Cell Biology & Molecular Genetics

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    Intrinsic DNA curvature in trypanosomes
    (Springer Nature, 2017-11-09) Smircich, Pablo; El-Sayed, Najib M.; Garat, Beatriz
    Trypanosoma cruzi and Trypanosoma brucei are protozoan parasites causing Chagas disease and African sleeping sickness, displaying unique features of cellular and molecular biology. Remarkably, no canonical signals for RNA polymerase II promoters, which drive protein coding genes transcription, have been identified so far. The secondary structure of DNA has long been recognized as a signal in biological processes and more recently, its involvement in transcription initiation in Leishmania was proposed. In order to study whether this feature is conserved in trypanosomatids, we undertook a genome wide search for intrinsic DNA curvature in T. cruzi and T. brucei. Using a region integrated intrinsic curvature (RIIC) scoring that we previously developed, a non-random distribution of sequence-dependent curvature was observed. High RIIC scores were found to be significantly correlated with transcription start sites in T. cruzi, which have been mapped in divergent switch regions, whereas in T. brucei, the high RIIC scores correlated with sites that have been involved not only in RNA polymerase II initiation but also in termination. In addition, we observed regions with high RIIC score presenting in-phase tracts of Adenines, in the subtelomeric regions of the T. brucei chromosomes that harbor the variable surface glycoproteins genes. In both T. cruzi and T. brucei genomes, a link between DNA conformational signals and gene expression was found. High sequence dependent curvature is associated with transcriptional regulation regions. High intrinsic curvature also occurs at the T. brucei chromosome subtelomeric regions where the recombination processes involved in the evasion of the immune host system take place. These findings underscore the relevance of indirect DNA readout in these ancient eukaryotes.
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    BOLT, AN AP2/ERF TRANSCRIPTION FACTOR, REGULATES ABIOTIC STRESS AND DEFENSE RESPONSES IN ARABIODPSIS THALIANA
    (2016) Bouten, Roxane; Chang, Caren; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Biotic and abiotic stresses negatively affect plant growth and development, hence decrease productivity. Many AP2/ERF family transcription factors in plants have important roles in stress response signaling although most have not yet been studied. Here I show that expression of an Arabidopsis thaliana AP2/ERF family member, which I call BOLT, is regulated by a MAPK pathway that includes MEKK1, MKK1, MKK2, and MPK4, and has roles in both biotic and abiotic stress response as well influencing growth and development. In this thesis, I examined BOLT’s gene expression pattern and protein localization, using GUS and YFP reporter genes respectively, measured its expression in response to biotic and abiotic stress and plant hormones using RT-qPCR, examined phenotypes by generating overexpressing and RNAi lines, and analyzed its effect on downstream gene expression using a microarray at time points after inducing BOLT expression. I found that BOLT is expressed in various plant tissues and the protein localizes to nuclear bodies as demonstrated in onion epidermal cells. Knockdown (RNAi) plants exhibit greater drought tolerance and are larger than wild type under low light conditions, while the overexpressors exhibit a dramatic early flowering phenotype and are small and weak under low light. Gene expression analysis suggests BOLT regulates genes involved in photosynthesis, hormone biosynthesis and signaling, and defense, many of which are also regulated in the MAPK pathway. Increased BOLT expression downregulates two discreet systems, cyclic electron flow and glycine cleavage, components of photosynthesis and photorespiration, respectively, which are two systems that are important under low light conditions. Taking these results together, I conclude that BOLT functions downstream of a stress responsive MAPK pathway and regulates a variety of growth- and stress-related genes necessary to balance growth and defense in response to biotic or abiotic stresses, or low light conditions.
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    Role of ubiquitination in Caenorhabditis elegans development and transcription regulation during spermatogenesis
    (2008-08-12) Kulkarni, Madhura D; Mount, Stephen; Smith, Harold; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Regulation of gene function can be achieved through a variety of mechanisms. In this dissertation, I present the genetic and molecular characterization of two genes involved in two distinct mechanisms of control. Each gene was initially identified by its functional role in sperm development in the model organism Caenorhabditis elegans. The first gene, uba-1, is an essential enzyme involved in protein turnover through ubiquitin-mediated proteolysis. A temperature-sensitive allele, (uba-1)it129, was isolated in a classical genetic screen for mutations that cause sperm-specific sterility. The second gene, spe-44, encodes a putative transcription factor. Its identification by microarray screening for sperm-enriched genes led to the cytological analysis of the deletion allele spe-44(ok1400), by reverse genetics approach. it129 encodes a conditional allele of uba-1, the sole E1 ubiquitin-activating enzyme in C. elegans. E1 functions at the apex of the ubiquitin-mediated conjugation pathway, and its activity is necessary for all subsequent steps in the reaction. Ubiquitin is covalently conjugated to various target proteins. Poly-ubiquitination typically results in target protein degradation, which provides an essential mechanism for the dynamic control of protein levels. Homozygous mutants of uba-1(it129) manifest pleiotropic phenotypes, and include novel roles for ubiquitination in sperm fertility, control of body size, and sex-specific development. We propose a model whereby proteins normally targeted for proteasomal degradation instead persist in uba-1(it129ts) and impair critical cellular processes. The second gene, spe-44, was identified as a putative sperm gene regulator in C. elegans based on its up-regulated expression during spermatogenesis and its significant sequence homology to the DNA-binding SAND domain. Genetic analysis of a deletion allele of spe-44(1400) has revealed its functional role during sperm development. Cytological analysis of spe-44(ok1400) showed developmental arrest of spermatocytes prior to spermatid differentiation. spe-44 mRNA is expressed in a narrow spatial and temporal window, just prior to spermatocyte differentiation, consistent with its functional role during spermatogenesis. Future study will be directed to find putative targets of spe-44 and the mechanisms that regulate gene expression using microarray analysis and yeast-one hybrid screens. These studies will help to understand transcriptional regulatory aspects of spermatogenesis in C. elegans.