RESTRICTION TOPOGRAPHY OF THE BACILLUS GENOME.

Title

Identifier

AAI8203333

identifier

8203333

Creator

TACKNEY, CHARLES THOMAS.

Contributor

Rivka Rudner

Date

1981

Language

English

Publisher

City University of New York.

Subject

Biology, General

Abstract

The organization of the Bacillus subtilis genome was studied with respect to proximity of various loci to restriction nuclease palindrome recognition sites. By employing a transformation analysis with mutant recipient competent cells, the relative transforming activity (RTA) of prototrophic donor loci were determined with respect to efficiency, relative susceptibility, linkage relationships and strain variation and evolution. The RTA values of EcoRI or HindIII digested donor DNA fell into distinct class ranks. B. subtilis prototrophic strain W23 loci for trpC2, metB10, thr-5 represent high, medium and low RTA categories respectively following EcoRI cleavage. Loci for thr-5 and trpC2 give high and low RTA capacities in mutant rescue following Hind III digestion. Strain 3610 (ATCC) gave anomalously high EcoRI generated trpC2 survival (30% RTA) as compared to the same locus in W23 (7% RTA). In addition, a HindIII sensitive site is inferred in close proximity to trpC2 of the W23 aromatic operon and is absent in this region of 3610 DNA. The entire trp aromatic cluster is intact following EcoRI cleavage of either W23 or 3610 DNA. Biological activity profiles of 168 donor trp information was similar to that of W23. Hind III digestion cleaves the W23 trp region such that the linkages trpE-hisB and trpC-hisB are interrupted while similar digestion of 3610 DNA leaves the entire trpD-A span intact. The linkage argA-leu-1 is interrupted by EcoRI cleavage in both 3610 and W23 with additional palindrome localization upstream and its proximity to arg and downstream distal to leu-1 sequences. Ligation generated repair (RTA L/R) was locus characteristic and distinctly inverted with low RTA members recovering biological activity to a much greater extent than high RTA representatives. In no case, however, could the trp-his linkage loss following Hind III digestion be improved. The relative susceptibilities of several loci to cleavage by EcoRI or Hind III were studied and a schematic organization relative to recognition palindromes presented.;A 2.4 x 10('6) M.W EcoRI fragment of B. pumilus trp('+) genomic DNA was cloned in vector pUB110 and employed as a specific trp homologous probe. The chimera encompassed trp operon cistrons E, D, C, F and was used, following nick translation and Southern blot hybridization analysis, to identify and map the trp operon region of B. pumilus chromosomal DNA as well as that of B. subtilis W23, 3610, 168 derivatives. A 5.7 and 9.8 Md. trp homologous Eco RI fragment was identified in B. subtilis W23 and 3610 respectively. Strain 168 derivatives had EcoRI fragment sizes resembling that of 3610. Hind III generated similar size fragments in both W23 and 3610 (3.0 Md). The W23 trp operon is interrupted by Hind III to generate a specific fragment of 3.0 while Eco/Hind double digestion gives 2.0 and 3.4 Md. fragments. Strain 3610 gives Hind III generated fragment sizes similar to W23. The RTA data and hybridization analysis allow the generation of organization schematics for the B. subtilis W23, 3610 and 168i('-) genomes as well as that of B. pumilus. These data indicate that the 168 (i('-)) derivatives commonly used as recipients in transformation analysis arose from a 3610 (NTCC; ATCC 6051) progenitor. Furthermore, it appears that the trp prototrophic sequences that reside in proficient (trp('+)) 168i('+) lines originated from strain W23 material. Subsequent mutation and drift of other loci have given rise to 168(i('+)) cells with strain variation of a microsequence nature. The history of the original Marburg organisms has been somewhat obscure and with the aid of biological and sequence specific probes, the "paragenotic" nature of W23 trp('+) sequences residing in a laterally evolving 3610 background has been traced. In addition, the relative structure and organization of several loci relative to restriction palindromes, as well as the feasibility of availability for cloning, has been demonstrated.

Type

dissertation

Source

PQT Legacy CUNY.xlsx

degree

Ph.D.

Program

Biology

Item