Amplification of a Circular Episome Carrying an Inverted Repeat of theDFR1Locus and Adjacent Autonomously Replicating Sequence Element ofSaccharomyces cerevisiae

ABSTRACT We report on the identification and first cloning of an autonomously replicating sequence element from the chromosome of an archaeon, the extreme halophile Halobacterium strain NRC-1. The putative replication origin was identified by association with the orc7 gene and replication ability in the host strain, demonstrated by cloning into a nonreplicating plasmid. Deletion analysis showed that sequences located up to 750 bp upstream of the orc7 gene translational start, plus the orc7 gene and 50 bp downstream, are sufficient to endow the plasmid with replication ability, as judged by expression of a plasmid-encoded mevinolin resistance selectable marker and plasmid recovery after transformation. Sequences located proximal to the two other chromosomally carried haloarchaeal orc genes (orc6 and orc8) are not able to promote efficient autonomous replication. Located within the 750-bp region upstream of orc7 is a nearly perfect inverted repeat of 31 bp, which flanks an extremely AT-rich (44%) stretch of 189 bp. The replication ability of the plasmid was lost when one copy of the inverted repeat was deleted. Additionally, the inverted repeat structure near orc7 homologs in the genomic sequences of two other halophiles, Haloarcula marismortui and Haloferax volcanii, is highly conserved. Our results indicate that, in halophilic archaea, a chromosomal origin of replication is physically linked to orc7 homologs and that this element is sufficient to promote autonomous replication. We discuss the finding of a functional haloarchaeal origin in relation to the large number of orc1-cdc6 homologs identified in the genomes of all haloarchaea to date.

Download Full-text

Organization of replication of ribosomal DNA in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.8.11.4927 ◽

1988 ◽

Vol 8 (11) ◽

pp. 4927-4935 ◽

Cited By ~ 197

Author(s):

M H Linskens ◽

J A Huberman

Keyword(s):

Saccharomyces Cerevisiae ◽

Ribosomal Dna ◽

Repeat Unit ◽

Replication Forks ◽

Sequence Element ◽

Autonomously Replicating Sequence ◽

Nontranscribed Spacer ◽

Precursor Rna ◽

Mapping Techniques ◽

Region Ii

Using recently developed replicon mapping techniques, we have analyzed the replication of the ribosomal DNA in Saccharomyces cerevisiae. The results show that (i) the functional origin of replication colocalizes with an autonomously replicating sequence element previously mapped to the nontranscribed spacer region, (ii) only a fraction of the potential origins are utilized in a single S phase, and (iii) the replication forks moving counter to the direction of transcription of the 37S precursor RNA stop at or near the termination site of transcription. Consequently, most ribosomal DNA is replicated unidirectionally by forks moving in the direction of transcription and most replicons are larger than the repeat unit. The significance of this finding for the replication of abundantly transcribed genes is discussed.

Download Full-text

Organization of replication of ribosomal DNA in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.8.11.4927-4935.1988 ◽

1988 ◽

Vol 8 (11) ◽

pp. 4927-4935

Author(s):

M H Linskens ◽

J A Huberman

Keyword(s):

Saccharomyces Cerevisiae ◽

Ribosomal Dna ◽

Repeat Unit ◽

Replication Forks ◽

Sequence Element ◽

Autonomously Replicating Sequence ◽

Nontranscribed Spacer ◽

Precursor Rna ◽

Mapping Techniques ◽

Region Ii

Using recently developed replicon mapping techniques, we have analyzed the replication of the ribosomal DNA in Saccharomyces cerevisiae. The results show that (i) the functional origin of replication colocalizes with an autonomously replicating sequence element previously mapped to the nontranscribed spacer region, (ii) only a fraction of the potential origins are utilized in a single S phase, and (iii) the replication forks moving counter to the direction of transcription of the 37S precursor RNA stop at or near the termination site of transcription. Consequently, most ribosomal DNA is replicated unidirectionally by forks moving in the direction of transcription and most replicons are larger than the repeat unit. The significance of this finding for the replication of abundantly transcribed genes is discussed.

Download Full-text

Faculty Opinions recommendation of The chaperonin TRiC blocks a huntingtin sequence element that promotes the conformational switch to aggregation.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.2365962.1994061 ◽

2010 ◽

Author(s):

Martin Gruebele ◽

Simon Ebbinghaus

Keyword(s):

Sequence Element ◽

Conformational Switch

Download Full-text

Active and inactive transplacement of the M26 recombination hotspot in Schizosaccharomyces pombe.

Genetics ◽

10.1093/genetics/141.1.33 ◽

1995 ◽

Vol 141 (1) ◽

pp. 33-48

Author(s):

J B Virgin ◽

J Metzger ◽

G R Smith

Keyword(s):

Schizosaccharomyces Pombe ◽

Meiotic Recombination ◽

Plasmid Dna ◽

Context Effect ◽

Intragenic Recombination ◽

Multicopy Plasmid ◽

Chromosomal Locus ◽

Sequence Element ◽

Recombination Hotspot ◽

Recombination Hotspots

Abstract The ade6-M26 mutation of the fission yeast Schizosaccharomyces pombe creates a meiotic recombination hotspot that elevates ade6 intragenic recombination approximately 10-15-fold. A heptanucleotide sequence including the M26 point mutation is required but not sufficient for hotspot activity. We studied the effects of plasmid and chromosomal context on M26 hotspot activity. The M26 hotspot was inactive on a multicopy plasmid containing M26 embedded within 3.0 or 5.9 kb of ade6 DNA. Random S. pombe genomic fragments totaling approximately 7 Mb did not activate the M26 hotspot on a plasmid. M26 hotspot activity was maintained when 3.0-, 4.4-, and 5.9-kb ade6-M26 DNA fragments, with various amounts of non-S. pombe plasmid DNA, were integrated at the ura4 chromosomal locus, but only in certain configurations relative to the ura4 gene and the cointegrated plasmid DNA. Several integrations created new M26-independent recombination hotspots. In all cases the non-ade6 DNA was located > 1 kb from the M26 site, and in some cases > 2 kb. Because the chromosomal context effect was transmitted over large distances, and did not appear to be mediated by a single discrete DNA sequence element, we infer that the local chromatin structure has a pronounced effect on M26 hotspot activity.

Download Full-text

Yeast autonomously replicating sequence (ARS): Identification, function, and modification

Engineering in Life Sciences ◽

10.1002/elsc.202000085 ◽

2021 ◽

Author(s):

Xiao‐Yu Tan ◽

Xiao‐Le Wu ◽

Ming‐Zhe Han ◽

Li Wang ◽

Li Xu ◽

...

Keyword(s):

Autonomously Replicating Sequence ◽

Identification Function

Download Full-text

Formation of Large Palindromic DNA by Homologous Recombination of Short Inverted Repeat Sequences in Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/161.3.1065 ◽

2002 ◽

Vol 161 (3) ◽

pp. 1065-1075

Author(s):

David K Butler ◽

David Gillespie ◽

Brandi Steele

Keyword(s):

Homologous Recombination ◽

Inverted Repeat ◽

Excision Repair ◽

Strand Break ◽

Double Strand Break ◽

Repeat Sequence ◽

Inverted Repeat Sequence ◽

Repeat Sequences ◽

Dna Double Strand Break ◽

Intermolecular Reaction

Abstract Large DNA palindromes form sporadically in many eukaryotic and prokaryotic genomes and are often associated with amplified genes. The presence of a short inverted repeat sequence near a DNA double-strand break has been implicated in the formation of large palindromes in a variety of organisms. Previously we have established that in Saccharomyces cerevisae a linear DNA palindrome is efficiently formed from a single-copy circular plasmid when a DNA double-strand break is introduced next to a short inverted repeat sequence. In this study we address whether the linear palindromes form by an intermolecular reaction (that is, a reaction between two identical fragments in a head-to-head arrangement) or by an unusual intramolecular reaction, as it apparently does in other examples of palindrome formation. Our evidence supports a model in which palindromes are primarily formed by an intermolecular reaction involving homologous recombination of short inverted repeat sequences. We have also extended our investigation into the requirement for DNA double-strand break repair genes in palindrome formation. We have found that a deletion of the RAD52 gene significantly reduces palindrome formation by intermolecular recombination and that deletions of two other genes in the RAD52-epistasis group (RAD51 and MRE11) have little or no effect on palindrome formation. In addition, palindrome formation is dramatically reduced by a deletion of the nucleotide excision repair gene RAD1.

Download Full-text

The chloroplast genome evolution of Venus slipper (Paphiopedilum): IR expansion, SSC contraction, and highly rearranged SSC regions

BMC Plant Biology ◽

10.1186/s12870-021-03053-y ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Yan-Yan Guo ◽

Jia-Xing Yang ◽

Ming-Zhu Bai ◽

Guo-Qiang Zhang ◽

Zhong-Jian Liu

Keyword(s):

Gene Order ◽

Inverted Repeat ◽

Single Copy ◽

Gene Content ◽

Comparative Genomic ◽

Substitution Rates ◽

Plastome Evolution ◽

Chloroplast Genomes ◽

Ideal System ◽

Small Single Copy

Abstract Background Paphiopedilum is the largest genus of slipper orchids. Previous studies showed that the phylogenetic relationships of this genus are not well resolved, and sparse taxon sampling documented inverted repeat (IR) expansion and small single copy (SSC) contraction of the chloroplast genomes of Paphiopedilum. Results Here, we sequenced, assembled, and annotated 77 plastomes of Paphiopedilum species (size range of 152,130 – 164,092 bp). The phylogeny based on the plastome resolved the relationships of the genus except for the phylogenetic position of two unstable species. We used phylogenetic and comparative genomic approaches to elucidate the plastome evolution of Paphiopedilum. The plastomes of Paphiopedilum have a conserved genome structure and gene content except in the SSC region. The large single copy/inverted repeat (LSC/IR) boundaries are relatively stable, while the boundaries of the inverted repeat and small single copy region (IR/SSC) varied among species. Corresponding to the IR/SSC boundary shifts, the chloroplast genomes of the genus experienced IR expansion and SSC contraction. The IR region incorporated one to six genes of the SSC region. Unexpectedly, great variation in the size, gene order, and gene content of the SSC regions was found, especially in the subg. Parvisepalum. Furthermore, Paphiopedilum provides evidence for the ongoing degradation of the ndh genes in the photoautotrophic plants. The estimated substitution rates of the protein coding genes show accelerated rates of evolution in clpP, psbH, and psbZ. Genes transferred to the IR region due to the boundary shift also have higher substitution rates. Conclusions We found IR expansion and SSC contraction in the chloroplast genomes of Paphiopedilum with dense sampling, and the genus shows variation in the size, gene order, and gene content of the SSC region. This genus provides an ideal system to investigate the dynamics of plastome evolution.

Download Full-text

A 160-bp Palindrome Is a Rad50·Rad32-Dependent Mitotic Recombination Hotspot inSchizosaccharomyces pombe

Genetics ◽

10.1093/genetics/161.1.461 ◽

2002 ◽

Vol 161 (1) ◽

pp. 461-468 ◽

Cited By ~ 6

Author(s):

Joseph A Farah ◽

Edgar Hartsuiker ◽

Ken-ichi Mizuno ◽

Kunihiro Ohta ◽

Gerald R Smith

Keyword(s):

Secondary Structure ◽

Schizosaccharomyces Pombe ◽

Inverted Repeat ◽

Mitotic Recombination ◽

Genome Integrity ◽

Recombination Hotspot ◽

Nuclease Domain ◽

Palindromic Sequences

AbstractPalindromic sequences can form hairpin and cruciform structures that pose a threat to genome integrity. We found that a 160-bp palindrome (an inverted repeat of 80 bp) conferred a mitotic recombination hotspot relative to a control nonpalindromic sequence when inserted into the ade6 gene of Schizosaccharomyces pombe. The hotspot activity of the palindrome, but not the basal level of recombination, was abolished by a rad50 deletion, by a rad50S “separation of function” mutation, or by a rad32-D25A mutation in the nuclease domain of the Rad32 protein, an Mre11 homolog. We propose that upon extrusion of the palindrome the Rad50·Rad32 nuclease complex recognizes and cleaves the secondary structure thus formed and generates a recombinogenic break in the DNA.

Download Full-text

Completion of Replication Map of Saccharomyces cerevisiae Chromosome III

Molecular Biology of the Cell ◽

10.1091/mbc.12.11.3317 ◽

2001 ◽

Vol 12 (11) ◽

pp. 3317-3327 ◽

Cited By ~ 54

Author(s):

Arkadi Poloumienko ◽

Ann Dershowitz ◽

Jitakshi De ◽

Carol S. Newlon

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Replication ◽

Complete Analysis ◽

Replication Origins ◽

Chromosomal Dna ◽

Chromosomal Replication ◽

Autonomously Replicating Sequence ◽

Eukaryotic Chromosome ◽

Ars Elements ◽

Chromosome Iii

In Saccharomyces cerevisiae chromosomal DNA replication initiates at intervals of ∼40 kb and depends upon the activity of autonomously replicating sequence (ARS) elements. The identification of ARS elements and analysis of their function as chromosomal replication origins requires the use of functional assays because they are not sufficiently similar to identify by DNA sequence analysis. To complete the systematic identification of ARS elements onS. cerevisiae chromosome III, overlapping clones covering 140 kb of the right arm were tested for their ability to promote extrachromosomal maintenance of plasmids. Examination of chromosomal replication intermediates of each of the seven ARS elements identified revealed that their efficiencies of use as chromosomal replication origins varied widely, with four ARS elements active in ≤10% of cells in the population and two ARS elements active in ≥90% of the population. Together with our previous analysis of a 200-kb region of chromosome III, these data provide the first complete analysis of ARS elements and DNA replication origins on an entire eukaryotic chromosome.

Download Full-text