Identification of an essential core element and stimulatory sequences in a Kluyveromyces lactis ARS element, KARS101

A Kluyveromyces lactis chromosomal sequence of 913 bp is sufficient for replication in Saccharomyces cerevisiae and K. lactis . This fragment contains a 12 bp sequence 5'-ATTTATTGTTTT-3' that is related to the S. cerevisiae ACS (ARS consensus sequence). This dodecamer was removed by site-directed mutagenesis and the effect on K. lactis and S. cerevisiae ARS (autonomous replicating sequence) activity was determined. The dodecamer is essential for S. cerevisiae ARS function but only contributes to K. lactis ARS activity; therefore, its role in K. lactis is unlikely to be the same as that of the essential S. cerevisiae ACS.
A 103 bp subclone was found to retain ARS activity in both yeasts, but the plasmid was very unstable in S. cerevisiae . Deletion and linker substitution mutagenesis of this fragment was undertaken to define the DNA sequence required for K. lactis ARS function and to test whether the sequence required for ARS activity in K. lactis and S. cerevisiae coincide. We found a 39 bp core region essential for K. lactis ARS function flanked by sequences that contribute to ARS efficiency. The instability of the plasmid in S. cerevisiae made a fine-structure analysis of the S. cerevisiae ARS element impossible. However, the sequences that promote high-frequency transformation in S. cerevisiae overlap the essential core of the K. lactis ARS element but have different end-points.     

Genetic analysis of an ARS element from the fission yeast Schizosaccharomyces pombe.

ARS (autonomously replicating sequence) elements are DNA fragments that can function as origins of DNA replication in yeast. We report the first fine-structure analysis of ars1, an ARS element of the fission yeast Schizosaccharomyces pombe. Characterization of a series of nested deletion mutations indicated that the minimal fragment of DNA encompassing ars1 is surprisingly large. No fragment < 650 bp retained significant ARS activity. Analysis of deletion and substitution mutations scanning the entire minimal ars1 identified a single essential 50 bp fragment (segment 1). Only one other 50 bp mutation reduced activity as much as 5-fold and most deletions were without effect. Thus, the minimal ars1 is composed of two general types of genetic elements, a small segment that is absolutely required for efficient ARS activity and a much larger region that is tolerant of internal structural alterations. Higher resolution analysis of segment 1 defined a critical 30 bp A/T-rich segment which appears to contain redundant genetic elements. Schizosaccharomyces pombe ars1 promoted high frequency transformation in the budding yeast S.cerevisiae but this heterologous activity was not dependent on segment 1. Our analysis indicates that the functional elements required for ARS function in S.pombe and S.cerevisiae are clearly different.     

Construction of a host-vector system in Candida maltosa by using an ARS site isolated from its genome.

To construct a host-vector system in an n-alkane-assimilating yeast, Candida maltosa, the isolation of an ARS site from its genome which replicates autonomously in C. maltosa was attempted. Leu- mutants of C. maltosa were transformed with a gene library prepared by using YEp13 (LEU2+) as a vector, and Leu+ transformants were obtained at a high frequency. A plasmid named pCS1 was isolated from the recipient cells. pCS1 contained a 6.3-kilobase (kb) fragment of the C. maltosa genome, and a 3.8-kb fragment with ARS activity was subcloned and designated the TRA (transformation ability) region. Vectors (pTRA1 and pTRA11) for C. maltosa J288 were constructed that contained this 3.8-kb fragment, pBR322, and the LEU2 gene of Saccharomyces cerevisiae. Transformation of C. maltosa J288 with these plasmids was successful by both spheroplast and lithium acetate methods. Southern blot analysis suggested that the copy number of pTRA1 in C. maltosa was between 10 and 20, and it was stably maintained during growth without selective pressure in the medium. It was also found that these vectors could transform S. cerevisiae leu2- to LEU2+, suggesting that the TRA region contained an ARS site(s) that was specific not only for C. maltosa but also for S. cerevisiae.     

Cloning and expression of a Saccharomyces diastaticus glucoamylase gene in Saccharomyces cerevisiae and Schizosaccharomyces pombe.

A recombinant plasmid pool of the Saccharomyces diastaticus genome was constructed in plasmid YEp13 and used to transform a strain of Saccharomyces cerevisiae. Six transformants were obtained which expressed amylolytic activity. The plasmids each contained a 3.9-kilobase (kb) BamHI fragment, and all of these fragments were cloned in the same orientations and had identical restriction maps, which differed from the map of the STA1 gene (I. Yamashita and S. Fukui, Agric. Biol. Chem. 47:2689-2692, 1983). The glucoamylase activity exhibited by all S. cerevisiae transformants was approximately 100 times less than that of the donor strain. An even lower level of activity was obtained when the recombinant plasmid was introduced into Schizosaccharomyces pombe. No expression was observed in Escherichia coli. The 3.9-kb BamHI fragment hybridized to two sequences (4.4 and 3.9 kb) in BamHI-digested S. diastaticus DNA, regardless of which DEX (STA) gene S. diastaticus contained, and one sequence (3.9 kb) in BamHI-digested S. cerevisiae DNA. Tetrad analysis of crosses involving untransformed S. cerevisiae and S. diastaticus indicated that the 4.4-kb homologous sequence cosegregated with the glucoamylase activity, whereas the 3.9-kb fragment was present in each of the meiotic products. Poly(A)+ RNA fractions from vegetative and sporulating diploid cultures of S. cerevisiae and S. diastaticus were probed with the 3.9-kb BamHI fragment. Two RNA species, measuring 2.1 and 1.5 kb, were found in both the vegetative and sporulating cultures of S. diastaticus, whereas one 1.5-kb species was present only in the RNA from sporulating cultures of S. cerevisiae.     

The aryldialkylphosphatase-encoding gene adpB from Nocardia sp. strain B-1: cloning, sequencing and expression in Escherichia coli.

Using degenerate oligodeoxyribonucleotides (oligos) derived from the N-terminal sequence of an aryldialkylphosphatase (ADPase) from Nocardia sp. strain B-1, an amplification reaction was used to isolate a DNA segment containing a 57-bp fragment from the adpB gene. Based on the nucleotide (nt) sequence of this fragment, a nondegenerate oligo was synthesized and used to screen a subgenomic library of strain B-1 DNA for fragments containing adpB. A 3.55-kb PstI fragment containing adpB was cloned into Escherichia coli, and the nt sequence of a 1600-bp region containing adpB was determined. Under control of the lac promoter of pUC19, adpB expression in E. coli cultures was approx. 15-fold higher than in strain B-1 under the native adpB promoter. Comparison of adpB with the Flavobacterium ADPase-encoding gene, opd, revealed no significant homology at the nt or aa levels.     

An autonomously replicating sequence of pSRI plasmid is effective in two yeast species, Zygosaccharomyces rouxii and Saccharomyces cerevisiae

The autonomously replicating sequences (ARSs) of pSR1, a cryptic circular DNA plasmid detected in a strain of Zygosaccharomyces rouxii, were delimited by subcloning and deletion analysis and by the isolation of nucleotide substitution mutations. A 30 base-pair (bp) sequence from inverted repeat 1 (IR1) and presumably the same region from IR2 of pSR1 functions as an ARS in the native host, Z. rouxii, and in a heterologous host, Saccharomyces cerevisiae. Thus, pSR1 has two ARSs per molecule, either of which is sufficient for replication of the plasmid molecule in both hosts. These hosts, however, respond differently to nucleotide substitutions in the 30 bp sequence, suggesting that the sequences required for ARS function in the two organisms are not exactly the same. In addition, a 137 bp sequence that overlaps the 30 bp sequence by 11 bp also functions as an ARS in Z. rouxii but not in S. cerevisiae. However, this 137 bp sequence enhances the stability of plasmids carrying the pSR1 ARS in S. cerevisiae. The 30 bp and 137 bp sequences each contain a single copy of the 11 bp ARS consensus sequence, which is essential for ARS function in S. cerevisiae. Small insertions between the 11 bp overlapping region and the 11 bp ARS consensus sequence showed that a proper distance between these two 11 bp sequences is essential for the ARS function of the 30 bp sequence. Point mutations that inactivate ARS function show that the ARS consensus sequence, as well as a short A:T segment in the overlapping sequence, is required for the ARS function of the 30 bp sequence.     

Development of a transformation system for the flavinogenic yeast Candida famata

Riboflavin-overproducing mutants of the flavinogenic yeast Candida famata are used for industrial riboflavin production. This paper describes the development of an efficient transformation system for this species. Leucine-deficient mutants have been isolated from C. famata VKM Y-9 wild-type strain. Among them leu2 mutants were identified by transformation to leucine prototrophy with plasmids YEp13 and PRpL2 carrying the Saccharomyces cerevisiae LEU2 gene. DNA fragments (called CfARSs) conferring increased transformation frequencies and extrachromosomal replication were isolated from a C. famata gene library constructed on the integrative vector containing the S. cerevisiae LEU2 gene as a selective marker. The smallest cloned fragment (CfARS16) has been sequenced. This one had high adenine plus thymine (A+T) base pair content and a sequence homologous to the S. cerevisiae ARS Consensus Sequence. Methods for spheroplast transformation and electrotransformation of the yeast C. famata were optimized. They conferred high transformation frequencies (up to 10(5) transformants per microg DNA) with a C. famata leu2 mutant using replicative plasmids containing the S. cerevisiae LEU2 gene as a selective marker. Riboflavin-deficient mutants were isolated from the C. famata leu2 strain and their biochemical identification was carried out. Using the developed transformation system, several C. famata genomic fragments complementing mutations of structural genes for riboflavin biosynthesis (coding for GTP cyclohydrolase, reductase, dihydroxybutanone phosphate synthase and riboflavin synthase, respectively) have been cloned.     

A reorganized Candida albicans DNA sequence promoting homologous non-integrative genetic transformation

In order to develop plasmids adequate for non-integrative genetic transformation of Candida albicans, a DNA fragment of 15.3 kb was cloned from this organism on the basis of its capacity to convert the integrative Saccharomyces cerevisiae vector YIp5 into a non-integrative one. Southern hybridization analysis, carried out with a labelled DNA probe of 3.6 kb derived from the cloned fragment, showed that it consisted of C. albicans DNA, the hybridization pattern indicating that the corresponding sequences were homologous to several chromosomal regions. The size of the C. albicans DNA promoting autonomous replication in S. cerevisiae was substantially reduced by subcloning. A 5.1 kb subfragment, defined by BamHI and SalI restriction sites, retained autonomous replication sequences (ARS) functional in the heterologous S. cerevisiae system and in C. albicans, when inserted in plasmid constructions that carried a S. cerevisiae trichodermin-resistance gene (tcm1) as selection marker. C. albicans transformants were both of the integrative and the non-integrative type and the plasmids recovered from the latter very often carried a reorganized ARS, indicating that recombination of the inserted ARS DNA had occurred in the homologous host. Successive reorganizations of the ARS insert in C. albicans eventually led to a more stable and much smaller fragment of 687 bp that was subsequently recovered unchanged from transformants. Sequence analysis of the 687 bp fragment revealed four 11-base blocks, rich in A+T, that carried the essential consensus sequence considered relevant for yeast ARS elements in addition to other features also described as characteristic of yeast replication origins.     

Identification of the sequences required for chromosomal replicator function in Kluyveromyces lactis

The analysis of replication intermediates of a Kluyveromyces lactis chromosomal autonomous replicating sequence (ARS), KARS101, has shown that it is active as a chromosomal replicator. KARS101 contains a 50 bp sequence conserved in two other K. lactis ARS elements. The deletion of the conserved sequence in KARS101 completely abolished replicator activity, in both the plasmids and the chromosome. Gel shift assays indicated that this sequence binds proteins present in K. lactis nuclear extracts, and a 40 bp sequence, previously defined as the core essential for K. lactis ARS function, is required for efficient binding. Reminiscent of the origin replication complex (ORC), the binding appears to be ATP dependent. A similar pattern of protection of the core was seen with in vitro footprinting. KARS101 also functions as an ARS sequence in Saccharomyces cerevisiae. A comparative study using S. cerevisiae nuclear extracts revealed that the sequence required for binding is a dodecanucleotide related to the S. cerevisiae ARS consensus sequence and essential for S. cerevisiae ARS activity.     

Sequence analysis of ARS elements in fission yeast.

Chromosomal DNA of Schizosaccharomyces pombe contains sequences with properties analogous to ARS elements of Saccharomyces cerevisiae. Following Sau3A fragmentation of the S. pombe genome we have recovered a number of such fragments in an M13-based shuttle vector, suitable for subsequent sequence analysis. The complete nucleotide sequence has been obtained for eight ARS+ inserts derived from the Sau3A cloning and for the ARS present in pFL20 isolated previously by Losson and Lacroute (Cell, 32, 371-377, 1983). The Sau3A clones are single fragments between 0.8 and 1.8 kb. No ARS+ clones smaller than this were recovered even though the average size Sau3A fragment in S. pombe is approximately 200-300 bp. The sequence analysis revealed that all clones are AT-rich (69-75% A + T residues), and all contain a particularly AT-rich 11 bp core element represented by the consensus sequence 5' (A/T)PuTT-TATTTA(A/T) 3'. Deletion mapping indicates that the consensus in all cases is in the vicinity of a functional ARS domain. However precise excision of the consensus by in vitro mutagenesis has little effect on ARS activity as judged by the transformation assay. We argue that the association of the consensus with the ARS domain occurs too reproducibly to be explained by chance alone. We suggest that although it may not be essential for the extrachromosomal maintenance of plasmids in S. pombe, the consensus does have a function in situ in the chromosome and thus is always present as a cryptic sequence in the isolated ARS element.     

Genetic complementation of the Saccharomyces cerevisiae leu2 gene by the Escherichia coli leuB gene.

The leucine operon of Escherichia coli was cloned on a plasmid possessing both E. coli and Saccharomyces cerevisiae replication origins. This plasmid, pEH25, transformed leuA, leuB, and leuD auxotrophs of E. coli to prototrophy; it also transformed leu2 auxotrophs of S. cerevisiae to prototrophy. beta-Isopropylmalate dehydrogenase was encoded by the leuB gene of E. coli and the leu2 gene of yeast. Verification that the leuB gene present on pEH26 was responsible for complementing yeast leu2 was obtained by isolating in E. coli several leuB mutations that resided on the plasmid. These mutant leuB- plasmids were no longer capable of complementing leu2 in S. cerevisiae. We conclude that S. cerevisiae is capable of transcribing at least a portion of the polycistronic leu operon of E. coli and can translate a functional protein from at least the second gene of this operon. The yeast Leu+ transformants obtained with pEH25, when cultured in minimal medium lacking leucine, grew with a doubling time three to four times longer than when cultured in medium supplemented with leucine.     

DNA sequence analysis of ARS elements from chromosome III of Saccharomyces cerevisiae: identification of a new conserved sequence.

Four fragments of Saccharomyces cerevisiae chromosome III DNA which carry ARS elements have been sequenced. Each fragment contains multiple copies of sequences that have at least 10 out of 11 bases of homology to a previously reported 11 bp core consensus sequence. A survey of these new ARS sequences and previously reported sequences revealed the presence of an additional 11 bp conserved element located on the 3' side of the T-rich strand of the core consensus. Subcloning analysis as well as deletion and transposon insertion mutagenesis of ARS fragments support a role for 3' conserved sequence in promoting ARS activity.     

Deletion mutations affecting autonomously replicating sequence ARS1 of Saccharomyces cerevisiae.

DNAs that contain specific yeast chromosomal sequences called ARSs transform Saccharomyces cerevisiae at high frequency and can replicate extrachromosomally as plasmids when introduced into S. cerevisiae by transformation. To determine the boundaries of the minimal sequences required for autonomous replication in S. cerevisiae, we have carried out in vitro mutagenesis of the first chromosomal ARS described, ARS1. Rather than identifying a distinct and continuous segment that mediates the ARS+ phenotype, we find three different functional domains within ARS1. We define domain A as the 11-base-pair (bp) sequence that is also found at most other ARS regions. It is necessary but not sufficient for high-frequency transformation. Domain B, which cannot mediate high-frequency transformation, or replicate by itself, is required for efficient, stable replication of plasmids containing domain A. Domain B, as we define it, is continuous with domain A in ARS1, but insertions of 4 bp between the two do not affect replication. The extent of domain B has an upper limit of 109 bp and a lower limit of 46 bp in size. There is no obvious sequence homology between domain B of ARS1 and any other ARS sequence. Finally, domain C is defined on the basis of our deletions as at least 200 bp flanking domain A on the opposite side from domain B and is also required for the stability of domain A in S. cerevisiae. The effect of deletions of domain C can be observed only in the absence of domain B, at least by the assays used in the current study, and the significance of this finding is discussed.     

The Saccharomyces cerevisiae chromosome III left telomere has a type X, but not a type Y'', ARS region.

A yeast Saccharomyces cerevisiae telomeric region was isolated by chromosome walking from HML alpha, the most distal known gene on the chromosome III left (IIIL) end. The terminal heterodisperse 3.3-kilobase (kb) SalI fragment on chromosome IIIL, 8.6 kb distal to HML alpha, was cloned in a circular vector to generate a telomeric probe. Southern hybridization and DNA sequencing analyses indicated that 0.6 kb (+/- 200 base pairs) of 5'-C1-3A-3' simple tandem repeat sequence, adjacent to a 1.2-kb type X ARS region, constitutes the telomere on the chromosome IIIL end, and no type Y' ARS region homologies exist between HML alpha and the IIIL terminus.     

Cloning of Lentinus edodes mitochondrial DNA fragment capable of autonomous replication in Saccharomyces cerevisiae

Mitochondrial (mt) DNA of the higher basidiomycetes Lentinus edodes with a molecular weight of about 69 kb was partially digested with Sau3AI, cloned with plasmid YIp32 (a hybrid of pBR322 and the yeast leu2 gene) and analyzed for sequences capable of autonomous replication (ARSs) in the eukaryote Saccharomyces cerevisiae. One recombinant plasmid was isolated which contained 3.2 kb fragment of the mtDNA with ARS activity. This plasmid (named pSK52) exhibited a high-frequency yeast transformation and was found to be maintained within the cell as an extrachromosomal element. The stability and copy number properties of pSK52 were similar to those of the recombinant plasmid of YIp32 and S. cerevisiae mt-ARS constructed as a reference. Subcloning experiments were carried out to assess the localization of ARS on the above 3.2 kb fragment, revealing that the fragment contains at least two ARSs.     

Characterization of PDR4, a Saccharomyces cerevisiae gene that confers pleiotropic drug resistance in high-copy number: identity with YAP1, encoding a transcriptional activator [corrected

PDR4 is a gene that confers pleiotropic drug resistance (pdr) to the yeast Saccharomyces cerevisiae when present in high copy number [Leppert et al., Genetics 125 (1990) 13-20]. Transposon insertion mutations had identified the active region of the gene as a 3.7-kb SalI-EcoRI restriction fragment of the 8-kb cloned fragment. We have confirmed this by showing that this fragment is sufficient to confer pdr, and have sequenced its entire 3761 bp. It contains a single complete open reading frame (ORF) extending from nucleotide (nt) position 1631-3580, coding for a protein of 650 amino acids (aa). A 2.7-kb fragment containing this ORF is also sufficient to confer pdr. The aa sequence contains no recognizable homologies or consensus sequences, so it is a novel protein of unknown function. It is apparently soluble, since no transmembrane-type sequences were predicted. A second, partial ORF was also found, on the opposite strand, extending from nt position 774 to past the SalI site, which is apparently unrelated to pdr.