Thesis (Ph. D.)--University of Chicago, Dept. of Molecular Genetics and Cell Biology, June 1999. / Includes bibliographical references. Also available in the Internet.

Accurate initiation of transcription by RNA polymerase II depends on general transcription factors (GTFs), which include the TATA-binding protein (TBP) and the transcription factors (TF) IIB, IIF, IIE and IIH. In order to reconstitute mouse transcription in vitro, we cloned the genes encoding mouse TFIIB, and both subunits of TFIIE and TFIIF from a mouse cDNA library. TBP and TFIIB were expressed in E.coli, while both subunits of TFIIE and the two subunits of TFIIF were expressed in a baculovirus system. All these factors were purified to > 90% homogeneity. The more complex transcription factors, TFIIH and RNA polymerase II, were purified more than 1000-fold and to near homogeneity, respectively, from tissue cultured mouse ascites cells. We have shown that the purified mouse transcription factors are active in a reconstituted RNA polymerase II in vitro transcription assay. The transcription reaction was inhibited by α-amanitine, and dependent on the addition of all the GTFs. Ribonucleotide reductase is a key enzyme in deoxyribonucleotide synthesis. It consists of two subunits, R1 and R2, which are both required for the enzyme activity. Transcription of the R1 and R2 genes is restiricted to the S-phase of the cell cycle, but the mechanisms that control this coordinated expression remain to be identified. We have studied initiation of transcription from the mouse R2 gene using a combination of in vivo reporter gene assays and in vitro transcription assays with crude nuclear extracts or with purified transcription factors. This promoter has an atypical TATA-box and a CCAAT-box that binds the transcription factor NF-Y. We found that a mutation in the R2 CCAAT-box had no effect on the transcription level in in vitro transcription assays reconstituted with pure transcription factors. However, it significantly decreased the level of transcription in similar experiments using crude nuclear extract. We also found that the sequence downstream from the R2 transcription start site (5´-UTR) (from +1 to +17 base pairs relative to transcription start site) is essential for initiation of transcription from this promoter. The presence of the wild type 5´-UTR made the R2 TATA-box redundant. On the other hand, the R2 5´-UTR had a repressing effect on transcription from the mouse R2 promoter. This region contains a palindrome sequence that covers 10 base pairs, and it is partially conserved in the human R2 promoter. Gel shift assays and in vitro transcription experiments using antibodies against mouse TAF4 (=TAF135) demonstrate that TAF4 is a component of the protein complex that interacts with this palindrome region, and suggest involvement of this component of the TFIID complex in negative regulation of the R2 promoter. The Adenovirus Major Late (AdML) promoter is commonly used as a model for studies of transcription initiation and regulation. It is a TATA-box dependent promoter, which also contains an initiator (Inr) element, a CCAAT-box interacting with transcription factor NF-Y, and an E-box binding the upstream stimulatory factor (USF). Using gel shift assays with recombinant NF-Y, USF, and immunopurified human TFIID, we show that binding of USF1 and NF-Y to DNA is not cooperative and that both factors independently facilitate binding of TFIID to the core promoter. The activation domains of NF-Y are expendable for this effect. Negative cofactor (NC2) comprises two subunits, which have a histone-fold structure similar to NF-Y, and represses transcription through formation of an inhibitory complex with TBP. Using an in vitro transcription system based on crude nuclear extracts, we show that NC2 has a negative effect on transcription in the presence of NF-Y or USF1, indicating that the two activators do not act as antirepressors. In vitro transcription using highly purified transcription factors efficiently reproduces repression of transcription by NC2. However, USF1 was inactive and NF-Y had a repressing effect in this system, which suggests that the activator functions of USF and NF-Y depend on cofactors.

Rhizopogon is a hypogeous fungal genus that forms ectomycorrhizae with genera of the Pinaceae. The greatest number and species of Rhizopogon are found in coniferous forests of the Pacific Northwestern United States, where members of the Pinaceae are also concentrated. Rhizopogon spp. are host-specific primarily with Pinus spp. and Pseudotsuga spp. and thus are an important component of these forest ecosystems. Rhizopogon includes over 100 species; however, the systematics of Rhizopogon have not been well understood. Currently the genus is placed in the Boletales, an order of ectomycorrhizal fungi that are primarily epigeous and have a tubular hymenium. Suillus is a stipitate genus closely related to Rhizopogon that is also in the Boletales and host specific with Pinaceae. I examined the relationship of Rhizopogon to Suillus and other genera in the Boletales. Infrageneric relationships in Rhizopogon were also investigated to test current taxonomic hypotheses and species concepts. Through phylogenetic analyses of large subunit and internal transcribed spacer nuclear ribosomal DNA sequences, I found that Rhizopogon and Suillus formed distinct monophyletic groups. Rhizopogon was composed of four distinct groups; sections Amylopogon and Villosuli were strongly supported monophyletic groups. Section Rhizopogon was not monophyletic, and formed two distinct clades. Section Fulviglebae formed a strongly supported group within section Villosuli. Taxonomic revisions were proposed. Suillus, Truncocolumella, and the Gomphidiaceae were transferred to the Rhizopogonaceae. In Rhizopogon, sections Amylopogon, Rhizopogon, and Villosuli were elevated to subgenera. Subgenus Roseoli was erected to accommodate the second section Rhizopogon Glade. In section Fulviglebae, Stirps Vinicolor, Rhizopogon ochraceisporus, R. subclavitisporus, and R. clavitisporus were transferred to subgenus Villosuli while the remaining species in section Fulviglebae were transferred to subgenus Rhizopogon. / Graduation date: 1999

Thesis (Ph. D.)--Rutgers University, 2009. / "Graduate Program in Microbiology and Molecular Genetics." Includes bibliographical references (p. 74-83).

published_or_final_version / abstract / toc / Clinical Oncology / Doctoral / Doctor of Philosophy

clk-1 encodes a hydroxylase that is necessary for ubiquinone (UQ) biosynthesis. clk-1 mutants do not synthesize UQ, but instead accumulate the precursor demethoxyubiquinone (DMQ). When fed on bacteria that synthesize UQ the mutants are viable but display slow development, behaviours and aging. However, they arrest development when fed on UQ synthesis-deficient bacteria. I have taken a genetic suppressor approach to investigate the causes of the various phenotypes as well as of the dietary requirements of the clk-1 mutants. / We identified two classes of mutants that suppress the defecation phenotypes of clk-1. All of these "dsc" mutants suppress the lengthened cycle of clk-1. Class I mutants also restore the ability to react normally to changes in temperature whereas the Class II mutants do not. The characterization of the Class I mutants suggests that part of the phenotype of clk-1 is due to an alteration of lipid metabolism, likely the level of lipid or lipoprotein oxidation. dsc-4 encodes the worm homolog of the Microsomal Triglyceride Transfer Protein (MTP), a protein required for the formation of low density lipoproteins (LDL) in vertebrates, and whose absence in people leads to abetalipoproteinemia. dsc-3 appears to be allelic to tat-2, which encodes a type IV P-type ATPase that is related to a family of human aminophospholipid transporters that includes ATP8B1/FIC1, whose inactivation results in cholestatic liver disease. dsc-3 and dsc-4 appear to affect distinct aspects of lipid metabolism. A general link between the Class II mutants and clk-1 remains elusive. dsc-1, a Class II gene, encodes a paired-like homeodomain transcription factor that is necessary for the GABA sensitivity of enteric muscles. / We also identified 9 clk-1(e2519)-specific suppressors, which suppress most Clk phenotypes, including their requirement for dietary UQ. Our analysis of these suppressors reveals that it is the lack of UQ rather than the presence of DMQ that is responsible for most phenotypes. In addition, they allowed us to show that most Clk phenotypes can be uncoupled from each other. We cloned six suppressors and all encode missense tRNA(Glu) suppressor genes. To my knowledge, these represent the first missense tRNA suppressors identified in any metazoan.

No description available.

In the following study, I have investigated aberrant integration events of the maize Activator/Dissociation ( Ac/Ds) family of transposable elements (TEs) in Arabidopsis. The purpose of the study was twofold: (i) to identify sequence modifications associated with aberrant transposition that are informative regarding the mechanism of Ac/Ds transposition; and (ii) to extend our understanding of the mechanisms by which class II TEs can influence genome structure. This work focuses on a large inversion identified on chromosome II. A lone Ds element comprises one breakpoint of the inversion and the second breakpoint is composed solely of Arabidopsis sequences. The analysis of the sequence modifications present at both breakpoints indicates that the event was precipitated by the abortive transposition of Ds. This is the first event of its kind identified for an Ac/Ds and the event defines a novel mechanism by which these TEs can induce change within a genome. Further, the presence of deletions at both termini of the implicated Ds suggests that the transposition of Ac/Ds involves fully excised intermediates. To obtain further support for this model, a population of Arabidopsis individuals harboring Ds excision events was screened for reintegrated elements. Several integrations were analyzed at the sequence level and compared to wild-type integration sites. While no genome rearrangements were detected, a number of integrations display small deletions within both the Ds termini and the DNA flanking the elements. These results are consistent with the presence of fully excised Ac/Ds intermediates. Further, the results suggest that dissolution of the transposase active complex at different points in the transposition process will result in the formation of distinct aberrant transposition products. During the characterization of the inversion, a novel Arabidopsis TE family, FARE, was identified. The FARE elements are foldback transposons, a heterogeneous and poorly characteri

In cell cycle checkpoint control, the Chk2 family protein kinases play a central role in mediating the cellular responses to DNA damage or DNA replication block. However, at the beginning of this project, there was no evidence for a Drosophila homologue of Chk2. loki was identified in a screen for serine/threonine protein kinases that are expressed in the ovary. Using a phylogenetic analysis, I showed that loki is a Drosophila chk2 orthologue. To characterize the checkpoint function of loki in Drosophila development, we created a loki null mutant and generated anti-Loki antibodies. Under normal laboratory conditions, loki null mutants display no apparent defect during the whole life span. Further functional analysis revealed that loki is not required for the meiotic pachytene checkpoint, the essential DNA replication checkpoint control in syncytial embryos and the DNA damage/replication checkpoint during the larval stage. However, in postblastoderm embryos, loki is required for the DNA damage checkpoint activated by gamma irradiation. Embryos lacking loki are not able to arrest the cell cycle in response to gamma irradiation.

When the Y chromosome of a Mus musculus domesticus mouse is placed onto the C57BL/6J (B6) inbred genetic background, the XY (B6.Y TIR) progeny develop only ovaries or ovotestes during fetal life. At puberty, while some of the hermaphroditic males become fertile, none of the XY sex-reversed females produce litters. The objective of my study was to clarify the cause of infertility in B6.YTIR females. We have previously demonstrated that the eggs ovulated from B6.YTIR ovaries undergo fertilization efficiently, but cannot develop beyond the 2-cell stage. In the present study, we collected oocytes directly from XY ovaries, and examined their maturation, fertilization and embryonic development in vitro. The results show that the majority of fertilized eggs fail to reach the blastocyst stage. To determine whether developmental incompetence of XY oocytes can be attributed to defects in the oocytes themselves or the surrounding XY somatic cells, we constructed female mouse chimera composed of B6.YTIR and XX BALB/c cells. All chimeric females produced progeny exclusively derived from XX oocytes. For comparison, most of XX ↔ XX chimeric females produced progeny derived from oocytes of either strain. / The ability of XY oocytes to regulate granulosa cell differentiation and functions was assessed in oocyte-cumulus complexes (OCC) in vitro . Microsurgical removal of oocytes prevented cumulus cell expansion and suppressed estradiol production while it promoted progesterone production. Coculture of the oocytectomized OCC with denuded oocytes from either XX or XY ovaries resumed cumulus expansion and the normal endocrine profile. Morphometric analyses indicated that XY oocytes were significantly smaller and their zona pellucida layer thinner than XX oocytes as early as the preantral stage. Furthermore, XY oocytes were attached with fewer cumulus cells in antral follicles. To determine whether developmental incompetence of the zygotes from XY ovaries resides in the nuclear or cytoplasmic component, we exchanged the pronuclei between the zygotes derived from B6.YTIR oocytes and those from XX oocytes and examined their development in vitro. The results indicate that both compartments are defective in the B6.YTIR oocyte. / In conclusion, the XY oocyte becomes cell-autonomously defective in both nuclear and cytoplasmic components during early oogenesis.