Host factors that affect Ty3 retrotransposition in Saccharomyces cerevisiae

Abstract

The retrovirus-like element Ty3 of Saccharomyces cerevisiae integrates at the transcription initiation region of RNA polymerase III. To identify host genes that affect transposition, a collection of insertion mutants was screened using a genetic assay in which insertion of Ty3 activates expression of a tRNA suppressor. Fifty-three loci were identified in this screen. Corresponding knockout mutants were tested for the ability to mobilize a galactose-inducible Ty3, marked with the HIS3 gene. Of 42 mutants tested, 22 had phenotypes similar to those displayed in the original assay. The proteins encoded by the defective genes are involved in chromatin dynamics, transcription, RNA processing, protein modification, cell cycle regulation, nuclear import, and unknown functions. These mutants were induced for Ty3 expression and assayed for Gag3p protein, integrase, cDNA, and Ty3 integration upstream of chromosomal tDNA(Val(AAC)) genes. Most mutants displayed differences from the wild type in one or more intermediates, although these were typically not as severe as the genetic defect. Because a relatively large number of genes affecting retrotransposition can be identified in yeast and because the majority of these genes have mammalian homologs, this approach provides an avenue for the identification of potential antiviral targets.

Figure 1.—

(A) Outline of the genetic assay for Ty3 retrotransposition screen. Leu⁺ transformants resulting from insertion mutagenesis were patched onto SD-His-Trp-Leu media and replica plated onto galactose-containing medium to induce expression of Ty3. These cultures were replicated onto appropriate selective medium (lacking adenine and/or lysine) to select for colonies in which suppressor activation by Ty3 insertion into the target plasmid allowed growth. (B) Patch assay of mutants isolated from the primary screen. Mutants were retested in replica patches (columns), and the wild type control is shown in the bottom row.

Figure 2.—

Genetic screen outline. Identification of host factors for Ty3. The number of candidate genes at each stage in the screen for host factors is shown. For details, see results.

Figure 3.—

Genetic assay using the HIS3-marked Ty3 element. Expression of the Ty3-HIS3 RNA (wavy line with arrow) was induced by growth on galactose-containing medium. Cells in the assay were replicated as described in materials and methods. Following reverse transcription, Ty3-HIS3 cDNA is integrated or recombined into the genome upstream of a tDNA (shaded triangle), leading to histidine prototrophy in the absence of Ty3 donor plasmid. Note that the HIS3 gene is antisense to Ty3.

Figure 4.—

Relative frequency of Ty3 transposition with the HIS3-marked Ty3 element. The frequency of Ty3-HIS3 transposition in deletion and reconstructed mutants was determined as described in materials and methods and expressed as the percentage of wild-type transposition (y-axis). The error bars represent standard deviation for each strain of three independent transformants.

Figure 5.—

Immunoblot analysis of Ty3 CA and IN proteins in BY4741 mutants. (A and B) Mutants that exhibit the Ty3 phenotype with two different genetic assays. Immunoblots with antibodies to Ty3 CA or IN proteins were performed on yeast extracts prepared from cultures induced for Ty3 expression. Two independent transformants were monitored for each mutant. Antibodies to CA recognize mature CA protein (26 kD) and precursor Gag3p (38 kD) as well as p31 (asterisk). Antibodies to IN recognize mature IN (61 kD) and three larger species (vertical line). These larger species have sizes consistent with Gag3-Pol3, Pol3, and RT-IN, but have not been definitely assigned. For gel-loading control, filters for IN immunoblots were reprobed with anti-Pgk1 polyclonal antibodies (Molecular Probes, Eugene, OR). A wild-type culture that was not induced for Ty3 expression [wt (raff)] was used as the negative control.

Figure 5.—

Immunoblot analysis of Ty3 CA and IN proteins in BY4741 mutants. (A and B) Mutants that exhibit the Ty3 phenotype with two different genetic assays. Immunoblots with antibodies to Ty3 CA or IN proteins were performed on yeast extracts prepared from cultures induced for Ty3 expression. Two independent transformants were monitored for each mutant. Antibodies to CA recognize mature CA protein (26 kD) and precursor Gag3p (38 kD) as well as p31 (asterisk). Antibodies to IN recognize mature IN (61 kD) and three larger species (vertical line). These larger species have sizes consistent with Gag3-Pol3, Pol3, and RT-IN, but have not been definitely assigned. For gel-loading control, filters for IN immunoblots were reprobed with anti-Pgk1 polyclonal antibodies (Molecular Probes, Eugene, OR). A wild-type culture that was not induced for Ty3 expression [wt (raff)] was used as the negative control.

Figure 6.—

cDNA production in BY4741 mutants expressing Ty3. (A) Southern blot analysis of Ty3 cDNA. Cells were induced for 6 hr for Ty3 expression and total DNA was extracted and cleaved with EcoRI. Southern blot analysis was performed with ³²P-labeled, Ty3-specific probe, which hybridizes to full-length cDNA of 5.4 kbp as well as to Ty3 donor plasmid. Two independent transformants were monitored for each mutant. (B) Relative amount of Ty3 cDNA in transposition mutants. Hybridization signals from Southern blots were quantitated using Quantity One software (Bio-Rad). Ty3 cDNA normalized to the plasmid was expressed as the percentge of wild type (y-axis). The bars represent the average of two independent transformants for each strain except fun30 for which one mutant showed no plasmid.

Figure 7.—

PCR analysis of Ty3 integration upstream of genomic tDNA^Val(AAC) genes. (A) Strategy for PCR detection of Ty3 integration into a target. Primers are specific to Ty3 (278) and to the 14 tDNA sequences. After insertion of Ty3, a diagnostic junction fragment can be amplified in PCR from the two primers. (B) Ty3 transposition into chromosomal tDNA^Val(AAC) loci. Total DNA was extracted from cultures induced for Ty3 expression for 6 hr, and different amounts of genomic DNA were used to establish the linear range of PCR detection. A total of 1–125 ng of DNA was used as the template for PCR. PCR products were separated on a 1.5% agarose gel and stained with ethidium bromide. PCR for integration (32 cycles, top); PCR templated by RAD52 to quantify starting DNA (20 cycles, bottom). (C) PCR analysis of Ty3 integration in mutant strains. PCR was performed as described for B using 25 and 5 ng of DNA for integration and RAD52, respectively. Two independent transformants were tested for each mutant. Data for 22 mutants are shown. For the negative control, 25 ng DNA was used from an uninduced culture (raff).