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.     

Plasmid pC present in Salmonella enterica serovar Enteritidis PT14b strains encodes a restriction modification system

Salmonella enterica serovar Enteritidis (S. Enteritidis) possesses plasmids of different sizes and roles. Besides the serovar-specific virulence plasmid present in most field strains, S. Enteritidis can harbour plasmids of low molecular mass whose biological role is poorly understood. We therefore sequenced plasmid pC present in S. Enteritidis strains belonging to phage type PT14b. The size of plasmid was determined to be 5,269 bp and it was predicted to encode four open reading frames (ORFs). The first two ORFs were found (initial 3,230 bp) to be highly homologous to rom and mbeA genes of ColE1 plasmid of Escherichia coli. Proteins encoded by the other two ORFs were 99% homologous to a restriction methylase and restriction endonuclease encoded by plasmid pECO29 of a field strain of E. coli. Using insertional mutagenesis we confirmed experimentally that the plasmid pC-encoded restriction modification system was functional and could explain the high resistance of S. Enteritidis PT14b strains to phage infection.     

Enhancement of UV survival, UV- and MMS-mutability, precise excision of Tn10 and complementation of umuC by plasmids of different incompatibility groups

29 conjugative resistance and colicin plasmids from 19 different incompatibility (Inc) groups were examined for their ability to enhance post-ultraviolet (UV) survival and UV- and methyl methanesulfonate(MMS)-induced mutability in Salmonella typhimurium LT2 strains. 14 Muc+ plasmids enhanced each of the survival and mutation-related properties tested, while 14 Muc- plasmids showed no enhancing effects in any tests. One Muc+ plasmid, pRG1251 (IncH1), enhanced post-UV survival and each of the mutation-related properties tested, except MMS-induced mutagenesis. Two further noteworthy plasmids, R391 (IncJ) and R394 (IncT), produced apparent strain-dependent effects in S. typhimurium which differed from those reported to have been found in Escherichia coli. Plasmid R391 enhanced post-UV survival in S. typhimurium, in contrast to its UV-sensitizing effects in E. coli. In both hosts plasmid R391 enhanced UV- and MMS-induced mutagenesis. Plasmid R394 had no enhancing effects on UV survival or UV- and MMS-induced mutagenesis in S. typhimurium, in contrast to its reported enhancement of MMS-induced mutagenesis in E. coli. Conjugal transfer of R394 to E. coli strain AB1157 and assays of mutagenesis-related traits supported results observed in S. typhimurium. Muc+ plasmids were found to enhance the frequency of precise excision of the transposon Tn10 when inserted within hisG or trpA in S. typhimurium strains. Precise excision could be further enhanced in S. typhimurium by UV-irradiation. Analysis of Tn10 mutants with altered IS10 ends indicated that intact inverted repeats at the ends of Tn10 were required for efficient enhancement of precise excision.     

New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites

We describe the production of new alleles of the LEU2, URA3 and TRP1 genes of Saccharomyces cerevisiae by in vitro mutagenesis. Each new allele, which lacks restriction enzyme recognition sequences found in the pUC19 multicloning site, was used to construct a unique series of yeast-Escherichia coli shuttle vectors derived from the plasmid pUC19. For each gene a 2 mu vector (YEplac), an ARS1 CEN4 vector (YCplac) and an integrative vector (YIplac) was constructed. The features of these vectors include (i) small size; (ii) unique recognition site for each restriction enzyme found in the pUC19 multicloning site; (iii) screening for plasmids containing inserts by color assay; (iv) high plasmid yield; (v) efficient transformation of S. cerevisiae. These vectors should allow greater flexibility with regard to DNA restriction fragment manipulation and subcloning.     

Expression of the Escherichia coli xylose isomerase gene in Saccharomyces cerevisiae

Transformation of Saccharomyces cerevisiae by yeast expression plasmids bearing the Escherichia coli xylose isomerase gene leads to production of the protein. Western blotting (immunoblotting) experiments show that immunoreactive protein chains which comigrate with the E. coli enzyme are made in the transformant strains and that the amount produced parallels the copy number of the plasmid. When comparable amounts of immunologically cross-reactive xylose isomerase protein made in E. coli or S. cerevisiae were assayed for enzymatic activity, however, the yeast protein was at least 10(3)-fold less active.     

Expression of the Escherichia coli xylose isomerase gene in Saccharomyces cerevisiae.

Transformation of Saccharomyces cerevisiae by yeast expression plasmids bearing the Escherichia coli xylose isomerase gene leads to production of the protein. Western blotting (immunoblotting) experiments show that immunoreactive protein chains which comigrate with the E. coli enzyme are made in the transformant strains and that the amount produced parallels the copy number of the plasmid. When comparable amounts of immunologically cross-reactive xylose isomerase protein made in E. coli or S. cerevisiae were assayed for enzymatic activity, however, the yeast protein was at least 10(3)-fold less active.     

Nucleotide sequence of the wild-type RAD4 gene of Saccharomyces cerevisiae and characterization of mutant rad4 alleles.

Shuttle plasmids carrying the wild-type RAD4 gene of Saccharomyces cerevisiae cannot be propagated in Escherichia coli (R. Fleer, W. Siede, and E. C. Friedberg, J. Bacteriol. 169:4884-4892, 1987). In order to determine the nucleotide sequence of the cloned gene, we used a plasmid carrying a mutant allele that allows plasmid propagation in E. coli. The wild-type sequence in the region of this mutation was determined from a second plasmid carrying a different mutant rad4 allele. We established the locations and characteristics of a number of spontaneously generated plasmid-borne RAD4 mutations that alleviate the toxicity of the wild-type gene in E. coli and of several mutagen-induced chromosomal mutations that inactivate the excision repair function of RAD4. These mutations are situated in very close proximity to each other, and all are expected to result in the expression of truncated polypeptides missing the carboxy-terminal one-third of the Rad4 polypeptide. This region of the gene may be important both for the toxic effect of the Rad4 protein in E. coli and for its role in DNA repair in S. cerevisiae.     

Chimeric plasmids for cloning of deoxyribonucleic acid sequences in Saccharomyces cerevisiae.

Two sets of plasmids, each carrying a Saccharomyces cerevisiae gene and a portion or all of the yeast 2-micron circle linked to the Escherichia coli plasmid pBR322, have been constructed. One of these sets contains a BamHI fragment of S. cerevisiae deoxyribonucleic acid that includes the yeast his3 gene, whereas the other set contains a BamHI fragment of S. cerevisiae that includes the yeast leu2 gene. All plasmids transform S. cerevisiae and E. coli with a high frequency, possess unique restriction endonuclease sites, and are retrievable from both host organisms. Plasmids carrying the 2.4-megadalton EcoRI fragment of the 2-micron circle transform yeast with 2- to 10-fold greater frequency than those carrying the 1.5-megadalton EcoRI fragment of the 2-micron circle. Restriction endonuclease analysis of plasmics retrieved from S. cerevisiae transformed with plasmics carrying the 2.4-megadalton EcoRI fragment showed that in 13 of 96 cases the original plasmic has acquired an additional copy of the 2-mcron circle. These altered plasmids appear to have arisen by means of an interplasmid recombination event while in S. cerevisiae. A clone bank of S. cerevisiae genes based upon one of these composite plasmids has been constructed. By using this bank and selecting directly in S. cerevisiae, the ura3, tyr1, and met2 genes have been cloned.     

IncP plasmids are most effective in mediating conjugation between Escherichia coli and streptomycetes

Mobilizable shuttle plasmids containing the origin of transfer (oriT) region of plasmid F (IncFI), ColIb-P9 (IncI1), and RP4/RP1 (IncPalpha) were constructed to test the ability of the cognate conjugation system to mediate gene transfer from Escherichia coli to Streptomyces. The conjugative system of the IncPalpha plasmids was shown to be most effective in conjugative transfer, giving peak values of (2.7 +/- 0.2) x 10(-2) S. lividans TK24 exconjugants per recipient cell. To assess whether the mating-pair formation system or the DNA-processing apparatus of the IncPalpha plasmids is crucial in conjugative transfer, an assay with an IncQ-based mobilizable plasmid (RSF1010) specifying its own DNA-processing system was developed. Only the IncPalpha plasmid mobilized the construct to S. lividans indicating that the mating-pair formation system is primarly responsible for the promiscuous transfer of the plasmids between E. coli and Streptomyces. Dynamic of conjugative transfer from E. coli to S. lividans was investigated and exconjugants starting from the first hour of mating were obtained.     

Replicative transformation of the filamentous fungus Ashbya gossypii with plasmids containing Saccharomyces cerevisiae ARS elements.

We have developed a transformation system for the filamentous ascomycete fungus Ashbya gossypii. Mycelial protoplasts were transformed to geneticin-resistance with plasmids containing the Escherichia coli kanamycin-resistance gene as a selectable marker and autonomously replicating sequences (ARS) from Saccharomyces cerevisiae (ARS1, 2 mu ARS). Transformation frequencies of up to 63 transformants per microgram of plasmid DNA were obtained. The transformants were unstable under nonselective conditions. Southern analysis of DNA separated by conventional and pulsed-field-gel electrophoresis showed that the transforming DNA was present as autonomously replicating plasmid. Plasmid integration into chromosomal DNA was not detected. We concluded that the S. cerevisiae ARS elements are functional in A. gossypii, since vectors lacking such elements did not yield transformants.     

Automatic elimination of unnecessary bacterial sequences from yeast vectors.

Most vectors for Saccharomyces cerevisiae are shuttle vectors which can be both propagated and selected in Escherichia coli. The DNA segments, however, which are required for propagation in E. coli are unnecessary and moreover toxic in S. cerevisiae. To delete these harmful DNA fragments from the vector after it is introduced into S. cerevisiae cells, we propose a specific gene conversion mechanism of a yeast plasmid, pSR1. Plasmid pSR1 has a pair of inverted repeats (IRs) that divides the plasmid molecule into two unique regions. Intramolecular recombination frequently occurs at a pair of specific recombination sites in IRs catalyzed by recombinase R, encoded by a pSR1 plasmid gene. This R-mediated recombination is often accompanied by gene conversion in IRs. Thus, a 2.1-kb pBR322 sequence for the E. coli host ligated into one of the IRs of a composite plasmid was automatically and effectively eliminated when the plasmid was introduced into S. cerevisiae cells.     

Cloning of a yeast gene coding for the glutamate synthase small subunit (GUS2) by complementation of Saccharomyces cerevisiae and Escherichia coli glutamate auxotrophs

A Saccharomyces cerevisiae glutamate auxotroph, lacking NADP-glutamate dehydrogenase (NADP-GDH) and glutamate synthase (GOGAT) activities, was complemented with a yeast genomic library. Clones were obtained which still lacked NADP-GDH but showed GOGAT activity. Northern analysis revealed that the DNA fragment present in the complementing plasmids coded for a 1.5kb mRNA. Since the only GOGAT enzyme so far purified from S. cerevisiae is made up of a small and a large subunit, the size of the mRNA suggested that the cloned DNA fragment could code for the GOGAT small subunit. Plasmids were purified and used to transform Escherichia coli glutamate auxotrophs. Transformants were only recovered when the recipient strain was an E. coli GDH-less mutant lacking the small GOGAT subunit. These data show that we have cloned the structural gene coding for the yeast small subunit (GUS2). Evidence is also presented indicating that the GOGAT enzyme which is synthesized in the E. coli transformants is a hybrid comprising the large E. coli subunit and the small S. cerevisiae subunit.     

Escherichia coli R-factors unstable in Salmonella typhi are deleted before being segregated in this host

Although resistant Salmonella typhi strains are found in the environment, many epidemiological data indicate that most isolates are multisensitive. Plasmids are not common in S. typhi, contrasting with other enterobacteria. Since S. typhi is able to receive R plasmids from other enterobacteria, such plasmidless condition may be due to destabilization of the plasmids acquired. The segregation process of three plasmids known to behave unstably in S. typhi strains was analyzed, and evidence was obtained that the three of them lost an antibiotic-resistance determinant due to small deletions before plasmid loss occurred. Once deleted, the plasmids entered a segregation phase with particular kinetic features in each case. Selective pressure prevented plasmid deletion and segregation. Like their parental plasmids, the deleted plasmids were unstable in S. typhi but stable in Escherichia coli, suggesting that deletions imply loss of genetic information relevant for a plasmid stability mechanism operative in S. typhi, but not in E. coli.     

Colonization factor antigen II (CFA/II) of enterotoxigenic Escherichia coli: molecular cloning of the CS3 determinant

The genes for the cell surface associated antigen CS3, produced by CFA/II type enterotoxigenic Escherichia coli, have been cloned in the plasmid vector pBR322 to produce a family of recombinant plasmids. These plasmids contain a series of HindIII fragments of which a fragment of 4.6 kb is common to all those expressing CS3. One of these plasmids, pPM474, has been subjected to mutagenesis with Tn1725 and deletions generated using Bal31. This has defined a minimum region of 3.75 kb necessary for the production of CS3 on the cell surface and implying genetic complexity as has been observed with other fimbrial antigens. Analysis of the plasmid encoded proteins in E. coli K-12 minicells has confirmed this complexity.     

Introduction of chloramphenicol resistance into the modified mouse mitochondrial genome: cloning of unstable sequences by passage through yeast

Despite increasing awareness of the importance of the mitochondrial genome in human pathology, very few attempts have been made so far toward genetic engineering of mitochondrial DNA (mtDNA). One of the reasons for this slow progress is the difficulty of cloning mtDNA in Escherichia coli, a trait in common with repetitive or palindromic sequences, and some viral sequences. We have previously made a construct containing the entire mouse mitochondrial genome and a cDNA sequence coding for human ornithine transcarbamylase in a yeast/bacterial shuttle vector, which can be stably maintained in E. coli. We wished to modify this vector for mitochondrial gene therapy by the addition of mitochondrial chloramphenicol resistance, conferred by a point mutation in the 16S rRNA gene. Attempts to modify this construct by a straightforward cloning approach in E. coli proved unsuccessful. Two successful strategies for modification of large unstable constructs in both E. coli and the yeast Saccharomyces cerevisiae are compared here.     

Transformation of Schwanniomyces occidentalis with an ADE2 gene cloned from S. occidentalis.

We have developed an efficient transformation system for the industrial yeast Schwanniomyces occidentalis (formerly Schwanniomyces castellii). The transformation system is based on ade2 mutants of S. occidentalis deficient for phosphoribosylaminoimidazole carboxylase that were generated by mutagenesis. As a selectable marker, we isolated and characterized the S. occidentalis ADE2 gene by complementation in an ade2 strain of Saccharomyces cerevisiae. S. occidentalis was transformed with the recombinant plasmid pADE, consisting of a 4.5-kilobase-pair (kbp) DNA fragment from S. occidentalis containing the ADE2 gene inserted into the S. cerevisiae expression vector pYcDE8 by a modification of the spheroplasting procedure of Beggs (J. D. Beggs, Nature [London] 275:104-108, 1978). Intact plasmids were recovered in Escherichia coli from whole-cell lysates of ADE+ transformants, indicating that plasmids were replicating autonomously. High-molecular-mass species of pADE2 were found by Southern hybridization analysis of intact genomic DNA preparations. The shift to higher molecular mass of these plasmids during electrophoresis in the presence ethidium bromide after exposure to shortwave UV suggests that they exist in a supercoiled form in the transformed host. Subclones of the 4.5-kbp insert indicated that ADE2-complementing activity and sequences conferring autonomous replication in S. occidentalis were located within a 2.7-kbp EcoRI-SphI fragment. Plasmids containing this region cloned into the bacterial vector pUC19 complemented ade2 mutants of S. occidentalis with efficiencies identical to those of the original plasmid pADE.     

Generation of various amino acids mutants in the trpR gene of Escherichia coli by site-directed mutagenesis

Tryptophan repressor (trpR) gene lacks various amino acid codons. To establish these codons in the trpR gene, we created the mutants by site-directed mutagenesis in the trpR gene of pHK1 plasmid. The interested regions of trpR gene were amplified, cloned in pT7-5 plasmid and transformed in to the cells harboring pGP1-2 plasmid. These plasmid products were labeled with (35)S Met, and following sequencing we observed the presence of mutants for cysteine, glycine, serine and lysine in the trpR gene of E. coli. Therefore, using these approach mutants in various genes of E. coli could be established and used as a tool to study translational bypassing in trpR gene of E. coli.     

Study of the transmissibility of multiple drug resistance and the capacity to ferment lactose in a clinical strain of Kl. pneumoniae]

The donor properties of K. pneumoniae PI 220 with multiple drug resistance were studied. It was shown that the above strain carried 2 plasmids, i.e. R-plasmid pPI 220 controling resistance to ampicillin, streptomycin, tetracycline, chloramphenicol, kanamycin and sulphanylamides and plasmid pPI 221 controlling lactose fermentation. Both plasmids can be transfered on conjugation to strain E. coli P678 at a temperture of 28 degrees C at a rate of 10(-5) for pPI 220 and 10(-4) for pPI 221. The drug resistance controlled by pPI 221 was transfered mainly in a "blocks" simultaneously to 6 drugs. Deletion of plasmid pPI 220 was observed rarely. The donor properties of the strain were defined by the conjugative plasmid pPI 220 controlling the self-transfer and mobilization of plasmid pPI 221 incapable of the self-transfer. E. coli P678 (pPI 220) (PPI 221) acquired the donor properties and transfered both plasmids to E. coli J62 on crossing simultaneously at a rate of 10(-2), as well as to S. typhimurium LT2 and P. rettgeri at a rate of 10(-5). In all the recipient strains studied the transfered plasmids were unstable and segregated also simultaneously at a rate being the highest for P. retgari PI 230. The clones with stable preservation of the plasmids could be obtained by selection.