Yeast telomere repeat sequence (TRS) improves circular plasmid segregation, and TRS plasmid segregation involves the RAP1 gene product.

Telomere repeat sequences (TRSs) can dramatically improve the segregation of unstable circular autonomously replicating sequence (ARS) plasmids in Saccharomyces cerevisiae. Deletion analysis demonstrated that yeast TRSs, which conform to the general sequence (C(1-3)A)n, are able to stabilize circular ARS plasmids. A number of TRS clones of different primary sequence and C(1-3)A tract length confer the plasmid stabilization phenotype. TRS sequences do not appear to improve plasmid replication efficiency, as determined by plasmid copy number analysis and functional assays for ARS activity. Pedigree analysis confirms that TRS-containing plasmids are missegregated at low frequency and that missegregated TRS-containing plasmids, like ARS plasmids, are preferentially retained by the mother cell. Plasmids stabilized by TRSs have properties that distinguish them from centromere-containing plasmids and 2 microns-based recombinant plasmids. Linear ARS plasmids, which include two TRS tracts at their termini, segregate inefficiently, while circular plasmids with one or two TRS tracts segregate efficiently, suggesting that plasmid topology or TRS accessibility interferes with TRS segregation function on linear plasmids. In strains carrying the temperature-sensitive mutant alleles rap1grc4 and rap1-5, TRS plasmids are not stable at the semipermissive temperature, suggesting that RAP1 protein is involved in TRS plasmid stability. In Schizosaccharomyces pombe, an ARS plasmid was stabilized by the addition of S. pombe telomere sequence, suggesting that the ability to improve the segregation of ARS plasmids is a general property of telomere repeats.

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Plasmid Copy Number and Plasmid Stability

New Trends and Developments in Biochemical Engineering - Advances in Biochemical Engineering/Biotechnology ◽

10.1007/b12440 ◽

2003 ◽

pp. 47-82 ◽

Cited By ~ 47

Author(s):

Karl Friehs

Keyword(s):

Copy Number ◽

Plasmid Stability ◽

Plasmid Copy Number ◽

Plasmid Copy

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Enhancement of telomere-plasmid segregation by the X-telomere associated sequence in Saccharomyces cerevisiae involves SIR2, SIR3, SIR4 and ABF1.

Genetics ◽

10.1093/genetics/136.3.757 ◽

1994 ◽

Vol 136 (3) ◽

pp. 757-767 ◽

Cited By ~ 2

Author(s):

S Enomoto ◽

M S Longtine ◽

J Berman

Keyword(s):

Binding Site ◽

Consensus Sequence ◽

Active Role ◽

Repeat Sequence ◽

Dependent Manner ◽

Enhancer Element ◽

Telomere Repeat ◽

Plasmid Segregation ◽

Telomere Function ◽

Associated Sequences

Abstract We have previously shown that circular replicating plasmids that carry yeast telomere repeat sequence (TG1-3) tracts segregate efficiently relative to analogous plasmids lacking the TG1-3 tract and this efficient segregation is dependent upon RAP1. While a long TG1-3 tract is sufficient to improve plasmid segregation, the segregation efficiency of telomere plasmids (TEL-plasmids) is enhanced when the X-Telomere Associated Sequence (X-TAS) is also included on the plasmids. We now demonstrate that the enhancement of TEL-plasmid segregation by the X-TAS depends on SIR2, SIR3, SIR4 and ABF1 in trans and requires the Abf1p-binding site within the X-TAS. Mutation of the Abf1p-binding site within the X-TAS results in TEL-plasmids that are no longer affected by mutations in SIR2, SIR3 or SIR4, despite the fact that other Abf1p-binding sites are present on the plasmid. Mutation of the ARS consensus sequence within the X-TAS converts the X-TAS from an enhancer element to a negative element that interferes with TEL-plasmid segregation in a SIR-dependent manner. Thus, telomere associated sequences interact with TG1-3 tracts on the plasmid, suggesting that the TASs have an active role in modulating telomere function.

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Cloning and functional characterization of Komagataella phaffii centromeres by a color-based plasmid stability assay

10.1101/433417 ◽

2018 ◽

Author(s):

Luiza Cesca Piva ◽

Janice Lisboa De Marco ◽

Lidia Maria Pepe de Moraes ◽

Viviane Castelo Branco Reis ◽

Fernando Araripe Gonçalves Torres

Keyword(s):

Copy Number ◽

Plasmid Stability ◽

Functional Characterization ◽

Plasmid Copy Number ◽

Plasmid Copy ◽

Komagataella Phaffii ◽

High Transformation ◽

Colony Color ◽

Stable Vectors

AbstractThe yeast Komagataella phaffii is widely used as a microbial host for heterologous protein production. However, molecular tools for this yeast are basically restricted to a few integrative and replicative plasmids. Four sequences that have recently been proposed as the K. phaffii centromeres could be used to develop a new class of mitotically stable vectors. In this work we designed a color-based genetic assay to investigate genetic stability in K. phaffii. Plasmids bearing K. phaffii centromeres and the ADE3 marker were evaluated in terms of mitotic stability in an ade2/ade3 auxotrophic strain which allows plasmid screening through colony color. Plasmid copy number was verified through qPCR. Our results confirmed that the centromeric plasmids were maintained at low copy number as a result of typical chromosome-like segregation during cell division. These features, combined with high transformation efficiency and in vivo assembly possibilities, prompt these plasmids as a new addition to the K. phaffii genetic toolbox.

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Effect of environmental growth conditions on plasmid stability, plasmid copy number, and catechol 2,3-dioxygenase activity in free and immobilizedEscherichia coli cells

Biotechnology and Bioengineering ◽

10.1002/bit.260330702 ◽

1989 ◽

Vol 33 (7) ◽

pp. 801-808 ◽

Cited By ~ 43

Author(s):

S. Sayadi ◽

M. Nasri ◽

J. N. Barbotin ◽

D. Thomas

Keyword(s):

Copy Number ◽

Plasmid Stability ◽

Growth Conditions ◽

Plasmid Copy Number ◽

Plasmid Copy ◽

Dioxygenase Activity

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Separate Roles of Escherichia coliReplication Proteins in Synthesis and Partitioning of pSC101 Plasmid DNA

Journal of Bacteriology ◽

10.1128/jb.181.24.7552-7557.1999 ◽

1999 ◽

Vol 181 (24) ◽

pp. 7552-7557 ◽

Cited By ~ 13

Author(s):

Christine Miller ◽

Stanley N. Cohen

Keyword(s):

Dna Replication ◽

Plasmid Dna ◽

Plasmid Copy Number ◽

Temperature Sensitive ◽

Replication Proteins ◽

Plasmid Copy ◽

Plasmid Partitioning ◽

Bacterial Replication ◽

Cell Membrane Binding ◽

Plasmid Replicons

ABSTRACT We report here that the Escherichia coli replication proteins DnaA, which is required to initiate replication of both the chromosome and plasmid pSC101, and DnaB, the helicase that unwinds strands during DNA replication, have effects on plasmid partitioning that are distinct from their functions in promoting plasmid DNA replication. Temperature-sensitive dnaB mutants cultured under conditions permissive for DNA replication failed to partition plasmids normally, and when cultured under conditions that prevent replication, they showed loss of the entire multicopy pool of plasmid replicons from half of the bacterial population during a single cell division. As was observed previously for DnaA, overexpression of the wild-type DnaB protein conversely stabilized the inheritance of partition-defective plasmids while not increasing plasmid copy number. The identification of dnaA mutations that selectively affected either replication or partitioning further demonstrated the separate roles of DnaA in these functions. The partition-related actions of DnaA were localized to a domain (the cell membrane binding domain) that is physically separate from the DnaA domain that interacts with other host replication proteins. Our results identify bacterial replication proteins that participate in partitioning of the pSC101 plasmid and provide evidence that these proteins mediate plasmid partitioning independently of their role in DNA synthesis.

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Genetic control of ColE1 plasmid stability that is independent of plasmid copy number regulation

Current Genetics ◽

10.1007/s00294-018-0858-0 ◽

2018 ◽

Vol 65 (1) ◽

pp. 179-192 ◽

Cited By ~ 2

Author(s):

Melissa S. Standley ◽

Samuel Million-Weaver ◽

David L. Alexander ◽

Shuai Hu ◽

Manel Camps

Keyword(s):

Genetic Control ◽

Copy Number ◽

Plasmid Stability ◽

Plasmid Copy Number ◽

Plasmid Copy ◽

Cole1 Plasmid ◽

Copy Number Regulation

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Influence of different origins of replication on transformation efficiency, plasmid copy number and plasmid stability in different industrial lactic acid bacterial strains

New Biotechnology ◽

10.1016/j.nbt.2009.06.289 ◽

2009 ◽

Vol 25 ◽

pp. S61

Author(s):

K. Spath ◽

S. Heinl ◽

E. Egger ◽

R. Grabherr

Keyword(s):

Lactic Acid ◽

Copy Number ◽

Transformation Efficiency ◽

Plasmid Stability ◽

Plasmid Copy Number ◽

Bacterial Strains ◽

Plasmid Copy ◽

Origins Of Replication ◽

Different Origins

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Structural Elements Required for Replication and Incompatibility of the Rhizobium etli Symbiotic Plasmid

Journal of Bacteriology ◽

10.1128/jb.182.11.3117-3124.2000 ◽

2000 ◽

Vol 182 (11) ◽

pp. 3117-3124 ◽

Cited By ~ 58

Author(s):

Miguel A. Ramírez-Romero ◽

Nora Soberón ◽

Angeles Pérez-Oseguera ◽

Juan Téllez-Sosa ◽

Miguel A. Cevallos

Keyword(s):

Plasmid Copy Number ◽

Intergenic Sequence ◽

Structural Elements ◽

Rhizobium Etli ◽

Plasmid Copy ◽

Plasmid Segregation ◽

Symbiotic Plasmid ◽

Conserved Genes ◽

Dna Strand ◽

Plasmid Replicons

ABSTRACT The symbiotic plasmid of Rhizobium etli CE3 belongs to the RepABC family of plasmid replicons. This family is characterized by the presence of three conserved genes, repA,repB, and repC, encoded by the same DNA strand. A long intergenic sequence (igs) between repBand repC is also conserved in all members of the plasmid family. In this paper we demonstrate that (i) the repABCgenes are organized in an operon; (ii) the RepC product is essential for replication; (iii) RepA and RepB products participate in plasmid segregation and in the regulation of plasmid copy number; (iv) there are two cis-acting incompatibility regions, one located in the igs (incα) and the other downstream ofrepC (incβ) (the former is essential for replication); and (v) RepA is a trans-acting incompatibility factor. We suggest that incα is acis-acting site required for plasmid partitioning and that the origin of replication lies within incβ.

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The OBF1 protein and its DNA-binding site are important for the function of an autonomously replicating sequence in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.9.7.2914 ◽

1989 ◽

Vol 9 (7) ◽

pp. 2914-2921 ◽

Cited By ~ 23

Author(s):

S S Walker ◽

S C Francesconi ◽

B K Tye ◽

S Eisenberg

Keyword(s):

Saccharomyces Cerevisiae ◽

Binding Site ◽

Binding Sites ◽

Plasmid Stability ◽

Wild Type ◽

Nucleotide Substitutions ◽

Mobility Shift ◽

Plasmid Copy ◽

Autonomously Replicating Sequence ◽

High Affinity

The autonomously replicating sequence ARS121 was cloned as a 480-base-pair (bp) long DNA fragment that confers on plasmids autonomous replication in Saccharomyces cerevisiae. This fragment contains two OBF1-binding sites (sites I and II) of different affinities, as identified by a gel mobility shift assay and footprint analysis. Nucleotide substitutions (16 to 18 bp) within either of the two sites obliterated detectable in vitro OBF1 binding to the mutagenized site. Linker substitution (6 bp) mutations within the high-affinity site I showed effects similar to those of the complete substitution, whereas DNA mutagenized outside the binding site bound OBF1 normally. We also tested the mitotic stability of centromeric plasmids bearing wild-type and mutagenized copies of ARS121. Both deletion of the sites and the extensive base alterations within either of the two OBF1-binding sites reduced the percentage of plasmid-containing cells in the population from about 88% to 50 to 63% under selective growth and from about 46% to 15 to 20% after 10 to 12 generations of nonselective growth. Furthermore, linker (6 bp) substitutions within site I, the high-affinity binding site, showed similar deficiencies in plasmid stability. In contrast, plasmids containing linker substitutions in sequences contiguous to site I displayed wild-type stability. In addition, plasmid copy number analysis indicated that the instability probably resulted not from nondisjunction during mitosis but rather from inefficient plasmid replication. The results strongly support the notion that the OBF1-binding sites and the OBF1 protein are important for normal ARS function as an origin of replication.

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