Replication properties ofARS1 plasmids inSaccharomyces cerevisiae: dependence on the carbon source

The replication behaviour of a number ofARS1-based plasmids was investigated on propagation inSaccharomyces cerevisiae grown with either glucose or galactose as carbon source. Growth on galactose results in reduced plasmid stability, as well as in reduced replication efficiency, when the entire 1.5-kbTRP1-ARS1 fragment is present on a plasmid. The galactose sensitivity is mediated by a 0.13-kb fragment harbouring part of theGAL3 promoter. This fragment exerts its effect when situated either 5 or 3 to the ARS core consensus at distances up to 0.9 kb. The endogenous 2 µm plasmid remained unaffected by the choice of carbon source.     

Regulated yeast promoters produced by DNA rearrangements selected in vivo

DNA rearrangements that activated a promoterless his3 gene were selected in vivo. DNA segments that promote the expression of his3 were identified in Ty1 DNA sequences and a variety of sites in the vector DNA. These elements appear to function when placed in either orientation relative to his3 but not when placed at the 3′ end of the his3 gene. Promoter elements regulated by carbon source, nitrogen source, pyrimidines or galactose were characterized. The assembly of more complex regulatory elements and transposons from these units is discussed.     

Replication of the Streptomyces plasmid pSN22 through single-stranded intermediates

The replication of the 11 kb conjugative multicopy Streptomyces plasmid pSN22 was analyzed. Mutation and complementation analyses indicated that the minimal region essential for plasmid replication was located on a 1.9 kb fragment of pSN22, containing a trans-acting element encoding a replication protein and a cis-acting sequence acting as a replication origin. Southern hybridization showed that minimal replicon plasmids accumulated much more single-stranded plasmid molecules than did wild-type pSN22. Only one strand was accumulated. A 500 by fragment from the pSN22 transfer region was identified which reduced the relative amount of single-stranded DNA, when added in the native orientation to minimal replicon plasmids. This 500 by DNA sequence may be an origin for second-strand synthesis. It had no effect on the efficiency of co-transformation, plasmid incompatibility, or stability. The results indicate that pSN22 replicates via single-stranded intermediates by a rolling circle mechanism.     

Application of the resident plasmid integration technique to construct a strain of Streptococcus godronii able to express the Bacillus circulans cycloisomaltooligosaccharide glucanotransferase gene,and secrete its active gene product

A novel transformation technique, resident plasmid integration, for the cloning of foreign DNA in oral streptococci was described recently (T. Shiroza and H. K. Kuramitsu, Plasmid, 1995, 34, 85–95). This technique is based on the integration of linearized foreign genes by recombination-proficient bacteria onto a resident plasmid, if an appropriate selection marker is flanked by the same anchor sites present in the resident plasmid. Since the transforming vehicles for this system included a pUC-derived replication origin, the high level expression in Escherichia coli cells hindered the cloning of certain genes. In the present study, new plasmids were constructed, two resident plasmids, four integration plasmids, and four cloning plasmids, all of which possess the medium-copy number replication origin, p15A ori, isolated from pACYC177. The resident plasmids consisted of the following three components: the p15A ori (0.65-kb BglII fragment), the pVA380-1 basic replicon functional in mutans streptococci (2.5-kb BamHI fragment), and either an erythromycin resistance or a spectinomycin resistance gene (0.9- or 1.1-kb BamHI fragment, respectively). Most of the basic replicon of pVA380-1, except for the 3′-portion of the 0.2-kb region, in the resident plasmid was replaced with a kanamycin resistance gene to construct the four integration plasmids. Therefore, the upstream and downstream anchor sites for the double cross-over event in this new system were 0.65-kb p15A ori and the 0.2-kb portion of the 3′-end of pVA380-1 replicon, respectively. This system was used to clone the gene coding for cycloisomaltooligosaccharide glucanotransferase which produces cycloisomaltooligosaccharide, a potent inhibitor of oral streptococcal glucosyltransferase, isolated from Bacillus circulans chromosome, into Streptococcus gordonii, and its gene product was successfully secreted into the culture media. Plasmids described here should be useful tools for introducing heterologous DNA into resident plasmids following integration in oral streptococci.     

A Short Region from the LEU2 Gene of Saccharomyces cerevisiae Functions as an ARS in the Yeast Saccharomyces exiguus Yp74L-3

We examined the autonomously replicating sequence (ARS) activity of some fragments derived from the LEU2 region of Saccharomyces cerevisiae onto Saccharomyces exiguus Yp74L-3. A DNA fragment functioning as an ARS in S. exiguus, but not in S. cerevisiae, was shown to exist. The ARS activity for S. exiguus was reduced by the 2-μm plasmid origin of S. cerevisiae when both elements coexisted on a single circular plasmid. Analysis of ARS activity with the PCR products from the fragment revealed that the ARS-acting sequence was located in the 3′-terminal area of the transcribed region of the LEU2 gene of S. cerevisiae. It is suggested that the ARS recognition system in S. exiguus is significantly different from that of S. cerevisiae. Received: 16 June 1998 / Accepted: 3 August 1998     

Novel Alterations in Plasmid DNA Associated with Aromatic Hydrocarbon Utilization by Pseudomonas putida R5-3

Subcultures of Pseudomonas putida R5-3 altered their plasmid DNA content in specific ways depending on the particular aromatic hydrocarbon utilized as the sole carbon source. Two indigenous plasmids, 115 and 95 kilobases (kb) in size, were observed in R5-3A, which was derived from R5-3 by growth on minimal medium containing p-methylbenzoate as the sole carbon source. When R5-3A was transferred to medium containing m-xylene or toluene, derivative strains were obtained in which the 95-kb plasmid was lost and a new plasmid of 50 or 60 kb appeared. Reversion to the original plasmid profile of R5-3A was observed when xylene- or toluene-grown cells were returned to medium containing p-methylbenzoate. Restriction enzyme analysis and Southern blot hybridizations of total plasmid DNA indicated deletions and rearrangements of DNA restriction fragments in the derivatives maintained on m-xylene and toluene when compared with the original R5-3A. In the derivatives which retrieved the original plasmid profile, the restriction enzyme fragment pattern was identical to that in the original R5-3A, in that the fragments which were missing after growth on m-xylene or toluene were again present. Southern blot hybridizations revealed that part of the plasmid DNA lost from the original plasmid profile was integrated into the chromosomal DNA of xylene-grown R5-3B and that these plasmid fragments were associated with aromatic hydrocarbon metabolism. Hybridization with pathway-specific DNA fragments from the TOL plasmid pWWO indicated that this 95-kb plasmid contains DNA homologous to the meta-fission pathway genes.     

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

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.     

Bi-directional replication and random termination

Two-dimensional (2D) agarose gel electrophoresis was used to study termination of DNA replication in a shuttle vector, YRp7′, when it replicated in Escherichia coli, Saccharomyces cerevisiae and Xenopus egg extracts. In E.coli, the 2D gel patterns obtained were consistent with uni-directional replication initiated at a specific site, the ColE1 origin. In consequence, termination also occurred precisely at the ColE1 origin. In Xenopus egg extracts, the particular shape of the bubble arc as well as the triangular smear detected to the left of the simple-Y pattern indicated random initiation and termination. In S.cerevisiae, initiation occurred at the ARS1 origin and replication proceeded in a bi-directional manner. However, termination did not always occur at a specific site 180° across from the origin, but almost all along the south hemisphere of the plasmid. Inversion, deletion or replacement of DNA sequences located throughout this hemisphere did not eliminate random termination. Analysis of the replication intermediates of another yeast plasmid bearing a different origin, ARS305, also exhibited random termination. We propose that the random termination events observed in S.cerevisiae could be due to an asynchronous departure of both forks from the bi-directional origin in addition to differences in the rate of fork progression. These observations could be extended to all bi-directional origins.     

Construction of a host-vector system in the osmophilic haploid yeast Zygosaccharomyces rouxii

A host-vector system for Zygosaccharomyces rouxii was developed. Chimeric plasmids useful as the Escherichia coli-Z. rouxii shuttle vector were constructed with a DNA fragment of pBR322, a fragment of pSR1 plasmid of Z. rouxii or the ARS1 sequence of Saccharomyces cerevisiae, and a fragment of the LEU2 gene of S. cerevisiae or a DNA fragment bearing Tn601 which confers G-418 resistance as a selective marker of the plasmid. For the hosts, wild-type strains of Z. rouxii were modified to give improved transformation frequencies or to mark a leucine-auxotrophic mutation which is complementable by the LEU2 DNA of S. cerevisiae. Transformation frequencies from several hundred to two thousand transformant clones per μg plasmid DNA samples were obtained.     

Influence of plasmid origin and promoter strength in fermentations of recombinant yeast

The effects of plasmid promoter strength and origin of replication on cloned gene expression in recombinant Saccharomyces cerevisiae have been studied in batch and continuous culture. The plasmids employed contain the Escherichia coli lacZ gene under the control of yeast promoters regulated by the galactose regulatory circuit. The synthesis of beta-galactosidase was therefore induced by the addition of galactose. The initial induction transients in batch culture were compared for strains containing plasmids with 2mu and ARS1 origins. As expected, cloned gene product synthesis was much lower with the ARS1 plasmid: average beta-galactosidase specific activity was an order of magnitude below that with the 2mu-based plasmid. This was primarily due to the low plasmid stability of 7.5% when the plasmid origin of replication was the ARS1 element. The influence of plasmid promoter strength was studied using the yeast GAL1, GAL10, and hybrid GAL10-CYC1 promoters. The rate of increase in beta-galactosidase specific activity after induction in batch culture was 3-5 times higher with the GAL1 promoter. Growth rate under induced conditions, however, was 15% lower than in the absence of lacZ expression for this promoter system. The influence of plasmid promoter strength on induction behavior and cloned gene expression was also studied in continuous fermentations. Higher beta-galactosidase production and lower biomass concentration and plasmid stability were observed for the strain bearing the plasmid with the stronger GAL1 promoter. Despite the decrease in biomass concentration and plasmid stability, overall productivity in continuous culture using the GAL1 promoter was three times that obtained with the GAL10-CYC1 promoter.     

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     

The Rep20 Replication Initiator From the pAG20 Plasmid of Acetobacter aceti

In the previously isolated pAG20 plasmid from the Acetobacter aceti CCM3610 strain, the Rep20 protein was characterized as a main replication initiator. The pAG20 plasmid origin was localized in the vicinity of the rep20 gene and contained two 21-nucleotide-long iteron sequences, two 13-nucleotide-long direct repeats, and a DnaA-binding site. Electrophoretic mobility shift assay and nonradioactive fragment analysis confirmed that the Rep20 protein interacted with two direct repeats (5′-TCCAAATTTGGAT′-3′) and their requirement during plasmid replication was verified by mutagenesis. Although the association could not be validated of the DnaA protein of from the host cells of Escherichia coli with the plasmid-encoded replication initiator that usually occurs during replication initiation, Rep20 was able to form dimeric structures by which it could bind the sequence of the rep20 gene and autoregulate its own expression. Targeted mutagenesis of the Rep20 protein revealed the importance of the third α-helix and ⁶³Lys, specifically during DNA binding. The second, closely adjacent β-sheet also took part in this process in which ⁵²Asn played a significant role.     

Fermentation of lactose by Zymomonas mobilis carrying a Lac+ recombinant plasmid

Lac⁺ recombinant plasmids encoding a β-galactosidase fused protein and lactose permease of Escherichia coli were introduced Zymomonas mobilis. The fused protein was expressed with 450 to 5,860 Miller units of β-galactosidase activity, and functioned as lactase. Raffinose uptake by Z. mobilis CP4 was enhanced in the plasmid-carrying strain over the plasmid-free strain, suggesting that the lactose permease was functioning in the organism. Z. mobilis carrying the plasmid could produce ethanol from lactose and whey, but could not grow on lactose as the sole carbon source. It was found that the growth of the organism was inhibited by either galactose of the galactose liberated from lactose.     

Isolation of replicational cue elements from a library of bent DNAs of Aspergillus oryzae

Two fragments that could function as replicational cue elements were isolated from a genomic DNA digest of Aspergillus oryzae on the basis of abnormal behavior in polyacrylamide gel electrophoresis. The vector used in this study contained a scaffold-associated region of the Drosophila melanogaster ftz gene to provide nuclear retention. Neither fragment contained a yeast ARS consensus sequence or an eukaryotic topoisomerase II binding sequence. One of the fragments showed sequence homology with the mitochondrial replication origin of Candida utilis and a portion of mitochondrial DNA of Aspergillus nidulans. This plasmid carrying the cue fragment could also replicate in HeLa and NIH3T3 cells.     

Replication of plasmids in mutants of Escherichia coli temperature-sensitive for initiation of deoxyribonucleic acid synthesis

Plasmid deoxyribonucleic acid (DNA) replication was studied in Escherichia coli hosts carrying temperature-sensitive (ts) initiation mutations. The replication of the R plasmid NR1 continues at the nonpermissive temperature in a ts dnaA mutant host but at a decreasing rate in proportion to the residual chromosome synthesis. The replication of NR1, as well as of the F plasmid F′lac, ceases immediately at the nonpermissive temperature in a ts dnaC mutant host. The ability to reinitiate R plasmid replication in the absence of protein or ribonucleic acid synthesis is accumulated at the nonpermissive temperature in a dnaC mutant host.     

Isolation of mutant promoters in the Escherichia coli galactose operon using local mutagenesis on cloned DNA fragments

We have worked out a system to obtain mutations that map in the promoter region of the Escherichia coli galactose operon. In order to easily detect small changes in gal promoter activity, we constructed a plasmid containing an operon fusion in which the lactose operon structural genes were controlled by the galactose operon promoter region. In cells harbouring this plasmid, even modest variations in the expression of the lac genes could be detected on MacConkey lactose indicator plates.Enrichment for mutations that map in the promoter segment of the galactose operon was achieved by mutagenesis in vitro of a small fragment of DNA covering the promoter region. After insertion of the mutagenized gal promoter fragment into the gal-lac fusion plasmid, lac^?1 cells were transformed and screened for an altered Lac⁺ phenotype on indicator plates. Several mutants were isolated due to lesions mapping in the small fragment covering the galactose promoter. In these mutants, the level of β-galactosidase was between 15 and 50% of the wild-type level.The mutant promoters were subsequently reinserted into a plasmid containing the intact galactose operon. Cells harbouring such plasmids, reconstituted with mutant galactose promoters, contained decreased levels of galactokinase that paralleled the decreases in β-galactosidase. The biochemical properties of these mutants are reported in the accompanying paper (Busby et al., 1982).     

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.