Isolation of temperature-sensitive mutants of 16 S rRNA in Escherichia coli

K Triman, E Becker, C Dammel, J Katz, H Mori, S Douthwaite, C Yapijakis, S Yoast, H F Noller

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

Resumé

Udgivelsesdato: 1989-Oct-20
OriginalsprogEngelsk
TidsskriftJournal of Molecular Biology
Vol/bind209
Udgave nummer4
Sider (fra-til)645-653
Antal sider8
ISSN0022-2836
StatusUdgivet - 20. okt. 1989

Fingeraftryk

Spectinomycin
rRNA Genes
Mutation
Hydroxylamine
Microbial Drug Resistance
DNA Sequence Analysis
Base Pairing
Alleles
Growth

Citer dette

Triman, K., Becker, E., Dammel, C., Katz, J., Mori, H., Douthwaite, S., ... Noller, H. F. (1989). Isolation of temperature-sensitive mutants of 16 S rRNA in Escherichia coli. Journal of Molecular Biology, 209(4), 645-653.
Triman, K ; Becker, E ; Dammel, C ; Katz, J ; Mori, H ; Douthwaite, S ; Yapijakis, C ; Yoast, S ; Noller, H F. / Isolation of temperature-sensitive mutants of 16 S rRNA in Escherichia coli. I: Journal of Molecular Biology. 1989 ; Bind 209, Nr. 4. s. 645-653.
@article{ac56a690dc0511dd9908000ea68e967b,
title = "Isolation of temperature-sensitive mutants of 16 S rRNA in Escherichia coli",
abstract = "Temperature-sensitive mutants have been isolated following hydroxylamine mutagenesis of a plasmid containing Escherichia coli rRNA genes carrying selectable markers for spectinomycin resistance (U1192 in 16 S rRNA) and erythromycin resistance (G2058 in 23 S rRNA). These antibiotic resistance alleles, originally identified by Morgan and co-workers, enable us to follow expression of cloned rRNA genes in vivo. Recessive mutations causing the loss of expression of the cloned 16 S rRNA gene were identified by the loss of the ability of cells to survive on media containing spectinomycin. The mutations were localized by in vitro restriction fragment replacement followed by in vivo marker rescue and were identified by DNA sequence analysis. We report here seven single-base alterations in 16 S rRNA (A146, U153, A350, A359, A538, A1292 and U1293), five of which produce temperature-sensitive spectinomycin resistance and two that produce unconditional loss of resistance. In each case, loss of ribosomal function can be accounted for by disruption of base-pairing in the secondary structure of 16 S rRNA. For the temperature-sensitive mutants, there is a lag period of about two generations between a shift to the restrictive temperature and cessation of growth, implying that the structural defects cause impairment of ribosome assembly.",
keywords = "Ampicillin, Culture Media, Drug Resistance, Erythromycin, Escherichia coli, Mutation, Operon, Penicillin Resistance, Phenotype, RNA, Ribosomal, RNA, Ribosomal, 16S, Spectinomycin, Temperature, Transformation, Bacterial",
author = "K Triman and E Becker and C Dammel and J Katz and H Mori and S Douthwaite and C Yapijakis and S Yoast and Noller, {H F}",
year = "1989",
month = "10",
day = "20",
language = "English",
volume = "209",
pages = "645--653",
journal = "Journal of Molecular Biology",
issn = "0022-2836",
publisher = "Heinemann",
number = "4",

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Triman, K, Becker, E, Dammel, C, Katz, J, Mori, H, Douthwaite, S, Yapijakis, C, Yoast, S & Noller, HF 1989, 'Isolation of temperature-sensitive mutants of 16 S rRNA in Escherichia coli', Journal of Molecular Biology, bind 209, nr. 4, s. 645-653.

Isolation of temperature-sensitive mutants of 16 S rRNA in Escherichia coli. / Triman, K; Becker, E; Dammel, C; Katz, J; Mori, H; Douthwaite, S; Yapijakis, C; Yoast, S; Noller, H F.

I: Journal of Molecular Biology, Bind 209, Nr. 4, 20.10.1989, s. 645-653.

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

TY - JOUR

T1 - Isolation of temperature-sensitive mutants of 16 S rRNA in Escherichia coli

AU - Triman, K

AU - Becker, E

AU - Dammel, C

AU - Katz, J

AU - Mori, H

AU - Douthwaite, S

AU - Yapijakis, C

AU - Yoast, S

AU - Noller, H F

PY - 1989/10/20

Y1 - 1989/10/20

N2 - Temperature-sensitive mutants have been isolated following hydroxylamine mutagenesis of a plasmid containing Escherichia coli rRNA genes carrying selectable markers for spectinomycin resistance (U1192 in 16 S rRNA) and erythromycin resistance (G2058 in 23 S rRNA). These antibiotic resistance alleles, originally identified by Morgan and co-workers, enable us to follow expression of cloned rRNA genes in vivo. Recessive mutations causing the loss of expression of the cloned 16 S rRNA gene were identified by the loss of the ability of cells to survive on media containing spectinomycin. The mutations were localized by in vitro restriction fragment replacement followed by in vivo marker rescue and were identified by DNA sequence analysis. We report here seven single-base alterations in 16 S rRNA (A146, U153, A350, A359, A538, A1292 and U1293), five of which produce temperature-sensitive spectinomycin resistance and two that produce unconditional loss of resistance. In each case, loss of ribosomal function can be accounted for by disruption of base-pairing in the secondary structure of 16 S rRNA. For the temperature-sensitive mutants, there is a lag period of about two generations between a shift to the restrictive temperature and cessation of growth, implying that the structural defects cause impairment of ribosome assembly.

AB - Temperature-sensitive mutants have been isolated following hydroxylamine mutagenesis of a plasmid containing Escherichia coli rRNA genes carrying selectable markers for spectinomycin resistance (U1192 in 16 S rRNA) and erythromycin resistance (G2058 in 23 S rRNA). These antibiotic resistance alleles, originally identified by Morgan and co-workers, enable us to follow expression of cloned rRNA genes in vivo. Recessive mutations causing the loss of expression of the cloned 16 S rRNA gene were identified by the loss of the ability of cells to survive on media containing spectinomycin. The mutations were localized by in vitro restriction fragment replacement followed by in vivo marker rescue and were identified by DNA sequence analysis. We report here seven single-base alterations in 16 S rRNA (A146, U153, A350, A359, A538, A1292 and U1293), five of which produce temperature-sensitive spectinomycin resistance and two that produce unconditional loss of resistance. In each case, loss of ribosomal function can be accounted for by disruption of base-pairing in the secondary structure of 16 S rRNA. For the temperature-sensitive mutants, there is a lag period of about two generations between a shift to the restrictive temperature and cessation of growth, implying that the structural defects cause impairment of ribosome assembly.

KW - Ampicillin

KW - Culture Media

KW - Drug Resistance

KW - Erythromycin

KW - Escherichia coli

KW - Mutation

KW - Operon

KW - Penicillin Resistance

KW - Phenotype

KW - RNA, Ribosomal

KW - RNA, Ribosomal, 16S

KW - Spectinomycin

KW - Temperature

KW - Transformation, Bacterial

M3 - Journal article

VL - 209

SP - 645

EP - 653

JO - Journal of Molecular Biology

JF - Journal of Molecular Biology

SN - 0022-2836

IS - 4

ER -

Triman K, Becker E, Dammel C, Katz J, Mori H, Douthwaite S et al. Isolation of temperature-sensitive mutants of 16 S rRNA in Escherichia coli. Journal of Molecular Biology. 1989 okt 20;209(4):645-653.