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D J Garfinkel

Publications and source records attributed to D J Garfinkel.

At least 19 recordsLinked to original sources
PubMed1992

Disruption of a silencer domain by a retrotransposon.

A galactose-inducible Ty element carrying the HIS3 gene has been used as an insertional mutagen to generate alpha-factor resistant mutants. This collection of Ty-induced mutations includes insertions into the gene for the alpha-factor receptor (STE2), several nonspecific STE genes, and mutations that lead to the expression of the normally silent HML alpha locus. The hml alpha "on" mutations fall into two classes, those that disrupt trans-acting regulators involved in silencing HML alpha and a novel class of mutations that activate HML alpha by insertion at that locus. The hml alpha::Ty "on" mutations illustrate the unusual ability of these retrotransposons to activate genes by overcoming gene silencing mechanisms. The hml alpha::Ty "on" mutations include examples of multimeric Ty arrays. Single Ty and solo delta insertion derivatives of these Ty multimers restore the ability of the silencing mechanism to repress HML alpha.

Blotting, Southern↗
PubMed1992

Ty element-induced temperature-sensitive mutations of Saccharomyces cerevisiae.

Temperature-sensitive mutants of Saccharomyces cerevisiae were isolated by insertional mutagenesis using the HIS3 marked retrotransposon TyH3HIS3. In such mutants, the TyHIS3 insertions are expected to identify loci which encode genes essential for cell growth at high temperatures but dispensable at low temperatures. Five mutations were isolated and named hit for high temperature growth. The hit1-1 mutation was located on chromosome X and conferred the pet phenotype. Two hit2 mutations, hit2-1 and hit2-2, were located on chromosome III and caused the deletion of the PET18 locus which has been shown to encode a gene required for growth at high temperatures. The hit3-1 mutation was located on chromosome VI and affected the CDC26 gene. The hit4-1 mutation was located on chromosome XIII. These hit mutations were analyzed in an attempt to identify novel genes involved in the heat shock response. The hit1-1 mutation caused a defect in synthesis of a 74-kD heat shock protein. Western blot analysis revealed that the heat shock protein corresponded to the SSC1 protein, a member of the yeast hsp70 family. In the hit1-1 mutant, the TyHIS3 insertion caused a deletion of a 3-kb DNA segment between the delta 1 and delta 4 sequences near the SUP4 locus. The 1031-bp wild-type HIT1 DNA which contained an open reading frame encoding a protein of 164 amino acids and the AGG arginine tRNA gene complemented all hit1-1 mutant phenotypes, indicating that the mutant phenotypes were caused by the deletion of these genes. The pleiotropy of the HIT1 locus was analyzed by constructing a disruption mutation of each gene in vitro and transplacing it to the chromosome. This analysis revealed that the HIT1 gene essential for growth at high temperatures encodes the 164-amino acid protein. The arginine tRNA gene, named HSX1, is essential for growth on a nonfermentable carbon source at high temperatures and for synthesis of the SSC1 heat shock protein.

Base Sequence↗
PubMed1992

Posttranslational control of Ty1 retrotransposition occurs at the level of protein processing.

High-level expression of a transpositionally competent Ty1 element fused to the inducible GAL1 promoter on a 2 microns plasmid (pGTy1) overcomes transpositional dormancy in Saccharomyces cerevisiae. To investigate the mechanisms controlling the rate of Ty1 retrotransposition, we quantitated transposition and Ty1 gene products in cells induced and uninduced for expression of pGTy1. The increase in Ty1 transposition was 45- to 125-fold greater than the increase in Ty1 RNA effected by pGTy1 induction. Translational efficiency of Ty1 RNA was not altered in transposition-induced cells, since p190TYA1-TYB1 protein synthesis increased in proportion to steady-state Ty1 RNA levels. Therefore, expression of a pGTy1 element increases the efficiency of Ty1 transposition at a posttranslational level. Galactose induction of pGTy1 enhanced TYA1 protein processing and allowed detection of processed TYB1 proteins, which are normally present at very low levels in uninduced cells. When the ability of genomic Ty1 elements to complement defined mutations in HIS3-marked pGTy1 elements was examined, mutations in the protease domain or certain mutations in the integrase domain failed to be complemented, but mutations in the reverse transcriptase domain were partially complemented by genomic Ty1 elements. Therefore, the activity of Ty1 elements in yeast cells may be limited by the availability of Ty1 protease and possibly integrase. These results suggest that Ty1 transposition is regulated at the level of protein processing and that this regulation is overcome by expression of a pGTy1 element.

Base Sequence↗
PubMed1991

RNA-mediated recombination in S. cerevisiae.

The existence of pseudogenes and the observation of intron loss suggest that RNA can serve as an intermediate in recombination. We used a HIS3 reporter gene to show that RNA-mediated recombination occurs in yeast. His3+ prototroph formation required transcription and expression of the retrotransposon Ty. Two RNA-mediated recombination events were detected: homologous recombination between the cDNA and plasmid his3 sequences, resulting in intron loss, and insertion of the cDNA into the chromosome in the absence of HIS3 homology. The chromosomal His3+ prototrophs showed many hallmarks of naturally occurring pseudogenes. They integrated at novel sites in the chromosome, lacked introns, and possessed poly(A) tracts. Additionally, their 5' ends corresponded with the site of initiation of the GAL1 transcript.

Base Sequence↗
PubMed1991

Single-step selection for Ty1 element retrotransposition.

The yeast retrotransposon Ty1 has been tagged with a reporter gene that allows selection of RNA-mediated transposition events and is applicable to the study of retroelements in other organisms. The reporter gene is a yeast HIS3 gene interrupted by an artificial intron (AI) in the antisense orientation. The HIS3AI sequences were inserted into a Ty1 element such that the intron is on the sense strand of the Ty1 element; therefore, splicing and retrotransposition of marked Ty1 transcripts can give rise to His+ cells. Fusion of the Ty1-H3mHIS3AI element to the inducible GAL1 promoter resulted in a high frequency of histidine prototrophs upon galactose induction. Moreover, spontaneous His+ revertants derived from strains containing genomic TymHIS3AI elements are a result of retrotransposition. By using this assay, we estimated the Ty1 transposition rate to be between 3 x 10(-7) and 1 x 10(-5) transpositions per Ty1 element per generation. Variations in the transposition rate of individual Ty1 elements are correlated with the relative abundance of their transcripts.

Blotting, Northern↗
PubMed1991

Regulation of retrotransposition in Saccharomyces cerevisiae.

Retrotransposons are a widely distributed group of eukaryotic mobile genetic elements that transpose through an RNA intermediate. The element Ty (Transposon yeast), found in the yeast Saccharomyces cerevisiae, is a model system for the study of retrotransposons because of the experimental tools that exist to manipulate and detect transposition. Ty transposition can be elevated to levels exceeding one transposition event per cell when an element is expressed from an inducible yeast promoter. In addition, individual genomic Ty elements can be tagged with a retrotransposition indicator gene that allows transposition events occurring at a rate of 10(-5) to 10(-7) per element per cell division to be detected phenotypically. These systems are being used to elucidate the mechanism of Ty transposition and clarify how Ty transposition is controlled.

DNA Transposable Elements↗
PubMed1991

Proteolytic processing of pol-TYB proteins from the yeast retrotransposon Ty1.

Using antibodies directed against the TYB1 protein of the transpositionally competent retrotransposon Ty1-H3, we have identified three mature proteins of 23, 60, and 90 kDa and processing intermediates of 140 and 160 kDa that are derived from the 190-kDa TYA1-TYB1 polyprotein. Mature proteins and variable amounts of the precursors cofractionate with Ty viruslike particles. The map locations and precursor-product relationships of mature TYB1 polypeptides suggest that p23 is Ty1 protease, p90 is integrase, and p60 contains reverse transcriptase and RNase H. Immunoprecipitation and immunoblot analyses of Ty1 proteins show that p190 is cleaved to form p160. The p160 intermediate is cleaved to form p23 and p140, and p140 is cleaved to form p90 and p60. Processing of TYB1 proteins is dependent on Ty1 protease. Immunoblot analysis of TYB proteins from different Ty1 isolates reveal that correct processing of TYB1 proteins is a characteristic of functional Ty1 elements, whereas aberrant processing is a common defect found in transposition-incompetent elements.

Amino Acid Sequence↗
PubMed1990

Ty RNA levels determine the spectrum of retrotransposition events that activate gene expression in Saccharomyces cerevisiae.

To learn more about the variety of Ty elements capable of activating gene expression, we characterized 206 spontaneous Ty transpositions that activate the promoterless gene his3 delta 4. Most of the Ty elements appear to be full-length, although a few deleted elements were recovered. Over 95% of the insertions belong to the Ty1 family, and the rest are Ty2 elements. The excessive number of Ty1 transpositions was unexpected because there are only 2-fold more Ty1 than Ty2 elements in the yeast strains used in the selection. However, there is 20-fold more Ty1 than Ty2 RNA present in these yeast strains. This difference in RNA level explains the greater number of Ty1 verses Ty2 transpositions at his3 delta 4, because Ty elements transpose through an RNA intermediate. A similar association between the Ty transcript level and transpositional activation of his3 delta 4 is obtained in cells expressing GAL1-promoted Ty2-H556 or Ty2-917 elements, but only if the element does not contain a marker. Genetically marked Ty2-H556NEO and -917NEO elements transpose into and activate his3 delta 4 with the same efficiency as the previously characterized Ty1-H3NEO element, but are underrepresented relative to the levels of TyNEO transcript. We also found that chromosomal Ty transcripts are even more abundant than previously estimated and comprise about 1% of total cellular RNA.

Blotting, Northern↗
PubMed1990

Multimeric arrays of the yeast retrotransposon Ty.

We have identified a novel integrated form of the yeast retrotransposon Ty consisting of multiple elements joined into large arrays. These arrays were first identified among Ty-induced alpha-pheromone-resistant mutants of MATa cells of Saccharomyces cerevisiae which contain Ty insertions at HML alpha that result in the expression of that normally silent cassette. These insertions are multimeric arrays of both the induced genetically marked Ty element and unmarked Ty elements. Structural analysis of the mutations indicated that the arrays include tandem direct repeats of Ty elements separated by only a single long terminal repeat. The Ty-HML junction fragments of one mutant were cloned and shown to contain a 5-base-pair duplication of the target sequence that is characteristic of a Ty transpositional insertion. In addition, the arrays include rearranged Ty elements that do not have normal long terminal repeat junctions. We have also identified multimeric Ty insertions at other chromosomal sites and as insertions that allow expression of a promoterless his3 gene on a plasmid. The results suggest that Ty transposition includes an intermediate that can undergo recombination to produce multimers.

Bacteriophage lambda↗
PubMed1989

The biology and exploitation of the retrotransposon Ty in Saccharomyces cerevisiae.

Retrotransposons are a widely distributed group of eukaryotic mobile genetic elements that transpose through an RNA intermediate. The element is transcribed into RNA, and this RNA is reverse transcribed into a DNA copy capable of insertion into many different chromosomal locations. Maturation of proteins and reverse transcription take place within noninfectious intracellular viruslike particles. We have studied the element Ty, which is found dispersed in the genome of the yeast Saccharomyces cerevisiae. The frequency of Ty element transposition is normally quite low but can be greatly increased by expressing an element from a strong promoter. We have used the ability to control the level of Ty transposition to investigate the functions of Ty proteins, the regulation of Ty transposition, and the exploitation of Ty elements as insertional mutagens in yeast. The information gained from these experiments should be applicable to the study of retrotransposons found in multicellular organisms.

Cloning, Molecular↗
PubMed1988

Transposon tagging using Ty elements in yeast.

We have used the ability to induce high levels of Ty transposition to develop a method for transposon mutagenesis in Saccharomyces cerevisiae. To facilitate genetic and molecular analysis, we have constructed GAL1-promoted TyH3 or Ty917 elements that contain unique cloning sites, and marked these elements with selectable genes. These genes include the yeast HIS3 gene, and the plasmid PiAN7 containing the Tn903 NEO gene. The marked Ty elements retain their ability to transpose, to mutate the LYS2, LYS5, or STE2 genes, and to activate the promoterless his3 delta 4 target gene. Ty elements containing selectable genes are also useful in strain construction, in chromosomal mapping, and in gene cloning strategies.

Cloning, Molecular↗
PubMed1988

Functional organization of the retrotransposon Ty from Saccharomyces cerevisiae: Ty protease is required for transposition.

We used several mutations generated in vitro to further characterize the functions of the products encoded by the TyB gene of the transpositionally active retrotransposon TyH3 from Saccharomyces cerevisiae. Mutations close to a core protein domain of TyB, which is homologous to retroviral proteases, have striking effects on Ty protein processing, the physiology of Ty viruslike particles, and transposition. The Ty protease is required for processing of both TyA and TyB proteins. Mutations in the protease resulted in the synthesis of morphologically and functionally aberrant Ty viruslike particles. The mutant particles displayed reverse transcriptase activity, but did not synthesize Ty DNA in vitro. Ty RNA was present in the mutant particles, but at very low levels. Transposition of a genetically tagged element ceased when the protease domain was mutated, demonstrating that Ty protease is essential for transposition. One of these mutations also defined a segment of TyB encoding an active reverse transcriptase. These results indicate that the Ty protease, like its retroviral counterpart, plays an important role in particle assembly, replication, and transposition of these elements.

DNA Transposable Elements↗
PubMed1988

Transpositional competence and transcription of endogenous Ty elements in Saccharomyces cerevisiae: implications for regulation of transposition.

Transposition of Ty elements in the yeast Saccharomyces cerevisiae occurs through an RNA intermediate. Although Ty RNA accounts for 5 to 10% of the total polyadenylated RNA in a haploid cell, the transposition frequency is only 10(-7) to 10(-8) per gene. To determine whether Ty elements native to the yeast genome are transpositionally competent, two elements were fused to the GAL1 promoter and tested for their ability to transpose. These native elements, Ty1-588 and Ty2-117, transposed at high levels when the GAL1 promoter was induced. Three Ty's identified as spontaneous transpositions in specific target genes were also tested. Of these three, Ty2-917 and the previously characterized element Ty1-H3 were shown to be transpositionally competent. The third element, Ty1-H1, was transposition defective. In addition, we marked the chromosomal copy of Ty1-588 with the NEO gene and demonstrated that Ty1-588NEO was actively transcribed in yeast cells. Ty1-588NEO transcription was regulated by the SPT3 and MAT loci in the same manner as that observed for Ty's collectively. These results indicate that the yeast genome contains functional Ty elements. The presence of a transpositionally competent, actively transcribed element suggests that regulation of Ty transposition occurs at a posttranscriptional level.

DNA Transposable Elements↗
PubMed1986

Lung mechanics, radiography and 67Ga scintigraphy in experimental Pneumocystis carinii pneumonia.

Respiratory system pressure-volume (P-V) studies, 67Ga-citrate scans, and chest radiographs were made in the corticosteroid-treated rat model of Pneumocystis carinii pneumonia. The steroid treatment used to provoke Pneumocystis infection in this model resulted in a reduction in body weight, lung weight and lung volumes compared to age-matched controls but no change in the normalized pressure-volume curve. P. carinii infection was associated with increased lung wet weight and flattening of the respiratory system P-V curve when compared to either age matched controls or steroid-treated animals on trimethoprim-sulfamethoxazole prophylaxis for P. carinii. Radiographs were interpreted as positive in only three of 11 animals with P. carinii, whereas 10 of 12 animals showed positive gallium-67 lung scans. We conclude that both gallium uptake and altered lung mechanics occur in the rat with Pneumocystis carinii and may reflect increased alveolar permeability and surfactant abnormalities noted in other studies.

Animals↗
PubMed1985

Ty element transposition: reverse transcriptase and virus-like particles.

We have found reverse transcriptase activity and virus-like particles only in yeast cells that contain a galactose-promoted Ty element induced on galactose. The cofractionation of reverse transcriptase, genomic-length Ty RNA, and a Ty-specified protein antigen in a particulate fraction and the ability of this complex to synthesize specifically a product that is homologous to the entire Ty suggest that reverse transcription of Ty RNA takes place in the particle. The absence of appreciable levels of reverse transcriptase and particles in uninduced cells despite the presence of at least 35 copies of chromosomal Ty elements suggest that some of these elements may be defective. The numerous virus-like particles visible in thin sections of Ty transposition-induced cells appear not to be infectious. These particles resemble the intracisternal A-type particles of the mouse and copia particles of Drosophila. The results support the idea that Ty elements and retroviruses share a common origin.

Antigens, Fungal↗
PubMed1985

Ty elements transpose through an RNA intermediate.

We have followed Ty transposition with a donor Ty element, TyH3, whose expression is under the control of the GAL1 promoter. Sequence analysis reveals dramatic structural differences in TyH3 before and after transposition. If the donor TyH3 is marked with an intron-containing fragment, the intron is correctly spliced out of the Ty during transposition, suggesting that the Ty RNA is the intermediate for transposition. Furthermore, the pattern of sequence inheritance in progeny Ty insertions derived from the marked Ty follows the predictions of the model of retroviral reverse transcription. Comparison of marked Ty elements before and after movement shows that transposition is highly mutagenic to the Ty element. These results demonstrate that during transposition, Ty sequence information flows from DNA to RNA to DNA.

Base Sequence↗