The 8.6 kb kalilo linear mitochondrial plasmid of Neurospora intermedia was found to give rise to multiple transcripts of 8.6, 4.4, 4.0, 1.3, 1.2, and 0.9 kb. A transcription map has been generated which shows similarities to other linear plasmids. These transcripts are all transcribed from a single, unique promoter sequence reiterated near the ends of the terminal inverted repeats of the linear plasmid. The transcripts are not processed, but instead utilize optional transcription stop sites. An analysis of sub-cellular RNA fractions has confirmed the mitochondrial location of kalilo transcription. The strong association of kalilo-specific RNA with rRNA to yield RNA artifacts is reported. Kalilo-specific RNA appears to be selectively unstable in affected strains of N. intermedia; this may be a general consequence of linear plasmid RNA. The 5' RNA start site was determined by primer extension and RNA sequencing. The sequence in this region does not show homology to any known mitochondrial, plasmid, nor nuclear promoter, and may constitute a novel element. The transcription start site shares homology with the terminus of the linear plasmid, and marks the end of a long series of direct repeats; therefore, the plasmid RNA polymerase may be bifunctional, it may recognize sequences at the ends of the plasmid as well as at the promoter. The analysis of the insertional behaviour of the linear mitochondrial plasmid was studied in parallel subculture series of the organism. It was determined that insertion, per se is not the event required to kill the organism. Generation of inserts of kalilo in the mtDNA is necessary, but not sufficient, for death to occur in all cases. An analysis of insertion sites has found one new site and good agreement with previously published locations. Insertion does not always appear to be random, so cultures may inherit undetectable amounts of mtlS-kalDNA. The analysis of insertion sites in one strain has suggested a novel possible structure for the mtDNA. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

The purpose of this study was to characterize recombinant plasraids carrying Drosophila melanogaster tRNA genes. The two groups of recombinant plasmids studied were those which carried tRNA₄Val genes and those with tRNA₄,₇Ser genes. pDt92 and pDt120, both tRNA₄Val gene-carrying plasmids, were characterized initially to determine the number of inserts they contained and the size of the inserts. For plasmids containing multiple inserts, the insert which carried the tRNA₄Val gene was also determined. These characteristics were studied by HindIII digestion of the plasmid DNA, agarose gel electrophoresis, Southern transfer onto nitrocellulose filters and hybridization to [¹²⁵I] tRNA₄Val. It was found that both, pDt92 and pDt120 contained two inserts each of sizes 0.5kb and 1.7kb,and 2.0kb and 5.*fkb respectively, with the 0.5kb and 2.0kb fragments carrying the tRNA₄Val genes. pDt92 and pDt120 then were recloned so as to contain only the fragments which carried the tRNA₄Val genes, namely the 0.5kb and 2.0kb fragment respectively. pDt92RC and pDt120RC plus three other tRNA₄,₇Ser gene containing plasmids, pDt16, pDt17RC and pDt27RC were further characterized by the technique of in situ hybridization to study the organization of these tRNA genes on the Drosophila genome. Four of these plasmids with the exception of pDt17RC hybridized to only one site on the Drosophila chromosome. Both, pDt92RC and pDt120RC hybridized to the 90BC site on the right arm of the third chromosome; pDt16 and pDt27RC hybridized to the 12DE site on the first or the X chromosome. pDt17RC on the other hand hybridized predominantly to the 12DE site and to a lesser extent to 2}E (2L), 56D (2R), 62D (3L) and 64D (3L) sites. These in situ hybridization results when studied together with those reported by Dunn et al. (1979b) show that genes for a single species of tRNA are located on more than one site on the Drosophila genome. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

No description available.

Nineteen laboratory strains of Azotobacter vinelandii and two organisms of the same species isolated from water samples were screened for the presence of plasmid deoxyribonucleic acid (DNA). Three laboratory strains and both organisms isolated from water samples contained one plasmid each. The migration distances of the plasmids in agarose gel electrophoresis were different molecular weights. The plasmids were cured by SDS or ethidium bromide treatment of the cultures.

No description available.

Purified DNA from a nalidixic acid resistant derivative of a virulent serotype 0:3 clinical isolate of Yersinia enterocolitica was subjected to transpositional mutagenesis in an effort to construct avirulent mutants. The resulting transpositional mutants, as well as the wild-type virulent strain and its isogenic derivative that had been cured of the virulence plasmid, were analyzed for plasmid DNA content. The plasmid DNA content of each strain was further characterized by restriction endonuclease digestion and the transposon insertion sites for the mutants were located. All of the strains were then tested for pathogenicity by the following assays: calcium dependence, colonization of the mouse gastrointestinal tract, HEp-2 cell adherence and invasion, HEp-2 cell monolayer detachment, autoagglutination, serum resistance, outer membrane protein production and production of V antigen. In addition, the hydrophobic properties of each strain were examined by a rapid polystyrene plate method and hydrophobic interaction chromatography. The results of the tests were compared to plasmid DNA analyses for each strain in an attempt to identify any plasmid-associated genes that are related to virulence. The wild-type strain was virulent, or positive, by all of the assays employed for evaluation of pathogenicity. In contrast, its isogenic derivative that had been cured of the virulence plasmid was negative, or avirulent, for the same assays with one exception. The avirulent plasmidless strain still retained the ability to adhere to and invade HEp-2 cells, supporting the belief that these properties are probably encoded by the bacterial chromosome. In addition, three transpositional mutants were constructed that were no longer calcium dependent, capable of detaching HEp-2 cell monolayers or able to produce three unique outer membrane proteins. Restriction endonuclease analysis confirmed the presence of the transposon on the Hind III "A" fragment of the virulence plasmid and located the region responsible for the lost virulence properties. The gene or set of genes identified were designated cal and the respective calcium independent mutants Cal⁻. Furthermore, the assays for hydrophobicity indicated that the virulence plasmid, specifically the cal gene(s), codes for hydrophobic properties on the surface of the bacterium. The study demonstrated that a virulence-associated region, cal, is located on the virulence plasmid of Y. entrocolitica and responsible for calcium dependence, HEp-2 cell monolayer detachment, the production of the three plasmid-specified outer membrane proteins and cell-surface hydrophobicity.

The nucleotide sequence for the pDKl TOL plasmid region encoding toluate-1,2-dioxygenase (Xy1XYZ, TO) was determined. TO is the first enzyme in the meta-cleavage operon, responsible for the conversion of toluates and benzoates to their carboxy-substituted diols. DNA sequence analysis revealed the presence of three open reading frames (ORF). The three ORFs correspond to xylX (1353 bp), xylY (486 bp) and xylZ (1008 bp), encoding predicted protein products of 51370 Da, 19368 Da and 36256 Da, respectively.

TOL plasmids encode enzymes responsible for utilization of toluene and related aromatic compounds by Pseudomonas putida, ultimately converting them to central metabolic intermediates. The nucleotide sequence for the 5.6 kb xylGFJQK region of the pDK1 TOL meta operon was determined. DNA sequence analysis revealed the presence of five open reading frames corresponding to xylG (1458 bp), xylF (846 bp), xylJ (783 bp), xylQ (936 bp) and xylK (1047 bp), encoding predicted protein products of 51.6, 31.3, 27.8, 32.8, and 36.6 kDa in size, respectively. The average G+C content of the xylLTEGFJQK region was 65.7%, somewhat higher than the 58.9% seen in the immediately upstream xylXYZ region and substantially more than the 50% G+C content reported for the upper TOL operon of this plasmid. Homology comparisons were made with genes and proteins of related catabolic plasmids. The dmpCDEFG and pWWO xylGFJQK regions exhibit consistently high levels of nucleotide and amino acid homology to pDK1 xylGFJQK throughout the entire region. In contrast, although the nucleotide sequence homology of the Acinetobacter atdCDE region to xylGFJ is high, the homology of atdFG to xylQK is markedly less. Such radical changes in homology between corresponding regions of different operons, combined with variable base and codon usage patterns within and between operons, provides additional support for the idea that the upper and lower operons encoding enzymes of aromatic pathways have evolved independently of one another and that these operons have continued to exchange genetic material with homologous expression units through a series of recombination events. Recombinant plasmids were constructed for individual expression of each of the xylGFJQK genes. HMSD (XylG) and HMSH (XylF) were partially purified and characterized with respect to substrate specificity and kinetic mechanism. Evidence was obtained suggesting that the HMSD reaction occurs via a steady state ordered mechanism or a random mechanism where binding of the first substrate effects the enzyme's affinity for the second substrate.

Three overlapping DNA fragments from the lower operon of Pseudomonas putida TOL plasmid pDK1, covering the xy1IH genes and downstream flanking region, were cloned into pUC19. They include a 2.8 kbp XhoI fragment, a 2.7 kbp PstI fragment and a 2.0 kbp EcoRI-HindIII fragment. They were subjected to DNA sequence analysis. The xy1I (4-oxalocrotonate decarboxylase) and xy1H (4-oxalocrotonate tautomerase) genes were found to possess coding regions of 792 and 189 nucleotides, respectively. A possible transcriptional terminator resembling E. coli rho-independent terminators was identified downstream of the translational stop of xy1H. An additional stem and loop structure was found in the intergenic region between xy1I and xy1H. The individual ORF's of the oxalocrotonate branch (xy1G, xy1I and xy1H) have been cloned into pUC18/19. The expression of the xy1G gene in E. coli was successfully assayed spectrophotometrically.

No description available.