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  • 11
    Electronic Resource
    Electronic Resource
    Construction of new yeast vectors and cloning of the nif (nitrogen fixation) gene cluster of Klebsiella pneumoniae in yeast (1981)
    Springer
    Current genetics 3 (1981), S. 173-180 
    Details
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; Yeast vectors ; Cosmids ; nif genes
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Two vectors, termed pG63.11 (7.6 Kb) and pHCG3 (9.6 Kb), suitable for yeast transformation have been constructed. The pHCG3 vector has cosmid properties. Both vectors contain a single 3.3 Kb EcoRI-HindIII fragment of yeast origin which carries the yeast URA3 gene (1.1 Kb) and the origin of replication of the 2 µm plasmid (2.2 Kb). They confer ampicillin resistance and they contain 5 unique EcoRI,HpaI,HindIII,BamHI and SalI restriction sites. Cosmid pHCG3 was used to clone the nitrogen fixation (nif) gene cluster of Klebsiella pneumoniae carried by twoHindIII fragments of 17 and 26 Kb, respectively. The resulting cosmid, termed pGPC875 (53 Kb) which conferred a Nif+ phenotype to Escherichia coli, was introduced in yeast by transformation. No acetylene reduction activity was detectable in the transformants. However it was shown that the entire information for nitrogen fixation can be replicated and maintained intact in yeast for more than 50 generations of growth.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00429819
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  • 12
    Electronic Resource
    Electronic Resource
    Localization of the upstream regulatory sites of yeast iso2-cytochrome c gene (1985)
    Springer
    Molecular genetics and genomics 199 (1985), S. 117-122 
    Details
    ISSN: 1617-4623
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary In order to study the regulation of expression of the iso2-cytochrome c gene, we have constructed a fused gene between the 5′ flanking region of the gene coding for the yeast iso2-cytochrome c and the coding region of the E. coli beta-galactosidase lacZ gene. When introduced in yeast cells this hybrid gene is expressed and regulated like the production of iso2-cytochrome c: it is under the control of the general catabolic repression and of the unlinked trans-acting CYP1 gene whose CYP1-18 allele causes an overproduction of iso2-cytochrome c. The expression of hybrid genes whose upstream region has been progressively shortened or altered by internal deletions was studied either in wild-type CYP1 + cells or in cells carrying the CYP1-18 allele grown either on glucose or on glycerol. It appears that the expression and the regulation of the iso2-cytochrome c gene is controlled by an upstream regulatory site composed of a positive and a negative element. This site is the target of regulation by the CYP1 gene product and, directly or through this gene, of the control by the general catabolic repression.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00327520
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  • 13
    Electronic Resource
    Electronic Resource
    Regulation of the expression of iso 2-cytochrome c gene in S. cerevisiae: cloning of the positive regulatory gene CYP1 and identification of the region of its target sequence on the structural gene CYP3 (1985)
    Springer
    Molecular genetics and genomics 199 (1985), S. 524-533 
    Details
    ISSN: 1617-4623
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary CYP1 is a trans acting regulatory locus modulating both iso 1- and iso 2-cytochrome c synthesis. Genetical analysis of various mutated alleles has allowed us to identify the gene product as a positive regulatory element. The region of the target sequence of the CYP1 product on the iso 2-cytochrome c structural gene was located by molecular and genetic analysis of two cis acting mutations located at the CYP3 locus: CYP3-36 and CYP3-4, which have been shown to arise from the integration of TY1 elements near the promoter site. Determination of the amount of iso 2-cytochrome c synthesized by strains bearing various genetic constructions, in which the cis acting mutations were associated with different alleles of the CYP1 trans acting locus, showed that TY1 inserted into CYP3-36 extinguishes the activation function due to a mutated overproducer allele CYP1-18, while CYP3-4 amplifies this function. This result identifies at least a part of the target sequence of the CYP1 product within the region separating the two TY1 insertions. To clone the CYP1 gene, we took advantage of the iso 2-cytochrome c overproducer phenotype of the mutated allele CYP1-18, which confers a Lactate+ phenotype on an iso 1-cytochrome c-deficient strain. Such a phenotype allowed the isolation of a recombinant plasmid YEpJFM1 carrying the mutated allele, able to complement on lactate medium a lactate - recipient strain. The identity of the YEpJFM1 sequence with the chromosomal gene was confirmed by homologous recombination at the CYP1 locus.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00330769
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  • 14
    Electronic Resource
    Electronic Resource
    The promoter of the β-glucosidase gene from Kluyveromyces fragilis contains sequences that act as upstream repressing sequences in Saccharomyces cerevisiae (1988)
    Springer
    Molecular genetics and genomics 213 (1988), S. 150-154 
    Details
    ISSN: 1617-4623
    Keywords: β-Glucosidase ; Kluyveromyces fragilis ; Saccharomyces cerevisiae ; Upstream repressing sequence ; Gene expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The relationship between the promoter length of the Kluyveromyces fragilis β-glucosidase gene and the level of its expression in Saccharomyces cerevisiae was studied by gene fusion between deleted promoter fragments of various lengths and the promoterless β-galactosidase gene of Escherichia coli. The removal of a region from position-425 to-232 led to a tenfold increase in the expression of the gene. The same results were obtained for the reconstructed β-glucosidase gene with the same promoter length. It is likely that the deletion of this part of the promoter removes negative regulatory elements which are functional in Saccharomyces cerevisiae. This increase in activity is the main event which may explain the high increase in gene expression (60-fold) previously observed for an upstream deletion obtained during subcloning experiments of the β-glucosidase gene. It is also shown that the expression of the gene greatly depends upon the nature of the recipient strain, the growth phase of the cell and that of the vector carrying it.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00333412
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  • 15
    Electronic Resource
    Electronic Resource
    Stable yeast transformation with chimeric plasmids using a 2 μm-circular DNA-less strain as a recipient (1979)
    Springer
    Molecular genetics and genomics 176 (1979), S. 335-342 
    Details
    ISSN: 1617-4623
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary By using two chimeric plasmids containing yeast URA3 gene as a selection marker and 2 μm yeast DNA linked to the bacterial plasmid pCR1, a yeast strain devoid of any 2 μm DNA sequence was transformed. Recovery in E. coli of plasmids from yeast transformants showed that the 2 μm-less strain was able to maintain the chimeric plasmids as autonomous replicons, with very infrequent plasmid recombination. Hybridization experiments gave no evidence for integration of the URA3 DNA sequence in the chromosomal DNA. The transformed clones showed a high stability of the ura+ character during vegetative multiplication, even in the absence of selective pressure. The specific activity of orotidine 5′ monophosphate decarboxylase (coded by the URA3 gene) was 5 to 10 fold higher than in the wild type. These features should offer new possibilities for cloning with yeast.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00333095
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  • 16
    Electronic Resource
    Electronic Resource
    Isolation and physical characterization of streptomycete plasmids (1981)
    Springer
    Molecular genetics and genomics 182 (1981), S. 53-59 
    Details
    ISSN: 1617-4623
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Covalently closed circular DNA was isolated from a strain of Streptomyces coelicolor ATCC 10147 and from a strain of Streptomyces coelicolor subspecies flavus ATCC 19894, using two different methods. The two plasmids were of uniform monomer size: 8.9 kb for pS 10147, the plasmid from S. coelicolor ATCC 10147, and around 125 kb for the plasmid from S. coelicolor ATCC 19894. A restriction enzyme map was constructed for pS 10147, using seven enzymes. Four of the enzymes, (BamHI, Bgl,II, PvuII, and XhoI) cut pS 10147 once while PstI made two cuts. The GC content of this plasmid was calculated to be 72%. The possible utilisation of pS 10147 as a cloning vector in Streptomyces is discussed.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00422766
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  • 17
    Electronic Resource
    Electronic Resource
    A map of the restriction targets in yeast 2 micron plasmid DNA cloned on bacteriophage lambda (1976)
    Springer
    Molecular genetics and genomics 148 (1976), S. 287-294 
    Details
    ISSN: 1617-4623
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The 2 micron circular DNA from S. cerevisiae has been cloned on bacteriophage λ. The two forms of circular DNA which exist in equilibrium due to recombination between inverted repeat sequences were separated as stable clones, and a map of the targets for restriction endonucleases EcoRI, HindIII and HpaI was constructed. The circular DNAs isolated from a particular oligomycin resistant strain and its parent oligomycin sensitive strain were compared by restriction endonuclease analysis, and no difference was detected. The potential uses of cloned 2 micron DNA in determining the possible biological role of these plasmids are considered.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00332903
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  • 18
    Electronic Resource
    Electronic Resource
    Cloning and expression of the structural gene for β-glucosidase of Kluyveromyces fragilis in Escherichia coli and Saccharomyces cerevisiae (1984)
    Springer
    Molecular genetics and genomics 195 (1984), S. 108-115 
    Details
    ISSN: 1617-4623
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Cellobiose, the last product in cellulose degradation, is converted into two molecules of glucose by a β-glucosidase. S. cerevisiae does posses the structural gene for a β-glucosidase, but it is very poorly expressed; we thus decided to isolate and characterize that of Kluyveromyces fragilis. We constructed in E. coli HB101 strain a genomic library of the Kluyveromyces fragilis Y610 strain (ATCC 12424), a yeast able to grow on cellobiose and which constitutively produces the β-glucosidase. The structural gene for β-glucosidase was identified by its expression in E. coli. The initial isolated cosmid KF1 contained an insert of 35 Kb and by successive subcloning the insert size was reduced to 3.5 Kb (KF4). This cloned β-glucosidase gene introduced in S. cerevisiae by transformation is expressed at a level of about 500 times that of K. fragilis. We checked by Southern hybridization that the high expression level was not due to a rearrangement of K. fragilis DNA during the cloning experiments. Nevertheless to obtain yeast transformants able to grow on cellobiose a yeast strain whose permeability to sugar is increased must be used and this last point is discussed.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00332732
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  • 19
    Electronic Resource
    Electronic Resource
    Plasmids in different strains of Streptomyces ambofaciens: free and integrated form of plasmid pSAM2 (1984)
    Springer
    Molecular genetics and genomics 198 (1984), S. 35-41 
    Details
    ISSN: 1617-4623
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Five strains of Streptomyces ambofaciens were examined for their plasmid content. Among these strains, four belong to the same lineage (strains B) and the other was isolated independently (strain A). A large plasmid (ca. 80 kb), called pSAM1 in this paper and already described, was present in all B strains, and absent in strain A. A second plasmid, not described before, was found as covalently closed circular DNA in two of the four B strains. This plasmid with a size 11.1 kb was called pSAM2. A restriction map for 14 enzymes was established. Hybridization experiments showed that a unique sequence homologous to this plasmid is integrated in a larger replicon, which is not pSAM1 and is probably the chromosome, in all B strains and not in strain A. It seems probable that the integrated se1uence is the origin of the free plasmid found in two strains of the B family. It is noteworthy that the integrated form and the free plasmid may be found together. Transformation experiments proved that pSAM2 may be maintained autonomously in S. ambofaciens strain A and in S. lividans. pSAM2 is a self-transmissible plasmid, able to elicit the lethal zygosis reaction. pSAM2 was compared to the plasmids SLP1, pIJ110 and pIJ408, which all come from integrated sequences in three Streptomyces species and are found as autonomous plasmids after transfer to S. lividans. If pSAM2 resembles these plasmids in its origin, it does not appear to be related directly to them. Concerning their plasmid content, the two isolates of S. ambofaciens are very different. One of them contains neither pSAM1 not pSAM2. As this isolate produces spiramycin, these plasmids probably do not play an important role in spiramycin production. Apart from its intrinsic biological interest, pSAM2 may be useful in the construction of cloning vectors for S. ambofaciens. Very stable transformants might be obtained in certain strains of S. ambofaciens, because of the possibility of integration of the pSAM2 derivative vector.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00328697
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  • 20
    Electronic Resource
    Electronic Resource
    Site-specific integration of plasmid pSAM2 in Streptomyces lividans and S. ambofaciens (1988)
    Springer
    Molecular genetics and genomics 212 (1988), S. 432-439 
    Details
    ISSN: 1617-4623
    Keywords: Streptomyces ; Plasmid ; Integration ; Transfer ; Excision
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Streptomyces ambofaciens strain ATCC23877 contains the 11.1 kb plasmid pSAM2 stably integrated into its chromosome. This plasmidic sequence is able to loop out and to be transferred at high frequency to S. lividans where it is found simultaneously as both free and integrated plasmid. When a UV derivative of strain ATCC23877 (strain ATCC15154) is used, the resident copy of pSAM2 can be transferred to S. lividans, but only the integrated form is found in this strain. In both cases, the integration occurs at a unique chromosomal region through the same plasmidic integration site as that in strain ATCC23877. The resident copy of strain ATCC15154 can also be transferred at low frequency to S. ambofaciens DSM40697 (devoid of any pSAM2 sequence). In this case, as several copies of pSAM2 are integrated, the integration pattern is complicated. Integration of a complete pSAM2 sequence in this strain occurs in a region that hybridizes with the integration zones of S. lividans and of S. ambofaciens strain ATCC23877. Comparison of the cloned integration zone of S. lividans before and after the integration event showed that the restriction pattern of the resident pSAM2 in strain ATCC15154 is similar to that of the free form of pSAM2 found naturally in another UV derivative of strain ATCC23877 (strain JI3212).
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00330847
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