A DNA fragment containing the ADE2 gene from Schwanniomyces occidentalis can be maintained as an extrachromosomal element

A 4.05-kb DNA fragment containing the ADE2 gene from Schwanniomyces occidentalis was cloned into the pUC19 vector. When an ade2 strain of Sc. occidentalis was transformed with this plasmid, pADE-2 was found to integrate into the host chromosome and was also present in a variety of extrachromosomal species. These extrachromosomal elements were present in multiple copies, varied in molecular mass and were composed of polymerized forms of pADE-2. A fragment containing the ADE2 gene was used to transform a Sc. occidentalis ade2 mutant, as either a linear or circularized molecule. The linear form integrated into the host genome, whereas the circularized form was found as a stably maintained extrachromosomal element with no evidence of integration or detectable loss of the Ade+ phenotype upon subculturing of transformed yeast under nonselective conditions for 60 generations. The ratio of the number of extrachromosomal ADE2 genes to genomic ADE2 ranged from 3.8 to 6.6.     

Cloning and Expression of a Schwanniomyces occidentalis alpha-Amylase Gene in Saccharomyces cerevisiae

An alpha-amylase gene (AMY) was cloned from Schwanniomyces occidentalis CCRC 21164 into Saccharomyces cerevisiae AH22 by inserting Sau3AI-generated DNA fragments into the BamHI site of YEp16. The 5-kilobase insert was shown to direct the synthesis of alpha-amylase. After subclones containing various lengths of restricted fragments were screened, a 3.4-kilobase fragment of the donor strain DNA was found to be sufficient for alpha-amylase synthesis. The concentration of alpha-amylase in culture broth produced by the S. cerevisiae transformants was about 1.5 times higher than that of the gene donor strain. The secreted alpha-amylase was shown to be indistinguishable from that of Schwanniomyces occidentalis on the basis of molecular weight and enzyme properties.     

Molecular cloning of eucaryotic genes required for excision repair of UV-irradiated DNA: isolation and partial characterization of the RAD3 gene of Saccharomyces cerevisiae.

We describe the molecular cloning of a 6-kilobase (kb) fragment of yeast chromosomal DNA containing the RAD3 gene of Saccharomyces cerevisiae. When present in the autonomously replicating yeast cloning vector YEp24, this fragment transformed two different UV-sensitive, excision repair-defective rad3 mutants of S. cerevisiae to UV resistance. The same result was obtained with a variety of other plasmids containing a 4.5-kb subclone of the 6-kb fragment. The UV sensitivity of mutants defective in the RAD1, RAD2, RAD4, and RAD14 loci was not affected by transformation with these plasmids. The 4.5-kb fragment was subcloned into the integrating yeast vector YIp5, and the resultant plasmid was used to transform the rad3-1 mutant to UV resistance. Both genetic and physical studies showed that this plasmid integrated by homologous recombination into the rad3 site uniquely. We conclude from these studies that the cloned DNA that transforms the rad3-1 mutant to UV resistance contains the yeast chromosomal RAD3 gene. The 4.5-kb fragment was mapped by restriction analysis, and studies on some of the subclones generated from this fragment indicate that the RAD3 gene is at least 1.5 kb in size.     

Transformation system for Schwanniomyces occidentalis

Spheroplasts of Schwanniomyces occidentalis were used for a transformation system. A putative leu2 mutant of S. occidentalis was complemented with the LEU2 gene (YEp13) from Saccharomyces cerevisiae. The transformation efficiency was 10 3 transformants/mg DNA. Although low stability was obtained, YEp13 could be recovered from transformants and kept the same size and restriction enzyme cutting sites like the original one. The replicon of 2 mm plasmid is responsible for the replication of YEp13 in S. occidentalis.     

Integrative transformation of Candida albicans, using a cloned Candida ADE2 gene.

Candida albicans is a diploid dimorphic yeast with no known sexual cycle. The development of a DNA transformation system would greatly improve the prospects for genetic analyses of this yeast. Plasmids were isolated from a Candida Sau3A partial library which complements the ade2-1 and ade2-5 mutations in Saccharomyces cerevisiae. These plasmids contain a common region, part of which, when subcloned, produces ade2 complementation. Among the small number of auxotrophs previously isolated in C. albicans, red adenine-requiring mutants had been identified by several groups. In two of these strains, the cloned Candida DNA transformed the mutants to ADE+ at frequencies of 0.5 to 5 transformants per micrograms of DNA. In about 50% of the transformants, plasmid DNA sequences became stably integrated into the host genome and, in the several cases analyzed by Southern hybridization, the DNA was integrated at the site of the ADE2 gene in one of the chromosomal homologs.     

Cloning of the ADE2 gene of Saccharomyces cerevisiae and localization of the ARS-sequence

Mutational changes in ADE2 result in the accumulation of red pigment in cells, which serves as an indicator for the selection of mutants. This easily detectable phenotype of red-coloured colonies can account for the wide use of ade2 mutants in yeast genetics. ADE2 gene was cloned in a shuttle vector by complementing the ade2 mutation in the yeast. It was shown that the 2.2 kbp HindIII fragment of yeast DNA contains structural sequences of the ADE2 gene as well as the ARS sequence. Deletion analysis of the 5' end of the ADE2 gene showed the ARS sequence to be situated at the distal end of the 1 kbp HindIII fragment. Removal of the ARS sequence does not influence ADE2 gene complementation ability. Transformants containing the ADE2 gene comprised in their plasmids form white colonies. Loss of the plasmids results in colour change of colonies.     

Isolation of the ARO1 cluster gene of Saccharomyces cerevisiae.

The AROl cluster gene was isolated by complementation in Saccharomyces cerevisiae after transformation with a comprehensive yeast DNA library of BamHI restriction fragments inserted into the shuttle vector YEp13. Most of the transformants exhibited the expected episomal inheritance of the ARO+ phenotype; however, one stable transformant has been shown to be an integration of the AROl fragment and the vector YEp13 at the arol locus. The insert containing AROl is a 17.2-kilobase pair (kbp) BamHI fragment which complements both nonsense and missense alleles of arol. Subcloning by Sau3AI partial digestion further locates the AROl segment to a 6.2-kbp region. An autonomously replicating sequence (ars) was found on the 17.2-kbp fragment. Yeast arol mutants transformed with the AROl episome express 5 to 12 times the normal level of the five AROl enzyme activities and possess elevated amounts of the AROl protein. The yeast AROl fragment also complemented aroA, aroB, aroD, and aroE mutants of Escherichia coli. The expression of AROl in both S. cerevisiae and E. coli was independent of the orientation of the fragment with respect to the vector.     

Molecular cloning of the ADE1 gene of Saccharomyces cerevisiae and stability of the transformants

Plasmid YEp(ADE1)1a, containing a 2.7-kb Sau3A fragment of Saccharomyces cerevisiae DNA inserted at the BamHI site of the yeast shuttle vector pBTI-1 (Morris et al., 1981), results in high frequency, unstable transformation of ade1 yeast strains. A second plasmid, YRp(ADE1)2, containing adjacent 0.5-kb and 3.0-kb BamHI fragments in pBR322 gave three types of yeast transformants: (1) transformants carrying extrachromosomal copies of the plasmid which indicate the presence of a functional ars sequence, (2) transformants indistinguishable from ade1 strains by hybridization analyis, and (3) a transformant carrying a multimeric form of YRp(ADE1)2. Cells transformed with either of the plasmids are free of the red pigment characteristic of ade1 mutants and indicate potential for direct colour-based selection of yeast transformants using ADE1 plasmids.     

Cloning and physical mapping of the ADE1 gene of the yeast Saccharomyces cerevisiae

ADE1 gene of Saccharomyces cerevisiae codes for the primary structure of SAICAR-synthetase. Mutational changes of ADE1 gene result in the accumulation of red pigment in cells. Colour differences, thus, serve as a basis for the selection of mutants or transformants. ADE1 gene was cloned as a 4.0 kb HindIII fragment of yeast DNA in a shuttle vector by complementing the ade1 mutation in yeast. The study of ADE1 gene expression in Escherichia coli showed that the 4.0 kb fragment containing the ADE1 gene does not complement purC mutations in E. coli. However, prototrophic colonies appeared at a frequency of 10(-7)-10(-8) after incubating clones bearing the recombinant plasmid with ADE1 gene on selective media. The plasmid DNA isolated from such clones complements the purC mutation in E. coli and the ade1 mutation in S. cerevisiae. Structural analysis of the plasmid demonstrated that the cloned DNA fragment contained an additional insertion of the bacterial origin. Further restriction enzyme analysis proved the insertion to be the bacterial element IS1. Expression of the cloned ADE1 gene in S. cerevisiae is controlled by its own promoter, whereas in E. coli it is controlled by the IS1 bacterial element.     

Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: isolation of the ADE1 gene.

The ADE1 gene of Saccharomyces cerevisiae was isolated by complementation in S. cerevisiae from a yeast genomic DNA library carried on plasmid YEp13. Electron microscopy of R-loop-containing DNA indicated the location of the ADE1 gene on the plasmid insert. Gene disruption and gene replacement were used to demonstrate that the ade1-complementing sequence was the actual ADE1 gene that maps on chromosome I. ade1 strains which normally form red colonies form white ones when transformed with the cloned ADE1 gene. This property should be very useful, since it enables detection of plasmids carrying this gene under nonselective conditions.     

A potassium transporter of the yeast Schwanniomyces occidentalis homologous to the Kup system of Escherichia coli has a high concentrative capacity.

The yeast Schwanniomyces occidentalis has a high-affinity K+ uptake system with a high concentrative capacity, which is able to deplete the external K+ to < 0.03 microM. We have cloned the gene HAK1 of S.occidentalis which complements defective K+ uptake by trk1 and trk1 trk2 mutants of Saccharomyces cerevisiae. When HAK1 was expressed in a trk1 trk2 S.cerevisiae mutant, transport affinities for K+ and other alkali cations resembled those of S.occidentalis. The predicted amino acid sequence of the HAK1 protein shows significant homology with the hydrophobic region of the Kup transporter of Escherichia coli. In S.occidentalis HAK1 expresses in K(+)-limiting conditions. Our data indicate that in K(+)-starved cells the system encoded by HAK1 is the major K+ transporter of S.occidentalis.     

Mutagenesis on cloned yeast genes. The mutation of the yeast gene comprising the plasmid and chromosome

The cells of Saccharomyces cerevisiae were transformed by plasmid pYG-007 treated in vitro with o-methylhydroxylamine. The plasmid consists of a portion of the bacterial plasmid with genes of resistance to ampicillin, chloramphenicol and tetracycline, 2 mkm yeast DNA and yeast genes ADE2 and LEU2. The collection of mutants containing a mutant allele of ADE2 gene within the plasmid was obtained. Interallelic complementation and that induced by suppression were studied in these ade 2 mutants. It was shown that all these induced ade 2 mutations were base-pair substitutions. Using the mechanism of conversion we managed to transfer the plasmid ade 2 mutations into the chromosome. Three pairs of strains carrying similar mutation in plasmid and chromosome were created. Analysis of frequency of reversions induced by UV-light and hydroxylaminopurine in the mutant ade2 locus comprised in the plasmid and chromosome showed that the former induced reversions in plasmid alleles less effectively than the latter.     

Analysis of the alpha-amylase gene of Schwanniomyces occidentalis and the secretion of its gene product in transformants of different yeast genera

We have cloned and characterized the alpha-amylase gene (AMY1) of the yeast Schwanniomyces occidentalis. A cosmid gene library of S. occidentalis DNA was screened in Saccharomyces cerevisiae for alpha-amylase secretion. The positive clone contained a DNA fragment harbouring an open reading frame of 1536 nucleotides coding for a 512-amino-acid polypeptide with a calculated Mr of 56,500. The deduced amino acid sequence reveals significant similarity to the sequence of the Saccharomycopsis fibuligera and Aspergillus oryzae alpha-amylases. The AMY l gene was found to be expressed from its original promoter in S. cerevisiae, Kluyveromyces lactis and Schizo-saccharomyces pombe leading to an active secreted gene product and thus enabling the different yeast transformants to grow on starch as a sole carbon source.     

"Illegal" transformation of red adenine-dependent mutants of the yeast Pichia pinus by the pYE(ADE2)2 plasmid

The red adenine-dependent mutants ade1 of the yeast Pichia pinus blocked in the VI step of adenine biosynthesis (lack of AIR-carboxylase) and ade2 mutants blocked in the VII step of adenine biosynthesis (lack of SAIKAR-synthase) were transformed with the plasmid pYE(ADE2)2 containing ADE2 gene of Saccharomyces cerevisiae encoding AIR-carboxylase. The appearance of white Ade+ clones with the frequency 2-7.10(-8) (which is ten-fold higher than reversion frequency) was only observed in the case of ade2 transformation. Genetic analysis points to connection of the "illegitimate" transformants' appearance with the change in the mutant ade2 locus or in a locus closely linked to the former. Ade+ phenotype was stable during 20 generations of mitotic budding. Southern blotting assay of transformant chromosomal DNA indicates that reconstitution of ade2 defective gene is related with its "correction", owing to integration of pYE(ADE2)2 sequence in the vicinity of the mutant locus.     

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.