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  • 1
    Electronic Resource
    Electronic Resource
    Molecular analysis of KTI12/TOT4, a Saccharomyces cerevisiae gene required for Kluyveromyces lactis zymocin action (2002)
    Oxford, UK : Blackwell Science Ltd.
    Molecular microbiology 43 (2002), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: TOT, the putative Kluyveromyces lactis zymocin target complex from Saccharomyces cerevisiae, is encoded by TOT1–7, six loci of which are isoallelic to RNA polymerase II (RNAPII) Elongator genes (ELP1–6). Unlike TOT1–3 (ELP1–3) and TOT5–7 (ELP5, ELP6 and ELP4 respectively), which display zymocin resistance when deleted, TOT4 (KTI12) also renders cells refractory to zymocin when maintained in multicopy or overexpressed from the GAL10 promoter. Elevated TOT4 copy number results in an intermediate tot phenotype, which includes mild sensitivities towards caffeine, Calcofluor white and elevated growth temperature, suggesting that TOT4 influences TOT/Elongator function. Tot4p interacts with Elongator, as shown by co-immunoprecipitation, and cell fractionation studies demonstrate partial co-migration with RNAPII and Elongator. As Elongator subunit interaction is not affected by either deletion of TOT4 or multicopy TOT4, Tot4p may not be a structural Elongator subunit but, rather, may regulate TOT/Elongator in a fashion that requires transient physical contact with TOT/Elongator. Consistent with a regulatory role, the presence of a potential P-loop motif conserved between yeast and human TOT4 homologues suggests capability of ATP or GTP binding and P-loop deletion renders Tot4p biologically inactive.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1365-2958.2002.02794.x
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  • 2
    Electronic Resource
    Electronic Resource
    KTI11 and KTI13, Saccharomyces cerevisiae genes controlling sensitivity to G1 arrest induced by Kluyveromyces lactis zymocin (2002)
    Oxford, UK : Blackwell Science Ltd.
    Molecular microbiology 44 (2002), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The Kluyveromyces lactis zymocin and its γ-toxin subunit inhibit cell cycle progression of Saccharomyces cerevisiae. To identify S. cerevisiae genes conferring zymocin sensitivity, we complemented the unclassified zymocin-resistant kti11 and kti13 mutations using a single-copy yeast library. Thus, we identified yeast open reading frames (ORFs) YBL071w-A and YAL020c/ATS1 as KTI11 and KTI13 respectively. Disruption of KTI11 and KTI13 results in the complex tot phenotype observed for the γ-toxin target site mutants, tot1–7, and includes zymocin resistance, thermosensitivity, hypersensitivity to drugs and slow growth. Both loci, KTI11 and KTI13, are actively transcribed protein-encoding genes as determined by reverse transcriptase–polymerase chain reaction (RT–PCR) and in vivo HA epitope tagging. Kti11p is highly conserved from yeast to man, and Kti13p/Ats1p is related to yeast Prp20p and mammalian RCC1, components of the Ran–GTP/GDP cycle. Combining disruptions in KTI11 or KTI13 with a deletion in TOT3/ELP3 coding for the RNA polymerase II (RNAPII) Elongator histone acetyltransferase (HAT) yielded synthetic effects on slow growth phenotype expression. This suggests genetic interaction and possibly links KTI11 and KTI13 to Elongator function.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1365-2958.2002.02928.x
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  • 3
    Electronic Resource
    Electronic Resource
    Kluyveromyces lactis zymocin mode of action is linked to RNA polymerase II function via Elongator (2001)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 42 (2001), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The putative Kluyveromyces lactis zymocin target complex, TOT, from Saccharomyces cerevisiae comprises five Tot proteins, four of which are RNA polymerase II (RNAP II) Elongator subunits. Recently, two more Elongator subunit genes, ELP6 (TOT6) and ELP4 (TOT7), have been identified. Deletions of both TOT6 and TOT7 result in the complex tot phenotype, including resistance to zymocin, thermosensitivity, slow growth and hypersensitivity towards drugs, thus reinforcing the notion that TOT/Elongator may be crucial in signalling zymocicity. Mutagenesis of ELP3/TOT3, the Elongator histone acetyltransferase (HAT) gene, revealed that zymocin sensitivity could be uncoupled from Elongator wild-type function, indicating that TOT interacts genetically with zymocin. To test the possibility that zymocin functions by affecting RNAP II activity in a TOT/Elongator-dependent manner, global poly(A)+ mRNA levels were found to decline drastically on zymocin treatment. Moreover, cells overexpressing Fcp1p, the RNAP II carboxy-terminal domain phosphatase, acquired partial zymocin resistance, whereas cells underproducing RNAP II became zymocin hypersensitive. This suggests that zymocin may convert TOT/Elongator into a cellular poison toxic for RNAP II function and eventually leading to the observed G1 cell cycle arrest.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1365-2958.2001.02705.x
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  • 4
    Electronic Resource
    Electronic Resource
    A cytoplasmic gene-shuffle system in Kluyveromyces lactis: use of epitope tagging to detect a killer plasmid-encoded gene product (1996)
    Oxford BSL : Blackwell Science Ltd
    Molecular microbiology 19 (1996), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: A novel gene shuffle approach has been developed for investigating the functions of genes on the cytoplasmic linear DNA killer plasmids of Kluyveromyces lactis. By transplacing k2ORF5 from the larger plasmid pGKL2(k2) onto pGKL1(k1) we have shown this gene to be essential and functionally interchangeable between plasmids. Once transferred onto k1, k2ORF5 is fully able to complement a k2ORF50 deletion on k2 in trans, giving rise to yeast strains containing only the two recombinant plasmid forms. Additionally, the in vivo product of k2ORF5 has been identified as a 19.5 kDa protein by transplacing an epitope-tagged k2ORF5 allele from k2 to k1. The ease of detection of the tagged ORF5 product in comparison to TRF1, the gene product of k2ORF10, indicates that Orf5p is one of the most abundant k2 products, implying structural rather than regulatory function.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1365-2958.1996.402942.x
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  • 5
    Electronic Resource
    Electronic Resource
    Elongator's toxin-target (TOT) function is nuclear localization sequence dependent and suppressed by post-translational modification (2003)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 49 (2003), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The toxin target (TOT) function of the Saccharomyces cerevisiae Elongator complex enables Kluyveromyces lactis zymocin to induce a G1 cell cycle arrest. Loss of a ubiquitin-related system (URM1–UBA4 ) and KTI11 enhances post-translational modification/proteolysis of Elongator subunit Tot1p (Elp1p) and abrogates its TOT function. Using TAP tagging, Kti11p contacts Elongator and translational proteins (Rps7Ap, Rps19Ap Eft2p, Yil103wp, Dph2p). Loss of YIL103w and DPH2 (involved in diphtheria toxicity) suppresses zymocicity implying that both toxins overlap in a manner mediated by Kti11p. Among the pool that co-fractionates with RNA polymerase II (pol II) and nucleolin, Nop1p, unmodified Tot1p dominates. Thus, modification/proteolysis may affect association of Elongator with pol II or its localization. Consistently, an Elongator-nuclear localization sequence (NLS) targets green fluorescent protein (GFP) to the nucleus, and its truncation yields TOT deficiency. Similarly, KAP120 deletion rescues cells from zymocin, suggesting that Elongator's TOT function requires NLS- and karyopherin-dependent nuclear import.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1365-2958.2003.03632.x
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  • 6
    Electronic Resource
    Electronic Resource
    Protein interactions within Saccharomyces cerevisiae Elongator, a complex essential for Kluyveromyces lactis zymocicity (2002)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 45 (2002), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: mTn3-tagging identified Kluyveromyces lactis zymocin target genes from Saccharomyces cerevisiae as TOT1–3/ELP1–3 coding for the RNA polymerase II (pol II) Elongator histone acetyltransferase (HAT) complex. tot phenotypes resulting from mTn3 tagging were similar to totΔ null alleles, suggesting loss of Elongator's integrity. Consistently, the Tot1–3/Elp1–3 proteins expressed from the mTn3-tagged genes were all predicted to be C-terminally truncated, lacking ≈ 80% of Tot1p, five WD40 Tot2p repeats and two HAT motifs of Tot3p. Besides its role as a HAT, Tot3p assists subunit communication within Elongator by mediating Tot2–Tot4, Tot2–Tot5, Tot2–Tot1 and Tot4–Tot5 protein–protein interactions. TOT1 and TOT2 are essential for Tot4–Tot2 and Tot4–Tot3 interactions respectively. The latter was lost with a C-terminal Tot2p truncation; the former was affected by progressively truncating TOT1. Despite being dispensable for Tot4–Tot2 interaction, the extreme C-terminus of Tot1p may play a role in TOT/Elongator function, as its truncation confers zymocin resistance. Tot4p/Kti12p, an Elongator-associated factor, also interacted with pol II and could be immunoprecipitated while being bound to the ADH1 promoter. Two-hybrid analysis showed that Tot4p also interacts with Cdc19p, suggesting that Tot4p plays an additional role in concert with Cdc19p, perhaps co-ordinating cell growth with carbon source metabolism.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1365-2958.2002.03055.x
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  • 7
    Electronic Resource
    Electronic Resource
    Genetic manipulation of Kluyveromyces lactis linear DNA plasmids: gene targeting and plasmid shuffles (1999)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS microbiology letters 178 (1999), S. 0 
    Details
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Genetic manipulation of yeast linear DNA plasmids, particularly of k1 and k2 from the non-conventional dairy yeast Kluyveromyces lactis, has been advanced by the recent establishment of DNA transformation-mediated one-step gene disruption and allele replacement techniques. These methods provide the basis for a strategy for the functional analysis of plasmid genes and DNA elements. By use of double selection regimens, these single-gene procedures have been extended to effect disruption of individual genes on plasmid k2 and transplacement of a functional copy onto plasmid k1, resulting in the production of yeast strains with an altered plasmid composition. This cytoplasmic gene shuffle system facilitates the introduction of specifically modified alleles into k1 or k2 in order to study the function, expression (from UCS promoters) and regulation of cytoplasmic linear plasmid genes. Additionally, identification, characterization and localization of plasmid gene products of interest are made possible by shuffling GFP-, epitope- or affinity purification-tagged alleles between k2 and k1. The gene shuffle approach can also be used for vector development and heterologous protein expression in order to exploit the biotechnical potential of the K. lactis k1/k2 system in yeast cell factory research.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1574-6968.1999.tb08678.x
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  • 8
    Electronic Resource
    Electronic Resource
    Yeast killer plasmid pGKL2: molecular analysis of UCS5, a cytoplasmic promoter element essential for ORF5 gene function (1996)
    Springer
    Molecular genetics and genomics 250 (1996), S. 286-294 
    Details
    ISSN: 1617-4623
    Keywords: Key words Kluyveromyces lactis ; Linear plasmids ; Cytoplasmic promoter
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract  A k2/k1 plasmid gene shuffle system has been used to investigate linear plasmid promoter function in Kluyveromyces lactis. By transplacing various ORF5 deletion constructs from the larger plasmid k2 onto k1, and analysing trans-complementation of an ORF50 deletion on k2, a 40 bp k2 fragment, including the UCS motif of ORF5 (UCS5), has been identified as a cis-acting promoter element essential for ORF5 gene function. Qualitative and quantitative transcript analyses of a UCS5-ScLEU2 fusion gene using Northern blot analysis and phosphor image technology revealed a plasmid-dependent LEU2 transcript distinct in size (1.55 kb) and regulation from its nuclear counterpart (1.35 kb): cytoplasmic, UCS5-driven expression of the marker gene was non-repressible by leucine and reduced five- to eight-fold compared to fully derepressed nuclear KlLEU2 mRNA levels. Thus, the killer plasmids k2 and k1 appear to express low levels of transcript overall, when relative gene copy numbers (one for the nuclear allele versus 50–100 copies for the plasmid-borne LEU2 gene) are taken into account.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF02174386
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  • 9
    Electronic Resource
    Electronic Resource
    Kluyveromyces lactis killer plasmid pGKL2: Molecular analysis of an essential gene, ORF5 (1995)
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 11 (1995), S. 615-628 
    Details
    ISSN: 0749-503X
    Keywords: Kluyveromyces lactis ; killer plasmid ; gene disruption ; epitope-tagging ; baculovirus over-expression ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The ORF5 of Kluyveromyces lactis killer plasmid pGKL2 (k2) is capable of encoding a small neutral protein of 18 kDa of as yet unassigned function. Although this ORF is located between two larger ORFs, 4 and 6, which it overlaps, RNA analysis showed that it is transcribed monocistronically. One-step gene disruption of ORF5, via in vivo homologous recombination between native plasmid k2 and a transfer vector employing the Saccharomyces cerevisiae LEU2 gene fused to the k2 UCS5 element, yielded Leu+ transformants at high frequencies. The transformants were found to carry a new recombinant form of k2 with ORF5 replaced by the LEU2 marker, termed rk2, in addition to the wild-type plasmids k1 and k2. Northern analysis detected a plasmid-dependent LEU2 transcript distinct in size and regulation from its nuclear counterpart. Recombinant plasmid, rk2, was unable to displace native k2 during Leu+ selective growth; however rk2 was displaced by k2 during non-selective growth. Thus, ORF5 appears to be an essential gene for plasmid integrity and/or maintenance. The ORF5 product was detected by over-expression of an epitope-tagged allele in the baculovirus system. Western analysis using a monoclonal antibody specific for the epitope tag identified a protein band with apparent molecular weight of 20 kDa, corresponding in size to the predicted product.
    Additional Material: 8 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/yea.320110703
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