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.     

Isolation and nucleotide sequence of an autonomously replicating sequence (ARS) element functional in Candida albicans and Saccharomyces cerevisiae

Summary An 8.6-kb fragment was isolated from an EcoRI digest of Candida albicans ATCC 10261 genomic DNA which conferred the property of autonomous replication in Saccharomyces cervisiae on the otherwise non-replicative plasmid pMK155 (5.6 kb). The DNA responsible for the replicative function was subcloned as a 1.2-kb fragment onto a non-replicative plasmid (pRC3915) containing the C. albicans URA3 and LEU2 genes to form plasmid pRC3920. This plasmid was capable of autonomous replication in both S. cerevisiae and C. albicans and transformed S. cerevisiae AH22 (leu2 ^–) to Leu⁺ at a frequency of 2.15 × 10³ transformants per pg DNA, and transformed C. albicans SGY-243 (ura3) to Ura⁺ at a frequency of 1.91 × 10³ transformants per g DNA. Sequence analysis of the cloned DNA revealed the presence of two identical regions of eleven base pairs (5TTTTATGTTTT3) which agreed with the consensus of autonomously replicating sequence (ARS) cores functional in S. cerevisiae. In addition there were two 10/11 and numerous 9/11 matches to the core consensus. The two 11/11 matches to the consensus, CaARS1 and CaARS2, were located on opposite strands in a non-coding AT-rich region and were separated by 107 bp. Also present on the C. albicans DNA, 538 by from the ARS cores, was a gene for 5S rRNA which showed sequence homology with several other yeast 5S rRNA genes. A sub-fragment (494 bp) containing the 5S rRNA gene (but not the region containing the ARS cores) hybridized to genomic DNAs from a number of yeast species, including S. cerevisiae, C. tropicalis, C. pseudotropicalis, C. parapsilosis, C. kruseii, C. (Torulopsis) glabrata and Neurospora crassa. The 709-bp ARS element (but not the 5S rRNA gene) was necessary for high-frequency transformation and autonomous plasmid replication in both S. cerevisiae and C. albicans.EMBL/GenBank database accession number: X16634 (5S rRNA)     

Bending of DNA segments with Saccharomyces cerevisiae autonomously replicating sequence activity, isolated from basidiomycete mitochondrial linear plasmids

Summary Previous studies have indicated that DNA bending is a general structural feature of sequences (ARSs) from cellular DNAs of yeasts and nuclear and mitochondrial genomic DNAs of other eukaryotes that are capable of autonomous replication in Saccharomyces cerevisiae. Here we showed that bending activity is also tightly associated with S. cerevisiae ARS function of segments cloned from mitochondrial linear DNA plasmids of the basidiomycetes Pleurotus ostreatus and Lentinus edodes. Two plasmids, designated pLPO2-like (9.4 kb), and pLPO3 (6.6 kb) were isolated from a strain of P. ostreatus. A 1029 by fragment with high-level ARS activity was cloned from pLPO3 and it contained one ARS consensus sequence (A/T)TTTAT(A/G)TTT(A/T) indispensable for activity and seven dispersed ARS consensus-like (10/11 match) sequences. A discrete bent DNA region was found to lie around 500 by upstream from the ARS consensus sequence (T-rich strand). Removal of the bent DNA region impaired ARS function. DNA bending was also implicated in the ARS function associated with a 1430 by fragment containing three consecutive ARS consensus sequences which had been cloned from the L. edodes plasmid pLLE1 (11.0 kb): the three consecutive ARSs responsible for high-level ARS function occurred in, and immediately adjacent to, a bent DNA region. A clear difference exists between the two plasmid-derived ARS fragments with respect to the distance between the bent DNA region and the ARS consensus sequence(s).     

Yeast ARS function and nuclear matrix association coincide in a short sequence from the human HPRT locus

Summary A sequence that supports extrachromosomal replication of plasmids in yeast has been identified within the first intron of the human hypoxanthine-guanine phosphori bosyltransferase (HPRT) gene. This represents the first isolation of such an autonomously replicating sequence (ARS) from an exactly known position in the human genome. This ARS shares similarities of imparted yeast phenotype and DNA sequence with other heterologus ARSs. In addition, this sequence is found to be a matrix association region (MAR) on the basis of specific binding to nuclear matrices prepared from several mammalian cell types. It also exhibits anomalous electrophoretic behavior, characteristic of bent DNA, on polyacrylamide gels. The coincidence of these properties supports the possibility that this region may play a role in DNA replication within its normal chromosomal context.     

圆红冬孢酵母自主复制序列分离和利用游离型质粒进行基因敲除

【目的】与整合型表达载体相比,游离型表达载体通常具有更高的拷贝数以实现目标基因的高强度表达,并且对于DNA操作应用更加方便和灵活。然而,目前的研究尚未确定适用于圆红冬孢酵母的游离型质粒,该酵母外源基因的表达或者基于CRISPR/Cas9的基因组编辑都需要通过整合方式来完成,这也是对其遗传改造进展缓慢的一个重要原因。本研究目的是构建圆红冬孢酵母的游离型质粒,使得其外源基因的表达和基因组编辑更方便省时。【方法】首先对圆红冬孢酵母苯丙氨酸氨裂解酶基因(phenylalanine ammonia-lyase gene, PAL)中可能存在的自主复制序列(autonomously replicating sequences, ARSs)进行挖掘和表征,将该基因及其上下游序列进行分段扩增,构建到带有β-异丙基苹果酸脱氢酶基因(β-isopropyl malate dehydrogenase gene, LEU2)的质粒中,通过电转化的方法导入LEU2基因缺陷的圆红冬孢酵母中,根据转化效率高低鉴定了该酵母的一个ARS。其次,以编码香叶基香叶基焦磷酸合成酶(geranylgeranyl pyrophosphate synthase, GGPPS)的BTS1基因为敲除靶点,将其gRNA构建到基于ARS的游离型质粒中,通过转化子直观的颜色变化来验证该游离型质粒是否成功应用于圆红冬孢酵母的CRISPR/Cas9体系。【结果】本工作鉴定了圆红冬孢酵母的ARS,构建了基于ARS元件的游离型质粒,并将该质粒应用于圆红冬孢酵母CRISPR/Cas9体系,成功实现了基于游离型质粒的基因敲除。【结论】本研究丰富了圆红冬孢酵母现有的工具库,为圆红冬孢酵母的合成生物学应用提供了良好的研究基础和技术支持。     

The function of the nuclear matrix attachment region of silkworm rDNA as an autonomously replicating sequence in plasmid and chromosomal replication origin in yeast

Nuclear matrix attachment regions (MARs) play a crucial role in chromatin architecture, gene expression, and DNA replication. Although it is well known that yeast autonomously replicating sequences (ARSs) bind nuclear matrix and MARs also function as ARS elements in yeast, whether a heterologous MAR or ARS element acts as a replication origin in the chromosome has not been elucidated. We previously identified a MAR (rMAR) located in the nontranscribed spacer (NTS) of silkworm Attacus ricini rDNA. We report here that this rMAR contains 10 copies of ARS consensus sequence (ACS) and several DNA unwinding regions. The rMAR employs ARS activity in yeast and a rARS element locates in the 3(') region of the rMAR. Furthermore, we have also revealed that either the rMAR or the rARS element functions as a replication origin in the chromosome. Our results provide the first direct evidence to demonstrate that heterologous rMAR and rARS display chromosomal origin activity, suggesting that the chromosome structure and replication origin of rDNA reserve some common features during evolution.     

Cloning of DNA sequences from Methanococcus vannielii capable of autonomous replication in yeast

Total DNA of the archaebacterium Methanococcus vannielii was digested with BamHI or BamHI/HindIII, cloned with plasmid Yip5 and analyzed for sequences capable of autonomous replication (ARSs) in the eukaryote Saccharomyces cerevisiae. Two recombinant plasmids were isolated which contained 3.3 kb and 8 kb fragments of methanogen derived DNA with ARS activity. They exhibited low transformation efficiencies for yeast and promoted slow growth of yeast transformants.Abbreviations Ap ampicillin - ARS autonomously replicating sequence - EtBr ethidium bromide - kb kilobase(s) - Mc. Methanococcus - R resistance - RE replication enhancer - RS replication sequence - Tc tetracycline     

A Petunia hybrida chloroplast DNA region, close to one of the inverted repeats, shows sequence homology with the Euglena gracilis chloroplast DNA region that carries the putative replication origin

Summary Three distinct chloroplast (cp) DNA fragments from Petunia hybrida, which promote autonomous replication in yeast, were mapped on the chloroplast genome. Sequence analysis revealed that these fragments (called ARS A, B and C) have a high AT content, numerous short direct and inverted repeats and at least one yeast ARS consensus sequence 5A/TTTTATPuTTTA/T, essential for yeast ARS activity. ARS A and B also showed the presence of (semi-)conserved sequences, present in all Chlamydomanas reinhardii cpDNA regions that promote autonomous replication in yeast (ARS sequences) or in C. reinhardii (ARC sequences). A 431 bp BamHI/EcoRI fragment, close to one of the inverted repeats and adjacent to the ARS B subfragment contains an AT-rich stretch of about 100 nucleotides that show extensive homology with an Euglena gracilis cpDNA fragment which is part of the replication origin region. This conserved region contains direct and inverted repeats, stem-and-loop structures can be folded and it contains an ARS consensus sequence. In the near vicinity a GC-rich block is present. All these features make this cpDNA region the best candidate for being the origin of replication of P. hybrida cpDNA.     

Structure-function relationships in replication origins of the yeast Saccharomyces cerevisiae: higher-order structural organization of DNA in regions flanking the ARS consensus sequence

In order to better understand the involvement of the DNA molecule in the replication initiation process we have characterized the structure of the DNA at Autonomously Replicating Sequences (ARSs) in Saccharomyces cerevisiae. Using a new method for anti-bent DNA analysis, which allowed us to take into account the bending contribution of each successive base plate, we have investigated the higher-order structural organization of the DNA in the region which immediately surrounds the ARS consensus sequence (ACS). We have identified left- and right-handed anti-bent DNAs which flank this consensus sequence. The data show that this organization correlates with an active ACS. Analysis of the minimum nucleotide sequence providing ARS function to plasmids reveals an example where the critical nucleotides are restricted to the ACS and the right-handed anti-bent DNA domain, although most of the origins considered contained both left- and right-handed anti-bent DNAs. Moreover, mutational analysis shows that the right-handed form is necessary in order to sustain a specific DNA conformation which is correlated with the level of plasmid maintenance. A model for the role of these individual structural components of the yeast replication origin is presented. We discuss the possible role of the right-handed anti-bent DNA domain, in conjunction with the ACS, in the process of replication initiation, and potentialities offered by the combination of left- and right-handed structural components in origin function. Received: 29 October 1999 / Accepted: 14 March 2000     

Random AT library: autonomously replicating sequence (ARS) activity of chemically synthesized random sequences for transformation of nonconventional yeast species

In a search for sequences that confer on bacterial plasmids the capacity of autonomous replication in yeast cells, we chemically synthesized polynucleotides 80 bp in length from an equimolar mixture of A and T. The random AT-polymer population, W80, was inserted into the plasmid YIp5-Kan1 (which carries the markers URA3 and G418(R), but does not replicate in yeast) and amplified in Escherichia coli. This library, representing 10 000 different AT sequences, was transformed into three species of yeast: Saccharomyces cerevisiae, Kluyveromyces lactis and Torulaspora delbrueckii. The aim was to evaluate the frequency, if any, of autonomously replicating sequences (ARSs) in the random sequences. A large number of transformants were obtained from each species. Many of them showed a stable transformed phenotype. Several W80 sequences were found many times for a given species, suggesting that each species preferred particular sequences for ARS function, although they are diverse in their primary sequence. In view of the high frequency and stability of the replicative plasmids found in the different hosts, this small random AT library may be conveniently used as a source of replicative gene vectors for genetic manipulation of many nonconventional yeast species, in place of searching for species-specific chromosomal ARSs.     

Trichoderma reesei sequences that bind to the nuclear matrix enhance transformation frequency

Three DNA fragments, trs1, 2 and 3, were isolated from the Trichoderma reesei genome on the basis of their ability to promote autonomous replication of plasmids in Saccharomyces cerevisiae. Each trs element bound specifically to the isolated T. reesei nuclear matrix in vitro, and two of them bound in vivo, indicating that they are matrix attachment regions (MARs). A similar sequence previously isolated from Aspergillus nidulans (ans1) was also shown to bind specifically to the T. reesei nuclear matrix in vitro. The T. reesei MARs are AT-rich sequences containing 70%, 86% and 73% A+T over 2.9, 0.8 and 3.7 kb, respectively for trs1, 2 and 3. They exhibited no significant sequence homology, but were shown to contain a number of sequence motifs that occur frequently in many MARs identified in other eukaryotes. However, these motifs occurred as frequently in the trs elements as in randomly generated sequences with the same A+T content. trs1 and 3 were shown to be present as single copies in the T. reesei genome. The presence of the trs elements in transforming plasmids enhanced the frequency of integrative transformation of T. reesei up to five fold over plasmids without a trs. No evidence was obtained to suggest that the trs elements promoted efficient replication of plasmids in T. reseei. A mechanism for the enhancement of transformation frequency by the trs elements is proposed. Received: 1 March 1997 / Accepted: 13 May 1997     

Cloning of Petunia hybrida chloroplast DNA sequences capable of autonomous replication in yeast

Summary Sequences from Petunia hybrida chloroplast DNA which have the property to promote autonomous replication in Saccharomyces cerevisiae were cloned in vector YIp5. Seven cloned chloroplast DNA fragments are localized at one of two different sites on the chloroplast genome. One site, arsA was mapped on a 1.8 Kb fragment at position 27.0–28.8 Kb on the P. hybrida chloroplast genome. The plasmids containing this arsA are stable both in yeast and E. coli. The other site, arsB, was shown to be very unstable and is located either in the small single copy region close to the inverted repeat or just in the inverted repeat. The functioning of these sequences as a possible origin of replication in vivo is discussed.     

Characterization of a chloroplast DNA sequence from Chlorella ellipsoidea that promotes autonomous replication in yeast

Summary An EcoRI 2.7 kbp fragment from Chlorella ellipsoidea chloroplast DNA (cpDNA) cloned in YIp5 was shown to promote autonomous replication in Saccharomyces cerevisiae. The fragment was localized in the small single copy region close to the inverted repeat. The ARS activity (autonomously replicating sequences in yeast) was found to be confined within a subclone of a ca. 300 bp HindIII fragment. Sequence analysis of this fragment revealed its high AT content and the presence of several direct and inverted repeats and a few elements that were related to the yeast ARS consensus sequence. Electron microscopic studies revealed that this sequence did not coincide with the primary replication origin of chloroplast DNA. The functioning of this sequence as a possible origin of plasmid replication in vivo is discussed. This is the first report on Chlorella cpDNA sequence. re]19850821 rv]19851211 ac]19851216     

Homology between the ribosomal DNA of Escherichia coli and mitochondrial DNA preparations of maize is principally to sequences other than mitochondrial rRNA genes

E. coli ribosomal DNA has been used to probe maize mitochondrial DNA. It hybridizes primarily with chloroplast ribosomal DNA sequences and with fungal and bacterial sequences which may contaminate the mtDNA preparations. It also hybridizes to the chloroplast 16S ribosomal RNA gene sequence present in the mitochondrial genome (1) as well as to the mitochondrial 18S ribosomal RNA gene sequence. Weak sequence homology was detected between E. coli rDNA and the mitochondrial 26S ribosomal RNA gene.     

Recent origin for a thermostable alcohol dehydrogenase allele ofDrosophila melanogaster

Summary The nucleotide sequence of theFast-Chateau Douglas isolate of the thermostable alcohol dehydrogenase allele is compared with the sequences of theSlow andFast alleles ofDrosophila melanogaster. Conceptual translation of theFChD sequence indicates that the thermostable polypeptide has the diagnostic FAST amino acid replacement at residue 192 and an additional replacement of serine for proline at residue 214. This suggests aFast origin for the thermostableAdh allele. However, some of the biochemical properties of the FCHD protein resemble those of the SLOW rather than the FAST polypeptides. The serine for proline replacement confers upon the thermostable polypeptide substrate specificities and some kinetic parameters similar to the SLOW protein. The same replacement substitution within the third coding exon also appears to alter the ADH protein concentration to a level similar to the SLOW polypeptide and the probable effect is at the level of mRNA concentration. The low level of nucleotide sequence variation, other than that leading to the amino acid substitution, suggests a recent origin for the thermostable allele. The time since divergence of theFChD sequence fromFast is estimated to be approximately 260,000–470,000 years.     

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.     

Identification of a centromeric activity in the autonomously replicating TRA region allows improvement of the host-vector system for Candida maltosa

A centromeric activity was identified in the previously isolated 3.8 kb DNA fragment that carries an autonomously replicating sequence (ARS) from the yeast Candida maltosa. Plasmids bearing duplicated copies of the centromeric DNA (dicentric plasmids) were physically unstable and structural rearrangements of the dicentric plasmids occurred frequently in the transformed cells. The centromeric DNA activity was dissociated from the ARS, which is 0.2 kb in size, and was delimited to a fragment at least 325 by in length. The centromeric DNA region included the consensus sequences of CDEI (centromeric DNA element I) and an AT-rich CDEII-like region of Saccharomyces cerevisiae but had no homology to the functionally critical CDEIII consensus. A plasmid bearing the whole 3.8 kb fragment was present in 1–2 copies per cell and was maintained stably even under non-selective culture conditions, while a plasmid having only the 0.2 kb ARS was unstable and accumulated to high copy numbers. The high-copy-number plasmid allowed us to overexpress a gene to a high level, which had never been attained before, under the control of both constitutive and inducible promoters in C. maltosa.     

Extra sequences found at P element excision sites in Drosophila melanogaster.

Summary. We have previously established a transgenic Drosophila line with a highly transposable P element insertion. Using this strain we analyzed transposition and excision of the P element at the molecular level. We examined sequences flanking the new insertion sites and those of the remnants after excision. Our results on mobilization of the P element demonstrate that target-site duplication at the original insertion site does not play a role in forward excision and transposition. After P element excision an 8 by target-site duplication and part of the 31 by terminal inverted repeat (5–18 bp) remained in all the strains examined. Moreover, in 11 out of 28 strains, extra sequences were found between the two remaining inverted repeats. The double-strand gap repair model does not explain the origin of these extra sequences. The mechanism creating them may be similar to the hairpin model proposed for the transposon Tam in Antirrhinum majus.