (TDF). However, absence of SRY and apparently all other Y sequences in majority of XX males and XX true hermaphrodites, and its presence in majority of the XY females with no detectable SRY mutation hitherto remains unexplained. These studies strongly suggest the presence of at least one important gene in the testis-determining pathway which must be present on the X chromosome and subjected to X inactivation. By making use of his Bkm probe isolated from snake described earlier, Dr Singh and his associates have isolated a gene from human testis cDNA library which is present in both sexes but expressed specifically in the male (testis) irrespective of the nature of heterogamety (XX/XY or ZZ/ZW). It is also expressed in the male mouse embryos at the stage at which differentiation of testis begins. These and their other studies concerning its mapping, conservation and association with sex related abnormalities suggest that the gene ' TO' is an important gene, other than the SRY, which may be involved in the complex pathway of sex determination. This explains all hitherto unexplained familial cases of XX males, XX hermaphrodites and XY females. Exploitation of this gene in animal breeding programme has tremendous potential in producing farm animals of desired sex.
Sex and germ cell specific Bkm binding protein
Bkm sequences in snakes, Bkm-associated Y-specific sequences p 102 d (2) in human and M34 in mouse, are interspersed amongst other sequences along the entire length of the sex-determining chromosomes including the sex-determining region. The consensus sequence in these sex chromosome specific repeats is the conserved tetra nucleotide repeat GATA component of Bkm. The associated sequences are species specific. The sex-determining chromosome remains condensed in all somatic cells but decondenses extensively in the germ cells behaving like a supergene. This might be brought about in response to a specific signal present only in the germ cells of the heterogametic sex. Dr Singh and his associates have purified a sex and germ cell specific protein from snake ovary which specifically binds GATA repeats of Bkm. This may be the potential signal responsible for the decondensation (activation) of the sex-determining chromosome. This explains the evolution, universal heterochromatic nature and functional significance of molecular organization of the sex determining chromosomes.
Molecular characterization of silkworm races by using Bkm probe, and its potential use for marker assisted breeding and varietal identification
Dr Singh and his colleagues utilized Banded Krait minor satellite DNA (Bkm)-2(8) probe for the characterization of different silkworm genomes. They have demonstrated that our indigenously developed Bkm probe can be used in generating DNA fingerprints in all the 13 genotypes studied. Using the two representative adverse genotypes, Nistari and NB1, we also showed that the genotype specific fingerprints are inherited by their F1 hybrid offspring. The presence of bands specific to diapausing and non-diapausing strains and to particular genotypes indicate their potential use for marker-assisted breeding and varietal identification. Their this work has led to the initiation of Silkworm Genome Programme in the new "Centre for DNA Fingerprinting and Diagnostics" which is presently housed in CCMB.
Use of DNA fingerprinting in wildlife conservation (genetic variation in Asiatic lions and Indian tigers)
Previous reports have suggested that Asiatic lions and tigers in India are highly inbred and exhibit very low levels of genetic variation. Dr Singh and his colleagues' analyses on these species have shown much higher degree of polymorphism than reported. RAPD analysis of 38 Asiatic lions, which exist as a single population in the Gir Forest Sanctuary in India, with four primers revealed an average heterozygosity of 25.82%. In Indian tigers, microsatellite analysis of five CA repeat loci and multilocus fingerprinting using Bkm 2(8) probe on a population of 22 individuals revealed a heterozygosity of 22.65%. Microsatellite analysis at loci Fca 77 and Fca 126 revealed polymorphism amongst the Asiatic x African lion hybrids, which has enabled the identification of the pure Asiatic lions from the hybrids. Similar analysis enabled them to identify hybrids of Indian and Siberian tigers through PCR amplification of hair samples.
To ascertain the variation which existed before the population bottleneck at the turn of the present century, microsatellite analysis was performed on 50-125 years old skin samples from museum specimens. Their results show similar levels of genetic variability as in the present population (21.01%). This suggests that low genetic variability may be the characteristic feature of these species and not the result of intensive inbreeding. DNA fingerprinting studies of Asiatic lions and tigers have further helped in identifying individuals with high genetic variability which can be used for conservation breeding programmes.
Based on this contribution funded by the Central Zoo Authority, Government of India, a decision for setting up of a Centre for Wildlife Conservation in collaboration with the Nehru Zoological Park, Department of Forests, Government of Andhra Pradesh; Centre for Cellular and Molecular Biology; Centre for DNA Fingerprinting and Diagnostics; Central Zoo Authority of India; Department of Biotechnology, has been taken. This Centre will be set up close to the Nehru Zoological Park, Hyderabad.