High frequency FLP-independent homologous DNA recombination of 2 μ plasmid in the yeast Saccharomyces cerevisiae

Summary The purpose of this work is to identify and quantitate in vivo 2 plasmid FLP-independent recombination in yeast, using a nonselective assay system for rapid detection of phenotypic expression of the recombination events. A tester plasmid was constructed such that in vivo recombination between 2 direct repeat sequences produces the resolution of the plasmid into two circular DNA molecules. This recombinational event is detected as a phenotypic shift from red to white colonies, due to the mitotic loss of the plasmid portion containing the yeast ADE8 gene in a recipient ade1 ade2 ade8 genetic background. In the absence of the 2 FLP recombinase and/or its target DNA sequence, recombination is not abolished but rather continues at a high frequency of about 17%. This suggests that the FLP-independent events are mediated by the chromosomally-encoded general homologous recombination system. We therefore conclude that the totality of 2 DNA recombination events occurring in FLP⁺ cells is the contribution of both FLP-mediated and FLP-independent events.     

Comparative analysis of spontaneous mitotic recombination in [cir 0] and [cir +] strains of the yeast Saccharomyces cerevisiae

Summary The influence of the 2 m plasmid on homologous recombination in the right arm of chromosome XV of the yeast Saccharomyces cerevisiae has been examined. No differences between spontaneous mitotic recombination rates in [cir ⁰] and [cir ⁺] derivatives of two yeast diploid tester strains were detected. In the course of analysis an unusually high coincident conversion frequency at ADE2, HIS3, and two RFLP loci adjacent to ADE2, was observed. The character of coincident homozygotization of linked markers argues for a break-and-replicate mechanism underlying the coincident conversion events.     

Recombinational properties of the Saccharomyces cerevisiae FLP gene expressed in Escherichia coli

Summary The FLP gene from the 2-m DNA of Saccharomyces cerevisiae is shown to be functionally expressed in Escherichia coli leading to site-specific intramolecular as well as intermolecular recombination between IR sequences. The expression was achieved under control of a low expression as well as a high expression E. coli promoter. The FLP gene was found to complement in trans a Flp^– plasmid and promote its interconversion.By the use of a low Flp expression plasmid, it could be shown that the rate of interconversion of a Flp^– plasmid by complementation in trans, was lower than that of a Flp⁺ plasmid, suggesting that in addition to the IR sequences another cis-acting function exists.Expression of the FLP gene fused to the lac promoter in an in vitro system yielded two polypeptides with apparent molecular weights of 44,000 and 37,000. The 37,000 dalton polypeptide can also be produced from Flp^– plasmids and is generated from a translation start within the FLP gene. The 44,000 dalton polypeptide is considered to represent the FLP gene product.     

Loss of 2 um DNA from Saccharomyces cerevisiae transformed with the chimaeric plasmid pJDB219

Summary Two plasmids containing Saccharomyces cerevisiae 2 µm DNA sequences and the S. cerevisiae LEU2 gene have been found to display incompatibility with 2 µm DNA; in the presence of the LEU2 plasmids, 2 µm DNA can be lost. The LEU2 plasmids can be lost spontaneously after (and before) 2 µm DNA loss has occurred, so that strains completely lacking 2 µm DNA sequences can be obtained routinely.     

Curing of Saccharomyces cerevisiae 2-μm DNA by transformation

Summary A general procedure for the curing of 2-m in Saccharomyces cerevisiae is described. The method is based on the displacement of endogenous 2-m DNA by the recombinant plasmid pMP78-1, which carries the yeast leu2 gene and the 2 -m DNA replicon, but cannot be maintained stably in a yeast cell without endogenous 2-m DNA. After transformation with pMP78-1 cells are grown selectively to displace 2-m DNA. During the non-selective growth which follows, plasmid pMP78-1 is lost and up to 100% of the cells completely lack plasmids. In conjunction with a kanamycin resistance marker, as present in plasmid pMP81, this method should be applicable to cure any wild-type yeast strain. The stability of recombinant plasmids in cir ⁺ and cir ⁰ strains has been compared.     

Cosmids carrying Aspergillus terreus DNA can integrate into Saccharomyces cerevisiae chromosome XII via recombination between yeast and foreign DNAs

Summary A genome clonotheque consisting of 25- to 40-kb Sau3A1 fragments of Aspergillus terreus DNA was constructed in the episomal cosmid vector pES33 containing the yeastARG4 gene. From the 475 transformants of cir° yeast strain ESH-0, 23 stable Arg ⁺ transformants were independently selected. Genetic and Southern analysis of these stable transformants showed that 39% arose as a result of recombination between cloned A. terreus DNA sequences and yeast chromosome XII. The recombination events most likely occurred in the regions of homology within the rDNA clusters of A. terreus and Saccharomyces cerevisiae.     

Phenotypic differences between induced and spontaneous 2μ-plasmid-free segregants of Saccharomyces cerevisiae

Summary The maximum specific growth rates (_max) of 2 -plasmid-free ([cir°]) segregants of three haploid and one diploid strain of Saccharomyces cerevisiae have been determined and compared with the _max of their 2 -plasmid-containing ([cir ⁺]) progenitors. Two classes of [cir°] strains have been examined: those induced by transformation with a 2 -based recombinant plasmid according to the method of Dobson et al. (1980) and those isolated as spontaneous [cir°] segregants from glucose-limited continuous cultures. The _max of the spontaneous [cir°] segregants was not found to differ significantly from that of their [cir ⁺] parents. In all cases, however, the induced [cir°] strains had a _max which was significantly less than that of their [cir ⁺] counterparts. This effect was particularly marked in the case of the diploid strain where a 34% reduction in _max was observed. The implications of these results are discussed in terms of the effect of the transformation process on host yeast cells.     

A cyclin protein modulates mitosis in the budding yeast Saccharomyces cerevisiae

Summary For the budding yeast Saccharomyces cerevisiae the mitotic cell cycle is coordinated with cell mass at the regulatory step start. The threshold amount of cell mass (reflected as a critical size) necessary for start is proportional to nutrient quality. This relationship leads to a transient accumulation of cells at start, termed nutrient modulation, upon enrichment of nutrient conditions. Nutrient enrichment abruptly increases the critical size needed for start, causing the smaller cells, produced in the previous cell cycle, to be delayed at start while growing larger. Here we show that, in S. cerevisiae, a second cell-cycle step, at mitosis, also exhibits nutrient modulation, and is, therefore, another point of cell-cycle regulation. At both mitosis and start, nutrient modulation was found through mutation to be regulated by the activity of the cyclin-related WHI1 (CLN3) gene product.     

Expression of the cloned endo-1,3-1,4-β-glucanase gene of Bacillus subtilis in Saccharomyces cerevisiae

Summary A cloned endo-1,3-1,4--glucanase gene from the Gram-positive bacterium B. subtilis has been located by deletion analysis on a 1.4 kb PvuI-ClaI DNA fragment. This gene has been sub-cloned in the yeast LEU2 vector pJDB207 to produce a hybrid plasmid designated pEHB9. pEHB9 has been transformed to S. cerevisiae and shown to direct the synthesis of an endo-1,3-1,4--glucanase in yeast. The -glucanase activity was low and could only be detected in crude cell extracts of yeast harbouring pEHB9.     

Interactions between chromosomal omnipotent suppressors and extrachromosomal effectors in Saccharomyces cerevisiae

Summary Chromosomal omnipotent suppressor mutations recovered in ⁺ strains of Saccharomyces cerevisiae were brought into ^– cytoplasm. SUP46, SUP138 and SUP139 acted as dominant omnipotent suppressors in the ^– cytoplasm though their suppressor activity was substantially reduced. SUP46 and SUP138 conferred recessive thermosensitivity and antibiotic sensitivity in ^– cytoplasm as in ⁺ cytoplasm. On the other hand, sup111 through sup115, which acted as recessive omnipotent suppressors in the ⁺ cytoplasm, manifested no, or very low, suppressor activity in the ^– cytoplasm. They, however, still enhanced the efficiency of the SUP29 tRNA suppressor in ^– cytoplasm. A multicopy plasmid carrying the wild-type SUP35 gene enhanced the efficiency of sup111 in ^– cytoplasm.     

An improved isolation procedure for yeast two-micrometer minichromosomes

Summary Two micrometer minichromosomes from Saccharomyces cerevisiae were isolated without detergent using metrizamide gradients. 2 m minichromosomes showed a lower density in metrizamide gradients relative to genomic chromatin. Our results suggest a lower ratio of proteins to DNA in 2-m minichromosomes as compared with genomic chromatin. The procedure described herein yields minichromosomes free of cellular chromatin and ribosomal protein contamination. This method may be useful for the isolation and characterization of other yeast minichromosomes.     

Introduction of nonselectible 2μ plasmid into [cir°] cells of the yeast S. cerevisiae by DNA transformation and in vivo site-specific resolution

Summary The 2 DNA plasmid of the yeast Saccharomyces cerevisiae does not confer any known selectable phenotype to the host cell carrying it. Selection of cells transformed with purified 2 DNA therefore cannot be achieved, and the intracellular presence of 2 can only be assessed by molecular analysis of the DNA complement. In addition, 2 alone does not replicate in bacterial hosts, thus rendering its amplification by conventional methods impossible. We have isolated a shuttle plasmid, pBH-2L, generated by in vivo sites-pecific recombination between the endogenous 2 DNA plasmid and pRL, a pBR322 derivative containing the yeast LEU2 gene and one 2 repeat sequence associated with the origin of replication. This new shuttle plasmid has the property, when transformed into yeast, of undergoing site-specific recombinational resolution between its two direct repeat sequences. This releases 2 plasmid and pRL as individual molecules. The latter can undergo progressive mitotic loss during growth in nonselective medium, ultimately leaving leucine auxotrophic transformants that contain only 2 DNA plasmid. This system can be utilized to introduce 2 DNA alone into cells lacking it, thereby providing a novel means to study the biology and the molecular genetics of the plasmid and its potential practical applications as a vector.     

A high-frequency transformation system for the yeast Kluyveromyces lactis

Summary This communication describes conditions for Kluyveromyces lactis protoplast regeneration and transformation and shows that a high frequency of transformation can be obtained with recombinant plasmids containing one of a series of K. lactis DNA fragments that presumably carry autonomously replicating sequences (called KARS). The vector YRp7, which contains a Saccharomyces cerevisiae autonomously replicating sequence (ARS), only led to integrative transformation of K. lactis indicating substantial differences in specificity between the DNA replication mechanisms of both yeast species. It is further shown that 2-m DNA derived vectors giving high frequency transformation in S. cerevisiae can transform K. lactis only with low frequency.     

Involvement of the Rat Caudate Nucleus in the Immunostimulatory Effect of Dago

The involvement of the caudate nucleus, i.e., the terminal zone of the nigrostriatal dopaminergic system, in neuroimmunostimulation during the activation of opioid receptors by the highly specific agonist DAGO. Single doses of DAGO (100 g/kg) in sham-operated control Wistar rats induced significant increases in the numbers of direct IgM-antibody-forming and total rosette-forming cells at the peak of the immune response after immunization with sheet red blood cells. The experiments showed that bilateral electrolytic lesioning of the caudate nucleus in rats suppressed the immune response, demonstrating its involvement in neuroimmunomodulation. Since the effect of immunostimulation induced by DAGO disappeared when given to animals with caudate nucleus lesions, it was concluded that this structure is involved in activatory immunogenesis via opioid mechanisms.     

Construction of a Saccharomyces cerevisiae strain able to ferment cellobiose

Summary The bglA gene, encoding a -glucosidase from Bacillus polymyxa, has been expressed in Saccharomyces cerevisiae under control of the CYC-GAL promoter inducible by galactose. The expression of bglA-encoded activity in the strain used as a host was not sufficient to allow its growth with cellobiose as a carbon source. However, a recessive mutation in a gene designated cem1 has been obtained which, combined with the expression of -glucosidase activity, allows the growth of S. cerevisiae on cellobiose. The expression of the bglA gene in a cemt strain confers on S. cerevisiae the capability for an efficient fermentation of cellobiose, as detected by the formation of CO₂.     

Non-selective transformation of Saccharomyces cerevisiae

Summary Wild or industrial yeast strains cannot be transformed by most selective vectors due to a lack of auxotrophic mutations. To enable identification of transformants of such yeast species, we have developed a 2-µm DNA vector with an indicator gene that can be used without any additional marker. The Escherichia coli gene for -lactamase (bla) was placed under the control of the yeast promoter for the structural gene encoding ADHI. This increased the amount of -lactamase produced in Saccharomyces cerevisiae 100-fold giving an enzyme activity in transformant colonies which is high enough to be detected directly on indicator plates. Non-selectively, the transformation frequency is even higher than under selective conditions indicating that selection does not assist the establishment of new plasmids. Transformants isolated non-selectively were found to retain the endogenous 2-µm DNA. Under control of appropriate promoters, the bacterial bla gene may also provide a convenient marker for other eukaryotic transformation systems.     

Effects of the CDC2 gene on adaptive mutation in the yeast Saccharomyces cerevisiae

We have studied the influence of a temperature-sensitive cdc2-1 mutation in DNA polymerase on the selection-induced mutation occurring at the LYS-2 locus in the yeast Saccharomyces cerevisiae. It was found that in cells plated on synthetic complete medium lacking only lysine, the numbers of Lys⁺ revertant colonies accumulated in a time-dependent manner in the absence of any detectable increase in cell number. When cdc2-1 mutant cells, after selective plating, were incubated at the restrictive temperature of 37°C for 5 h daily for 7 days, the frequency of an adaptive reversion of lys ^- Lys⁺ was significantly higher than the frequency in cells incubated only at the permissive temperature, or in wild-type cells incubated either at 23°C or 37°C. Therefore, when the proof-reading activity of DNA polymerase is impaired under restrictive conditions, the frequency of adaptive mutations is markedly enhanced.     

A mutation affecting sexual agglutinability in MATα locus of Saccharomyces cerevisiae

Summary A temperature-sensitive non-agglutinative mutant of Saccharomyces cerevisiae was isolated and characterized. The mutation, sag2, affected sexual agglutination, conjugation and production of -mating pheromone at a restrictive temperature, but not the response to -mating pheromone. Genetic analyses showed that the mutation was recessive and in the MAT locus. The sag2 mutation complemented with mat2 but not with mat1 These results suggest that sag2 is in the MAT1 gene and that at a restrictive temperature the mutation, sag2, inactivated the MAT1 product, a positive regulator of -mating functions. The sag2 mutation is like mat1-5 in its retention of response to -mating pheromone. However, at 25 °C, sag2 cells were competent to mate, whereas mat1-5 cells were not. Hence, sag2 is regarded as a new allele in the MAT1 gene, which we designate mat1-11.     

Comparison of expression of the endo-β-1,3-1,4-glucanase gene from Bacillus subtilis in Saccharomyces cerevisiae from the CYC1 and ADH1 promoters

Summary The endo--1,3-1,4-glucanase gene from B. subtilis was placed under yeast promoter control in a number of different yeast expression vectors. The hybrid plasmids were transformed into S. cerevisiae where they directed the synthesis of varying amounts of active enzyme. The presence of B. subtilis DNA sequences 5 to the initiation codon for the B. subtilis -glucanase gene reduced expression of the gene in yeast. A 1,000-fold increase in the yield of -glucanase was obtained using the ADH1 promoter compared with the CYC1 promoter.