HYDROXYUREA GLOBALLY INHIBITS RNA SYNTHESIS, DESTABILIZES mRNAs, AND TRIGGERS MEC1-OPPOSED RNAPII ACCUMULATION AT 3’ ENDS OF GENES

My dissertation includes two projects. The first project is focused on the regulation of glycolysis and oxidative metabolism by inducing heme synthesis in cancer cells. The second project deals with the effect of hydroxyurea on RNA synthesis. Heme is an essential cofactor for enzymes of the electron transport chain (ETC) and ATP synthesis in mitochondrial oxidative phosphorylation (OXPHOS). Heme also binds to and destabilizes Bach1, a transcription regulator that controls expression of several groups of genes important for glycolysis, ETC, and metastasis of cancer cells. Since many cancers are characterized by a high glycolytic rate regardless of oxygen availability, targeting glycolysis, ETC, and OXPHOS have emerged as a potential therapeutic strategy. Here, we report that enhancing heme synthesis through exogenous supplementation of heme precursor 5-aminolevulinic acid (ALA) suppresses oxidative metabolism as well as glycolysis and significantly reduces proliferation of both ovarian and breast cancer cells. ALA supplementation also destabilizes Bach1 and inhibits migration of both cell types. Promoting heme synthesis by ALA supplementation may thus represent a promising new anti-cancer strategy, particularly in cancers that are sensitive to altered redox signaling. In the second project, we investigated transcription changes triggered by DNA damage response (DDR). These changes depend on the on the nature of DNA damage, activation of checkpoint kinases, and stage of cell cycle. The transcription changes can be localized and affect only damaged DNA, but they can also be global and affect genes that are not damaged. While the purpose of localized transcription inhibition is to avoid transcription of damaged genes and to make DNA accessible for repair, the purpose and mechanisms of global transcription inhibition of not damaged genes is less well understood. We show here that a brief cell treatment with hydroxyurea (HU) globally inhibits RNA synthesis and transcription by RNA polymerase I, II, and III (RNAPI, RNAPII, and RNAPIII). HU triggers RNAPII accumulation at the 3’ ends of genes, indicating defect in transcriptional termination and pre-mRNA 3’ end processing. Interestingly, we observe destabilization of mRNAs after HU treatment. Experiments with cells defective in individual stages of mRNA decay pathway suggest that mRNAs produced in the presence of HU contain shorter poly(A) tails that do not require deadenylation for subsequent degradation, or that a decay step subsequent to deadenylation is activated by HU. The HU-triggered RNAPII accumulation at the 3’ ends of genes is exacerbated when the checkpoint kinase Mec1p is inactivated, suggesting involvement of DNA damage checkpoint in transcriptional terminatio

Nitrate Accumulation and Utilization in Fodder Oats Varieties as Affected by Different Nitrogen Levels

Importance of fodder crops in agriculture needs no emphasis because of the fact that regular fodder availability is basic requirement for livestock production. The area under fodder in Punjab is 2.03 million hectares with total production of 45 million tons, which is not sufficient to meet the maintenance requirements of livestock. To improve the quality of milk production it is important that animals are fed with good quality of fodder. Nitrogen is an essential primary nutrient for plant growth and plays an important role in productivity of forage crops. The application of N at various growth stages is one of the ways to increase forage productivity of crops. The excessive use of nitrogen can lead to deteriorate soil health and accumulation of nitrate-N in fodders above the permissible limit (\u3e 5000 ppm) which is toxic to animals. Some of the crops such as Sudan grass, pearl millet and oats are potent accumulators of nitrate. Oats is the most important winter cereal crop grown in northern, western and central India. Oats is gaining importance throughout the world due to its uses as human food, animal feed and fodder crop. One of reasons of nitrate toxicity in oats is high input of fertilizer. When growing conditions are favorable, plants take up nitrogen in form of nitrate. The nitrate is rapidly converted into ammonia which is incorporated into the plant protein. Unfavorable growing conditions can interfere with nitrate use and cause it to accumulate in the plant. Nitrate toxicity arises when nitrate conversion into nitrite is faster than its utilization into ammonia Nitrate reductase (NR) is considered a key enzyme in nitrogen metabolism. It is not only rate limiting enzyme in inorganic nitrogen assimilation but also the major regulatory step in N metabolism (Young et al., 2009). NR is considered to catalyze the NO3 - assimilation because it initiates the reaction when NO3 - is available. NR activity is modified rapidly in response to level of nitrate, CO2, light, carbon skeletons and nitrogen metabolites. In the present study inter relationship between crude protein level, nitrate-N value and NR activity in relation to N inputs has been worked out

Creating variability through interspecific hybridization and its utilization for genetic improvement in mungbean [Vigna radiata (L.) Wilczek]

Interspecific hybridization is important for genetic enhancement of crop plants. The present study was conducted to study genetic variation in advanced interspecific lines of mungbean for yield and its component traits, to determine the association among different traits and their contribution towards seed yield through correlation and path coefficient analysis. A set of 64 genotypes including 51 advanced interspecific lines derived from mungbean (Vigna radiata L. Wilczek) × urdbean (Vigna mungo L. Hepper) and mungbean (Vigna radiata L. Wilczek) × ricebean (Vignaumbellata Thumb.) crosses and 13 parents (mungbean, urdbean and ricebean) was the experimental material for this study. The mean sums of squares for genotypes were highly significant for all the traits. Mean sum of squares for replications were also highly significant for all traits except days to 50 % flowering, days to maturity and harvest index at 1 % and 5 % level of significance. This indicated substantial magnitude of diversity and variability in the interspecific lines and parents under study, which could be further exploited. High to moderate PCV and GCV along with high heritability and genetic advance was observed for biological yield per plant, seed yield per plant and plant height, indicating that these traits could be easy targets for phenotypic selection and consequently, may be improved genetically via simple plant selection methods. On the basis of correlation studies, it could be concluded that all the traits under investigation except number of seeds per pod and harvest index were important for selection for yield improvement. Path analysis further revealed that harvest index could also be one of the criteria of selection for higher yield in these interspecific lines

Towards a novel content organisation in agriculture using semantic technologies: a study with topic maps as a tool

Agricultural information management needs to be responsive to the needs of varied stakeholders, and content repurposing to suit multiple audiences is a key requirement. A framework that combines the semantic web techniques of Topic Maps (TM) and a global agricultural thesaurus, the FAO AGROVOC, has been developed on a pilot basis for five crops. The framework when overlaid on a web-based collection of information objects reveals interesting possibilities, especially in presenting the repository content in noticeably different ways to a subject-matter specialist or to an extension worker. Results are discussed and opportunities for further development are briefly described

A historical and theological evaluation of the Sikh Gurdwaras Act, 1925

Gurdwara legislation, Gurdwara management and institutions of Sikh leadership affect all Sikhs and are in existence to uphold and perpetuate Sikh religious traditions and values. This study is a historical and theological evaluation of the Sikh Gurdwaras Act, which was a secular legislation introduced in 1925 by the British administration in India. It evolved as a response to the Gurdwara Reform Movement, which was a religiously motivated struggle, to provide a scheme of management for Gurdwaras. The study constructs a narrative which evaluates the historical context and factors leading to the creation of the Act. Through a theological evaluation which engages in the interpretation and application of the Sikh Sacred Scriptures, the study then highlights that there is a contradiction which should not exist between: the content and implementation of the Sikh Gurdwaras Act, 1925; the constitution and actions of Sikh leadership institutions (the SGPC and the SAD); and between the theological teachings of the Sikh Dharam. The study ultimately suggests that there is need for the Sikh Gurdwaras Act, 1925 to be redeveloped so that Gurdwara legislation, Gurdwara management and institutions of Sikh leadership epitomise and are a more authentic reflection of the teachings within the Sikh Sacred Scriptures

Hydroxyurea and inactivation of checkpoint kinase MEC1 inhibit transcription termination and pre-mRNA cleavage at polyadenylation sites in budding yeast

Abstract The DNA damage response (DDR) is an evolutionarily conserved process essential for cell survival. The transcription changes triggered by DDR depend on the nature of DNA damage, activation of checkpoint kinases, and the stage of cell cycle. The transcription changes can be localized and affect only damaged DNA, but they can be also global and affect genes that are not damaged. While the purpose of localized transcription inhibition is to avoid transcription of damaged genes and make DNA accessible for repair, the purpose and mechanisms of global transcription inhibition of undamaged genes are less well understood. We show here that a brief cell treatment with hydroxyurea (HU) globally inhibits RNA synthesis and transcription by RNA polymerase I, II, and III (RNAPI, RNAPII, and RNAPIII). HU reduces efficiency of transcription termination and inhibits pre-mRNA cleavage at the polyadenylation (pA) sites, destabilizes mRNAs, and shortens poly(A) tails of mRNAs, indicating defects in pre-mRNA 3′ end processing. Inactivation of the checkpoint kinase Mec1p downregulates the efficiency of transcription termination and reduces the efficiency of pre-mRNAs clevage at the pA sites, suggesting the involvement of DNA damage checkpoint in transcription termination and pre-mRNA 3′ end processing

Reciprocal Regulation of AMPK/SNF1 and Protein Acetylation

Adenosine monophosphate (AMP)-activated protein kinase (AMPK) serves as an energy sensor and master regulator of metabolism. In general, AMPK inhibits anabolism to minimize energy consumption and activates catabolism to increase ATP production. One of the mechanisms employed by AMPK to regulate metabolism is protein acetylation. AMPK regulates protein acetylation by at least five distinct mechanisms. First, AMPK phosphorylates and inhibits acetyl-CoA carboxylase (ACC) and thus regulates acetyl-CoA homeostasis. Since acetyl-CoA is a substrate for all lysine acetyltransferases (KATs), AMPK affects the activity of KATs by regulating the cellular level of acetyl-CoA. Second, AMPK activates histone deacetylases (HDACs) sirtuins by increasing the cellular concentration of NAD+, a cofactor of sirtuins. Third, AMPK inhibits class I and II HDACs by upregulating hepatic synthesis of α-hydroxybutyrate, a natural inhibitor of HDACs. Fourth, AMPK induces translocation of HDACs 4 and 5 from the nucleus to the cytoplasm and thus increases histone acetylation in the nucleus. Fifth, AMPK directly phosphorylates and downregulates p300 KAT. On the other hand, protein acetylation regulates AMPK activity. Sirtuin SIRT1-mediated deacetylation of liver kinase B1 (LKB1), an upstream kinase of AMPK, activates LKB1 and AMPK. AMPK phosphorylates and inactivates ACC, thus increasing acetyl-CoA level and promoting LKB1 acetylation and inhibition. In yeast cells, acetylation of Sip2p, one of the regulatory β-subunits of the SNF1 complex, results in inhibition of SNF1. This results in activation of ACC and reduced cellular level of acetyl-CoA, which promotes deacetylation of Sip2p and activation of SNF1. Thus, in both yeast and mammalian cells, AMPK/SNF1 regulate protein acetylation and are themselves regulated by protein acetylation