Research



Research Highlights
 
Basic Research

Human genetics 
Deletion of amelogenin gene on Y chromosome of normal males and its implication in forensic applications and prenatal diagnosis 

Human identification is an important forensic function. Forensic samples are generally very small in quantity. Therefore, various strategies have been adopted to utilize the minimum quantity of sample to detect maximum variability in a single Polymerase Chain Reaction. For gender identification, amelogenin gene is included along with the multiplex STRs, since it is present on the short arm of the X (AMEL X) and Y (AMEL Y) chromosome of human and other primates. Studies at the CCMB using profiler plus kit showed deletion of amelogenin in three males who possessed a normal SRY gene. But for the detection of presence of SRY gene and other Y-chromosome specific STRs (DYS19, DYS389, DYS390, DYS391 and DYS393) these individuals would have been identified as females. 

This would have serious consequences for forensic investigation and prenatal sex identification of foetus with known family history of genetic disorders affecting only the male child. CCMB coined a term for such males as deleted-amelogenin males (DAM). 

CCMB has suggested the use of additional Y chromosome markers such as SRY and/or Y-STRs for gender identification, considering the importance of sex-identification of forensic samples and prenatal diagnosis in cases with known history of genetic disorders, which affect only male children. 


Human genome diversity 
CCMB has undertaken a study on genetic diversity in various castes and tribal populations to construct haplotypes, which, in turn, are useful to construct the evolutionary tree. Samples from different caste and tribal populations including Great Andamanese and Nicobaris numbering 300 were analysed using various Y chromosome-specific STRs and biallelic markers, sequence variation at the hypervariable region (HVR) and the 9bp intergenic deletion of the mitochondrial DNA (mtDNA). Other studies have shown that the 9bp intergenic deletion is more predominant in Asian populations. However, only 7 out of 300 samples showed the 9bp deletion. The populations including Onges and Great Andamanese did not show the deletion, suggesting that the frequency of the 9bp deletion is very low among Indian populations. Analysis of HVRI of the mtDNA D-loop showed population-specific haplotypes. Two Great Andamanese showed a transversion at the nucleotide position 16,265, which is a characteristic feature of one of the major lineages in PNG Island and Melanesia. This observation is one of the proofs that the Andamanese represent one of the earliest human lineages.


Microbial biodiversity of Antarctica
Microbial biodiversity as a source of genetic material for biotechnology applications has been well recognised and has been in the forefront of research activity involving extremophiles. Extensive work carried out at the CCMB on soil, water and organic debris samples collected from Antarctica led to the identification of new species in addition to the known bacteria and yeasts. This was done using 16S rDNA sequence makeup, which is now increasingly being used for identification of families, genera and species of bacteria, and also to infer their phylogenetic relationships. 


Rice pathology and genetics
Xanthomonas oryzae pathovar oryzae (Xoo) causes bacterial leaf blight, a serious disease of rice. CCMB has been using the techniques of genetics and molecular biology to identify virulence functions, and to understand the epidemiology of Xoo. These results conclusively demonstrate that IS elements contribute to the phenomenon of stationary phase variation in Xoo.

Transposon insertions have also been isolated in a novel virulence gene of Xoo. It was shown that the N-terminal region of YadA, an adhesin produced by various Yersinia species was required for binding to collagen and causing virulence. The primary sequence of the Xoo protein (CCMB tentatively calls it XadA) indicates that there are at least three evenly spaced, imperfectly repeated regions each of which show homology to the same region of YadA. The predicted sequence of XadA has multiple membrane spanning domains with the repeat regions likely to be extruding into the extracellular space. The XadA protein also shows significant homology to another high molecular weight, outer membrane located "adhesin" protein (UspA1; ubiquitous surface protein A1) produced by the animal pathogen, Moraxella catarhallis. CCMB is attempting to standardize assays for adhesion of Xoo to rice tissues; these will then be used to determine if the xadA mutant is defective in adhesion to the host. 

It has long been recognized that siderophores (iron chelators) and their receptors are important virulence factors of animal pathogens. Recent evidence suggests that the same may apply to plant pathogens. CCMB has now found that a Xoo strain carrying a mutation in a gene that encodes the receptor for a phenolate type of iron siderophore is severely deficient for virulence following epiphytic inoculation of rice leaves. In this mode of infection, which is more akin to natural conditions, the bacterium has to survive (and possibly grow) on the leaf surface prior to entry into the plant through the hydathodal openings at the leaf edges. A more moderate defect in virulence was also observed following wound inoculation of rice leaves. This suggests that this iron siderophore receptor is not only likely to be important for survival/growth on the leaf surface but may also contribute (albeit to a lesser degree) to growth within the plant. 


Microbial resistance to plant antibiotics
The primary phytoalexins of leguminous plants called isoflavonoids are well known for antifungal effects. Some additional, novel effects of isoflavonoids on free-living soil amoebae (called dictyostelids or cellular slime moulds) were discovered at the CCMB. These effects represent a facet of a novel plant-microbe interaction wherein the amoebae continue feeding on bacteria in the vicinity of root lesions (the site of isoflavonoid production) and thus effectively disinfect the lesion from potentially pathogenic bacteria. 


Mechanism of homeotic gene control in Drosophila
CCMB scientists have shown for the first time that organizing centres of limbs are subjected to the regulation by the genes, which are involved in the positioning of the organs in the body of the fruit fly. They also cloned a new gene, which regulates growth during wing and limb development. 


Developmental biology of plant reproduction 
Apomixis - the formation of seeds without fertilization - occurs naturally in many plant species such as dandelions and parthenium. Engineering apomixis is of great interest in crop plants as it would be a very valuable tool in plant breeding. Genes that confer apomixis, act at the stage of female meiosis; hence understanding the control of female meiosis is necessary to ultimately engineer apomixis in crop plants. CCMB scientists have discovered a novel gene called DYAD, which is involved in the control of female meiosis in plants. 


Lamin gene regulation 
CCMB has demonstrated a new role for lamins in supporting nuclear compartments containing RNA splicing factors, also called nuclear speckles. These were identified by confocal microscopy of mammalian cells using monoclonal antibodies raised to recombinant lamin A and antibodies to the RNA splicing factors SC-35 and U5-116 kD in co-localisation experiments. These studies substantiate the proposal that the regulation of nuclear events by compartmentalization of essential factors is structurally dependent on the nucleoskeletal network of proteins. 


Eukaryotic transcription and chromatin structure 
In eukaryotic cells, chromatin structure and transcription are intimately linked with each other and a clear understanding of both is required to learn details of any one of them. CCMB has been studying different aspects of both and the focus during the year was on the mechanism behind transcriptional activation when the central factor, TATA-binding protein (TBP) is the target. The most interesting findings had been that transcriptional activation could affect the TBP dimer levels and significantly enhance the binding of TBP, specially, with weaker TATA boxes. TBP binding to TATA box and dimer levels showed direct correlation to the levels of the divalent cation magnesium. Dimerization and in vitro activity of yeast TBP are sensitive to ionic and protein concentration and the TBP was found to be largely in an active monomeric conformation in the presence of magnesium. The results suggested strong requirement for direct interaction of TBP and activator in the case of weak TATA boxes for TBP-TATA complex formation. 


Studies on the regulation of cell growth 
The tumour suppressor protein p53 plays an important role in mediating response to stress such as induced by DNA damage and hypoxia resulting in either growth arrest or apoptosis. It is a sequence-specific DNA binding protein and its biological responses are mediated by transcriptional activation of various target genes. CCMB has found that human caspase-1 is a transcriptional target of p53, which contributes in part to p53-mediated apoptosis. In addition, the CCMB observations provide a link between p53 and inflammation, since the primary role of caspase-1 is in the production of pro-inflammatory cytokines interleukin 1beta and IL-18. 

One of the pathways leading to the induction of programmed cell death (apoptosis) involves release of cytochrome C from mitochondria into the cytosol where cytochrome C mediates activation of various proteases including caspase-9 and caspase-3. CCMB has found that hematopoietic cell kinase Hck induces apoptosis through its SH3 and unique domains, which requires caspase-3 for release of mitochondrial cytochrome C and apoptosis. Thus, the Centre suggests a role of caspase-3 upstream of release of mitochondrial cytochrome C, in addition to the well-known role of caspase-3 downstream of cytochrome C release. 


Regulation and function of sex-specific secretory proteins of salivary and lacrimal glands 
Earlier work at the CCMB has identified abundant, secretory and sex hormone-repressed 24 and 20.5 kDa male-specific proteins in submandibular salivary glands (SMG) and a female-specific 20 kDa protein in lacrimal gland (LG) of adult hamsters. The work done during the year involved cloning of the cDNA encoding SMG male-specific proteins. The deduced amino acid sequences indicated that they are new members of the lipocalin protein family which have a close similarity with rat odorant-binding protein of nasal mucosa. Further studies indicated that the sex-specific SMG and LG proteins were products of similar, if not, identical genes, which showed an unusual repression at the transcriptional level with both androgens and estrogens with subtle differences. The results of homology search and the male and lactation-specific presence of SMG proteins in adult saliva and urine suggested the possibility of their involvement in olfaction-mediated chemical communication in hamsters. 


Chaperone-like activity of a lens protein 
Alpha -crystallin, a major protein of the eye lens, exhibits molecular chaperone-like activity, which is important in the maintenance of eye lens transparency. It is a multimeric protein composed of two types of subunits, alpha A and alpha B crystallins, homologous to the proteins of the sHSP family. The homology of these two proteins is particularly high in the C-terminal region, which contains the highly conserved 'alpha -crystallin domain'. CCMB has generated two chimeric proteins, alpha ANBC and alpha BNAC, in which the N-terminals of alpha A and alpha B crystallins have been swapped. Swapping the N-terminal domain between human alpha A and alpha B crystallins makes a more effective chaperone in the case of alpha BNAC chimera, whereas alpha ANBC chimera loses its protective abilities completely. This study provides a promising protein engineering approach to enhance the chaperone-like activity of alpha -crystallin, which may give useful leads to design strategies to tackle chaperone-diseases such as cataract. 


Peptide membrane interactions 
CCMB has established the identity of antibacterial peptides from the skin secretions of the amphibian species Rana tigrina by extensive use of protein sequencing techniques and mass spectrometry and confirmed it by synthesis and biological activity. The peptides are composed of 12 amino acids and have the motif CXXXXXXXC where X are either hydrophobic or cationic amino acids and C is cysteine. The motif and sequences are unique among host-defense amphibian antibacterial peptides. Circular dichroism studies and theoretical calculations suggest that the peptides adopt beta -turn structures. The peptides appear to exert their activity by permeabilizing bacterial membranes. 


Mathematical modelling of the spatiotemporal organization in biological systems 
Theoretical analyses of the complex circuitry of biochemical pathways in cells have shown how the understanding of the nature of controls and their interactions in genetic and metabolic reactions decide the dynamic behaviour of pathway functioning. Work at the CCMB provides methods that give specific conditions to correct pathological states in metabolic regulation, design new pathways, predict conditions for overproduction of biochemicals (e.g., amino acids) and also throw light on the evolution of metabolic pathways. CCMB has shown that a minimal model can describe the multiplicity of dynamics observed in cellular signalling processes such as, the frequency and amplitude variation in calcium oscillations "sparks" and "puffs". Studies on the noise on the evolution of the pathway and the collective effects of a large number of cells exhibiting a particular behaviour are in progress. 

Ability to control the spatial pattern and temporal dynamics in biological systems has very important implications in correcting pathological states. CCMB has developed a simple and useful method to control the dynamics in discrete population models and spatially extended systems which is not only applicable to control complex dynamics that may arise in a stable system (cardiac arrhythmia) but can also be used to induce complexity in a situation where diseased state is characterized by reduced complexity (e.g., in neurophysiology). 


Computational Biology & Bioinformatics
Theoretical approaches are increasingly being used in modern biology to model and uncover information contained in the genome and protein sequences and structures, and predict behaviour of biological systems. This allows study of the function and evolution of these biological molecules, and other higher order systems, such as the intra-cellular biochemical pathways, cell-cell interactions, ecological and epidemiological systems.

Several groups in the CCMB are involved in computational modelling of such phenomena, along with generating data to test or supplement their models. Of the two posters here, one PDF summarizes work in the areas of Genome Analysis, Structural Biology, Protein Structure-Function and Bioinformatics, Molecular Modelling, and Computational Analysis in Human Evolutionary Studies. The other poster PDF describes the work done in the Mathematical Modelling and Computational Biology Group, which is working on theoretical studies of different areas in biology, to understand and identify the generic mechanisms that underlie the formation and evolution of spatiotemporal patterns at different levels of biological organization - from ecological to genetic.