Role of transcription in plasmid maintenance in the hpr1Delta mutant of Saccharomyces cerevisiae

Abstract

The Saccharomyces cerevisiae hyperrecombination mutation hpr1Delta results in instability of sequences between direct repeats that is dependent on transcription of the repeat. Here it is shown that the HPR1 gene also functions in plasmid stability in the presence of destabilizing transcription elongation. In the hpr1Delta mutant, plasmid instability results from unchecked transcription elongation, which can be suppressed by a strong transcription terminator. The plasmid system has been used to examine in vivo aspects of transcription in the absence of Hpr1p. Nuclear run-on studies suggest that there is an increased RNA polymerase II density in the hpr1Delta mutant strain, but this is not accompanied by an increase in accumulation of cytoplasmic mRNA. Suppression of plasmid instability in hpr1Delta can also be achieved by high-copy expression of the RNA splicing factor SUB2, which has recently been proposed to function in mRNA export, in addition to its role in pre-mRNA splicing. High-copy-number SUB2 expression is accompanied by an increase in message accumulation from the plasmid, suggesting that the mechanism of suppression by Sub2p involves the formation of mature mRNA. Models for the role of Hpr1p in mature mRNA formation and the cause of plasmid instability in the absence of the Hpr1 protein are discussed.

FIG. 1.

A yeast chromosomal insert confers differential plasmid stability in an hpr1Δ background, dependent on its orientation in the plasmid. pRM101 and pRM102 differ in the orientation of a 1.7-kb yeast chromosomal insert. The PET56 and DED1 genes are both oriented away from HIS3 and are designated pet56 and ded1 solely to indicate that they are truncations. The photograph above each plasmid diagram gives a qualitative representation of its respective stability in hpr1Δ cells. hpr1Δ cells containing the plasmid were taken from selective (SC-Trp) plates, streaked onto nonselective rich medium (YPD) plates, and then replica plated back onto selective (SC-Trp) plates. An example of a colony that has lost the plasmid is indicated by the white arrow.

FIG. 2.

Analysis of transcription through the bacterial ori-amp sequence on both pRM102 and pRM101. Northern analysis was performed to identify transcripts originating from the DED1 promoter (pRM102, lanes 1 through 6) and the PET56 promoter (pRM101, lanes 7 through 12) with a DNA probe from the bacterial ori sequence on the plasmid. RNA was isolated from three independent cultures grown to mid-log phase in selective (SC-Trp) medium for both wild-type (wt) and hpr1Δ strains each containing either pRM102 or pRM101. The arrow points to the major transcript (approximately 2.5 kb) that is generated from the DED1 promoter on pRM102 (lanes 1 to 6). This major transcript is not observed when the PET56 promoter is oriented toward the bacterial ori-amp sequence (pRM101, lanes 7 to 12). Relative actin mRNA levels from each culture are shown at the bottom

FIG. 3.

Early termination of transcription from the DED1 promoter suppresses plasmid instability in hpr1Δ cells. CYC1 transcription terminators were inserted either directly after the ded1 sequence on pRM102 (pRM110), directly in front of the CEN6 sequence (pRM111), or between the ori and amp sequences (pRM112) (see Materials and Methods). Given are qualitative assessments of the relative stability of each plasmid in an hpr1Δ background.

FIG. 4.

Quantitation of the major transcript produced from the DED1 promoter on pRM102 in both wild-type (wt) and hpr1Δ strains. (A) Northern analysis of transcription from the DED1 promoter present on pRM102 in both wild-type and hpr1Δ stains. RNA was isolated from three independent cultures of both wild-type and hpr1Δ strains, each containing pRM102, grown to mid-log phase in liquid SC-Trp. The transfers were probed with the same bacterial ori probe as in Fig. 2. The arrow points to the major transcript (approximately 2.5 kb) that is generated from the DED1 promoter on pRM102. (B) Southern analysis to determine the relative amount of plasmid present in each strain. DNA was isolated from each of the same cultures used for the above Northern assay. The DNAs were digested with BamHI, and equal amounts were loaded onto a 0.9% agarose gel. The transferred materials were simultaneously probed for plasmid (with the bacterial ori probe), as well as a single-copy yeast gene (ACT1). (C) Quantitation of the DED1 promoter transcript levels present in hpr1Δ cells relative to those in the wild type. Relative levels of transcript produced from the DED1 promoter were normalized against relative actin mRNA levels, as well as the relative amount of plasmid present in each strain. The relative amount of plasmid was determined by comparing the plasmid levels against that of a single-copy chromosomal sequence (ACT1).

FIG. 5.

Quantitative RT-PCR analysis of transcription from the DED1 promoter of pRM102. (A) Schematic of the locations of the five RT primers used to analyze the DED1 promoter transcript. (B). Southern analysis of the RT-PCR products of the five reactions from wild-type and hpr1Δ cells carrying pRM102. The position of the dnaB transcript used as a PCR efficiency control is indicated. Relative levels of plasmid and total mRNAs present in each reaction were determined by Southern and Northern analyses, respectively. (C). Quantitated RT-PCR levels of hpr1Δ(pRM102) cells relative to those of wild-type cells carrying pRM102 are shown. The results are from three independent experiments for each reaction. The error bars indicate standard deviations. Absolute RT-PCR levels were corrected for the amount of plasmid present in each strain and for the total amount of mRNA. chrom., chromosome.

FIG. 6.

High-copy-number SUB2 suppresses plasmid instability and increases the relative amount of the major transcript produced from the DED1 promoter on pRM1115 in hpr1Δ cells. pRM115 is identical to pRM102 except that 630 bp of the E. coli dnaB sequence is inserted directly 3′ to the ded1 sequence so that the plasmid DED1 promoter transcript could be uniquely probed in the presence of the high-copy-number YEp-SUB2 plasmid (YEp351 backbone). (A) Northern analysis of transcription from the DED1 promoter present on pRM115 in both wild-type (wt) and hpr1Δ stains in the presence of high-copy-number YEp-SUB2. RNA was isolated from three independent cultures grown to mid-log phase in liquid SC-Trp-Leu. The same amount of RNA was loaded onto each lane, and transfers were probed with the bacterial dnaB probe. The blots were subsequently stripped and reprobed for actin mRNA. (B) Southern analysis to determine the relative amount of plasmid pRM115 present in each strain. DNA was isolated from each of the same cultures used for the above Northern assay. DNAs were digested with EcoRI. The transfer was simultaneously probed for pRM115 (dnaB probe) and a single-copy yeast gene (ACT1). (C) Quantitation of the DED1 promoter transcript levels present in hpr1Δ cells relative to those in wild-type cells. Relative levels of transcript produced from the DED1 promoter were normalized against relative actin mRNA levels, as well as the relative amount of plasmid present in each strain.

FIG. 7.

Transcriptional run-on analysis of the plasmid DED1 promoter transcript in hpr1Δ and wild-type cells. Cells were grown under selection in liquid medium (SC-Trp) to mid-log phase prior to run-on analysis. (A) Radiolabeled RNA from both wild-type (wt) and hpr1Δ cells, each containing pRM102, bound to each probe. Transcription of rRNA was used to normalize transcription levels. The arrow indicates the direction of transcription. The location of each probe from the plasmid is shown in panel C. The bar graph shows the average level of transcription across each probe from four independent experiments for hpr1Δ cells relative to wild-type cells, normalized against both the total amount of RNA (25S rRNA) and the relative amount of plasmid present in each strain (B). Error bars represent the standard deviations. (B) Southern analysis to determine the relative amount of plasmid pRM102 present in each strain. DNA was isolated from each of the same cultures used for the above run-on analysis. DNAs were digested with EcoRI. The transfer was simultaneously probed for pRM102 (bacterial ori probe), as well as a single-copy yeast gene (ACT1). chrom., chromosome.