This dissertation focuses on pre-mRNA 3'-end processing in eukaryotes, a crucial step in defining the 3'end of most protein-coding mRNAs. In vertebrates, two distinct molecular machines are involved: the canonical machinery, consisting of a Cleavage Factor (CF) module, Polyadenylation Specificity (PSF) module, Cleavage Stimulation Factor, and other complexes, and the U7 snRNP machinery (U7 machinery), which consist of a core U7 snRNP complex and the Histone Cleavage Complex (HCC). U7 snRNP is involved in replication-dependent histone pre-mRNA 3'-end processing. Interestingly, the cleavage modules of the canonical and U7 machinery share an endonuclease, CPSF73, that catalyzes the cleavage reaction for 3’-end processing of pre-mRNAs. CPSF73 also possesses 5’-3’ exonuclease activity in the U7 machinery. CPSF73 has been identified as a potential target for anticancer and antimalarial small-molecule inhibitors. Traditionally, CPSF73 nuclease activity has been demonstrated using a gel-based end-point assay, using radio-labeled or fluorescently labeled RNA substrates. In Chapter Two (Ch. 2) of this dissertation introduces a novel, real-time fluorescence assay to investigate CPSF73 nuclease activity. This efficient and high-throughput assay holds potential for identifying new CPSF73 inhibitors. Chapter Three (Ch. 3) of this dissertation delves into the structural characterization of the mammalian PSF (mPSF) module in complex with the second most frequent PAS variants, AUUAAA. Structure studies have revealed the molecular mechanism underlying mPSF recognition of the most common PAS sequence, AAUAAA. This study presents a cryo-EM structure of mPSF in complex with AUUAAA. While the binding modes remain highly similar between the two PAS variants, we observed conformational differences in the A1 and U2 nucleotides in AUUAAA compared to the A1 and A2 of AAUAAA. Furthermore, CPSF30 displayed conformational changes near the U2 nucleotide of AUUAAA. Attempts to explore the binding modes of two rare PAS sequences, AAGAAA and GAUAAA, were inconclusive due to a lack of RNA density in the EM maps. An atomic model of the ternary structure (CPSF160, WDR33, CPSF30) was produced using the EM map of the AAGAAA sample. The ternary structure revealed PAS recognizing residues to be disordered in CPSF30 (ZF2 and ZF3) and WDR33. Overall, this dissertation provides insights into the intricate mechanisms of pre-mRNA 3'-end processing in mammals, laying the groundwork for future studies and potentially leading to the development of novel inhibitors targeting CPSF73.

Os herbicidas ametrina e clomazone são amplamente utilizados na cultura de cana-deaçúcar, apresentam degradação essencialmente microbiana e são considerados contaminantes de solos e águas. A exposição dos microrganismos a estes xenobióticos pode resultar em dano oxidativo devido ao aumento na produção de espécies reativas de oxigênio (EROs). Este trabalho teve como objetivo analisar respostas bioquímicas e fisiológicas de dois isolados bacterianos tolerantes aos herbicidas ametrina e clomazone. As bactérias foram isoladas de solos agrícolas e a partir de estudos fisiológicos, crescimento e formação de halo, foram selecionadas duas que apresentaram maior tolerância aos herbicidas. Esses microrganismos foram cultivados em meio de cultura ágar nutriente, a 30°C, por 14 horas na presença dos herbicidas ametrina (25 mM), clomazone (9 mM), e em meio composto pelos dois herbicidas: ametrina (20 mM) + clomazone (20 mM). Essas concentrações representam as dosagens utilizadas para o cultivo de cana-deaçúcar para a aplicação pré e pós-emergência. Por meio da análise filogenética baseada no gene 16S ribossômico, o isolado bacteriano CC07 mostrou-se próximo a Pseudomonas aeruginosa, enquanto que o isolado 4C07 ficou próximo à Pseudomonas fulva. A peroxidação lipídica foi observada somente para o isolado CC07 na presença dos herbicidas em mistura. O perfil protéico foi avaliado em SDS-PAGE, o qual revelou a indução de uma nova proteína de aproximadamente 60 KDa para o isolado 4C07 na presença dos herbicidas, porém, não foram observadas diferenças significativas para o isolado CC07. A enzima superóxido dismutase (SOD) apresentou aumento significativo da atividade para ambos isolados quando expostos aos herbicidas, entretanto, diferenças na atividade da enzima catalase (CAT) não foram observadas. Para o isolado 4C07 na presença dos herbicidas foi observado aumento significativo no conteúdo de glutationa reduzida (GSH), o qual foi acompanhado pela indução de uma nova isoforma de GR (I), que respondeu especificamente ao clomazone e os herbicidas em mistura. A glutationa-S-transferase (GST) também apresentou acréscimos de atividade quando o isolado 4C07 foi exposto aos herbicidas. Porém, para o isolado CC07 as enzimas GR, GST, assim, como a GSH não apresentaram respostas significativas a presença dos herbicidas. Assim, foi possível observar que o isolado 4C07 possui um sistema antioxidante mais eficaz, quando comparado com o isolado CC07. Além disso, os resultados sugerem que as enzimas SOD, GR, GST e o composto GSH podem estar relacionados com o mecanismo de tolerância do isolado 4C07 aos herbicidas ametrina e clomazone, o que pode demonstrar uma provável adaptação aos ambientes estressantes. / The herbicides ametrina and clomazone are widely used in the sugar cane cultivation, showing essential microbial degradation, besides being considered contaminants of soil and water. The exposure of microorganisms to xenobiotics can result in oxidative damage due to increased production of reactive oxygen species (ROS). This study aimed to analyze biochemical and physiological responses of two bacterial strains tolerant to the herbicides ametrina and clomazone. The bacteria strains were isolated from agricultural soils and according to physiological studies, growth and halo formation, two of them with the most herbicide tolerance were selected. These microorganisms were grown in nutrient agar plates at 30 ° C for 14 hours in the presence of herbicides ametrina (25 mM), clomazone (9 mM), and in a medium composed by two herbicides: ametrina (20 mM) + clomazone (20 mM). These concentrations represent the concentrations used for growing sugar cane for pre and post-emergence. Through the phylogenetic analysis based on 16S ribosomal gene, the CC07 strain was close to Pseudomonas aeruginosa, while the 4C07 strain was close to Pseudomonas fulva. According to the results obtained, lipid peroxidation was only observed for CC07 strain in a medium composed by two herbicides. The protein profile was evaluated by SDS-PAGE, which revealed the induction of a new protein of approximately 60 kDa for 4C07 strain in the presence of the two herbicides, differently to that observed for CC07 strain. The activity of the enzyme superoxide dismutase (SOD) increased significantly for both strains when exposed to the herbicides, however, no differences were observed for catalase (CAT) activity. The 4C07 strain showed increase in content of reduced glutathione (GSH) in the presence of the herbicides, which was accompanied by the induction of a new isoform of GR (I). Similarly, the 4C07 strain exhibited an increase in Glutathione-S-transferase (GST) activity to herbicides exposure. However, for the CC07 strain, the GR and GST enzymes, as well as the GSH content did not exhibit differences. Thus, we observed that the 4C07 strain may have an antioxidant system more effective when compared to the CC07 strain. Moreover, the results suggest that the enzymes SOD, GR, GST and GSH content may be related to the mechanism of tolerance of 4C07 strain to ametrina and clomazone herbicides, showing the tendency to deal better and adapt to stressful environments.

G-quadruplexes (G4s) are nucleic acid structures formed from π-stacked planar sets of four Hoogsteen hydrogen bonded guanine bases. G4s emerged as potential therapeutic targets based on their ability to modulate gene expression and inhibit the ability of telomerase to elongate chromosomal telomeres. Raman spectroscopy, polarized Raman spectroscopy, surface-enhanced Raman spectroscopy (SERS), and other optical spectroscopic techniques were used to characterize the G4s formed by four different DNA sequences: human telomeric (HT), thrombin-binding aptamer (TBA), nuclease hypersensitive element III1 region of the c- Myc gene promoter (Myc), and a single loop-isomer of Myc (MycL1). / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2015. / FAU Electronic Theses and Dissertations Collection

Nuclear pre-mRNA splicing proceeds via two mechanistically conserved consecutive trans-esterification reactions catalyzed by the spliceosome. The ordered coalescence of spliceosomal snRNPs and splicing factors on the pre-mRNA, coupled with essential spliceosomal rearrangements poise the splice-sites in proximity for the two catalytic reactions, ensuring intron removal and exon ligation to yield functional mRNA (reviewed in Will and Lührmann, 2006). Scope of the study The S. cerevisiae splicing factors Prp18 and Slu7 and their human homologs function during second catalytic reaction. In S. cerevisiae, Slu7 is essential, whereas Prp18 is dispensable at temperatures <30°C (Vijayraghavan et al., 1989; Vijayraghavan and Abelson, 1990; Frank et al., 1992; Horowitz and Abelson, 1993b; reviewed in Umen and Guthrie, 1995). Slu7 acts in concert with Prp18 and their direct interaction is required for their stable spliceosomal association (Zhang and Schwer, 1997; James et al., 2002). In vitro studies indicate that both the factors are dispensable for splicing of introns with short distances between branch nucleotide to 3’ splice-site (Brys and Schwer, 1996; Zhang and Schwer, 1997). Furthermore, mutational analyses of Slu7 and Prp18 have defined their functional domains/motifs (Frank and Guthrie, 1992; Bacíková and Horowitz, 2002; James et al., 2002). In this study, we have examined functions for the predicted homologs of Slu7 and Prp18 in fission yeast; an evolutionarily divergent organism where splicing mechanisms are not well understood and whose genome harbors genes with predominantly multiple introns with degenerate splice-junction sequences. Towards this goal, a combinatorial approach employing genetic and biochemical methods was undertaken to understand splicing functions and interactions of SpSlu7 and SpPrp18. Our mutational analysis of these protein factors provided an overview of the domains/motifs critical for their in vivo functions. Lastly our analysis of components of the budding yeast Cef1p-associated complex show novel interactions and splicing functions for two uncharacterized, yet evolutionarily conserved proteins. Conserved fission yeast splicing proteins SpSlu7 and SpPrp18 are essential for pre-mRNA splicing but have altered spliceosomal associations and functions Analyzing conserved splicing factors in evolutionarily divergent organisms is an important means to gain deeper functional insights on splicing mechanisms in genomes with varied gene architecture. We initiated our analysis of the ‘predicted’ S. pombe second-step splicing factors: spprp18+ and spslu7+, by genetically depleting these factors. We find spprp18+ is essential for viability, unlike budding yeast PRP18; while SLU7 is essential in both yeasts. The complete essentiality of both these fission yeast factors, prompted us to create conditional-lethal thiamine repressible ‘switch-off’ strains to probe their splicing functions. Through semi-quantitative RT-PCR and northern blot analysis we demonstrate splicing defects for tfIId+ pre-mRNAs upon metabolic depletion of spprp18+ or spslu7+, thus linking their essentiality to a role in pre-mRNA splicing. Further we examined whether their requirement as splicing factors is governed by specific intronic features. We find both factors are required in vivo for removal of several introns. However, for the introns tested, their functions are not strictly correlated with intron length, number, position or the branch-nucleotide to 3’ splice-site distance. The latter features dictate the need for their S. cerevisiae homologs. Strikingly the lack of either one of these essential proteins, arrests splicing before the first catalytic step; implicating possible functions early in spliceosome assembly even before any catalytic event, as opposed to budding yeast Slu7 and Prp18, which are second-step factors assembling late onto the spliceosome after the first splicing reaction. Given the different splicing arrest point, on depletion of SpSlu7 and SpPrp18, we investigated through yeast two-hybrid and co-immunoprecipitation assays whether the direct interaction between these proteins is conserved. We find despite being nuclear localized these proteins do not interact in either of the assays employed. A structural basis for the lack of interaction was provided by our homology modeling of SpPrp18, that was based on the crystal structure of S. cerevisiae Prp1879 (Jiang et al., 2000). Together these data raise the possibility of contextual functions and interactions for these conserved proteins that varies with changes in gene architecture. This likelihood is strengthened by our reciprocal genetic complementation tests; wherein we find that SpSlu7 and SpPrp18 cannot complement the corresponding S. cerevisiae null alleles and vice versa. Additionally, the human homologs, hSlu7 and hPrp18 also failed to rescue null alleles for spslu7+ and spprp18+. To understand the likely point of coalescence of SpSlu7 and SpPrp18 on assembling spliceosomes, we probed their snRNP associations through co-immunoprecipitation analysis. Our data revealed interaction of SpSlu7 with the U2, U5 and U6 snRNPs at moderate salt concentrations with the interaction with U5 snRNP being retained at higher salt conditions. SpPrp18, on the other hand, showed only a very weak association with U5 snRNP. Our analysis thus indicates that the assembly and step of action for “predicted” late-acting splicing factors in fission yeast differs from that in budding yeast, implicating novel interactions and functions for these fission yeast splicing factors. Mutational analysis of fission yeast SpPrp18 and SpSlu7 identifies functional domains To examine the protein domains/motifs critical for the functions of SpPrp18 and SpSlu7, we have performed a mutational study. This analysis was important after our findings that these factors are early acting and do not interact. The data gathered would shed light on the contribution of different domains/motifs in the functional diversification of these factors. Guided by the findings of Bacíková and Horowitz (2002); site-specific missense mutants were created in the highly conserved carboxyl-terminus (CR domain and helix 5) of SpPrp18. Additionally, site-specific missense mutants were generated in a conserved amino-terminus domain that is absent in budding yeast Prp18. Our data showed mutants in the highly conserved helix 5 and the CR domain of SpPrp18 to be recessive and non-functional, despite being stably expressed. This contrasts with the temperature-sensitivity conferred by similar mutants in homologous residues in budding yeast Prp18 (Bacíková and Horowitz, 2002). We speculate that the essentiality of the CR domain and helix 5 mutants of SpPrp18 arises due to a defect in spliceosomal association. However, the mutants in conserved residues in the protein’s amino-terminal domain are phenotypically wild type at various growth temperatures tested, suggesting redundant functions for these residues. Our data, based on analysis of a single missense mutant in the highly conserved zinc knuckle motif of SpSlu7, ascribes essential functions for the zinc knuckle motif. We find the mutant to be recessive and non-functional despite stable expression and normal cellular localization of the mutant protein. This contrasts with the behavior of zinc knuckle mutants in budding yeast and human Slu7. Budding yeast Slu7 mutants are functionally wild type and human Slu7 mutants have an altered cellular localization (Frank and Guthrie, 1992; James et al., 2002; Shomron et al., 2004). Possible roles for the zinc knuckle motif of SpSlu7 could be in facilitating interaction of SpSlu7 with U5 snRNA or even with some protein factor. Functional analysis of budding yeast Cef1p-associated complex SpSlu7 and its budding yeast homolog ScSlu7 co-purify with Cdc5/Cef1 in a complex of ~30 proteins together with U2, U5 and U6 snRNAs (Gavin et al., 2002; Ohi et al., 2002). Functional characterization of six proteins of the budding yeast Cef1p complex: Ydl209c (Cwc2/Ntc40), Ycr063w (Cwc14/Bud31), Yju2 (Cwc16), Ygr278w (Cwc22), Ylr424w (Spp382/Ntr1) and Ygl128c (Cwc23) was initiated using a combination of genetic and biochemical approaches. We probed direct protein-protein interactions between members of the Cef1p-associated complex by yeast two-hybrid assays. We also examined the pre-mRNA splicing roles for an essential factor, Yju2/Cwc16 and for a non-essential factor, Ycr063w/Cwc14. Our data reveals direct interaction between Yju2 and early acting factors, Syf1/Ntc90 and Clf1/Ntc77. Similarly interaction of Ydl209c/Cwc2 with early acting splicing factors, Prp19, Syf1/Ntc90 and Clf1/Ntc77 was noted. We created a temperature-sensitive expression strain for YJU2 using a temperature-sensitive Gal4 transcription trans-activator (Chakshusmathi et al., 2004; Mondal et al., 2007) to interrogate the splicing functions of YJU2. RT-PCR and northern blot assays show that depletion of YJU2 causes splicing defects for intron containing pre-mRNAs. We predict early splicing functions for YJU2 as is known for its interacting partners. Furthermore, we find that genetic depletion of the non-essential factor YCR063w causes temperature-sensitivity as has been reported for a few other factors (for e.g. Prp17, Lea1, Snt309/Ntc25, Ecm2) of Cef1p-associated complex (Jones et al, 1995; Chen et al., 1998). Although our yeast two-hybrid data does not reveal any direct interactions between Ycr063w and other proteins of the Cef1p-associated complex, we probed its functions through in vitro splicing assays. Splicing extracts from ycr063w/ycr063w cells show compromised second-step splicing at higher temperatures, thereby implying an auxiliary function for Ycr063w in stabilizing some functionally critical interactions during splicing. These studies employing complementary genetic and biochemical approaches implicate functional divergence of conserved predicted ‘second-step’ fission yeast factors, SpSlu7 and SpPrp18, suggesting co-evolution of splicing factors with changes in genome architecture and intron-exon structure. Our studies on Cef1p-associated complex show novel interactions and implicate pre-mRNA splicing functions for two previously uncharacterized proteins.

Restriction endonucleases occur ubiquitously among bacteria, archaea and in viruses of certain unicellular algae, and they are usually accompanied by a modification enzyme of identical specificity; together, the two activities form a restriction-modification (R-M) system- the prokaryotic equivalent of an immune system. More than 3,800 R-M enzymes have been characterized so far and they manifest 262 unique recognition specificities. These enzymes represent the largest family of functionally related enzymes. Based on the number and organization of subunits, cofactor requirements, catalytic mechanism, and sequence specificity, restriction enzymes have been classified into different types, Types I, II, III, and IV. R-M systems are important model systems for studying highly specific DNA-Protein interactions and serve as excellent systems for investigating structure-function relationship and for understanding the evolution of functionally similar enzymes with highly dissimilar sequence. In bacteria, DNA methyltransferases (MTases) associated with R-M systems protects the host DNA from cleavage by the cognate restriction endonuclease recognizing the same sequence and provides the integrity of host cell genome against foreign DNA invasion. The modification MTases catalyses the addition of a methyl group to one nucleotide in each strand of the recognition sequence using S-adenosyl-L-methionine (AdoMet) as the methyl group donor. Based on the chemistry of the methylation reaction catalyzed, DNA MTases are classified as C5 enzymes (endocyclic MTases), which transfer the methyl group to C5 position of cytosine, and N6 and N4 enzymes (exocyclic amino MTases), which transfer the methyl group to the exocyclic amino group of adenine or cytosine, respectively. DNA MTases of all three types contain conserved regions, which are responsible for catalysis and AdoMet binding, and variable regions known as target recognition domains (TRD), which determine the substrate specificity of a particular enzyme. Ten conserved amino acid motifs (I–X) are found in C5 MTases. Exocyclic DNA MTases are subdivided further into six groups (namely α, β, γ, ζ, δ and ε), according to the linear arrangements of three conserved motifs, the AdoMet-binding domain (FXGXG), the TRD (target recognition domain) and the catalytic domain (D/N/S)PP(Y/F). Base flipping has been proposed as a general mechanism used by all MTases in which the target base to be methylated is rotated 180º out of the DNA into a catalytic domain (motif IV). EcoP15I restriction enzyme (R.EcoP15I) belongs to the Type III restriction-modification (R-M) family. These enzymes are composed of two subunits, Res (Restriction) and Mod (Modification). The Mod subunit alone functions as a DNA methyltransferase in presence of AdoMet and magnesium and determines the specificity for restriction and methylation, whereas restriction activity requires the cooperation of both the Res and Mod subunits. EcoP15I methyltransferase (M.EcoP15I), a homodimeric enzyme catalyzes the transfer of a methyl group from AdoMet to the second adenine residue in the recognition sequence, 5’-CAGCAG-3’, in presence of magnesium ions. M.EcoP15I belongs to the β-subfamily of N6-adenine methyltransferases. In addition to the two highly conserved sequence motifs, FXGXG (motif 1) involved in AdoMet binding and DPPY (motif IV) involved in catalysis, the amino acid residues of the region 355-377 contains a PD(X)n(D/E)XK-like motif involved in metal binding. A Mutation in the Mod Subunit of EcoP15I Restriction Enzyme Converts the DNA Methyltransferase to a Site-Specific Endonuclease An interesting aspect of M.EcoP15I is that the methylation requires magnesium and magnesium binding to the PD(X)n(D/E)XK-like motif participates in base flipping. The PD-(D/E)XK superfamily of Mg2+-dependent nucleases were initially identified in structurally characterized Type II REases and later found in many enzymes involved in DNA replication, recombination and repair. The charged residues from the catalytic triads are implicated in metal ion mediated DNA cleavage. In EcoP15I DNA methyltransferase, a PD(X)n(D/E)XK like motif is present in which the partially conserved proline is replaced by methionine (MD(X)18(D/E)XK). Using site-directed mutagenesis methionine at 357 was changed to proline (M357P), which resulted in the formation of a Mg2+ binding/catalytic motif similar to several Mg2+-dependent endonucleases. Substitution of methionine at position 357 by proline converts EcoP15I DNA methyltransferase to a site-specific endonuclease. The mutant protein specifically binds to the recognition sequence 5’-CAGCAG-3’ and cleaves DNA in presence of Mg2+. The engineered EcoP15I-M357P is an active, sequence-dependent restriction endonuclease that cleaves DNA 10/1 nucleotide away from its recognition sequence in the presence of Mg2+. Unlike the holoenzyme, R.EcoP15I, the engineered endonuclease neither requires AdoMet or ATP nor requires two sites in the inverted orientation for DNA cleavage. It is of potential interest to use such an engineered enzyme as a genetic manipulation tool. Dimerisation of EcoP15I DNA Methyltransferase is Required for Sequence Recognition and Catalysis In the cell, after each round of replication, substrate for any DNA MTase is hemimethylated DNA and therefore, only a single methylation event restores the fully methylated state. This is in agreement with the fact that most of the DNA MTases studied exist as monomers in solution. The peculiar feature of M.EcoP15I is that it methylates only one strand of the DNA, at the N6-position of the adenine residue. Earlier studies using gel filtration and glutaraldehyde cross-linking demonstrated that M.EcoP15I exists as dimer in solution. However, the significance of dimerisation in the reaction mechanism of EcoP15I MTase is not clear. Therefore, experiments have been performed to determine whether M.EcoP15I could function as a monomer and the significance of dimerisation, if any, in catalysis. Towards this a homology model of the M.EcoP15I was generated by “FRankenstein monster” approach. Residues D223, V225, and V392, the side chains of which are present in the putative dimerisation interface in the model were targeted for site-directed mutagenesis. These residues were mutated to lysine and their importance was studied. Methylation and in vitro restriction assays showed that the triple mutant was catalytically inactive. Interestingly, the mutations resulted in weakening of the interaction between the monomers leading to both monomeric and dimeric species. M.EcoP15I was inactive in the monomeric form and therefore, dimerisation might be the initial step in its function. This must be required for positioning of the target base of the DNA in the active-site pocket of the M.EcoP15I. A part of this interface may be involved in site-specific DNA binding. Dimerisation of M.EcoP15I is, therefore, a prerequisite for the high-affinity substrate binding needed for efficient catalysis. Understanding the role(s) of Amino and Carboxyl-terminal Domains of EcoP15I DNA Methyltransferase in DNA Recognition and Catalysis N-terminal and C- terminal domains (NTD and CTD) of proteins are known to play many important roles such as folding, stability, dimerisation, regulation of gene expression, enzyme activity and substrate binding. From the modeled dimeric structure of M.EcoP15I, it was hypothesized that N- and C-termini are in close proximity with each other. In addition, it was predicted that each monomer can bind to AdoMet and DNA. Towards understanding the role(s) of the N- and C-terminal domains of M.EcoP15I in its structure and function, N-, and C-terminal deletions were created. Interestingly, deletion of N-terminal 53 amino acids and C-terminal 127 amino acids from of EcoP15I MTase converted the dimeric enzyme to a stable, monomeric protein that was structurally stable but enzymatically inactive. Each monomer could bind single-stranded DNA but dimerisation was required for double-stranded DNA binding and methylation. This indicated that amino acids at the N- and C-termini are important for maintaining a proper dimeric structure for M.EcoP15I functions. Therefore, it can be proposed that in a complex three-dimensional structure, the NTD and CTD should be properly maintained in order to execute its function, including dimerisation and DNA binding. However, since the 3D structure of M.EcoP15I has not yet been determined, the biochemical, biophysical and bioinformatics approaches may serve to provide useful information on the relative contributions of the electrostatic forces and hydrophobic contacts to the structural stability. Understanding the structural organization and folding of M.EcoP15I is crucial to elucidation of the mechanism of action.

DNA repair pathways have evolved to protect the genome from damage caused by intrinsic and extrinsic factors. Although numerous DNA repair mechanisms have been studied and reported, information regarding how they coordinate with the necessary changes in chromatin structure is scarce. Smc (structural maintenance of chromosomes) proteins are a conserved, essential family of proteins required for chromosome organization and accurate segregation. The budding yeast, Saccharomyces cerevisiae has three Smc-protein complexes: Smc1/3 complex (cohesin), Smc2/4 complex (condensin) and the Smc5/6 complex, required for sister chromatid cohesion, condensation and DNA repair, respectively. The chromatin associated Smc5/6 complex consists of Smc5, Smc6 and six non-smc elements (Nse1-Nse6). Smc5 and Smc6 are required for stability of repetitive chromosomal regions and sister chromatid recombination-mediated repair of double-strand breaks. Mms21/Nse2, a subunit of the Smc5/6 complex, is a SUMO E3-ligase, which conjugates SUMO (small ubiquitin-like modifier) to Smc5 and Yku70 (DNA repair protein) and its SUMO ligase activity protects the cells from extrinsic DNA damage. To address the role of Nse2 SUMO ligase in cellular events, we isolated mutants (nse2∆sl and nse2C221A) defective in the E3-ligase domain of Nse2 and found that these mutants are sensitive to genotoxic agents, for example MMS, UV or bleomycin, as expected. We found that cysteine 221 present in the SP-RING domain of Nse2 is required in the function of Nse2 in resisting genotoxic stress. We found that nse2∆sl cultures are slow growing and show increased abundance of cells having 2N DNA content (indicative of a G2-M cell cycle delay or arrest) relative to wild type cells. The DNA damage checkpoint pathway is activated to a limited extent in unchallenged nse2∆sl mutant cells indicating that cells lacking the SUMO ligase activity of Nse2 incur spontaneous DNA damage. Furthermore nse2∆sl cells are exquisitely sensitive to caffeine, an agent known to override the DNA damage checkpoint in a number of organisms by inhibiting the DNA damage checkpoint transducer ATR (Homo sapiens), Mec1 (Saccharomyces cerevisiae) and Rad3 (Schizosaccharomyces pombe). In order to investigate the importance of the DNA damage checkpoint pathway for nse2∆sl cells, we employed a genetic approach. We found that nse2∆sl exhibits synthetic sick interaction with mec1∆ but not tel1∆ (defective in Mec1 or Tel1 PI kinases) or mrc1∆ (defective in Mrc1 or mediator of replication checkpoint 1) indicating that the DNA damage induced Mec1 dependent checkpoint pathway is selectively required but the replication stress checkpoint pathway is dispensable for optimal growth of unchallenged nse2∆sl cells. In order to further investigate the role of Nse2 in S phase events, we used camptothecin (CPT), a drug that induces S phase specific double strand breaks. CPT inhibits topoisomerase I by trapping the covalent Top1-DNA intermediate. Collision of a DNA replication fork with such a complex results in double-strand and single-strand breaks in DNA. We found that nse2∆sl is CPT-sensitive and that nse2∆sl top1-8 has a synthetic sick phenotype. Thus, our chemical and genetic interaction studies suggest that the SUMO ligase activity of Nse2 may be required when Top1 function is compromised. Interestingly, human and yeast Top1 proteins are known to be sumoylated. Our findings suggest that MMS-induced enhancement of Top1 sumoylation in budding yeast is partially dependent on SUMO ligase activity of Nse2. Since both sumoylation and Top1 play a role in telomere maintenance, we also examined the telomere length in single as well as double mutants and found that there is slight telomere lengthening in nse2∆sl top1-8 double mutant. To gain further insight into the genetic interaction between Nse2 and other proteins which affect DNA topology, we also investigated genetic interaction of Nse2 with other topoisomerases. We found that top3-2 nse2∆sl exhibited a synthetic sick phenotype but nse2∆sl top2-4 showed partial rescue of temperature sensitivity. In order to investigate whether chromosome integrity is compromised in nse2∆sl cells we employed a YAC (yeast artificial chromosome) based assay to examine GCRs (gross chromosomal rearrangements). We found elevated levels of GCR in nse2∆sl cells compared to wild type cells. Furthermore, deletion of DNA Topoisomerase1 in nse2∆sl background selectively destabilizes a longer YAC relative to shorter YACs. We also examined the effect of varying origin number on YAC stability in nse2∆sl as well as top1∆ and nse2∆sl top1∆ cells. We found that a YAC having fewer origins is not destabilized in nse2∆sl and top1∆ single mutants but is destabilized in the nse2∆sl top1∆ double mutant. Since Nse2 is a non-SMC member of the Smc5/6 complex, we also investigated the effect of varying origin number on YAC stability in smc6-56 and smc656 top1∆ mutants. We found that the stability of a YAC is modestly compromised in the smc6-56 mutant but its derivative having fewer origins is not further destabilized, rather it seems to be stabilized. In order to gain molecular insights into the involvement of the SUMO ligase activity of Nse2 in maintenance of chromosome integrity, we examined sumoylation of specific substrates following a candidate approach. Smc5 and Yku70 are known targets of Nse2dependent sumoylation. We found that Smc6 is also sumoylated and that the MMS-induced enhancement of Smc6 sumoylation in budding yeast is partially dependent on Nse2. To understand the functional significance of Smc5 sumoylation, we mutated lysine residues of all the four predicted sumoylation sites ψKXE/D, individually as well as all four together. We found that all the single as well as quadruple mutants were weakly sensitive to MMS suggesting that these putative sumoylation sites of Smc5 may contribute towards countering MMS-induced DNA damage. Interestingly, we found that Smc5 sumoylation is enhanced when treated with MMS (methyl methane sulfonate) but not significantly with HU (hydroxyurea) and CPT (camptothecin). We also generated putative ATP-binding defective mutants in Smc5. Previous studies suggest that the ATPase motif is required for the essential function of some Smc proteins (for example, Smc1 and Smc6). We found that smc5K75E and smc5K75Q, having a mutation in the lysine residue of the conserved GXGKS motif present in the Walker A type box at the Nterminus exhibited a null phenotype implying that this conserved lysine residue is required for essential function of Smc5. In this study, employing genetic and biochemical methods, we have characterized the Nse2 SUMO ligase defective mutant and analyzed its role in the unperturbed mitotic cell cycle and in genome maintenance. We have also employed genetic methods to study the involvement of both Nse2 and DNA Topoisomerase I in maintaining genomic stability. Lastly, we have addressed the functional significance of Lysine residues of putative sumoylation sites and the conserved ATP-binding motif of Smc5 by mutational analysis. In conclusion, our study highlights an important role for the SUMO ligase activity of Nse2 in maintaining genomic stability and suggests that sumoylation of Smc5 may be important for resisting MMS-induced genotoxic stress.

Breast Cancer susceptibility gene 1 (BRCA1) & Breast Cancer Breast cancer is one of the most common malignancies affecting women worldwide. About 5-10% of all cases are estimated to be familial. Mutations in the BRCA1 (Breast Cancer susceptibility gene 1) gene account for about 15-20% of inherited breast cancer cases and 60-80% of families predisposed to both breast and ovarian cancer. BRCA1 mutations also result in susceptibility to early-onset breast and ovarian cancer. The human BRCA1 gene encodes a multi-domain 1,863 amino acid nuclear protein that is expressed in a wide variety of adult human tissues. The N-terminal end of BRCA1 contains a RING-finger domain. Exon 11 of BRCA1 contains two nuclear localization signals towards its N-terminal for targeting BRCA1 to the nucleus. The carboxyl terminus contains two BRCT (BRCA1 C-terminal) domains and a transcriptional activation domain. This study was carried out to functionally characterize BRCA1 and to find out the percentage in which BRCA1 gene is mutated in Indian familial breast and/or ovarian cancer families. The work has been divided into three sections: 1. Identification & characterization of a BRCA1 Associated Protein 2 (BAP2). 2. Germ-line BRCA1 mutation Analysis in Indian Breast and/or Ovarian Cancer Families. 3. Characterization of a novel missense mutation (E116K) in BRCA1. BRCA1 is known to interact with large number of proteins and is involved in various cellular functions like tumorigenesis, transcription, DNA damage repair, cell-cycle control, ubiquitinylation, genetic stability, cell growth and apoptosis. The interacting partners of BRCA1 have given a lot of clue about the functions of this complex protein. In the first project, we used the yeast two-hybrid system to identify novel interacting proteins of BRCA1. We used the 1-500 amino acid region of BRCA1 as bait in library screen and picked up a novel clone (clone 89) showing interaction with BRCA1. Clone 89 contains approximately 2.3 Kb long cDNA sequence. Using the nucleotide blast search, we obtained a full-length cDNA of approximately 5.4 Kb (KIAA0657) that is located on chromosome 2, 2q36.1 region. We have named this new protein BRCA1 Associated Protein 2 (BAP2). Translation of this coding sequence gave a protein that has homology to Titin protein. This protein, which has 1,236 amino acids, contains 9 Immunoglobulin like domains. The homologues of this protein exists in many other organisms but the function is not known. We have confirmed the interaction between BRCA1 and c89 using in vitro GST pull-down assay. We have studied the influence of BAP2 on various functions of BRCA1 like transcription, colony suppression and cell cycle. In the transcription assays, BAP2 activated p21 promoter activity perhaps by using endogenous BRCA1 as simultaneous ectopic expression of truncated BRCA1 (containing aa 1-500) abolished this activity. Further, BAP2 also increased the ability of BRCA1 to activate p21 promoter suggesting that BAP2 may act as a co-activator of BRCA1 functions. Surprisingly, we observed that BAP2 inhibited p53-mediated transcription both in the absence and presence of BRCA1. BAP2 failed to inhibit colony growth by itself as well as in combination with BRCA1. In the cell-cycle study, we found that BAP2 did not have any significant effect on cell cycle profile by itself. However, it drastically augmented the G2/M arrest mediated by BRCA1. Thus we conclude that we have identified a novel interacting protein of BRCA1 that regulates certain functions of BRCA1. Detection of mutations is of central importance in the study of genetic and malignant diseases. Mutation detection helps us in understanding the protein structure, function and expression. More than that, it is also important for pre-symptomatic/antenatal diagnosis, confirmation of the genetic cause of the disease and the mode of inheritance of a disease in a particular family, the prediction of clinical phenotype and the potentiation of diagnostic analysis in the case of families with incomplete pedigrees or with new mutations. Therefore, the importance of direct mutation analysis cannot be understated. The second project deals with screening of mutations in BRCA1 gene in 50 familial breast and/or ovarian cancer families using the technique of Conformation Sensitive Gel Electrophoresis (CSGE). CSGE can be used to detect mismatches in DNA heteroduplexes that contain one strand of wild type and one strand of mutated DNA. In a collaborative study with Kidwai Memorial Hospital for Oncology, Bangalore, we screened 50 families suffering from breast and/or ovarian cancer. We detected 13 mutations in this study out of which 3 are novel and 10 have already been reported earlier (Breast Information Core). All the mutations obtained in our study result in truncation of the BRCA1 protein either because of non-sense mutation or frame-shift mutation. Interestingly, 8 of the mutations detected are 185delAG mutations – the most commonly occurring mutation in Ashkenazi Jewish population. From this study, we conclude that BRCA1 is mutated in 26% of familial breast and/or ovarian cancer cases in India. Genetic testing in individuals with family history of breast, ovarian or both has become very common. It is difficult to interpret the result of genetic screen if a DNA change in the gene does not result in truncation of the protein. Rare missense changes of unknown functional and pathogenic significance are called unclassified variants. It is important to study the functional implications of these unclassified variants in order to determine the risk associated with the presence of such variations. The third project deals with characterization of one such missense variation. In an earlier mutation analysis study for BRCA1 gene in breast cancer samples, we found a novel missense variation resulting in Glu116Lys (E116K) change. In order to determine if this variant is a disease associated missense mutation or a benign sequence alteration; we introduced this variation into full length BRCA1 cDNA and studied its effect on the known functions of BRCA1, namely, transcription, colony suppression and cell cycle. We found that E116K is defective for activating transcription. However, it continued to inhibit growth in colony formation assay and arrest cells in G2/M phase of cell cycle. We conclude that E116K mutation results in loss of transactivation function of BRCA1 but has no effect on colony formation and cell cycle regulation; thus it can be categorized as a novel missense mutation.

Phylogenetics is the study of evolution of organisms. Evolution occurs due to mutations of DNA sequences. The reasons behind these seemingly random mutations are largely unknown. There are many algorithms that build phylogenetic trees from DNA sequences. However, there are certain uncertainties associated with these phylogenetic trees. Fine level analysis of these phylogenetic trees is both important and interesting for evolutionary biologists. In this thesis, we try to model evolutions of DNA sequences using Cellular Automata and resolve the uncertainties associated with the phylogenetic trees. In particular, we determine the effect of neighboring DNA base-pairs on the mutation of a base-pair. Cellular Automata can be viewed as an array of cells which modifies itself in discrete time-steps according to a governing rule. The state of the cell at the next time-step depends on its current state and state of its neighbors. We have used cellular automata rules for analysis and predictions of DNA sequence transformations on Computational grids. In the first part of the thesis, DNA sequence evolution is modeled as a cellular automaton with each cell having one of the four possible states, corresponding to four bases. Phylogenetic trees are explored in order to find out the cellular automata rules that may have guided the evolutions. Master-client paradigm is used to exploit the parallelism in the sequence transformation analysis. Load balancing and fault-tolerance techniques are developed to enable the execution of the explorations on grid resources. The analysis of the sequence transformations is used to resolve uncertainties associated with the phylogenetic trees namely, intermediate sequences in the phylogenetic tree and the exact number of time-steps required for the evolution of a branch. The model is further used to find out various statistics such as most popular rules at a particular time-step in the evolution history of a branch in a phylogenetic tree. We have observed some interesting statistics regarding the unknown base pairs in the intermediate sequences of the phylogenetic tree and the most popular rules used for sequence transformations. Next part of the thesis deals with predictions of future sequences using the previous sequences. First, we try to find out the preserved sequences so that cellular automata rules can be applied selectively. Then, random strategies are developed as base benchmarks. Roulette Wheel strategy is used for predicting future DNA sequences. Though the prediction strategies are able to better the random benchmarks in most of the cases, average performance improvement over the random strategies is not significant. The possible reasons are discussed.

Transforming growth factor-β (TGF-β) is the proto-type member of a super family of secreted proteins comprised of several structurally related, but functionally divergent proteins like the BMP, activin, inhibin, mullerian inhibitory substance etc. TGF-β was originally identified as a secreted factor, which in the presence of EGF was capable of transforming normal rat kidney fibroblasts. Studies over the years have shown that this protein is multifunctional that influences several processes including development, immune function, epithelial cell growth and motility, wound healing etc. TGF-β plays important role in the normal physiology as well as in pathological conditions in mammals. There are three mammalian isoforms that are involved in several developmental processes as has been shown by the knockout mice models. An important role for TGF-β has been implicated in several disease processes like fibrotic disorders (of liver, lung, kidney), inflammatory disorders (rheumatoid arthritis), autoimmune disorders (systemic lupus erythematosus) and cancer. TGF-β has a dual role in carcinogenesis. Initially it acts as a tumor suppressor and causes growth arrest of epithelial cells and cells in the early stages of cancer. But in an established tumor, TGF-β exerts an effect which is favorable for the survival, progression and metastasis of the tumor by promoting epithelial-mesenchymal transition (EMT), angiogenesis and escape from immune surveillance. Studies using mouse models have shown that an intact TGF-β signaling is essential for the metastasis of breast cancer. These observations indicate that the normal epithelial cells show differential response to TGF-β as compared to the tumor they give rise to. Supporting this, it has been shown that prostate tumor cells show invasive behavior in response to TGF-β and not non-tumorigenic cells. Most actions of TGF-β are brought about by regulation of gene expression and differential gene expression mediated by TGF-β has been reported in tumor cells and normal cells. For example, in response to TGF-β, tumorcells show increase in the production of proteases like uPA, MMPs etc and down regulation of the inhibitors of proteases TIMP isoforms, whereas this is not observed in the normal cells. However, there is no clear understanding of the mechanism (s) responsible for differential responses of various cell types to TGF-β. Since a role for TGF-β has been established in several pathological conditions particularly cancer and fibortic disorders, this pathway are a very attractive target for therapeutic intervention. Hence, if the TGF-β pathway has to be targeted for therapy of any disease, it becomes essential to identify the targets of TGF-β in different cell-types and their mechanism of regulation, particularly in un-transformed and transformed cells. Over the past few years, there have been several independent transcriptome analyses of cells in response to TGF-β treatment in various cell types such as HaCaT, fibroblasts, corneal epithelial cells etc. From a comparison of these studies, it is noted that TGF-β regulates genes in a cell type specific manner. Considering the dual role of TGF-β on normal and transformed cells, identification of genes and/or biochemical pathways regulated by TGF-β in these cells may allow identification of therapeutic targets for diseases involving TGF-β signaling pathway. With this background, the following objectives were set for the current investigation: 1. Identification of targets of TGF-β in normal and tumor cells and also the genes differentially regulated by TGF-β 2. Understand the mechanism of regulation of a few selected genes 3. Characterize novel targets of TGF-β with respect to their regulation by TGF-β and also their function Towards the aim of identification of targets of TGF-β in different cell-lines, expression profiling of genes in response to TGF-β was performed in a lung adenocarcinoma cell line (A549) and a matched immortalized lung epithelial cell line (HPL1D). Our data showed similar regulation of 267 genes in HPL1D and A549 cells by TGF-β. This suggests that the genes commonly regulated in both HPL1D and A549 are not tumor specific. Some of these genes were also reported to be regulated by TGF-β in other studies using micro array in various cell types. While 1757 genes are exclusively regulated by TGF-β in A549, only 733 genes are exclusively regulated in HPL1D cells. The reasons for this differential response are not known. However, some of the genes exclusively regulated in A549 such as Integrin αV, thrombospondin 1 have been shown to aid tumor survival, maintenance and metastasis. In contrast, in HPL1D, TGF-β regulates tumor suppressor genes like WT1, ECM proteins like collagen which are responsible for arrest of cell growth and apoptosis. This differential gene regulation in normal and tumor cells may explain the dual role of TGF-β in carcinogenesis. The differences in the effects of TGF-β on these two cell-lines could be due to the phenotypic properties of these cells, HPL1D being a non-transformed cell-line and A549 being a transformed cell-line. It is also possible that the differences are due to cell-type specific effects. In order to address this question, expression profiling in response to TGF-β was carried out using another cell-line namely HaCaT, which is an immortalized skin keratinocyte cell-line. When the expression profiles of the three celllines namely HPL1D, HaCaT and A549 in response to TGF-β treatment were compared, it was found that the genes regulated by TGF-β can be divided into seven categories based on the cell-line in which they are regulated. In this comparison, it was seen that there were several genes which were regulated by TGF-β in A549 and HaCaT despite the fact that these two cell-lines have little in common. The reason for these two celllines to show similarities in their gene expression profile in response to TGF-β is unclear. When the genes regulated by TGF-β in the three cell-lines were categorized based on their annotated functions using the DAVID tool, it was found that signaling pathways like MAP kinas, focal adhesion, Wnt signaling are regulated by TGF-β in all the celllines. On the other hand, Integrin αV was found to be regulated in A549 and HaCaT cells and very marginal regulation was seen in HPL1D cells. This could be one of the reasons for the similarities between A549 and HaCaT. There are studies which show the role of Integrin αV in some of the TGF-β mediated actions although the mechanism by which Integrin signaling modulates gene expression is not well understood. Our data shows that indeed thrombospondin 1 which is regulated by TGF-β in A549 and HaCaT is regulated through the integrin signaling pathways as blocking this pathway partially blocks the induction of this gene by TGF-β. TGF-β actions on cells are to a large extent are carried out by the phosphorylation of SMAD 2/3 by activated TGF-β type I receptor upon TGF-β signaling. Several genes that are transcriptionally regulated by TGF-β contain a SMAD complex binding element (SBE). However, over the last few years, evidences have accumulated which suggest that some actions of TGF-β could be independent of SMADs, mediated by the other signaling pathways like the MAP kinas, PKC and others. In order to understand the mechanism of regulation of a few selected genes by TGF−β, inhibitors for the three MAP kinas pathways (p38, ERK and JNK) were used prior to treatment with TGF-β. The expression of these genes was assessed by qRT-PCR analyses. These studies showed that most of the genes regulated by TGF-β require one or more of the MAP kinas pathways. In HaCaT and A549, the number of genes dependent on the MAP kinas pathways is more compared to HPL1D. Based on our data, we propose that activated MAP kinas pathway could be one of the essential determining factors for the various differential actions of TGF-β in tumor cells. However, the reason for the behaviour of HaCaT cells, which are untransformed cells in a manner similar to the A549 cells, is still unclear. One of the reasons for the similarity could be the activation of the integrin signaling pathway as described before. The expression profiling data identified several novel targets of TGF-β. One such target is S100A2, a calcium binding protein containing an EF hand motif that has been implicated in cancer. A progressive reduction in the expression of this gene has been reported with increasing grade of the tumor. Our studies show that this gene is regulated by TGF-β in HaCaT and HPl1D, but not in A549 cells. The induction of S100A2 by TGF-β in HaCaT cells is likely to be transcriptional as it is sensitive to actinomycin treatment. We further investigated role of other signaling pathways in the regulation of S100A2 by TGF-β and found that the regulation of this gene by TGF-β depends on the ERK and also the integrin signaling pathways. In order to characterize this gene with respect to its functions, A549 cells were chosen as they have very low endogenous expression of S100A2. Hence, in order to explore if there is any role for the loss of S100A2 expression in the progression of A549 cells, we cloned the DNA of S100A2 in a mammalian expression vector, transected A549 cells with this and isolated clones stably expressing this gene. We performed assays to assess cell proliferation, cell migration and potential to form colonies in soft agar. The data suggests phenotypic differences in the colonies that formed in soft agar and no major differences in other assays. Overall, our data has identified several novel targets regulated by TGF-β other than S100A2 like IGFBP7, FGFR1, and SPUVE etc. Further, regulation of several genes was found to be in a cell type specific manner involving MAP kinase and integrin signaling pathways. This study also identified major differences in the genes regulated by TGF-β in transformed and non-transformed lung epithelial cells.

Tuberculosis remains one of the oldest diseases known to mankind but still persists as a very major risk. Discovery of several antimycobacterials was marked by a steady decline in the active cases of pulmonary tuberculosis. However, in the recent past there has been a surge in its clinical incidence. The reasons for this failure are the emergence of multi drug resistance and the ability of the organism to survive under extreme condition or for a very long period of time known as ‘persistence’. The latter one established itself as a major hindrance in the effective control of tuberculosis. The latent bacilli are confined for a very long period after the infection in caseous cavities, called granulomma, inside the host and gets reactivated any time when the host becomes immuno-compromised. Latency has been successfully simulated in vitro by various models which mimic the in vivo condition by depleting O2 (Wayne, 1977), nutrients (Nyka, 1974) or the carbon source (Ojha et al., 2000). Stationary phase is exemplary of a stage in bacterial growth where the organism is exposed to various stresses like nutrient starvation, accumulation of toxic metabolites, low temperature, high osmolarity and acidity to name a few. Some evidences suggest that cells survive in nutrient deprived stationary phase. The present investigation was pursued with an objective to further our understanding on the mechanism of adaptation that the persistent mycobacterium may undertake to survive during the stationary phase of growth. The fast growing M.smegmatis, a nonpathogenic member in the non-tuberculous genera, however, with a genetic and metabolic similarity to M.tuberculosis has been used as a model for this study. Chapter 1 introduces the challenges in tuberculosis therapy and discusses the reason for drug tolerance and persistence of M.tuberculosis and M.avium complexes that were uncovered recently. It focuses on the intricate lipid rich cell wall which forms the first barrier for drug delivery with an emphasis on the cell surface antigenic glycolipids, the glycopeptidolipids. A detail account of their structure, biosynthetic pathway, intracellular function and their implications on biofilm formation has been provided. The evolution of the genetic approaches currently used in mycobacterial research is highlighted. The transcription apparatus and the regulation of gene expression in mycobacteria at different environmental condition and stages of growth are also discussed. The need for a detail investigation of the stationary phase RNAP in mycobacteria is stressed. Chapter 2 observes the changes in the cell surface of M.smegmatis at different ambience of growth. It focuses on the composition of glycopeptidolipid, one of the non-covalently attached free lipids and the mycolic-acids which are covalently attached to the inner layer of the cell wall. Composition of the mycobacterial cell wall with respect to the glycopeptidolipids and mycolic acids during biofilm mode of growth is also addressed. This chapter examines the conditional synthesis of a novel class of polar glycopeptidolipid in carbon starved cultures of M.smegmatis and determines their molecular structure. Chapter 3 revisits the biosynthetic pathway of the glycopeptidolipids and justifies a need for a fresh perspective. It identifies a glycosyltransferase responsible for the transfer of an extra rhamnose to the existing rhamnose linked to the terminal alaninol in the new polar glycopeptidolipid. This chapter also identifies a conserved Polyketide synthase in the glycopeptidolipid biosynthetic cluster. Characterization of the domains present in its two distinct modules with a correlation to the structure of the fatty acylchain together with the formation of a hydroxy fatty acyl chain is delineated. Chapter 4 deals with the construction of a suicide vector which when recombines to the mc2155 genome, incorporates a hexa-histidine tag at the C’ of the β΄ subunit of the RNAP. It details the strategy for the construction of the strain and established the genetic exchange event both genotypically and phenotypically. A single step procedure for purification of the holo-RNAP is also described in this chapter. In chapter 5 the role of the mycobacterial principal likes sigma factor, SigB, at the stationary phase of growth is highlighted. An approach of expression proteomics involving differential display of the total protein was undertaken to investigate the genes that are under the SigB regular during the stationary phase of growth. This chapter also examines the stationary phase induced changes in the RNAP. Various proteins that interact with the assembly are identified and their role in conferring rifampicin resistance to the RNAP is described. Appendix 1 details the preparation of the partially methylated deoxy monosaccharide using the esoteric reactions of organic synthesis. They were used extensively for glycosyl linkage analysis of the glycopeptidolipids by mass spectrometry, where they acted as standards.