Enhanced expression of EGFP gene in CHSE-214 cells by an ARS element from mud loach (Misgurnus mizolepis)

The origins of replication are associated with nuclear matrices or are found in close proximity to matrix attachment regions (MARs). In this report, fish MARs were cloned into an autonomously replicating sequence (ARS) cloning vector and were screened for ARS elements in Saccharomyces cerevisiae. Sixteen clones were isolated that were able to grow on the selective plates. In particular, an ARS905 that shows high efficiency among them was selected for this study. Southern hybridization indicated the autonomous replication of the transformation vector containing the ARS905 element. DNA sequences analysis showed that the ARS905 contained two ARS consensus sequences as well as MAR motifs, such as AT tracts, ORI patterns, and ATC tracts. In vitro matrix binding analysis, major matrix binding activity and ARS function coincided in a subfragment of the ARS905. To analyze the effects of ARS905 on expression of a reporter gene, an ARS905(E1158) with ARS activity was inserted into pBaEGFP(+) containing mud loach beta-actin promoter, EGFP as a reporter gene, and SV40 poly(A) signal. The pBaEGFP(+)-ARS905(E1158) was transfected into a fish cell line, CHSE-214. The intensity of EGFP transfected cells was a 7-fold of the control at 11days post-transfection. These results indicate that ARS905 enhances the expression of the EGFP gene and that it should be as a component of expression vectors in further fish biotechnological studies.     

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.     

Drosophila ARSs contain the yeast ARS consensus sequence and a replication enhancer.

A number of restriction fragments that function as autonomously replicating sequences (ARSs) in yeast have been isolated from Drosophila melanogaster DNA. The behaviour in yeast of plasmids containing Drosophila ARS elements was studied and compared to that exhibited by the archetypal yeast ARS-1 plasmid. ARS functions were localised by subcloning and BAL-31 deletion analysis. These studies demonstrated the structural and functional complexity of Drosophila ARSs. Each Drosophila ARS element has at least two domains, one essential for replication (the replication sequence, RS) and a second (the replication enhancer, RE) which is essential for maximum function of the RS. The RS of three Drosophila ARSs was shown to contain a sequence identical to an 11 bp yeast ARS consensus sequence (5' A/T TTTATPuTTT A/T 3'). These observations lend support to the hypothesis that heterologous ARS elements may be of biological significance.     

Functional conservation of multiple elements in yeast chromosomal replicators.

Replicators that control the initiation of DNA replication in the chromosomes of Saccharomyces cerevisiae retain their function when cloned into plasmids, where they are commonly referred to as autonomously replicating sequences (ARSs). Previous studies of the structure of ARS1 in both plasmid and chromosome contexts have shown that it contains one essential DNA element, A, that includes a match to the ARS consensus sequence (ACS), and three additional elements, B1, B2, and B3, that are also important for ARS function. Elements A and B3 are bound by a candidate initiator protein called the origin recognition complex and ARS-binding factor 1, respectively. Although the A and B3 elements have been found in other ARSs, sequence comparisons among ARSs have failed to identify B1- and B2-like elements. To assess the generality of the modular nature of yeast replicators, linker substitution mutagenesis of another yeast chromosomal replicator, ARS307, was performed. Three DNA sequence elements were identified in ARS307, and they were demonstrated to be functionally equivalent to the A, B1, and B2 elements present in ARS1. Despite the lack of DNA sequence similarity, the B1 and B2 elements at each ARS were functionally conserved. Single-base substitutions in the core of the ARS1 B1 and B2 elements identified critical nucleotides required for the function of the B1 element. In contrast, no single-point mutations were found to affect B2 function. The results suggest that multiple DNA sequence elements might be a general and conserved feature of replicator sequences in S. cerevisiae.     

The basidiomycete Lentinus edodes linear mitochondrial DNA plasmid contains a segment exhibiting a high autonomously replicating sequence activity in Saccharomyces cerevisiae.

A linear DNA plasmid, designated pLLE1, has been isolated from a mitochondrial fraction of a strain of Lentinus edodes. pLLE1(11.0 kbp) was sensitive to the 3'----5'-acting exonuclease III and resistant to the 5'----3'-acting lambda exonuclease. It showed no homology with mitochondrial and nuclear genomic DNAs of plasmidless strain as well as the pLLE1-harboring host strain of L. edodes. The 1434-bp fragment (sequences) capable of autonomous replication in the yeast Saccharomyces cerevisiae (ARSs) was cloned from pLLE1 DNA with YIp32 (pBR322 containing yeast LEU2 DNA), which displayed a high ARS activity. The cloned 1434-bp fragment was shown to lie near to the end of pLLE1 DNA (nucleotides about 800-2200) and contained three consecutive ARS consensus sequences (A/T)TTTAT(A/G)TTT(A/T) of S. cerevisiae and dispersive eight ARS consensus-like sequences. The subcloned 366-bp fragment containing the three ARSs retained original ARS activity of the 1434-bp fragment.     

ARS binding factor I of the yeast Saccharomyces cerevisiae binds to sequences in telomeric and nontelomeric autonomously replicating sequences.

We have analyzed various autonomously replicating sequences (ARSs) in yeast nuclear extract with ARS-specific synthetic oligonucleotides. The EI oligonucleotide sequence, which is derived from HMRE-ARS, and the F1 oligonucleotide sequence, which is derived from telomeric ARS120, appeared to bind to the same cellular factor with high specificity. In addition, each of these oligonucleotides was a competitive inhibitor of the binding of the other. Binding of the ARS binding factor (ABF) to either of these oligonucleotides was inhibited strongly by plasmids containing ARS1 and telomeric TF1-ARS. DNase I footprinting analyses with yeast nuclear extract showed that EI and F1 oligonucleotides eliminated protection of the binding site of ARS binding factor I (ABFI) in domain B of ARS1. Sequence analyses of various telomeric (ARS120 and TF1-ARS) and nontelomeric ARSs (ARS1 and HMRE-ARS) showed the presence of consensus ABFI binding sites in the protein binding domains of all of these ARSs. Consequently, the ABFI and ABFI-like factors bind to these domain B-like sequences in a wide spectrum of ARSs, both telomeric and nontelomeric.     

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).     

DNA structures associated with autonomously replicating sequences from plants

DNA fragments capable of conferring autonomous replicating ability to plasmids inSaccharomyces cerevisiae were isolated from four different plant genomes and from the Ti plasmid ofAgrobacterium tumefaciens. The DNA structure of these autonomously replicating sequences (ARSs) as well as two from yeast were studied using retardation during polyacrylamide gel electrophoresis and computer analysis as measures of sequence-dependent DNA structures. Bent DNA was found to be associated with the ARS elements. An 11 bp ARS consensus sequence required for ARS function was also identified in the elements examined and was flanked by unusually straight structures which were rich in A+T content. These results show that the ARS elements from genomes of higher plants have structural and sequence features in common with ARS elements from yeast and higher animals.Supported by Grant 1RO1-GM41708-O1 from the National Institute of Health.     

Suppression of transgene silencing by matrix attachment regions in maize: a dual role for the maize 5' ADH1 matrix attachment region

Matrix attachment regions (MARs) are DNA sequences that bind an internal nuclear network of nonhistone proteins called the nuclear matrix. Thus, they may define discrete gene-containing chromatin loops in vivo. We have studied the effects of flanking transgenes with MARs on transgene expression levels in maize callus and in transformed maize plants. Three MAR elements, two from maize (Adh1 5' MAR and Mha1 5' MAR) and one from yeast (ARS1), had very different effects on transgene expression that bore no relation to their affinity for the nuclear matrix in vitro. In callus, two of the MAR elements (Adh1 5' MAR and ARS1) reduced transgene silencing but had no effect on the variability of expression. In transgenic plants, Adh1 5' MAR had the effect of localizing beta-glucuronidase expression to lateral root initiation sites. A possible model accounting for the function of Adh1 5' MAR is discussed.     

烟草MARs的分离及其功能分析

从烟草基因组中克隆到两条新的MAR片段（M14和M17）,序列分析表明,它们具有90%AT-box,A-box,T-box,碱基非配对区域,拓扑异构酶Ⅱ识别位点,弯曲DNA序列,复制起始序列和ATATTT等典型的MAR序列特征,并与原有MAR序列的特征不同。将它们分别构建到植物表达载体pCAMBIA2301 GUS基因（uidA）表达盒一侧及两侧,通过农杆菌介导转化烟草。组织化学染色法定性检测GUS活性表明,带有M14和M17的uidA基因在转基因烟草中稳定表达。GUS活性的定量检测表明,表达载体上uidA基因一端或两端连接有MAR的转化烟草中,GUS的表达水平与对照相比都有了明显提高,而uidA基因两侧连有MAR的载体提高表达水平的效果优于一端连有MAR的载体,可使GUS活性增强3.14倍,但不同转化个体之间表达水平的差异仍然明显。上述结果表明,所得DNA序列为两条新的MAR片段,并且具有提高转基因表达水平的功能。     

Isolation and characterization of an autonomously replicating sequence from Ustilago maydis.

DNA fragments that function as autonomously replicating sequences (ARSs) have been isolated from Ustilago maydis. When inserted into an integrative transforming vector, the fragments increased the frequency of U. maydis transformation several-thousandfold. ARS-containing plasmids were transmitted in U. maydis as extrachromosomal elements through replication. They were maintained at a level of about 25 copies per cell but were mitotically unstable. One ARS characterized in detail, which we called UARS1, was localized to a 1.7-kilobase fragment. UARS1 contained a cluster of active sequences. This element could be reduced further into three separate subfragments, each of which retained ARS activity. The smallest one was 383 base pairs (bp) long. Although not active itself in yeast, this small fragment contained seven 8-bp direct repeats, two contiguous 30-bp direct repeats, and five 11-bp units in both orientations with sequences similar but not identical to the consensus sequence found to be crucial for ARS activity in Saccharomyces cerevisiae.     

A yeast replication origin consists of multiple copies of a small conserved sequence

Autonomously replicating sequences (ARSs) of the yeast S. cerevisiae function as replication origins on plasmids and probably also on chromosomes. ARS function requires a copy of the ARS core consensus (5'-[A/T]TTTAT[A/G]TTT[A/T]-3') and additional sequences 3' to the T-rich strand of the consensus. Our analysis of an ARS from chromosome III, the C2G1 ARS, suggests that ARS function depends on the presence of an exact match to the core consensus and the presence of additional near matches in the 3' flanking region. We have demonstrated that ARS function can be mediated by multiple matches to the core consensus by constructing synthetic ARS elements from oligonucleotides containing copies of the consensus sequence. We find that two copies of the core consensus are sufficient for ARS activity and that an artificial ARS as efficient as a natural chromosomal ARS can be constructed from multiple core consensus elements in a specific orientation.     

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.     

Yeast centromeric plasmids as shuttle vectors between Escherichia coli, Bacillus subtilis and Saccharomyces cerevisiae

A number of hybrid plasmids which can autonomously replicate in E. coli, B. subtilis and S. cerevisiae was constructed. Replication of these plasmids both in yeast and in B. subtilis starts on a sequences originating from Staphylococcus aureus plasmids pC194 and pE194. In yeast these hybrids are unstable like those yeast vectors which contain eukaryotic ARSs, but their stability has been increased by addition of yeast centromeric sequence. Both pC194 and pE194 DNAs contain sequences which reveal strong similarities with the yeast ARS consensus. Nevertheless the replication efficiences of these plasmids in yeast are different.     

Isolation and characterization of sequences from mouse chromosomal DNA with ARS function in yeasts.

Fragments of chromosomal DNA from a variety of eucaryotes can act as ARSs (autonomously replicating sequence) in yeasts. ARSs enable plasmids to be maintained in extrachromosomal form, presumably because they function as initiation sites for DNA replication. We isolated eight different sequences from mouse chromosomal DNA which function as ARSs in Saccharomyces cerevisiae (bakers' yeast). Although the replication efficiency of the different mouse ARSs in yeasts appears to vary widely, about one-half of them functions as well as the yeast chromosomal sequence ARS1. Moreover, five of the ARSs also promote self replication of plasmids in Schizosaccharomyces pombe (fission yeast). Each of the ARSs was cloned into plasmids suitable for transformation of mouse tissue culture cells. Plasmids were introduced into thymidine kinase (TK)-deficient mouse L cells by the calcium phosphate precipitation technique in the absence of carrier DNA. In some experiments, the ARS plasmid contained the herpes simplex virus type 1 TK gene; in other experiments (cotransformations), the TK gene was carried on a separate plasmid used in the same transformation. In contrast to their behavior in yeasts, none of the ARS plasmids displayed a significant increase in transformation frequency in mouse cells compared with control plasmids. Moreover, only 1 of over 100 cell lines contained the original plasmid in extrachromosomal form. The majority of cell lines produced by transformation with an ARS TK plasmid contained multiple copies of plasmid integrated into chromosomal DNA. In most cases, results with plasmids used in cotransformations were similar to those for plasmids carrying TK. However, cell lines produced by cotransformations with plasmids containing any one of three of the ARSs (m24, m25, or m26) often contained extrachromosomal DNAs.     

Anatomy and dynamics of DNA replication fork movement in yeast telomeric regions

Replication initiation and replication fork movement in the subtelomeric and telomeric DNA of native Y' telomeres of yeast were analyzed using two-dimensional gel electrophoresis techniques. Replication origins (ARSs) at internal Y' elements were found to fire in early-mid-S phase, while ARSs at the terminal Y' elements were confirmed to fire late. An unfired Y' ARS, an inserted foreign (bacterial) sequence, and, as previously reported, telomeric DNA each were shown to impose a replication fork pause, and pausing is relieved by the Rrm3p helicase. The pause at telomeric sequence TG(1-3) repeats was stronger at the terminal tract than at the internal TG(1-3) sequences located between tandem Y' elements. We show that the telomeric replication fork pause associated with the terminal TG(1-3) tracts begins approximately 100 bp upstream of the telomeric repeat tract sequence. Telomeric pause strength was dependent upon telomere length per se and did not require the presence of a variety of factors implicated in telomere metabolism and/or known to cause telomere shortening. The telomeric replication fork pause was specific to yeast telomeric sequence and was independent of the Sir and Rif proteins, major known components of yeast telomeric heterochromatin.     

Prediction of Saccharomyces cerevisiae replication origins

Background  

Autonomously replicating sequences (ARSs) function as replication origins in Saccharomyces cerevisiae. ARSs contain the 17 bp ARS consensus sequence (ACS), which binds the origin recognition complex. The yeast genome contains more than 10,000 ACS matches, but there are only a few hundred origins, and little flanking sequence similarity has been found. Thus, identification of origins by sequence alone has not been possible.     

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.     

Replicator dominance in a eukaryotic chromosome.

Replicators are genetic elements that control initiation at an origin of DNA replication (ori). They were first identified in the yeast Saccharomyces cerevisiae as autonomously replicating sequences (ARSs) that confer on a plasmid the ability to replicate in the S phase of the cell cycle. The DNA sequences required for ARS function on a plasmid have been defined, but because many sequences that participate in ARS activity are not components of chromosomal replicators, a mutational analysis of the ARS1 replicator located on chromosome IV of S. cerevisiae was performed. The results of this analysis indicate that four DNA elements (A, B1, B2 and B3) are either essential or important for ori activation in the chromosome. In a yeast strain containing two closely spaced and identical copies of the ARS1 replicator in the chromosome, only one is active. The mechanism of replicator repression requires the essential A element of the active replicator. This element is the binding site for the origin recognition complex (ORC), a putative initiator protein. The process that determines which replicator is used, however, depends entirely upon flanking DNA sequences.     

Isolation of an autonomously replicating sequence (ARS) from satellite DNA of Drosophila melanogaster

Summary Autonomously replicating sequences (ARSs) were cloned from the 1.688 satellite DNA of D. melanogaster using YIp5, consisting of pBR322 and the yeast ura3 gene, as the cloning vector and YNN27, a Ura^– yeast strain as the recipient. Three out of six clones contained an ARS and the average frequncy of the occurrence of ARS was thus calculated to be approximately one per 14 kbp of the satellite DNA. A 500 bp ARS fragment (BgHS500) was obtained from one of the resultant clones (pYDS57). BgHS500 does not hybridize with the major repeating unit (370 bp) but it does with the minor unique sequence of the satellite. The sequence of BgHS500 was determined and found to be rich in AT and to contain the sequence, 5AAAACATAAAA3, a sequence common to yeast ARSs. However, a smaller fragment (150 bp) isolated from BgHS500 and containing the 11 bp sequence did not exhibit the characteristics of an ARS. The average copy number in the transformants of pBgHS500, a recombinant molecule of BgHS500 and YIp5, ranged from 0.05–0.5, while that of the parent plasmid, pYDS57, was about 2–10. On the basis of these results, it is postulated that the sequence 5AAAACATAAAA3 may possibly consiitute the core of ARSs and certain other sequences may also be necessary to insure that the ARS consistently undergoes at least one complete replication in each cell cycle. The role of ARSs in the genome of D. melanogaster is discussed.