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.

No description available.

No description available.

The aim of this project was to clone the gene encoding the catalytic subunit of the rat DNA replication enzyme, DNA polymerase alpha. A strategy was adopted in which cDNA clones expressing the catalytic subunit sequences would be identified using anti-DNA polymerase antibodies. DNA polymerase alpha was partially purified from regenerating rat liver and exponentially growing rat Y3 myeloma cells. The catalytic subunit was identified as a 170-180kD polypeptide by activity gel analysis of partially purified Y3 cell fractions. The catalytic subunit was found to be susceptible to degradation but without loss of polymerase activity. Glycerol gradient analysis indicated a two stage degradation of DNA polymerase in vivo. Sera were collected from mice immunised with partially purified DNA polymerase alpha from regenerated rat liver. These sera cross-reacted with Western-blotted Y3 cell fractions; removed polymerase activity from solution in plate binding assays and bound alpha polymerase activity (140-180kD) on an immuno-adsorption column cDNA was synthesised using size selected mRNA from exponentially growing Y3 cells and cloned into the expression vectors pUC8 and ?gtll, both of which utilise the lac Z gene to express cloned DNA sequences. Immunoscreening of the ?gtll library was frustrated by non-specific binding of the serum. This non-specific binding was overcome by pre-adsorbing the serum against a lysate of E. coli JM 83. Screening of the pUC8 library revealled 27 out of 2.25x104 colonies which bound pre-adsorbed anti-DNA polymerase alpha serum.

No description available.

The ability of Escherichia coli to use the expression signals of mycobacterial genes was tested by inserting fragments of M. bovis BCG DNA into the E. coli promoter-probe plasmid pKK232-8. Comparison with the promoter activity achieved following insertion of restriction fragments of the E. coli host into. pKK232-8 revealed that a significant proportion of M. bovis BCG promoters were functional in E. coli. These results confirmed the suitability of E. coli as a host for the cloning and expression of mycobacterial genes. Using a variety of E. coli cloning vectors (pBR322, pUC13, EMBL4 and gtll), M. bovis BCG and M. leprae DM gene libraries were prepared. Recombinant M. bovis BCG clones were screened with rabbit antiserum and clones expressing M. bovis BCG antigens were identified. A pBR322/M. bovis BCG clone, expressing a 65KD molecule, was isolated and this antigen was shown to be cross-reactive with a 65KD M. leprae antigen. Recombinant gtll clones, expressing antigenic M. bovis BCG molecules, were also detected and a partial DM sequence was determined for one of these molecules. Moreover, recombinant gtll clones expressing (i) an 85KD biotinylated M. leprae molecule and (ii) an 85KD biotinylated M. bovis BCG molecule were also detected. In an attempt to test the feasibility of diagnosing leprosy by the presence of antibodies to specific antigens, antisera samples from leprosy patients and their contacts were screened for antibodies to mycobacterial antigens. Although only a small number of antisera were tested, a number of candidate antigens were identified.

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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.

No description available.