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Titolo:
A SUICIDE VECTOR FOR ALLELIC RECOMBINATION INVOLVING THE GENE FOR GLUTAMATE 1-SEMIALDEHYDE AMINOTRANSFERASE IN THE CYANOBACTERIUM SYNECHOCOCCUS PCC-7942
Autore:
ALLISON G; GOUGH K; ROGERS L; SMITH A;
Indirizzi:
UNIV WALES ABERYSTWYTH,INST BIOL SCI,PLANT ALGAL RES GRP,GEORGE STAPLEDON BLDG CEREDIGION SY23 3DD WALES UNIV WALES ABERYSTWYTH,INST BIOL SCI,PLANT ALGAL RES GRP CEREDIGION SY23 3DD WALES
Titolo Testata:
MGG. Molecular & general genetics
fascicolo: 4, volume: 255, anno: 1997,
pagine: 392 - 399
SICI:
0026-8925(1997)255:4<392:ASVFAR>2.0.ZU;2-8
Fonte:
ISI
Lingua:
ENG
Soggetto:
DELTA-AMINOLEVULINIC-ACID; GREENING BARLEY LEAVES; ESCHERICHIA-COLI; SEMIALDEHYDE AMINOTRANSFERASE; TETRAPYRROLE BIOSYNTHESIS; CHLAMYDOMONAS-REINHARDTII; ANACYSTIS-NIDULANS; BACILLUS-SUBTILIS; TRANSFER RNA; GABACULINE;
Keywords:
DELTA-AMINOLEVULINIC ACID; GLUTAMATE 1-SEMI-ALDEHYDE AMINOTRANSFERASE; SUICIDE VECTOR; GABACULINE RESISTANCE; ALLELIC RECOMBINATION;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Science Citation Index Expanded
Citazioni:
33
Recensione:
Indirizzi per estratti:
Citazione:
G. Allison et al., "A SUICIDE VECTOR FOR ALLELIC RECOMBINATION INVOLVING THE GENE FOR GLUTAMATE 1-SEMIALDEHYDE AMINOTRANSFERASE IN THE CYANOBACTERIUM SYNECHOCOCCUS PCC-7942", MGG. Molecular & general genetics, 255(4), 1997, pp. 392-399

Abstract

Gabaculine (2,3-dihydro 3-amino benzoic acid) is a potent inhibitor of tetrapyrrole biosynthesis in organisms that use the C-5 pathway for the synthesis of 6-aminolaevulinic acid. Glutamate semialdehyde aminotransferase (GSA-AT), the enzyme catalysing the formation of this key precursor of tetrapyrroles, is normally inhibited by concentrations of gabaculine in the order of 5 mu M However, in Synechococcus 6301 strain GR6, a cyanobacterium that is resistant to 100 mu M gabaculine, thisenzyme has undergone two changes in structure: a deletion of three amino acids from positions 5 to 7 and the substitution of isoleucine formethionine at position 248. To establish the effect in vivo of these specific changes in the gene for GSA-AT (hemL), a suicide vector (pHS7) containing an antibiotic cassette was constructed to achieve the replacement, by homologous recombination, of the wild-type hemL gene in the chromosome by a modified form of the gene. Recombinant strains of Synechococcus 7942 obtained using pHS7-hemL(GR6) were indistinguishablefrom Synechococcus 6301 GR6 in terms of the resistance of growth and of chlorophyll accumulation to high concentrations of gabaculine, while a wild-type recombinant produced using pHS7-hemL(WT) had retained its sensitivity. Southern hybridisation using gene probes for hemL, amp(r) and cm(r) confirmed that chromosomal integration of the plasmids had occurred in both WT and GR6 recombinants. Growth and chlorophyll accumulation in equivalent strains with the hemL gene containing either the deletion or the transition characteristic of Synechococcus 6301 GR6were inhibited by 10 mu M gabaculine. Consequently, resistance in vivo to high concentrations of this compound is dependent on both the changes in gene/enzyme structure. This investigation has established the effectiveness of the suicide vector pHS7 for studying the effect in vivo of specific changes in the hemL gene. It has also demonstrated thatreplacement of the wild-type gene by that from Synechococcus 6301 GR6is sufficient to confer resistance in vivo to high concentrations of gabaculine.

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Documento generato il 19/09/20 alle ore 12:56:23