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  • 1
    Electronic Resource
    Electronic Resource
    Springer
    Journal of industrial microbiology and biotechnology 14 (1995), S. 355-364 
    ISSN: 1476-5535
    Keywords: Transformation ; Fungi ; Yeast ; Genetics ; Biotechnology
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Summary The genetic investigation of fungi has been extended substantially by DNA-mediated transformation, providing a supplement to more conventional genetic approaches based upon sexual and parasexual processes. Initial transformation studies with the yeastSaccharomyces cerevisiae provided the model for transformation systems in other fungi with regard to methodology, vector construction and selection strategies. There are, however, certain differences betweenS. cerevisiae and filamentous fungi with regard to type of genomic insertion and the availability of shuttle vectors. Single-site linked insertions are common in yeast due to the high level of homology required for recombination between vectored and genomic sequences, whereas mycelial fungi often show a high frequency of heterologous and unlinked insertions, often in the form of random and multiple-site integrations. While extrachromosomally-maintained or replicative vectors are readily available for use with yeasts, such vectors have been difficult to construct for use with filamentous fungi. The development of vectors for replicative transformation with these fungi awaits further study. It is proposed that replicative vectors may be inherently less efficient for use with mycelial fungi relative to yeasts, since the mycelium, as an extended and semicontinuous network of cells, may delimit an adequate diffusion of the vector carrying the selectable gene, thus leading to a high frequency of abortive or unstable transformants.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 0192-253X
    Keywords: Translation elongation factor 1α (EF-1α) ; developmental regulation ; oogenesis ; microinjection ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Transcription of the Xenopus laevis EF-1αS gene commences at the mid-blastula stage of embryonic development and then continues constitutively in all somatic tissues. The EF-1αS promoter is extremely active in the early Xenopus embryo where EF-1αS transcripts account for as much as 40% of all new polyadenylated transcripts. We have isolated the Xenopus EF-1αS gene and used microinjection techniques to identify promoter elements responsible for embryonic transcription. These in vivo expression studies have identified an enhancer fragment, located approximately 4.4 kb upstream of the transcription start site, that is required for maximum expression from the EF-1αS promoter. The enhancer fragment contains both an octamer and a G/C box sequence, but mutation studies indicate that the octamer plays no significant role in regulation of EF-1αS expression in the embryo. The presence of a G/C element in the enhancer and of multiple G/C boxes in the proximal promoter region suggests that the G/C box binding protein, Spl, plays a major role in the developmental regulation of EF-1αS promoter activity. © 1995 Wiley-Liss, Inc.
    Additional Material: 9 Ill.
    Type of Medium: Electronic Resource
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  • 3
    ISSN: 0749-503X
    Keywords: Yeast ; messenger RNA ; translation ; codon bias ; RNA secondary-structure ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The effects of poor codon bias and secondary structure formation upon the translation of the pyruvate kinase (PYK1) mRNA have been investigated in Saccharomyces cerevisiae. Following insertion mutagenesis at the 5′-end of the PYK1 coding region, the gene was transformed into yeast, and translation assessed directly in vivo by determining the distribution of the modified PYK1 mRNAs across polysomes fractionated by sucrose density gradient centrifugation. The chromosomally-encoded (wild-type) PYK1 mRNA, and the actin, ribosomal protein L3 and glyceraldehyde-3-phosphate dehydrogenase mRNAs were used to control for minor differences between polysome preparations. An insertion containing 13 non-preferred codons at the 5′-end of the coding region was found to have no significant effect upon PYK1 mRNA translation. In contrast, translation was inhibited by an insertion which increased the formation of secondary structures at the 5′-end of the mRNA (overall ΔG = -36·6 kcal/mol). Control insertions were also analysed to exclude the possibility that alterations to the amino acid sequence of pyruvate kinase affect the translation of its mRNA. These insertions, which introduced preferred codons or restored wild-type levels of secondary structure formation, did not significantly influence PYK1 mRNA translation.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
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  • 4
    ISSN: 0749-503X
    Keywords: Schizosaccharomyces pombe ; fission yeast ; glycosyl phosphatidylinositol anchors ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The Schizosaccharomyces pombe gpi1+ gene was cloned by complementation of the Saccharomyces cerevisiae gpi1 mutant, which has temperature-sensitive defects in growth and glycosyl phosphatidylinositol (GPI) membrane anchoring of protein, and which is defective in vitro in the first step in GPI anchor assembly, the formation of N-acetylglucosaminyl phosphatidylinositol (GlcNAc-PI). S. pombe gpi1+ encodes a protein with 29% identity to amino acids 87-609 of the S. cerevisiae protein, and is the functional homolog of the S. cerevisiae Gpi1 protein, for it restores [3H]inositol-labelling of protein and in vitro GlcNAc-PI synthetic activity to both S. cerevisiae gpi1 and gpi1::URA3 cells. Disruption of gpi1+ is lethal. Haploid Δgpi1+::his7+ spores germinate, but proceed through no more than three rounds of cell division, many cells ceasing growth as binucleate, septate cells with thickened septa. These results indicate that GPI synthesis is an essential function in fission yeast, and suggest that GPI anchoring is also required for completion of cytokinesis. The nucleotide sequence reported will appear in the GenBank Nucleotide Sequence database under the Accession Number U77355.©1997 John Wiley & Sons, Ltd.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
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