Characterisation of an autonomously replicating sequence from the fission yeast Schizosaccharomyces pombe

Summary A DNA sequence has been isolated from Schizosaccharomyces pombe which promotes high frequency transformation of plasmids in the same organism. It is closely linked to the DNA ligase gene CDC17 and has therefore been named ARS17 although in structure it differs substantially from ARS elements in Saccharomyces cerevisiae. ARS17 spans some 1.8 kb of DNA and deletion of any part of this region affects activity. Moreover, there does not appear to be any short sequence which is, by itself, sufficient for high frequency transformation. ARS17 lies between and partly overlaps two divergently transcribed genes and it is extremely AT rich. It lacks the consensus sequence found in S. cerevisiae ARSs and it has no ARS activity in S. cerevisiae.     

Nucleotide sequences and stability of a Nicotiana nuclear DNA segment possessing autonomously replicating ability in yeast

The nucleotide sequence of a tobacco (Nicotiana tabacum) chromosomal DNA segment(t3-ars) capable of replication in yeast (ars: autonomously replicating sequences) is presented. The subcloned region (618 bp) contained 11 bp consensus (5 A/TTTTATP_uTTTA/T 3) essential for several yeast ars, and 73% A and T. Unique 70 bp repetitive sequences resided next to this sequence. Thirty-two bp AT repeats were also seen in the neighbourhood of the repetitive sequence. The hybrid plasmid containing t3-ars was mitotically stabilized by the help of yeast centromere (CEN4).     

An autonomously replicating sequence from HeLa DNA shows a similar organization to the yeast ARS1 element

Summary A HeLa DNA fragment, which may function as an anchorage point to the nuclear matrix for human chromosomes 1 and 2, also functions as an autonomously replicating sequence (ARS) in the yeast Saccharomyces cerevisiae. In the present report we show that this DNA fragment contains both bent DNA and an A-T rich region which appear to be associated with the ARS function. More interestingly, DNA sequence analysis shows that the spatial distribution of these features is strikingly similar to that found in the yeast ARS1 element.     

Murine genomic DNA sequences replicating autonomously in mouse L cells

Plasmids that replicate autonomously in mouse L cells were constructed by inserting random genomic DNA fragments from Ltk- cells into a plasmid containing the HSV-1 thymidine kinase gene with a truncated low-efficiency promoter. HAT resistance was used as a selective marker. The presence of free plasmids in the DNA of transformants was demonstrated by hybridization with a specific plasmid probe, by electron microscopic visualization of circular DNA, and by recovering these plasmids by E. coli transformation. Nineteen different DNA fragments were isolated. They were characterized as murine autonomously replicating sequences by Mbol restriction endonuclease sensitivity, by bromodeoxyuridine substitution, by copy number determination, and by segregation analysis. Sequence analysis of the inserts of nine plasmids revealed a conserved element of 12 bp (CTCATGAGAGGCCAA) in five out of nine autonomously replicating sequences.     

Sequence analysis of three fragments of maize nuclear DNA which replicate autonomously in yeast

There maize nuclear DNA fragments were isolated on the basis of their ability to confer replication on chimeric plasmids in yeast. These Eco RI fragments of 2.5, 2.8 and 5.5 kb are repeated elements within the maize genome. The 2.5 and 2.8 kb fragments represent a family of elements repeated 11 000 times in the maize haploid genome, while the 5.5 kb fragment is part of another family of 28 000 elements. These fragments were subcloned to further define the unique region of ARS activity. The sequence of each 550–650 bp ARS subclone is reported here, and compared to the flanking regions which do not show ARS activity. The ARS elements are 65–70% A+T as compared to 50–55% for the maize genome as a whole. There is approximately 15% sequence divergence, as well as variation of ARS efficiency, among family members. ARS subclones contain the proposed yeast consensus sequence.     

四种DNA复制起始方式的比较

近年来有关DNA复制的机制研究取得一些重要进展,但现有高等生物学相关专业的《生物化学》、《分子生物学》和《基因工程》等教材中DNA复制机理尤其是复制起始相关章节的内容更新不够。结合最新的研究进展,本文综述了四种DNA复制方式:双向复制(原核大肠杆菌和真核生物)、单起点单向(质粒ColE1)、哺乳动物线粒体DNA(取代环)以及最近发现的不需要引物的DNA聚合酶起始的DNA复制方式,丰富了教材相关的内容,强调复制的不同方式是不同的复制起始复合物装备的结果。文末对四种不同的复制方式异同进行了比较,根据复制原点在复制复合物装备中的重要作用,对教材中复制原点的概念进行了进一步的剖析。本文内容将有利于学生跳出课程的框架,将相关的知识点融会贯通,夯实理论基础并在将来能有所应用。     

Classes of autonomously replicating sequences are found among early-replicating monkey DNA

Thirteen new independent clones of origin-enriched sequences (ors) that are capable of autonomous replication have been identified from a library of 100 ors i clones that had been previously isolated from early replicating monkey (CV-1) DNA. Autonomous replication was assayed by transient episomal replication in transfected HeLa cells; ors-plasmid DNA was isolated at various times after transfection and screened by the DpnI resistance assay and the bromodeoxyuridine (BrdUrd) substitution assay to differentiate between input and newly replicated DNA. Four of the autonomously replicating clones were identified by screening the ors-library with probes of ors 3, 8, 9 and 12, previously shown to be capable of autonomous replication (Frappier and Zannis-Hadjopoulos, Proc. Natl. Acad. Sci. USA (1987) 84, 6668-6672). The other nine functional ors clones were identified among 18 randomly chosen ones, which were similarly screened for autonomous replication. Nucleotide sequence analyses of 11 of the newly identified functional ors plasmids revealed, in most of them, features similar to those present in other viral or eukaryotic replication origins, notably the presence of AT-rich regions and inverted repeats. Pairwise comparisons between the newly identified ors showed no extensive sequence homologies, other than the presence of the alpha-satellite repetitive sequence family in three ors and of the repetitive Alu sequence family in one ors. The results suggest that there exist different classes of mammalian replication origin, highly or moderately repetitive and unique, and that their activation is most probably dependent on the presence of structural determinants rather than on a particular sequence.     

Two autonomously replicating sequences near oli-1 gene of yeast mitochondrial DNA

We cloned two autonomously replicating sequences from a short segment of mtDNA of an oligomycin-resistant petite yeast, O-111, into a vector pYleu 12 constructed from yeast LEU 2 gene and pBR 322. These plasmids, pYmit 4 and pYmit 1, had frequencies of transformation of yeast as high as that of YEp 13, having a replicator of 2 mu DNA. They were maintained as plasmids in yeast under selective conditions and shuttled from yeast to E. coli. No evidence was obtained that these plasmids were incompatible with the wild-type mitochondrial genome. These sequences were located in intergenic regions.     

Mapping autonomously replicating sequence elements in a 73-kb region of chromosome II of the fission yeast, Schizosaccharomyces pombe

Autonomously replicating sequence (ARS) elements are the genetic determinants of replication origin function in yeasts. They can be easily identified as the plasmids containing them transform yeast cells at a high frequency. As the first step towards identifying all potential replication origins in a 73-kb region of the long arm of fission yeast chromosome II, we have mapped five new ARS elements using systematic subcloning and transformation assay. 2D analysis of one of the ARS plasmids that showed highest transformation frequency localized the replication origin activity within the cloned genomic DNA. All the new ARS elements are localized in two clusters in centromere proximal 40 kb of the region. The presence of at least six ARS elements, including the previously reported ars727, is suggestive of a higher origin density in this region than that predicted earlier using a computer based search.     

Biochemical regulation of the initiation of DNA replication

Abstract

The initiation of DNA replication is a key step in the cell division cycle and in DNA endoreduplication. Initiation of replication takes place at specific places in chromosomes known as replication origins. These are subject to temporal regulation within the cell cycle and may also be regulated as a function of plant development. In yeast, replication origins are recognised and bound by three different groups of proteins at different stages of the cell cycle. Of these, the MCM proteins are the most likely to be involved in activating the origins in order to facilitate initiation. MCM-like proteins also occur in plants, but have not been characterised in detail. Other proteins which bind to origins have been identified, as has a protein with a strong affinity for ds-ss junctions in DNA molecules.     

Micronuclear DNA of Oxytricha nova contains sequences with autonomously replicating activity in Saccharomyces cerevisiae.

Oxytricha nova is a hypotrichous ciliate with micronuclei and macronuclei. Micronuclei, which contain large, chromosomal-sized DNA, are genetically inert but undergo meiosis and exchange during cell mating. Macronuclei, which contain only small, gene-sized DNA molecules, provide all of the nuclear RNA needed to run the cell. After cell mating the macronucleus is derived from a micronucleus, a derivation that includes excision of the genes from chromosomes and elimination of the remaining DNA. The eliminated DNA includes all of the repetitious sequences and approximately 95% of the unique sequences. We cloned large restriction fragments from the micronucleus that confer replication ability on a replication-deficient plasmid in Saccharomyces cerevisiae. Sequences that confer replication ability are called autonomously replicating sequences. The frequency and effectiveness of autonomously replicating sequences in micronuclear DNA are similar to those reported for DNAs of other organisms introduced into yeast cells. Of the 12 micronuclear fragments with autonomously replicating sequence activity, 9 also showed homology to macronuclear DNA, indicating that they contain a macronuclear gene sequence. We conclude from this that autonomously replicating sequence activity is nonrandomly distributed throughout micronuclear DNA and is preferentially associated with those regions of micronuclear DNA that contain genes.     

Transformation of Aspergillus parasiticus using autonomously replicating plasmids from Aspergillus nidulans

Abstract A genetic transformation system for the aflatoxin-producing fungus Aspergillus parasiticus using two autonomously replicating plasmids from A. nidulans (ARp1 and pDHG25) is reported. Transformation frequencies using the plasmid pDHG25 were from 5 × 10² to 2.5 × 10⁴ transformants per 10⁶ viable protoplasts and μg DNA. The stability of the plasmids in the transformants was also studied. This transformation system offers a new opportunity to clone genes related to aflatoxin production using appropriate aflatoxin-defective mutants.     

New yeast vectors containing the autonomously replicating sequences from Candida maltosa genome

Two different DNA sequences from the yeast Candida maltosa confer the ability to replicate autonomously to the yeast integrative vector pLD700 on which they are cloned. The recombinant plasmids pLD701 and pLD702 with autonomously replicating sequences (ARS) from Candida maltosa and LEU2 gene from Saccharomyces cerevisiae transform the auxotrophic strain S. cerevisiae DC5 with the efficiency 3-5 x 10(3) per microgram of DNA. Like other yeast vectors harbouring ARS, these plasmids are not stable in yeast cells. Restriction and hybridization analyses have revealed the pLD701 plasmid to contain ARS from chromosomal DNA of C. maltosa. Plasmid pLD701 appears to be a useful vector for yeast transformation.     

Genomic organization of two families of highly repeated nuclear DNA sequences of maize selected for autonomous replicating activity in yeast

Maize nuclear DNA sequences capable of promoting the autonomous replication of plasmids in yeast were isolated by ligating Eco RI-digested fragments into yeast vectors unable to replicate autonomously. Three such autonomously replicating sequences (ARS), representing two families of highly repeated sequences within the maize genome, were isolated and characterized. Each repetitive family shows hybridization patterns on a Southern blot characteristic of a dispersed sequence. Unlike most repetitive sequences in maize, both ARS families have a constant copy number and characteristic genomic hybridization pattern in the inbred lines examined. Larger genome clones with sequence homology to the ARS-containing elements were selected from a lambda library of maize genomic DNA. There was typically only one copy of an ARS-homologous sequence on each 12–15 kb genomic fragment.     

The Mcm467 complex of Saccharomyces cerevisiae is preferentially activated by autonomously replicating DNA sequences

We have analyzed the role of single-stranded DNA (ssDNA) in the modulation of the ATPase activity of Mcm467 helicase of the yeast Saccharomyces cerevisiae. The ATPase activity of the Mcm467 complex is modulated in a sequence-specific manner and that the ssDNA sequences derived from the origin of DNA replication of S. cerevisiae autonomously replicating sequence 1 (ARS1) are the most effective stimulators. Synthetic oligonucleotides, such as oligo(dA) and oligo(dT), also stimulated the ATPase activity of the Mcm467 complex, where oligo(dT) was more effective than oligo(dA). However, the preference of a thymidine stretch appeared unimportant, because with yeast ARS1 derived sequences, the A-rich strand was as effective in stimulating the ATPase activity, as was the T-rich strand. Both of these strands were more effective stimulators than either oligo(dA)( )()or oligo(dT). The DNA helicase activity of Mcm467 complex is also significantly stimulated by the ARS1-derived sequences. These results indicate that the ssDNA sequences containing A and B1 motifs of ARS1, activate the Mcm467 complex and stimulate its ATPase and DNA helicase activities. Our results also indicate that the yeast replication protein A stimulated the ATPase activity of the Mcm467 complex.     

Isolation of polysaccharide-free DNA from plants

A quick procedure for the isolation of polysaccharide-free DNA from different plant species and cell suspension or callus cultures is described. The originality of the method lies in the use of a mixture of glycoside hydrolases that leads, after phenol and chloroform extraction, to the isolation of pure DNA without any polysaccharide contamination. The highly purified DNA can be used for nucleotide analysis by HPLC, RFLP analysis and PCR amplification.     

In addition to the ARS core, the ARS box is necessary for autonomously replicating sequences

Summary Autonomously replicating sequences (ARSs) were cloned from nuclear and mitochondrial DNA of D. melanogaster using YIp5, which is composed of pBR322 and the yeast ura3 gene, as the cloning vector and YNN27, a Ura^- yeast strain as the recipient. The nucleotide sequences of six ARSs, two from nuclear bulk, two from the nuclear 1.688 satellite, and two from mitochondorial DNA, were determined. The relationship between the transformation frequency and the inclusion of the ARS core, 5 _T ^A TT-TAT _A ^G TTT _T ^A 3, of these fragments was analysed. All the ARSs contained an ARS core or a single base change of it. However, not all the fragments that contained a single base change of the ARS core were able to transform the recipient cells, suggesting that certain bases in the ARS core were not exchangeable. It is suggested by transformation experiments with subfragments that in addition to an ARS core, an ARS box which is located within 25 bp upstream of the ARS core and whose sequence is composed of 5TNT _G ^A AA 3, is necessary for autonomous replication.     

Enriched autonomously replicating sequences in a nuclear matrix-DNA complex isolated from synchronized HeLa cells

Nucleoids isolated from either synchronized or exponentially growing HeLa cells were digested with restriction enzymes to separate a nuclear matrix-bound DNA component from the rest. Partial libraries were constructed by inserting DNA fragments from both components into a yeast-bacteria plasmid vector. A random sample from these libraries was tested for ARS activity by a standard yeast transformation assay. We found that synchronization for DNA replication results in an enrichment for autonomously replicating sequences in the library constructed with the DNA component bound to the nuclear matrix.