1887

Abstract

chromosomal DNA cloned into the vector plasmid pBR322 in is able to complement the metabolic defect of an aspartate-semialdehyde dehydrogenase (EC 1.2.1.11) gene () deletion in the host strain. We constructed two Asd recombinant plasmids, pYA570 and pYA571, containing 4·7 and 4·5 kilobases, respectively, of chromosomal DNA inserted into the dIII restriction endonuclease site of pBR322 in the same orientation. The UAB62 Asd DNA did not hybridize with DNA which contained an intact gene, but did hybridize with UAB62 chromosomal DNA. Derivative Asd plasmids were then constructed from pYA570. One, pYA574, had a 4·5 kilobase insert DNA in the opposite direction from pYA570. In another, pYA575, the insert DNA was reduced in size to 1·3 kilobases. It was seen that the orientation of the DNA fragment inserted into the promoter region of the pBR322 tetracycline resistance (Tc) gene affected expression of Tc. Orientation of the insert also affected the stability of the plasmid in certain strains. Restriction maps for pYA570, pYA571, pYA574 and pYA575 using the endonucleases RI, HI, dIII, I and I were determined. Asd plasmid-directed protein synthesis was studied in minicells. The plasmids pYA570, pYA574 and pYA575 each produced large amounts of a protein, with a monomeric molecular weight of about 45000, that was distinct from both pBR322 and specified protein; this protein is the gene product. Smaller derivatives of recombinant plasmid pYA575 that were Asd allowed the location of the gene promoter and the direction of transcription to be determined.

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1982-05-01
2021-10-21
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