MAPPING OF RIBOSOMAL RNA GENE SETS WITH INTEGRABLE PLASMIDS IN BACILLUS SUBTILIS.
Item
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Title
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MAPPING OF RIBOSOMAL RNA GENE SETS WITH INTEGRABLE PLASMIDS IN BACILLUS SUBTILIS.
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Identifier
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AAI8801728
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identifier
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8801728
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Creator
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LAFAUCI, GIUSEPPE.
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Contributor
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Rivka Rudner
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Date
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1987
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Language
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English
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Publisher
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City University of New York.
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Subject
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Biology, Molecular
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Abstract
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Integrable plasmids, containing different cloned rDNA fragments from within the transcriptional unit of the rRNA gene set of Bacillus subtilis, were constructed using the bifunctional vector plasmid pJH101 (Ferrari et al., 1983). All constructs were able to transform B. subtilis strains to chloramphenicol resistance (Cm{dollar}\sp{lcub}\rm r{rcub}{dollar}) at low efficiencies. Plasmids pGR102 and pWR103, both containing cloned 16S, abutment, and 23S sequences, had the highest transforming efficiencies. Southern hybridization of BclI restricted chromosomal DNAs revealed that each Cm{dollar}\sp{lcub}\rm r{rcub}{dollar}-transformant lost a parent-type rDNA band and gained one or more larger band(s), indicating that one or more copies of a plasmid integrated in a given rRNA gene set. Southern hybridization analysis of DNA double restricted with BclI and SalI proved that in all strains the entire plasmid (either a monomer or a multimer) is inserted into a rRNA gene set. Plasmid insertion occurred by homologous recombination between cloned rDNA sequences and a given rRNA gene set (Campbell-like integration). The comparison of the rRNA BclI patterns obtained with DNAs of parental and Cm{dollar}\sp{lcub}\rm r{rcub}{dollar}-transformant strains proved that nine of the ten gene sets present in the chromosome of the bacterium were involved in plasmid integration. PBSl transduction crosses with the nine mapping kit strains of Dedonder et al., 1977, revealed that the plasmids can integrate in five different regions of the chromosome: (1) purA-cysA; (2) cysA-aroI; (3) dal-1-purB33; (4) tre-12-glyB133; (5) aroG-thr-5. Additional transduction crosses allowed the mapping of three unassigned rRNA gene sets: rrnK (4.8Kb BclI rrn-homolog) between cysA and amyE; rrnE (6.6Kb BclI rrn-homolog) between pha-1 and furB; and rrnD (5.4Kb BclI rrn-homolog) between glyB133 and tre-12. Nine mapped rRNA gene sets were assigned to individual BclI rrn-homologs. The physical and genetic data indicate that four gene sets (rrnH, rrnI, rrnG, and rrnK) are located in the region of the chromosome between cysA and aroI. These genes form two distinct clusters each containing two rRNA gene sets: rrnH-rrnI (4.9 and 5.8Kb BclI rRNA homologs, respectively), and rrnG-rrnK (5.5 and 4.8Kb BclI rRNA homologs, respectively). Laboratory strains of B. subtilis having only nine rRNA gene sets are described. The deleted gene sets are either rrnK (BD170, trpC2, thr-5) or rrnI (CU420, trpC2 leuB6, ilvC4). In six out of twenty-seven transformants, plasmid integration into a clustered rRNA gene set was associated with the deletion of the adjacent gene set. It is postulated that deletions could result from two simultaneous crossover events between a multimer plasmid and two clustered rRNA gene sets (rrnH-rrnI or rrnG-rrnK). Models showing the possible mechanism are presented.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
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Program
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Biology
