The major evolutionary transition from single-celled to multicellular life is believed to have occurred independently of the main metazoan lineages in the cellular slime moulds, of which Dictyostelium discoideum is the best-studied species. Unusually, in this case multicellular development is a facultative trait and part of an asexual life cycle. It is triggered by starvation and involves aggregation of hitherto independent and possibly unrelated free-living cells. The consequences of multicellularity in D.discoideum are strongly influenced by the environment and meaningful external perturbations are easily carried out. This makes the organism ideally suited to a study of epigenetic factors that regulate development. In an attempt to understand how conserved epigenetic pathways are integrated within the developmental framework, two likely players were chosen for investigation - heat shock protein 90 (Hsp90) and histone deacetylases (HDACs). Hsp90 has been implicated in diverse biological processes such as protein folding, cell cycle control, signal transduction, and morphological evolution. The role of Hsp90 in D.discoideum life cycle was studied using a specific inhibitor, geldanamycin. Inhibition of Hsp90 function in D.discoideum caused a delay in aggregation and an arrest of development at the ‘mound’ stage. A reduction in Hsp90activity in starving cells of D.discoideum resulted in the generation of a range of phenotypes. The study suggests that Hsp90 is required for a specific developmental transition of the social amoeba and is important in generating a reliable outcome of the developmental process. Histone acetylation regulates gene expression and leads to the establishment and maintenance of cellular phenotypes during development of plants and animals. To study the roles of HDACs in D.discoideum, biochemical, pharmacological and genetic approaches were employed. The inhibition of HDAC activity by trichostatin A resulted in histone hyperacetylation and a delay in cell aggregation and differentiation. Cyclic AMP oscillations were normal in starved amoebae treated with trichostatin A but the expression of a subset of cAMP-regulated genes was delayed. Bioinformatic analysis indicated that there are four genes encoding putative HDACs in D.discoideum. One of these four genes, hdaB, was found to be dispensable for growth and development under laboratory conditions; but formed spores with lower efficiency than the wild type in chimeras. The work shows that HDAC activity is important for regulating two aspects of multicellular development: (a) heterochrony, namely the relative timing of developmental events, and (b) modulating the behaviour of single cells in a manner that is sensitive to their social environment.

Il sorgo zuccherino utilizzato per produrre cibo, mangimi e carburante con limitato impiego di risorse, risponde ai criteri dell’agricoltura moderna ed è impiegato nella produzione di bioenergia. È una pianta C4 adattata agli ambienti semi-aridi, caratteristica che dovrebbe essere mantenuta e migliorata nel processo di ottenimento di nuovi genotipi. Per comprendere le basi genetiche e fisiologiche della tolleranza alla siccità, genotipi di sorgo (IS 19453, Mpwekwa, SDS19483, IS33350, BR505, BR501) sono stati valutati in camera di crescita e in serra. Lo stress idrico è iniziato quando le piante avevano consumato l'80% di acqua disponibile. L’RNA totale è stato estratto da piante irrigate e non a diversi livelli di stress idrico. L'analisi di espressione genica è stata eseguita attraverso l’uso delle tecniche microarray e q-RT PCR. Il numero dei geni differenzialmente espressi aumentava all’aumentare del livello di stress. Gran parte dei geni sovra espressi erano coinvolti nei meccanismi di difesa, di trasporto, di regolazione genica, e nel metabolismo lipidico, proteico e degli zuccheri. Nelle piante non irrigate, al più alto livello di stress, i geni sovra-regolati presentavano livelli di espressione di 2-5 volte superiori rispetto ai campioni di controllo. Questi risultati serviranno all’identificazione di ”single nucleotide polymorphisms” nelle sequenze dei geni candidati e al loro impiego come marcatori nel processo di miglioramento genetico assistito. / Sweet sorghum providing food, feed and fuel with a limited use of resources, responds to the criteria of sustainable bioenergy production. Sorghum is a C4 plant adapted to semi-arid environments, characteristic that should be maintained and further improved in the process of breding new genotypes for bioenergy production. To understand the genetic and physiological basis of drought tolerance, sorghum genotypes (IS 19453, Mpwekwa, SDS19483, IS33350, BR505, BR501) were evaluated in growth chamber and greenhouse experiments. Drought stress started when plants had consumed 80% of transpirable soil water. Total RNA was extracted from irrigated and not irrigated plants at different levels of water stress, and gene expression analysis was carried out using microarray and q-RT PCR techniques. The number of differentially expressed genes increased with the stress level. Most of the up regulated genes were involved in cell rescue, transport, nucleic acid binding, and in lipid, protein and sugar metabolism. In non-irrigated plants, at the higher stress level, up-regulated genes presented levels of expression 2-5 fold higher compared to control samples. These preliminary results will be useful for the identification of single nucleotide polymorphisms in candidate genes sequences in order to use them as markers for assisted breeding.

Prokaryotic cells such as Escherichia coli use glucose as their preferred carbon source. In the absence of glucose, these cells resort to other sources to generate glucose and this process of de novo synthesis of glucose is termed gluconeogenesis. Phosphoenolpyruvate carboxykinase (Pck) is one of the three enzymes important in regulating gluconeogenesis. It converts oxaloacetic acid (OAA) from the Krebs cycle to phosphoenolpyruvate (PEP), a glycolytic intermediate. The Pck structural gene (pckA) is regulated by catabolite repression. There is a 100-fold induction of pckA-lacZ fusions at the onset of stationary phase concurrent with induction of glycogen synthesis. Mutants affecting the expression of pckA were analysed to shed some light on the mechanism of its genetic regulation.<p>Spontaneous mutants isolated with Pck- (lack of PEP carboxykinase activity) and Suc- (inability to utilise succinate as carbon source) phenotypes were previously characterised as atpG mutants defective in the ã subunit of ATP synthase.<p>In this work we find by reverse transcriptase and real time quantitative PCR that levels of pckA mRNA are normal in the atpG mutants and that the defects in expression of pckA are therefore likely at the level of translation, protein assembly and/or protein degradation. As expected, ATP synthase activity and proton pumping in inside-out membrane vesicles were defective in these atpG mutants. It is likely that one of these defects is affecting regulation or expression of the pckA gene. It was observed that atpG mutants were defective in calcium-dependent transformation although they could be made competent for electroporation. The atpG mutants were also defective for growth of P1 bacteriophage although they could serve as recipients for P1-dependent generalised transduction. These latter phenotypes are also likely due to defects in energy metabolism.

Lambdoid phages suppress transcription termination to fully express their genes. Antitermination of early gene expression in most lambdoid phages is mediated by an interaction between the N protein and a number of host-encoded factors. Bacteriophage HK022 does not rely on a protein for antitermination. To promote full expression of early phage genes, the transcripts of the HK022 put sites interact directly with RNA polymerase to convert it to a termination resistant form. Bacteriophage HK639 also uses RNA-mediated antitermination. However, it only possesses a single put-like element in its left operon. Because most lambdoid phages, including HK022, have antiterminator elements in each of their early operons, the presence of a single antitermination site in HK639 was unexpected. We have shown that host genes involved in promoting protein-mediated antitermination are not required for HK639 growth. We have also shown that expression of the left operon is essential for lytic growth. Replacement of the left operon promoter, PL, and the putL antitermination sequence prevented HK639 phage release. A similar construct that only replaced putL also prevented phage release. These results suggest that antitermination is required for HK639 excision and/or lytic growth. To distinguish between a defect in phage excision versus a defect in lytic growth, the mutations were crossed onto lytically growing phage. Recombinant phages could not be recovered which suggests a defect in lytic growth is preventing phage release. Additional replacements of left operon sequences suggest that antitermination is not the only requirement for lytic growth. A 2,161bp deletion (HK639 genome coordinates 30,888-33,048) and a 1,736bp deletion (HK639 genome coordinates 29,152- 30,887) downstream of the HK639 putL site also prevented phage release, whereas a 1,746bp deletion (HK639 genome coordinates 29,151-27,406) did not. These results suggest that the deleted HK639 left operon sequences are required for lytic growth. BLAST analysis did not provide insight into the function of the deleted genes. Although the function of many of the HK639 left operon genes is unknown, their importance in phage growth can now be verified by complementation analysis. Our results suggest that HK639 may use a novel mechanism to control the expression of its early genes.

Prokaryotic cells such as Escherichia coli use glucose as their preferred carbon source. In the absence of glucose, these cells resort to other sources to generate glucose and this process of de novo synthesis of glucose is termed gluconeogenesis. Phosphoenolpyruvate carboxykinase (Pck) is one of the three enzymes important in regulating gluconeogenesis. It converts oxaloacetic acid (OAA) from the Krebs cycle to phosphoenolpyruvate (PEP), a glycolytic intermediate. The Pck structural gene (pckA) is regulated by catabolite repression. There is a 100-fold induction of pckA-lacZ fusions at the onset of stationary phase concurrent with induction of glycogen synthesis. Mutants affecting the expression of pckA were analysed to shed some light on the mechanism of its genetic regulation.<p>Spontaneous mutants isolated with Pck- (lack of PEP carboxykinase activity) and Suc- (inability to utilise succinate as carbon source) phenotypes were previously characterised as atpG mutants defective in the ã subunit of ATP synthase.<p>In this work we find by reverse transcriptase and real time quantitative PCR that levels of pckA mRNA are normal in the atpG mutants and that the defects in expression of pckA are therefore likely at the level of translation, protein assembly and/or protein degradation. As expected, ATP synthase activity and proton pumping in inside-out membrane vesicles were defective in these atpG mutants. It is likely that one of these defects is affecting regulation or expression of the pckA gene. It was observed that atpG mutants were defective in calcium-dependent transformation although they could be made competent for electroporation. The atpG mutants were also defective for growth of P1 bacteriophage although they could serve as recipients for P1-dependent generalised transduction. These latter phenotypes are also likely due to defects in energy metabolism.

Candida albicans, a commensal of gastrointestinal and uro-vaginal tract can cause superficial as well as life threatening disseminated infections under conditions of lowered immunity of the host such as HIV infection, drug induced immune suppression [given during organ transplantation to prevent rejection] and radiation therapy [head and neck cancer patients] (Odds, 1988; Fidel and Sobel, 1996). Candida albicans shows a range of morphologies, it can switch from budding yeast morphology to pseudohyphae (chains of elongated cells with visible constrictions at the sites of septa) and hyphae (linear filaments without visible constrictions at the septa) (Mitchell, 1998). The various factors that contribute to its virulence include its ability to undergo yeast to hyphal transition, formation of biofilms, adhesion and secretion of aspartyl proteinases. Hyphae are considered to be involved in invasive growth as they are frequently identified in infected tissues and strains defective in morphological transition (yeast to hyphal) are avirulent (Leberer et al., 1996; Lo et al., 1997; Stoldt et al., 1997). Morphological switching is not only necessary for successful establishment of infection but important for evading components host defense system like macrophages or dendritic cells. A network of signaling pathways that operate in C. albicans continuously assess the nutrient availability, cell density and other environmental conditions. The integrated output of these pathways determine the response of C. albicans under given set of environmental/media conditions and eventually determines the gene expression and morphogenic transition (Liu., 2001). C. albicans utilizes at least two major signaling pathways besides others for regulating the morphological transition. One of these two pathways uses Cph1 as transcription factor and is the homolog of Ste12 in S. cerevisiae which is shown to be involved in Pseudohyphal growth and mating. The other pathway includes Efg1 (homolog of Phd1 in S. cerevisiae) as transcription factor. Biofilm formation by Candida species is an important virulence factor and has gained considerable interest recently as these specialized survival structures are found in implanted devices such as indwelling catheters and prosthetic heart valves (Hawser and Douglas, 1994; Douglas, 2003). These biofilms lead to the failure of implants besides providing multiple drug resistance (Baillie and Douglas, 1999). A better understanding of the C. albicans interaction with the host at the site of infection and with the components of immune system will help in identifying new potential drug targets. (a) Genome wide expression profile of Candida albicans from patient samples and characterization of CaRPB4/7: To get a better insight in C. albicans response at the site of infection we were interested in mapping the expression profile of Candida albicans in active state of human infections. Patients suffering from head and neck cancer undergoing radiation therapy have high risk of C. albicans infection. We identified five such patients with heavy oral thrush infections and C. albicans samples were collected from them. Candida albicans was confirmed in these samples by various microbiological tests following which the samples were used for RNA isolation. The whole genome expression analysis leads to the identification of 188 up regulated and 88 down regulated genes in patient samples. Our data analysis revealed that Protein Kinase A pathway and many downstream genes of the same were differentially expressed. Analysis of saliva (saliva is known for antifungal and antibacterial activity) from these patients showed that unlike healthy individuals, the patient saliva favours yeast to hyphal transition of C. albicans cells. This might be a reason for high risk of infection. A major class of upregulated genes is found to be functionally involved in transcription which includes some RNA polymeraseII and III subunits. CaRPB4, the forth largest subunit of RNA polymeraseII, was found to be upregulated in patient samples. RPB4 has been shown to form sub complex with RPB7, the seventh largest subunit of RNA polymeraseII, and both subunits are known to play a role in a variety of stress conditions and pseudohyphal development in Saccharomyces cerevisiae. We characterized the CaRPB4 and CaRPB7 (homolog in Candida albicans) for their ability to complement their S. cerevisiae counterparts. CaRPB4 and CaRPB7 were able to complement majority of the phenotypes associated with these subunits in S. cerevisiae. Overexpression of CaRPB7 in S. cerevisiae enhances pseudohyphal growth. Considering the high degree of conservation of signaling pathways between S. cerevisiae and C. albicans it can be speculated that CaRPB7 might be involved in pseudohyphal development in C. albicans. We found that over expression of CaRPB4 in Candida albicans shows enhanced agar invasive growth which can be thought analogous to tissue invasion in host and hence might contribute for establishment of infection. This suggests that both the RNA polII subunits have a role to play in the virulence of C. albicans. (b) Characterization of UDP-Galactose 4-Epimerase (GAL10) from Candida albicans and their role in virulence. Enzyme UDP-Galactose-4-Epimerase [GAL10] is responsible for conversion of UDP-galactose to UDP-glucose which then gets metabolized by the cells through glycolysis and TCA cycle. The enzyme catalyzes a reversible reaction and can convert glucose to galactose in the absence of galactose as shown in Trypanosoma brucei and also involved in its virulence. In this study, we have identified the functional homolog of GAL10 in Candida albicans. S. cerevisiae and C. albicans GAL10 homologs are similar in their domainal organization as the proteins have a mutarotase and an epimerase domain. The former is responsible for conversion of ﾟ-D-galactose to a-D-galactose and the latter for epimerization of UDP-galactose to UDP-glucose. The synteny of galactose metabolizing structural genes is conserved among some fungi. To study the importance of CaGAL10 we generated deletion mutant of the gene in C. albicans. Our studies show that CaGAL10 [C. albicans GAL10] is involved in cell wall organization and in oxidative stress response. The mutant strain of GAL10 is hyperfilamentous in Lee’s and spider medium and the biofilm formed is morphologically different from the wild type strain. These set of results suggests that CaGAL10 plays an important role in organization/integrity of cell wall in C. albicans and speculate that it might be involved in virulence. (c) Study of Candida albicans-macrophage interaction and identification of transcriptional regulator of genes encoding proteins of translation machinery: Macrophages serve as the effector cells of cell mediated immunity in the control of infections. They are considered to be important for resistance to muco-cutaneous and systemic candidiasis. Our studies were aimed at understanding the response of Candida albicans cells to the presence of macrophages for extended period of time. The response was monitored using microarrays. Specifically genes involved in galactose, protein and lipid metabolism and stress response undergo concerted changes in their transcript levels. We analyzed the promoters of coregulated genes to identify common DNA elements present in them which might be involved in their transcriptional regulation. Promoter analysis of differentially expressed genes revealed presence of CPH1 and EFG1 transcription factor binding sites. Besides identifying CPH1 and EFG1 Binding sites, we identified two novel DNA elements in promoters of coregulated gene. A conserved motif TGAAAAGGAAG was identified in the promoters of genes involved in energy generation. Another 18 mer consensus palindromic sequence TAGGGCTNTAGCCCTAAT was identified in the promoters of about 48 genes. Majority of these genes encode ribosomal proteins. With the help of techniques like EMSA (Electophoretic Mobility Shift Assay) and south-western we had shown the presence of a protein of ~66 KDa molecular weight binding to the sequence with high specificity.

PTB (HnRNP I) is a multifunctional RNA binding protein which participates in a variety of RNA metabolic processes put together called as post transcriptional gene regulation. It interacts with shuttling hnRNPs L, K and E2 of the spliceosomal machinery and also with other RNA binding proteins like PSF, Raver1 and Raver2, which assists PTB in splicing. Based on the complexity of these processes and multifunctional nature of PTB, we hypothesized that; it might interact with various additional proteins not identified till date. Keeping this objective in mind, we set out to screen the custom made 18 day old mouse testes cDNA library in pGAD10 vector available in the laboratory, to hunt for novel interacting partners of PTB using the Clontech’s Matchmaker Gal4 yeast two hybrid system III. PTB1, the prototype of PTB was chosen and the above mentioned cDNA library was screened for novel PTB interacting partners. Twenty five large scale library transformations (spanning 8*106 independent clones) were performed and 99 putatives were obtained. By re-transformation of these library plasmids with bait construct to check for the interaction phenotype and eliminating bait independent activation of reporter genes and elimination of known false positives, only 5 clones were consistent with the interaction phenotype. All these library plasmids were sequenced with vector specific primers, ORF was identified and BLAST analysis for the identification of insert was done. Two of these clones encoded the partial CDS of mouse Protein Inhibitor of Activated STAT3-PIAS3. One of these encoded the partial CDS of mouse TOLL Interacting Protein-TOLLIP. The other two encoded the partial CDS of mouse importin-α and mouse hnRNP K, both of which were already known interacting partners of PTB. GST pull down assay and mammalian matchmaker co-immunoprecipitation was used for confirming the in vitro one to one physical interaction between PTB and these newly identified protein partners. Indirect Immunofloresence was used for demonstrating the co-localization of PTB and PIAS3 in Gc1Spg mouse spermatogonial cell line. The fact that PIAS3 an E3 SUMO ligase was picked up as an interacting partner of PTB was interesting and we hypothesized that PTB might be a sumoylation substrate. Towards this, we first resorted to the prediction of sumoylation consensus motif by using SUMOPLOT. PTB indeed was found to have sumoylation consensus sites. Subsequently, in vivo sumoylation of PTB was demonstrated, where in over expression of donor protein [SUMO-1] and acceptor protein [PTB] in RAG-1 mouse kidney cell line had resulted in the identification of an approximately 67 kDa slow moving SUMO modified myc tagged PTB band apart from the bulk of unmodified 57 kDa myc-PTB. This confirmed the fact that PTB is SUMO modified only at a single consensus target site in vivo and attempts are made to map this site of modification. SUMOylation regulates diverse biological processes in vivo ranging from nucleo- cytoplasmic shuttling, alteration of protein-protein interaction, DNA protein interaction etc. PTB shuttles rapidly between the nucleus and cytoplasm in a transcription sensitive manner and the translocation of PTB to the cytoplasm, happens under the conditions of cell stress, viral infections, apoptosis and exposure of cells to genotoxic agents like doxorubicin. Phosphorylation of PTB at Ser-16 residue has been shown to modulate the nucleo-cytoplasmic shuttling of PTB, albeit shuttling can also occur irrespective of this modification. Interaction of PTB with an E3 SUMO ligase-PIAS3 and the fact that it is SUMOylated in vivo, we hypothesize that K-47 residue present in the NLS/NES might be the most probable site of this SUMO modification and SUMOylation of PTB by PIAS3 might regulate the nucleo-cytoplasmic shuttling of PTB.

Spermatogenesis is a complex process of male germ cell development in which the diploid spermatogonia undergo series of mitotic divisions and differentiation steps culminating into the preleptotene spermatocytes which then enter into the meiotic prophase following a single replication cycle. This phase is characterized by meiotic recombination and is followed by reduction division resulting in haploid round spermatids. These cells then undergo extensive morphological and nuclear changes to form a unique cell, spermatozoa. This entire germ cell differentiation process occurs in a unique environment present inside the seminiferous tubules which is created by the Sertoli cells, the somatic cells in the tubules by forming junctions with each other thus providing unique milieu to the developing germ cells. Within the tubule, the germ cells are also arranged in an orderly manner called stages of spermatogenesis indicating a complex mechanism of germ cell differentiation. This complex differentiation process is a consequence of developmentally and precisely regulated differential gene expression (Eddy, 2002). Unraveling the molecular mechanisms involved in the male germ cell development is an uphill task due to the complexity of the cyto-architecture existing in the tubules and further complicated by unavailability of established germ cell lines and lack of cell culture systems that facilitate the germ cell differentiation in vitro. Comparative gene expression analysis of spermatogenesis in nematodes, flies and rodents revealed highly conserved transcriptomes and have provided some insights into its regulation (Schlecht and Primig, 2003). However, these data fail to represent the genetic and biological complexity of human spermatogenesis. In the present study, an attempt has been made to identify the genes that are differentially expressed in human tetraploid and haploid germ cells and to investigate the mechanism of regulation of the genes expressed in the post-meiotic germ cells. To identify the cell type specific genes, expression profiling of the human tetraploid and haploid germ cells was carried out using cDNA microarray. These cells were purified by centrifugal elutriation (Meistrich et al., 1981; Shetty et al., 1996) from the human testicular tissues obtained from the patients undergoing orchidectomy as treatment for prostate cancer. Purity of the enriched population of the germ cells was ascertained by DNA flow cytometry and by RT-PCR analysis using the known cell-specific markers and ruling out contamination of the somatic cells such as the Sertoli cells and the Leydig cells. Microarray experiments were carried out with the RNA isolated from each cell type and labeling the cDNA with Cy3/Cy5-dUTP and hybridizing to the human 19K array chip (University Health Network, Toronto, Canada) containing 19,200 ESTs. Two independent hybridizations were carried out using the germ cells isolated from two individuals and the microarray data were analyzed using Avadis 3.1 software (Strand Life Sciences, India). Analysis of the microarray data following normalization revealed that 723 transcripts showed higher expression in the meiotic cells whereas 459 transcripts showed higher expression in the post-meiotic germ cells. Microarray data were validated further by RT-PCR analysis of some of the differentially regulated genes. The DAVID analysis (Database for Annotation, Visualization and Integrated Discovery; http://david.abcc.ncifcrf.gov/) of these genes revealed that many genes associated with diverse functions and pathways appeared to be differentially expressed in both cell types. It is known that many biological systems exhibit distinct temporal gene expression profiles during different processes related to cell cycle, stress response and differentiation. Similarly, there are sets of genes, which respond to specific stimuli, appear to be synchronized in their expression. Such ‘synexpressed’ genes have been shown to be regulated by common transcription regulatory processes and have similar upstream transcription factor binding sites (Niehrs and Pollet, 1999). And therefore, having identified genes that appeared to be differentially expressed in the haploid and the tetraploid germ cells, attempt was made to analyze transcription factor binding sites in the promoter of those genes. In silico promoter analysis of several genes showing higher post-meiotic expression was carried out in order to identify the common regulatory motifs. Analysis of the annotated promoters (available from Eukaryotic Promoter Database; http://www.epd.isb-sib.ch/) of about forty genes highly expressed in the post-meiotic germ cells using TFSEARCH program (http://www.cbrc.jp/ research/db/TFSEARCH.html) confirmed that many genes had common transcription factor binding sites. Interestingly, almost all of the analyzed genes harbored SRY (Sex determining Region in Y)/SOX (SRY-box containing) binding motifs. In addition, the promoters of genes such as Protamine 1 and 2, Transition protein 1 and 2, A kinase (PRKA) anchor protein 4 that are known to be expressed post-meiotically, also harbor SRY binding sites suggesting that SRY may be one of the key regulators of the post-meiotic gene expression. SRY is a HMG-box containing member of Sox-family of architectural transcription factors. SRY is encoded by the Y chromosome and was first discovered as the testis-determining factor in mammals (Koopman et al., 1991). SRY HMG-box is eighty amino acids conserved motif that binds to the minor groove of the DNA in a sequence-dependent manner resulting in its bending and thus regulating the gene expression. The RT-PCR analysis of the human haploid and tetraploid germ cells showed very high expression of SRY in the post-meiotic cells further suggesting key role of SRY in the post-meiotic gene regulation. Role of SRY in the post-meiotic gene expression was investigated by determining the effect of SRY on human Protamine 1 (PRM1) promoter, a gene known to be exclusively expressed in the round spermatids and as indicated above, harbors many SRY binding sites in its promoter. SRY cDNA was cloned into the mammalian expression vector, pcDNA3.1 and the PRM1 promoter was cloned into the promoter-less pGL3 Basic vector upstream of the Luciferase reporter gene. Co-transfection of both constructs led to up-regulation of PRM1 promoter activity in both HeLa cells and LNCaP cells in a dose-dependent manner clearly demonstrating the role of SRY in PRM1 gene expression. Sequential deletion of the SRY binding sites in the PRM1 promoter led to the identification of the critical SRY binding motif important for SRY-mediated upregulation of PRM1 gene expression. This was confirmed by demonstrating in vitro binding of SRY to its critical binding site in the PRM1 promoter by gel shift assay using the nuclear extract of the HeLa cells transfected with FLAG-tagged SRY. The human SRY is an atypical transcription factor that binds DNA through its HMG, but unlike the mouse Sry and other Sox proteins, lacks the trans-activation domain and therefore requires other factors for its actions. Recently, the glutamine-rich, zinc-finger containing transactivator, Specificity protein 1 (Sp1) has been identified as one such interacting partner (Wissmuller et al., 2006). RT-PCR analysis showed that human SP1 is highly expressed in the haploid germ cells and could up-regulate PRM1 expression which harbors two SP1 binding sites in its promoter. When co-transfected, SRY and SP1 up-regulated PRM1 promoter in co-operative manner suggesting that SP1 may act in coordination with SRY in regulating PRM1. All these data taken together clearly signifies a critical role of SRY in post-meiotic germ cell gene expression. Recent reports suggest that SRY is also expressed in the adult human brain and prostate. However, its role in these tissues is not clearly understood. The Y chromosome has been shown to be frequently lost in prostate cancer and has also been shown to suppress the tumorigenicity of the PC-3 prostate cancer cells suggesting that the Y chromosome encoded genes may be involved in tumor suppression. SRY can physically interact with the androgen receptor (AR) and thereby interfere in its downstream signaling (Yuan et al., 2001). Since the prostate tumors show initial androgen-dependency, it was interesting to look at the role of SRY in the prostate cancer. To decipher the effect of SRY on the androgen-responsive LNCaP cells, stable clones of LNCaP expressing human SRY were generated. These clones showed significant decrease in growth in response to 5α-dihydrotestosterone (DHT) compared to the vector transfected or the parental LNCaP cells. In the soft agar colony formation assay, the SRY expressing LNCaP formed smaller colonies as compared to the controls in presence of DHT. Preliminary experiments in male athymic nude mice demonstrated that one of the SRY expressing clones showed reduced tumor growth compared to control cells suggesting that SRY may play a role in prostate cancer progression by decreasing the sensitivity to DHT. To summarize, the present study has identified several genes differentially expressed in the human haploid and tetraploid germ cells and further showed that SRY may be one of the key regulators of the post-meiotic gene expression.

Complex organization is found in many biological systems. For example, biopolymers could possess very hierarchic structure, which provides their functional peculiarity. Understating such, complex organization allows describing biological phenomena and predicting molecule functions. Besides, we can try to characterize the specific phenomenon by some probabilistic quantities (variances, means, etc), assuming the primary biopolymer structure to be randomly formed according to some statistical distribution. Such a formulation is oriented toward evolutionary problems.Artificially constructed biological network is another common object of statistical physics with rich functional properties. A behavior of cells is a consequence of complex interactions between its numerous components, such as DNA, RNA, proteins and small molecules. Cells use signaling pathways and regulatory mechanisms to coordinate multiple processes, allowing them to respond and to adapt to changing environment. Recent theoretical advances allow us to describe cellular network structure using graph concepts to reveal the principal organizational features shared with numerous non-biological networks.The aim of this thesis is to develop bunch of methods for studying statistical and dynamic objects of complex architecture and, in particular, scale-free structures, which have no characteristic spatial and/or time scale. For such systems, the use of standard mathematical methods, relying on the average behavior of the whole system, is often incorrect or useless, while a detailed many-body description is almost hopeless because of the combinatorial complexity of the problem. Here we focus on two problems.The first part addresses to statistical analysis of random biopolymers. Apart from the evolutionary context, our studies cover more general problems of planar topology appeared in description of various systems, ranging from gauge theory to biophysics. We investigate analytically and numerically a phase transition of a generic planar matching problem, from the regime, where almost all the vertices are paired, to the situation, where a finite fraction of them remains unmatched.The second part of this work focus on statistical properties of networks. We demonstrate the possibility to define co-expression gene clusters within a network context from their specific motif distribution signatures. We also show how a method based on the shortest path function (SPF) can be applied to gene interactions sub-networks of co-expression gene clusters, to efficiently predict novel regulatory transcription factors (TFs). The biological significance of this method by applying it on groups of genes with a shared regulatory locus, found by genetic genomics, is presented. Finally, we discuss formation of stable patters of motifs in networks under selective evolution in context of creation of islands of "superfamilies".

Overexpression of Phosphoenolpyruvate carboxykinase - cytosolic (PEPCK, encoded by Pck-1 gene) has been found to be associated with the prevalence of hyperglycemia in Type 2 Diabetes Mellitus (T2DM) patients. The Pck-1 enzyme catalyzes the rate limiting step in endogenous glucose production. The aims of this study are to develop a Pck-1:eGFP reporter zebrafish and validate it as a potential tool for the screening of novel anti-diabetic compounds. 3.6 kb zebrafish Pck-1 promoter fragment was cloned and a Pck-1:eGFP expression vector was constructed. After DNA microinjection, we generated Pck-1:eGFP reporter zebrafish with strong eGFP expression in developing liver. Validation studies confirmed that Pck-1:eGFP zebrafish embryos responded to treatment of glucose, cAMP and dexamethasone, metformin and rosiglitazone similarly to that of humans. This novel Pck-1:eGFP reporter fish line can serve as a tool for the screening and development of novel anti-diabetic drugs that may have potential in the treatment of T2DM.