scholarly journals The phasmid as a tool for plasmid genetics: II. Isolation of point mutations that affect replication of a ColE1-related plasmid

1982 ◽  
Vol 40 (3) ◽  
pp. 233-247 ◽  
Author(s):  
Gianni Cesareni ◽  
Luisa Castagnoli ◽  
Sydney Brenner
Keyword(s):  
Copy Number ◽  
Point Mutations ◽  
Plasmid Replication ◽  
Single Base ◽  
Lambdoid Phage ◽  
Copy Number Plasmid ◽  
High Copy Number Plasmid ◽  
Single Base Pair ◽  
Related Plasmid ◽  
Single Base Pair Change

SUMMARYThe insertion of a high-copy-number plasmid into a lambdoid phage chromosome which lacks a functional repressor gene confers on the hybrid ‘phasmid’ the capacity to grow on an immune lysogen. This was found to be due to titration of repressor because of plasmid replication. We have exploited this property in order to isolate mutants that affect plasmid replication. These mutants have been mapped in a region that was previously characterized as necessary for plasmid replication and incompatibility properties. Some of the mutations could revert at frequencies characteristic of single-base-pair change mutations.

scholarly journals Point mutations implicate repeated sequences as essential elements of the CYC7 negative upstream site in Saccharomyces cerevisiae.

1985 ◽  
Vol 5 (11) ◽  
pp. 2951-2958 ◽  
Author(s):  
C F Wright ◽  
R S Zitomer
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Sequences ◽  
Base Pair ◽  
Point Mutations ◽  
Regulatory Elements ◽  
Upstream Site ◽  
Single Base ◽  
Negative Element ◽  
Single Base Pair ◽  
Single Base Pair Change

The transcription of the CYC7 gene of Saccharomyces cerevisiae, encoding the iso-2-cytochrome c protein, is controlled by two upstream regulatory elements, a positive element and a negative element. The nature of the DNA sequences in the negative element were investigated in a two-part approach. The first involved the construction of a CYC7-galK fusion gene which placed the coding sequence of the Escherichia coli galactokinase gene under the regulation of the CYC7 upstream sequences. This fusion allowed the quantitation by galactokinase enzyme assays of the effects on gene expression of a variety of previously isolated deletion mutations within the negative site. The results suggested that the negative site contained three related sequences. This hypothesis was tested in the second part of these studies, the selection of point mutations within the region of the negative site which led to increased CYC7 expression. Point mutations were introduced by a technique which induced mutations within a localized region at high efficiency. All but one of the mutations involved more than a single base-pair change. The mutations followed the pattern that multiple base-pair changes occurred in one repeat or single base-pair changes occurred in two repeats, with the exception of one mutant, which had a single base-pair change in one repeat. This pattern of mutations and the base pairs that were altered strongly supported the hypothesis that the repeats are integral elements of the negative site.

Point mutations implicate repeated sequences as essential elements of the CYC7 negative upstream site in Saccharomyces cerevisiae

1985 ◽  
Vol 5 (11) ◽  
pp. 2951-2958
Author(s):  
C F Wright ◽  
R S Zitomer
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Sequences ◽  
Base Pair ◽  
Point Mutations ◽  
Regulatory Elements ◽  
Upstream Site ◽  
Single Base ◽  
Negative Element ◽  
Single Base Pair ◽  
Single Base Pair Change

The transcription of the CYC7 gene of Saccharomyces cerevisiae, encoding the iso-2-cytochrome c protein, is controlled by two upstream regulatory elements, a positive element and a negative element. The nature of the DNA sequences in the negative element were investigated in a two-part approach. The first involved the construction of a CYC7-galK fusion gene which placed the coding sequence of the Escherichia coli galactokinase gene under the regulation of the CYC7 upstream sequences. This fusion allowed the quantitation by galactokinase enzyme assays of the effects on gene expression of a variety of previously isolated deletion mutations within the negative site. The results suggested that the negative site contained three related sequences. This hypothesis was tested in the second part of these studies, the selection of point mutations within the region of the negative site which led to increased CYC7 expression. Point mutations were introduced by a technique which induced mutations within a localized region at high efficiency. All but one of the mutations involved more than a single base-pair change. The mutations followed the pattern that multiple base-pair changes occurred in one repeat or single base-pair changes occurred in two repeats, with the exception of one mutant, which had a single base-pair change in one repeat. This pattern of mutations and the base pairs that were altered strongly supported the hypothesis that the repeats are integral elements of the negative site.

scholarly journals Nucleotide sequence of a high copy number plasmid fromHalobacteriumstrain GRB

1990 ◽  
Vol 18 (11) ◽  
pp. 3408-3408 ◽  
Author(s):  
Neil R. Hackett ◽  
Mark P. Krebs ◽  
Shiladitya DasSarma ◽  
Werner Goebel ◽  
Uttam L. RajBhandary ◽  
...  
Keyword(s):  
Nucleotide Sequence ◽  
Copy Number ◽  
High Copy Number ◽  
Copy Number Plasmid ◽  
High Copy Number Plasmid

scholarly journals Contribution of Aggregation-Promoting Factor to Maintenance of Cell Shape in Lactobacillusgasseri 4B2

2003 ◽  
Vol 185 (11) ◽  
pp. 3288-3296 ◽  
Author(s):  
Ivana Jankovic ◽  
Marco Ventura ◽  
Valerie Meylan ◽  
Martine Rouvet ◽  
Marina Elli ◽  
...  
Keyword(s):  
Cell Division ◽  
Copy Number ◽  
Promoter Region ◽  
Cell Shape ◽  
Down Regulation ◽  
Copy Number Plasmid ◽  
Recombinant Cells ◽  
High Copy Number Plasmid ◽  
Induced Changes ◽  
First Time

ABSTRACT Aggregation-promoting factor (APF) was originally described as a protein involved in the conjugation and autoaggregation of Lactobacillus gasseri 4B2, whose corresponding apf gene was cloned and sequenced. In this report, we identified and sequenced an additional apf gene located in the region upstream of the previously published one. Inactivation of both apf genes was unsuccessful, indicating that APF function may be essential for the cell. Overproduction of APF proteins caused drastic alteration in the cell shape of this strain. These cells were irregular, twisted, enlarged, and tightly bound in unbreakable clumps of chains. Down-regulation of APF synthesis was achieved by cloning of the apf2 promoter region on a high-copy-number plasmid, which recruited a putative apf activator. As a consequence, the shape of the corresponding recombinant cells was elongated (filamentous) and cell division sites were no longer visible. None of the induced changes in APF production levels was clearly correlated with modifications of the aggregation phenotype. This report shows, for the first time, that APF proteins are mainly critical for L. gasseri 4B2 cell shape maintenance.

Deletion mutant analysis of the Staphylococcus aureus plasmid pI258 mercury-resistance determinant

1991 ◽  
Vol 37 (8) ◽  
pp. 624-631 ◽  
Author(s):  
Kenneth Babich ◽  
Mike Engle ◽  
Jeffery S. Skinner ◽  
Richard A. Laddaga
Keyword(s):  
Staphylococcus Aureus ◽  
Ion Transport ◽  
Copy Number ◽  
Deletion Mutant ◽  
Resistance Determinant ◽  
Mercury Resistance ◽  
Mutant Analysis ◽  
Mer Operon ◽  
Copy Number Plasmid ◽  
High Copy Number Plasmid

Deletion mutant analysis of the mercury-resistant determinant (mer operon) from the Staphylococcus aureus plasmid pI258 was used to verify the location of the merA and merB genes and to show the existence of mercuric ion transport gene(s). ORF5 was confirmed to be a transport gene and has an amino acid product sequence homologous to the merT gene products from several gram-negative bacteria and a Bacillus species. Deletion analysis established that inactivation of merA on a broad-spectrum mer resistance determinant resulted in a mercury-hypersensitive phenotype. Gene dosage had no apparent effect on the level of resistance conferred by the intact mer operon or on the expression of an inducible phenotype, except that when the intact pI258 mer operon was on a high copy number plasmid, uninduced cells possessed a volatilization rate that was at most only 3.5-fold less than that observed for induced cells. There was no need for mercury ion transport proteins for full resistance when the mer operon was expressed in a high copy number plasmid. Key words: mercury resistance, Staphylococcus aureus plasmid.

Development of Small High-Copy-Number Plasmid Vectors for Gene Expression in Caulobacter crescentus

Plasmid ◽  
2001 ◽  
Vol 46 (1) ◽  
pp. 37-46 ◽  
Author(s):  
Elizabeth Umelo-Njaka ◽  
John F. Nomellini ◽  
Harry Yim ◽  
John Smit
Keyword(s):  
Gene Expression ◽  
Copy Number ◽  
Caulobacter Crescentus ◽  
High Copy Number ◽  
Plasmid Vectors ◽  
Copy Number Plasmid ◽  
High Copy Number Plasmid
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