Heterogeneous functional Ty1 elements are abundant in the Saccharomyces cerevisiae genome.

Abstract Despite the abundance of Ty1 RNA in Saccharomyces cerevisiae, Ty1 retrotransposition is a rare event. To determine whether transpositional dormancy is the result of defective Ty1 elements, functional and defective alleles of the retrotransposon in the yeast genome were quantitated. Genomic Ty1 elements were isolated by gap repair-mediated recombination of pGTy1-H3(delta 475-3944) HIS3, a multicopy plasmid containing a GAL1/Ty1-H3 fusion element lacking most of the gag domain (TYA) and the protease (PR) and integrase (IN) domains. Of 39 independent gap repaired pGTyHIS3 elements isolated, 29 (74%) transposed at high levels following galactose induction. The presence of restriction site polymorphisms within the gap repaired region of the 29 functional pGTyHIS3 elements indicated that they were derived from at least eight different genomic Ty1 elements and one Ty2 element. Of the 10 defective pGTyHIS3 elements, one was a partial gap repair event while the other nine were derived from at least six different genomic Ty1 elements. These results suggest that most genomic Ty1 elements encode functional TYA, PR and IN proteins. To understand how functional Ty1 elements are regulated, we tested the hypothesis that a TYB protein associates preferentially in cis with the RNA template that encodes it, thereby promoting transposition of its own element. A genomic Ty1 mhis3AI element containing either an in-frame insertion in PR or a deletion in TYB transposed at the same rate as a wild-type Ty1mhis3AI allele, indicating that TYB proteins act efficiently in trans. This result suggests in principle that defective genomic Ty1 elements could encode trans-acting repressors of transposition; however, expression of only one of the nine defective pGTy1 isolates had a negative effect on genomic Ty1 mhis3AI element transposition in trans, and this effect was modest. Therefore, the few defective Ty1 elements in the genome are not responsible for transpositional dormancy.

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Identification and characterization of the centromere from chromosome XIV in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.5.11.2887-2893.1985 ◽

1985 ◽

Vol 5 (11) ◽

pp. 2887-2893

Author(s):

M Neitz ◽

J Carbon

Keyword(s):

Saccharomyces Cerevisiae ◽

Restriction Fragment ◽

Dna Sequences ◽

Yeast Genome ◽

Ura3 Gene ◽

Dna Restriction ◽

Meiotic Analyses ◽

The Right ◽

In Cis

A functional centromere located on a small DNA restriction fragment from Saccharomyces cerevisiae was identified as CEN14 by integrating centromere-adjacent DNA plus the URA3 gene by homologous recombination into the yeast genome and then by localizing the URA3 gene to chromosome XIV by standard tetrad analysis. DNA sequence analysis revealed that CEN14 possesses sequences (elements I, II, and III) that are characteristic of other yeast centromeres. Mitotic and meiotic analyses indicated that the CEN14 function resides on a 259-base-pair (bp) RsaI-EcoRV restriction fragment, containing sequences that extend only 27 bp to the right of the element I to III region. In conjunction with previous findings on CEN3 and CEN11, these results indicate that the specific DNA sequences required in cis for yeast centromere function are contained within a region about 150 bp in length.

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A deletion that includes the segment coding for the signal peptidase cleavage site delays release of Saccharomyces cerevisiae acid phosphatase from the endoplasmic reticulum

Molecular and Cellular Biology ◽

10.1128/mcb.6.2.723-729.1986 ◽

1986 ◽

Vol 6 (2) ◽

pp. 723-729

Author(s):

R Haguenauer-Tsapis ◽

M Nagy ◽

A Ryter

Keyword(s):

Saccharomyces Cerevisiae ◽

Endoplasmic Reticulum ◽

Acid Phosphatase ◽

Cleavage Site ◽

Ultrastructural Localization ◽

Signal Peptidase ◽

Multicopy Plasmid ◽

Wild Type ◽

Sugar Chains ◽

Pho5 Gene

We studied ultrastructural localization of acid phosphatase in derepressed Saccharomyces cerevisiae cells transformed with a multicopy plasmid carrying either the wild-type PHO5 gene or a PHO5 gene deleted in the region overlapping the signal peptidase cleavage site. Wild-type enzyme was located in the cell wall, as was 50% of the modified protein, which carried high-mannose-sugar chains. The remaining 50% of the protein was active and core glycosylated, and it accumulated in the endoplasmic reticulum cisternae. The signal peptide remained uncleaved in both forms. Cells expressing the modified protein exhibited an exaggerated endoplasmic reticulum with dilated lumen.

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An unusual genomic position effect on Drosophila white gene expression: pairing dependence, interactions with zeste, and molecular analysis of revertants.

Genetics ◽

10.1093/genetics/130.1.125 ◽

1992 ◽

Vol 130 (1) ◽

pp. 125-138 ◽

Cited By ~ 2

Author(s):

T Hazelrigg ◽

S Petersen

Keyword(s):

Gene Expression ◽

Regulatory Element ◽

Position Effect ◽

Insertion Site ◽

White Gene ◽

Chromosome 2 ◽

Wild Type ◽

Gene Copies ◽

In Trans ◽

In Cis

Abstract The white gene in the AR4-24 P[white,rosy] insertion on chromosome 2 has a novel expression pattern, in which it is repressed in the dorsal half of the eye. X-ray mutagenesis led to the isolation of six revertants mapping to chromosome 2, which are wild type in a zeste+ background, and three extreme derivatives, in which white gene expression is repressed in ventral regions of the eye as well. By Southern blot analyses the breakpoints of five of the revertants and one of the extreme derivatives were mapped in the flanking DNA bordering each side of the AR4-24 insertion. The revertants show some dorsal repression of white in the presence of z1, and by this criterion each is only a partial revertant. The extreme derivatives act not only in cis, but also in trans to repress expression of AR4-24 and its various derivatives. We provide evidence that these trans effects are proximity-dependent effects, possibly mediated by pairing of gene copies, as they do not extend to copies of the white gene located elsewhere in the genome. We show that one extreme derivative, E1, is a small deletion spanning the insertion site at the 5' end of the white gene, and propose that the distance between a negative regulatory element in the 5' flanking DNA and the white promoter influences the degree of the repression.

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Trans-dimerization of JAM-A regulates Rap2 and is mediated by a domain that is distinct from the cis-dimerization interface

Molecular Biology of the Cell ◽

10.1091/mbc.e14-01-0018 ◽

2014 ◽

Vol 25 (10) ◽

pp. 1574-1585 ◽

Cited By ~ 15

Author(s):

Ana C. Monteiro ◽

Anny-Claude Luissint ◽

Ronen Sumagin ◽

Caroline Lai ◽

Franziska Vielmuth ◽

...

Keyword(s):

Epithelial Cell Proliferation ◽

Wild Type ◽

Transfected Cells ◽

Force Microscopy ◽

Atomic Force ◽

Unique Site ◽

Dimerization Interface ◽

In Trans ◽

In Cis ◽

Common Cell

Junctional adhesion molecule-A (JAM-A) is a tight junction–associated signaling protein that regulates epithelial cell proliferation, migration, and barrier function. JAM-A dimerization on a common cell surface (in cis) has been shown to regulate cell migration, and evidence suggests that JAM-A may form homodimers between cells (in trans). Indeed, transfection experiments revealed accumulation of JAM-A at sites between transfected cells, which was lost in cells expressing cis- or predicted trans-dimerization null mutants. Of importance, microspheres coated with JAM-A containing alanine substitutions to residues 43NNP45 (NNP-JAM-A) within the predicted trans-dimerization site did not aggregate. In contrast, beads coated with cis-null JAM-A demonstrated enhanced clustering similar to that observed with wild-type (WT) JAM-A. In addition, atomic force microscopy revealed decreased association forces in NNP-JAM-A compared with WT and cis-null JAM-A. Assessment of effects of JAM-A dimerization on cell signaling revealed that expression of trans- but not cis-null JAM-A mutants decreased Rap2 activity. Furthermore, confluent cells, which enable trans-dimerization, had enhanced Rap2 activity. Taken together, these results suggest that trans-dimerization of JAM-A occurs at a unique site and with different affinity compared with dimerization in cis. Trans-dimerization of JAM-A may thus act as a barrier-inducing molecular switch that is activated when cells become confluent.

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sir2 mutants of Kluyveromyces lactis are hypersensitive to DNA-targeting drugs.

Molecular and Cellular Biology ◽

10.1128/mcb.14.7.4501 ◽

1994 ◽

Vol 14 (7) ◽

pp. 4501-4508 ◽

Cited By ~ 26

Author(s):

X J Chen ◽

G D Clark-Walker

Keyword(s):

Saccharomyces Cerevisiae ◽

Protein Function ◽

Kluyveromyces Lactis ◽

Functional Equivalence ◽

Striking Difference ◽

Multicopy Plasmid ◽

Wild Type ◽

Dna Targeting ◽

Mating Efficiency ◽

Type Gene

A Kluyveromyces lactis mutant, hypersensitive to the DNA-targeting drugs ethidium bromide (EtBr), berenil, and HOE15030, can be complemented by a wild-type gene with homology to SIR2 of Saccharomyces cerevisiae (ScSIR2). The deduced amino acid sequence of the K. lactis Sir2 protein has 53% identity with ScSir2 protein but is 108 residues longer. K. lactis sir2 mutants show decreased mating efficiency, deficiency in sporulation, an increase in recombination at the ribosomal DNA locus, and EtBr-induced death. Some functional equivalence between the Sir2 proteins of K. lactis and S. cerevisiae has been demonstrated by introduction of ScSIR2 into a sir2 mutant of K. lactis. Expression of ScSIR2 on a multicopy plasmid restores resistance to EtBr and complements sporulation deficiency. Similarly, mating efficiency of a sir2 mutant of S. cerevisiae is partially restored by K. lactis SIR2 on a multicopy plasmid. Although these observations suggest that there has been some conservation of Sir2 protein function, a striking difference is that sir2 mutants of S. cerevisiae, unlike their K. lactis counterparts, are not hypersensitive to DNA-targeting drugs.

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The GTS1 gene, which contains a Gly-Thr repeat, affects the timing of budding and cell size of the yeast Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.14.8.5569 ◽

1994 ◽

Vol 14 (8) ◽

pp. 5569-5578 ◽

Cited By ~ 41

Author(s):

K Mitsui ◽

S Yaguchi ◽

K Tsurugi

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acid ◽

Cell Size ◽

Single Cells ◽

Exponential Growth Phase ◽

Restrictive Temperature ◽

Multicopy Plasmid ◽

Wild Type ◽

Yeast Saccharomyces Cerevisiae ◽

Mean Cell Volume

A gene with an open reading frame encoding a protein of 417 amino acid residues with a Gly-Thr repeat was isolated from the yeast Saccharomyces cerevisiae by using synthetic oligonucleotides encoding three Gly-Thr dimers as probes. The deduced amino acid sequence showed partial homology to the clock-affecting gene, per, of Drosophila melanogaster in the regions including the GT repeat. The function of the gene, named GTS1, was examined by characterizing the phenotypes of transformants with different copy numbers of the GTS1 gene produced either by inactivating the GTS1 gene by gene disruption (TM delta gts1) or by transformation with multicopy plasmid pPER119 (TMpGTS1). They grew at similar rates during the exponential growth phase, but the lag phases were shorter for TM delta gts1 and longer for TMpGTS1 cells than that for the wild type. Analyses of their cell cycle parameters using synchronized cells revealed that the unbudding period changed as a function of gene dosage; that is, the periods of TM delta gts1 and TMpGTS1 were about 20% shorter and longer, respectively, than that of the wild-type. Another significant change in the transformants was detected in the distribution of the cell size. The mean cell volume of the TM delta gts1 cells in the unbudded period (single cells) was 27% smaller than that of single wild-type cells, whereas that of single TMpGTS1 cells was 48% larger. Furthermore, in the temperature-sensitive cdc4 mutant, the GTS1 gene affected the timing of budding at the restrictive temperature. Thus, the GTS1 gene product appears to modulate the timing of budding to obtain an appropriate cell size independent of the DNA replication cycle.

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A rare tRNA-Arg(CCU) that regulates Ty1 element ribosomal frameshifting is essential for Ty1 retrotransposition in Saccharomyces cerevisiae.

Genetics ◽

10.1093/genetics/135.2.309 ◽

1993 ◽

Vol 135 (2) ◽

pp. 309-320 ◽

Cited By ~ 7

Author(s):

K Kawakami ◽

S Pande ◽

B Faiola ◽

D P Moore ◽

J D Boeke ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Protein Processing ◽

Ribonuclease H ◽

Wild Type ◽

Ribosomal Frameshifting ◽

Translational Frameshifting ◽

Rate Limiting Step ◽

Limiting Step ◽

Ty1 Retrotransposition ◽

Rate Limiting

Abstract Translation of the yeast retrotransposon Ty1 TYA1(gag)-TYB1(pol) gene occurs by a +1 ribosomal frameshifting event at the sequence CUU AGG C. Because overexpression of a low abundance tRNA-Arg(CCU) encoded by the HSX1 gene resulted in a reduction in Ty1 frameshifting, it was suggested that a translational pause at the AGG-Arg codon is required for optimum frameshifting. The present work shows that the absence of tRNA-Arg(CCU) affects Ty1 transposition, translational frameshifting, and accumulation of mature TYB1 proteins. Transposition of genetically tagged Ty1 elements decreases at least 50-fold and translational frameshifting increases 3-17-fold in cells lacking tRNA-Arg(CCU). Accumulation of Ty1-integrase and Ty1-reverse transcriptase/ribonuclease H is defective in an hsx1 mutant. The defect in Ty1 transposition is complemented by the wild-type HSX1 gene or a mutant tRNA-Arg(UCU) gene containing a C for T substitution in the first position of the anticodon. Overexpression of TYA1 stimulates Ty1 transposition 50-fold above wild-type levels when the level of transposition is compared in isogenic hsx1 and HSX1 strains. Thus, the HSX1 gene determines the ratio of the TYA1 to TYA1-TYB1 precursors required for protein processing or stability, and keeps expression of TYB1 a rate-limiting step in the retrotransposition cycle.

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Mutations in Residues Involved in Zinc Binding in the Catalytic Site of Escherichia coli Threonyl-tRNA Synthetase Confer a Dominant Lethal Phenotype

Journal of Bacteriology ◽

10.1128/jb.00439-07 ◽

2007 ◽

Vol 189 (19) ◽

pp. 6839-6848 ◽

Cited By ~ 3

Author(s):

Joël Caillet ◽

Monique Graffe ◽

Flore Eyermann ◽

Pascale Romby ◽

Mathias Springer

Keyword(s):

Escherichia Coli ◽

Trna Synthetase ◽

Dominant Negative ◽

Zinc Binding ◽

Regulatory Function ◽

Dominant Negative Effect ◽

Multicopy Plasmid ◽

Wild Type ◽

Zinc Binding Site ◽

Negative Effect

ABSTRACT Escherichia coli threonyl-tRNA synthetase is a homodimeric protein that acts as both an enzyme and a regulator of gene expression: the protein aminoacylates tRNAThr isoacceptors and binds to its own mRNA, inhibiting its translation. The enzyme contains a zinc atom in its active site, which is essential for the recognition of threonine. Mutations in any of the three amino acids forming the zinc-binding site inactivate the enzyme and have a dominant negative effect on growth if the corresponding genes are placed on a multicopy plasmid. We show here that this particular property is not due to the formation of inactive heterodimers, the titration of tRNAThr by an inactive enzyme, or its misaminoacylation but is, rather, due to the regulatory function of threonyl-tRNA synthetase. Overproduction of the inactive enzyme represses the expression of the wild-type chromosomal copy of the gene to an extent incompatible with bacterial growth.

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Identification and characterization of the centromere from chromosome XIV in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.5.11.2887 ◽

1985 ◽

Vol 5 (11) ◽

pp. 2887-2893 ◽

Cited By ~ 11

Author(s):

M Neitz ◽

J Carbon

Keyword(s):

Saccharomyces Cerevisiae ◽

Restriction Fragment ◽

Dna Sequences ◽

Yeast Genome ◽

Ura3 Gene ◽

Dna Restriction ◽

Meiotic Analyses ◽

The Right ◽

In Cis

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P-Body Components Are Required for Ty1 Retrotransposition during Assembly of Retrotransposition-Competent Virus-Like Particles

Molecular and Cellular Biology ◽

10.1128/mcb.00251-09 ◽

2009 ◽

Vol 30 (2) ◽

pp. 382-398 ◽

Cited By ~ 52

Author(s):

Mary Ann Checkley ◽

Kunio Nagashima ◽

Stephen J. Lockett ◽

Katherine M. Nyswaner ◽

David J. Garfinkel

Keyword(s):

Saccharomyces Cerevisiae ◽

Immunofluorescence Microscopy ◽

Wild Type ◽

Virus Like Particles ◽

P Bodies ◽

Cellular Processes ◽

Body Component ◽

Body Components ◽

Ty1 Retrotransposition

ABSTRACT Ty1 is a retrovirus-like retrotransposon whose replication is influenced by diverse cellular processes in Saccharomyces cerevisiae. We have identified cytoplasmic P-body components encoded by DHH1, KEM1, LSM1, and PAT1 as cofactors that posttranscriptionally enhance Ty1 retrotransposition. Using fluorescent in situ hybridization and immunofluorescence microscopy, we found that Ty1 mRNA and Gag colocalize to discrete cytoplasmic foci in wild-type cells. These foci, which are distinct from P-bodies, do not form in P-body component mutants or under conditions suboptimal for retrotransposition. Our immunoelectron microscopy (IEM) data suggest that mRNA/Gag foci are sites where virus-like particles (VLPs) cluster. Overexpression of Ty1 leads to a large increase in retrotransposition in wild-type cells, which allows VLPs to be detected by IEM. However, retrotransposition is still reduced in P-body component mutants under these conditions. Moreover, the percentage of Ty1 mRNA/Gag foci and VLP clusters and levels of integrase and reverse transcriptase are reduced in these mutants. Ty1 antisense RNAs, which have been reported to inhibit Ty1 transposition, are more abundant in the kem1Δ mutant and colocalize with Ty1 mRNA in the cytoplasm. Therefore, Kem1p may prevent the aggregation of Ty1 antisense and mRNAs. Overall, our results suggest that P-body components enhance the formation of retrotransposition-competent Ty1 VLPs.

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