The polyhomeotic (ph) locus of Drosophila melanogaster has been characterized genetically. Early studies showed that ph is a member of the Polycomb (Pc) group. These genes have similar phenotypes and are required for normal segment determination. Recent analyses of amorphic ph mutations show that the ph locus is complex, has a strong maternal effect and plays a role in cuticular development. To test the function of ph at the molecular level, the cloning of the ph locus was undertaken. One strain had been shown to contain a P element insertion near ph. A genomic library was prepared from this strain and a recombinant phage containing this P element insertion was isolated by transposon tagging. The DNA flanking the insertion was used as a starting point for a chromosomal walk. A series of overlapping phage spanning 170 kilobases was isolated. Southern blot analysis was used to determine the locations of important deficiency breakpoints within the region covered by the walk. A distance of approximately 35 kb was shown to separate the two deficiency breakpoints which include ph. This interval was found to contain rearrangements in four of the seven ph alleles which were examined by Southern blot analysis. The interval also contains a repeated sequence. The relationship between the genetic and molecular structure of ph is discussed. / Science, Faculty of / Zoology, Department of / Graduate

No description available.

In this thesis, we describe the development of a method to delete and to clone specific large regions from the 1700 kilobase pExo megaplasmid of Rhizobium meliloti. In principal, the region to be cloned is flanked by FRT sites, which direct site specific recombination by the Flp recombinase. Targeting constructs were designed to include part of the IS50 from Tn5, the FRT site, an origin of transfer (oriT), and a replication origin from RK2 or the F plasmid. These constructs were directed to known Tn5-derivative insertion sites in the pExo megaplasmid. A plasmid which expresses the Flp recombinase constitutively in R. meliloti was made, and the transfer of this plasmid to FRT-flanked megaplasmid regions was shown to result in the deletion of the intervening DNA. We demonstrated that the pExo megaplasmid DNA regions could be captured in Escherichia coli, however in this case, the megaplasmid excision event appears to be directed by the oriT sites rather than the FRT sites. We present strong evidence that a specific 50 kb region contains the oriV of the pExo megaplasmid; R. meliloti strains deleted for this region could not be isolated, and this region was found to replicate autonomously in Agrobacterium tumefaciens. Preliminary sequence analysis has revealed strong homology within this region to genes encoding the RepABC replication proteins of several Rhizobium and Agrobacterium plasmids. / Thesis / Master of Science (MS)

RNA has many diverse functions in living organisms, from serving as genome for many viruses, to regulating DNA replication, transcription, translation and other metabolic processes. Some RNA, like protein, has been shown to have catalytic activity. The great proportion of the mass of RNA in living cells, in the form of ribosomal RNA (rRNA), transfer RNA (tRNA) and messenger RNA (mRNA), constitutes the machinery of protein synthesis, the remainder (approximately 2%) consists of many heterogeneous RNA species of relatively small size, loosely termed "small RNAs", the functions of many of which are completely unknown. In an attempt to understand some of these functions, three hitherto undescribed small RNAs of the budding yeast Saccharomyces cerevisiae were identified and their genes were cloned. These three small RNAs, which lack polyadenylation at their 3' ends, appear to represent the three most abundant RNA species in this organism after rRNA and tRNA. The most abundant of the three was found to be mainly cytoplasmic and was therefore called "small cytoplasmic RNA 1" (scR1). The other two RNAs, named snR17 and snR30, were found to be enriched in nuclear fractions and to possess trimethyl guanosine cap structures at their 5 ends, identifying them as belonging to the ubiquitous class of "U" small nuclear RNAs (U snRNAs), of which several are required for the endonucleolytic cleavage and splicing reactions in the maturation pathways of nuclear precursor mRNAs (pre-mRNA). Whereas scR1 and snR30 are both encoded by single genes, snR17 is the only yeast small RNA found so far to be encoded by two genes. SnR17 was found to be essential: haploid yeast strains lacking intact copies of one or other of the genes appeared to grow normally, but strains lacking both genes were inviable. The nucleotide sequences of the snR17 genes were determined, and the primary and predicted secondary structures of the RNA, 328 nucleotides in length, were found to show significant similarities to those of U3 snRNA, an abundant U snRNA, the function of which is not known. SnR17 belongs to a family of S. cerevisiae snRNAs which, unlike those involved in pre-mRNA splicing, are located in the nucleolus hydrogen-bonded to pre-rRNA, and are associated with antigenic protein that is recognized by human antibodies specific for a 36 kD polypeptide of the U3 small nuclear ribonucleoprotein (U3 snRNP) in mammals. U3 snRNA is also nucleolar and associated with pre-rRNA. Given their structural similarities, snR17 and U3 snRNA are presumably homologous. Yeast snRNAs associated with the anti-(U3)RNP antigen share with U3 snRNAs a conserved nucleotide sequence element. This sequence element alone, however, when injected into Xenopus oocytes, was not sufficient to direct binding of the antigen. The association of snRNAs with pre-rRNA suggests that they function in ribosomal biogenesis.

No description available.

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

published_or_final_version / Zoology / Doctoral / Doctor of Philosophy

published_or_final_version / Zoology / Master / Master of Philosophy

In order to characterize the protein product of the major capsid protein gene of Bovine Papillomavirus Type 2, 93% of the L1 open reading frame was cloned into two different expression vectors. This coding sequence produced a hybrid product when cloned into the expression vector pKK233-2, which interacted weakly with anti-BPV antisera and proved unable to elicit neutralizing antibodies. When the sequence was cloned into the expression vector pRA10, a more native form of the major capsid protein was produced, which interacted well with anti-BPV sera. Antisera raised against this cloned product was able to neutralize BPV in a tissue culture transformation assay. The 3' end of the L1 open reading frame was cloned into pBA10 in order to characterize the immunogenic potential of the carboxy terminus of the major capsid gene. The carboxy terminus proved to have no greater ability to interact with anti-BPV antisera, showing that the immunogenic epitopes of the protein are probably evenly distributed along the linear sequence.

No description available.