In the first part of this thesis, I describe the expression of C. fimi cellulase genes in the closely related Brevibacterium lactofermentum by generating a shuttle vector able to replicate selectively in the latter and carrying full length cellulase-encoding genes. The expression of those genes apparently originated from some unpredicted regulatory sequences, possibly located within the vector itself. The enzymatic activity was mostly found in the culture medium in B. lactofermentum indicating that the organism secreted the enzymes. The putative C. fimi promoter sequences did not function in B. lactofermentum, making difficult the analysis of their roles in expression of C. fimi cellulase genes. In the second part of this thesis, I describe the characterization of a recombinant C. fimi exo-ϐ-1,4-glucosidase (CbgA) expressed in E. coli. The purified enzyme had a Mr of 183 kDa and hydrolysed various ϐ-glucosides with a preference for cello-oligosaccharides in the order C5>C4>C3>C2. The intact CbgA polypeptide was not required for enzymatic activity since removal of about 700 residues from the amino terminus did not reduce activity. The purified enzyme was used to raise polyclonal antibodies which in turn were used to identify the corresponding enzyme in C. fimi. During the fractionation of C. fimi ϐ-glucosidases, several enzymes hydrolyzing various ϐ-glucosides were isolated together with the native CbgA, which was present in the culture medium as part of a protein aggregate. Part of the nucleotide sequence of the 7.2 kb insert was determined. Alignments of the N-terminal amino acid sequences of the purified CbgA and truncated polypeptides with the partial nucleotide sequence of the cloned C. fimi DNA showed that precise excision was responsible for the appearance of a truncated form of CbgA. Alignment of the amino-terminal sequence of a CbgA:CexCBD fusion peptide indicated that the pre-mature CbgA starts with a putative leader sequence of 49 amino acids which is followed by a region rich in Pro and Ala residues. Two GTG translational initiation codons followed by sequences resemblingprokaryotic ribosome binding sites and separated by a large open reading frame were identified from data obtained after in vitro site-directed mutagenesis of the most upstream initiation codon suggesting that internal re-initiation may occur and that upstream regulatory sequences had not been isolated. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

Drosophila melanoqaster individuals trisomic for an entire chromosome arm can survive to late stages of pupal development. To examine gene expression in these hyperploids, the levels of five enzymes whose structural genes are located on the left arm of chromosome two have been examined both in aneuploid and in diploid strains. Elevated levels of enzyme activity were observed in larvae possessing small segmental duplications for these genes. However, in 2L trisomies, the three distally mapping loci showed compensated levels of expression close to that observed in the diploid strains. Analysis of electrophoretic variants revealed that for one of these compensated loci all three alleles were expressed in trisomies. Two proximally located genes displayed dose-dependent levels of enzyme activity. For most genes, autosomal compensation appears to be very discrete: either the expression of the gene is repressed or it is not. To extend these observations, and to determine if autosomal compensation was peculiar to the left arm of chromosome two, trisomies for the X, for 2R, and for 3L also were examined. Compensating and non-compensating loci were also found on 3L, whereas all loci examined in X-chromosomal trisomies were dosage compensated. This suggests that X-chromosomal and autosomal trisomies are not necessarily analagous. Dosage compensation in X-chromosomal trisomies (metafemales) may occur exclusively or partially by the mechanism that operates between euploid males and females. However, some compensation in X trisomies may occur by regulatory controls distinct from male-female dosage compensation as indicated by the following results. The expression of LSP-1aT a gene that normally escapes complete dosage compensation in diploid males, was fully compensated in trisomic-X larvae. Possibly, compensation of this gene in these individuals was mediated by regulatory mechanisms other than those controlling male-female dosage compensation. As such, loci that normally do not reside on the X chromosome, but which have been transposed to this chromosome, might be expected to escape compensation in metafemales. This appears to be the case; an Adh gene that had been transposed from the second to the X chromosome was expressed at a similar level (per gene) in metafemales and females. In addition, a native X-chromosomal locus appeared to be compensated between males and females, but was not compensated in X-chromosomal trisomies. Thus, some X-linked loci escape regulation by dosage compensation in metafemales. It is possible that some of the regulatory systems operating in X-chromosomal and autosomal trisomies are analagous, and reflect a common form hyperploid compensation. The level at which compensation occured was investigated by measuring the quantities of RNA produced by several genes in whole-arm trisomies. For the heat-shock gene, hsp 85. compensation for protein levels appeared to be Post-transcriptionally regulated. However, measurements of RNA synthesis on salivary gland polytene chromosomes revealed that for most of the genes compensation was transcriptionally regulated. Dosage compensation on the autosomes probably reflects the existence of a system that normally operates in diploids to control gene expression by negative regulation. / Science, Faculty of / Zoology, Department of / Graduate

Indiana University-Purdue University, Indianapolis / This dissertation examines the molecular mechanisms regulating Rac2 gene expression during cell differentiation and identification of a minimal cis-element required for the induction of Rac2 gene expression during K562 cell differentiation. The Rho family GTPase Rac2 is expressed in hematopoietic cell lineages and is further up-regulated upon terminal myeloid cell differentiation. Rac2 plays an important role in many hematopoietic cellular functions, such as neutrophil chemotaxis, superoxide production, cytoskeletal reorganization, and stem cell adhesion. Despite the crucial role of Rac2 in blood cell function, little is known about the mechanisms of Rac2 gene regulation during blood cell differentiation. Previous studies from the Skalnik lab determined that a human Rac2 gene fragment containing the 1.6 kb upstream and 8 kb downstream sequence directs lineage-specific expression of Rac2 in transgenic mice. In addition, epigenetic modifications such as DNA methylation also play important roles in the lineage-specific expression of Rac2. The current study investigated the molecular mechanisms regulating human Rac2 gene expression during cell differentiation using chemically induced megakaryocytic differentiation of the human chronic myelogenous leukemia cell line K562 as the model system. Phorbol 12-myristate 13-acetate (PMA) stimulation of K562 cells resulted in increased Rac2 mRNA expression as analyzed by real time-polymerase chain reaction (RT-PCR). Luciferase reporter gene assays revealed that increased transcriptional activity of the Rac2 gene is mediated by the Rac2 promoter region. Nested 5’- deletions of the promoter region identified a critical regulatory region between -4223 bp and -4008 bp upstream of the transcription start site. Super shift and chromatin immunoprecipitation assays indicated binding by the transcription factor AP1 to three distinct binding sites within the 135 bp minimal regulatory region. PMA stimulation of K562 cells led to extensive changes in chromatin structure, including increased histone H3 acetylation, within the 135 bp Rac2 cis-element. These findings provide evidence for the interplay between epigenetic modifications, transcription factors and cis-acting regulatory elements within the Rac2 gene promoter region to regulate Rac2 expression during K562 cell differentiation.

Horizontal gene transfer (HGT), a mechanism that facilitates exchange of genetic material between organisms from different lineages, has a profound impact on prokaryotic evolution. To infer HGT, we first developed a comparative genomics-based tool, APP, which can perform phyletic pattern analysis using completely sequenced genomes to identify genes are unique to a genome or have sporadic distribution in its close relatives. Performance assessment against currently available tools on a manually created 18-genome dataset and 2 benchmarking datasets revealed the superior accuracy of APP over other methods. We then utilized a parametric method to construct a gene exchange network. The composition-based method, Jenson-Shannon Codon Bias (JS-CB), groups genes into clusters based on similar codon usage bias. These clusters were analyzed using APP and examined for the enrichment HGT associated marker genes, then annotated as of native or alien origin based on these multiple lines of evidence. Intergenome clustering enabled identification of genes mobilized across alien components of the genomes (alien-alien transfer) and from native components of donor genomes to the recipient genomes (native-alien transfer). Functional classification of alien gene clusters revealed that metabolism associated genes are most frequently mobilized, in concurrence with previous reports, and additionally, a large number of genes with yet unknown functions were found to have been horizontally transferred, a important finding that needs to be further investigated.

The Genome Reverse Compiler (GRC) is an annotation tool for prokaryotic genomes. Its name and philosophy are based on analogy with a high-level programming language compiler. In this analogy, the genome is a program in a certain low-level language that humans cannot understand. Given the sequence of any prokaryotic genome, GRC produces its corresponding "high-level program"--its annotation. GRC works in a completely automatic manner, using standard input and output formats. The goal is to provide an open-source, easy-to-run, very efficient annotation program. / Master of Science

No description available.

Resistance to aminoglycosides in the organisms that produce them is often ascribed to the well characterised and clinically important antibiotic modifying enzymes. However, at least three aminoglycoside producing actinomycetes, namely Micromonospora purpurea, Streptomyces tenjimariensis, and Streptomyces tenebrarius possess ribosomes that are refractory to some members of this class of drugs. In these cases, resistance is due to methylation of rRNA of the small ribosomal subunit. This study supports the possibility that this mechanism might be more widespread than hitherto suspected. Two of the methylase genes have been analysed at the nucleotide level and their transcripts mapped. The gentamicin resistance methylase gene (kgmA) from M. purpurea codes for a 36 kDa protein consisting of 249 amino acids. Like most actinomycete genes, kgmA is not expressed in E. coli from its own promoter, although the determinant was expressed in this Gram-negative host as a result of DNA rearrangement. Sequence analysis of the mutated plasmid suggested that the methylase was expressed as a translational fusion with the lacZ' gene of pUC18, a view that was later confirmed. Transcript mapping revealed that kgmA is probably read from a single promoter but that it might be part of a polycistron. The second gene examined confers resistance to kanamycin and apramycin, and originated in S. tenjimariensis. This determinant (kamA) was shown to encode a predicted protein of 155 amino acids with a molecular weight of 19 kDa. Unlike kgmA, this gene could not be expressed as either a transcriptional or translational fusion in E. coli. Transcription of kamA is directed by tandem promoters and is a monocistron since the the transcript terminates only 160 bp downstream of the stop codon.

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No description available.