Thesis (M.S. in biochemistry)--Washington State University, December 2009. / Title from PDF title page (viewed on Jan. 4, 2010). "School of Molecular Biosciences." Includes bibliographical references (p. 18-20).

Thesis (Ph. D.)--University of Chicago, Department of Statistics, August 2003. / Includes bibliographical references. Also available on the Internet.

abstract / Biochemistry / Doctoral / Doctor of Philosophy

Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 5 uppsatser.

Thesis (Ph. D.) -- California Institute of Technology, 2010. / Title from home page (viewed 04/05/10). Advisor and committee chair names found in the thesis' metadata record in the digital repository. Includes bibliographical references.

Thesis (Ph.D.) - University of Queensland, 2003. / Includes bibliography.

No description available.

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