Identification of Functionally Related Genes That Stimulate Early Meiotic Gene Expression in Yeast

Meiosis and spore formation in the yeast Saccharomyces cerevisiae are associated with increased expression of sporulation-specific genes. One of these genes, IME2, encodes a putative protein kinase that is a positive regulator of other sporulation-specific genes. We have isolated mutations that cause reduced expression of an ime2-lacZ fusion gene. We found mutations in IME1, a known positive regulator of IME2, and MCK1, a known positive regulator of IME1. We also isolated recessive mutations in 12 other genes, which we designate RIM (Regulator of IME2) genes. Our analysis indicates that the defects in rim1, rim8, rim9 and rim13 mutants are a consequence of diminished IME1 expression and can be suppressed by expression of IME1 from the heterologous ACT1 promoter. These rim mutations also reduced expression of an ime1-HIS3 fusion, in which the HIS3 gene is expressed from the IME1 promoter, and caused reduced levels of IME1 RNA. Although the rim1, rim8, rim9 and rim13 mutant phenotypes are similar to those of mck1 mutants, we found that the defects in ime2-lacZ expression and sporulation of the mck1 rim double mutants were more severe than either single mutant. In contrast, the defects of the rim rim double mutants were similar to either single mutant. The rim1, rim8, rim9 and rim13 mutants also display slow growth at 17° and share a smooth colony morphology that is not evident in mck1 mutants or isogenic wild-type strains. We suggest that RIM1, RIM8, RIM9 and RIM13 encode functionally related products that act in parallel to MCK1 to stimulate IME1 expression.     

Positive control of sporulation-specific genes by the IME1 and IME2 products in Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, meiosis and spore formation require the induction of sporulation-specific genes. Two genes are thought to activate the sporulation program: IME1 and IME2 (inducer of meiosis). Both genes are induced upon entry into meiosis, and IME1 is required for IME2 expression. We report here that IME1 is essential for expression of four sporulation-specific genes. In contrast, IME2 is not absolutely essential for expression of the sporulation-specific genes, but contributes to their rapid induction. Expression of IME2 from a heterologous promoter permits the expression of these sporulation-specific genes, meiotic recombination, and spore formation in the absence of IME1. We propose that the IME1 and IME2 products can each activate sporulation-specific genes independently. In addition, the IME1 product stimulates sporulation-specific gene expression indirectly through activation of IME2 expression.     

A transcriptional cascade governs entry into meiosis in Saccharomyces cerevisiae.

Two signals activate meiosis in yeast: starvation and expression of the a1 and alpha 2 products of the mating-type locus. Prior studies suggest that these signals stimulate expression of an activator of meiosis, the IME1 (inducer of meiosis) product. We have cloned a gene, IME2, with properties similar to those of IME1: both genes are required for meiosis, and both RNAs are induced in meiotic cells. Elevated dosage of IME1 or IME2 stimulates the meiotic recombination pathway without starvation; thus, the IME products may be part of the switch that activates meiosis. IME1 was found to be required for IME2 expression, and a multicopy IME2 plasmid permitted meiosis in an ime1 deletion mutant. Accordingly, we propose that the IME1 product stimulates meiosis mainly through activation of IME2 expression.     

Bipartite structure of an early meiotic upstream activation sequence from Saccharomyces cerevisiae.

Diploid a/alpha Saccharomyces cerevisiae cells cease mitotic growth and enter meiosis in response to starvation. Expression of meiotic genes depends on the IME1 gene product, which accumulates only in meiotic cells. We report here an analysis of the regulatory region of IME2, an IME1-dependent meiotic gene. Deletion and substitution studies identified a 48-bp IME1-dependent upstream activation sequence (UAS). Activity of the UAS also requires the RIM11, RIM15, and RIM16 gene products, which are required for expression of the chromosomal IME2 promoter and for meiosis. Through a selection for suppressors that permit UAS activity in an ime1 deletion mutant, we identified recessive mutations in three genes, SIN3 (also called RPD1, UME4, and SDI1), RPD3, and UME6 (also called CAR80), that were previously known as negative regulators of other early meiotic genes. Mutational analysis of the IME2 UAS reveals two critical sequence elements: a G+C-rich sequence (called URS1), previously identified at many meiotic genes, and a newly described element, the T4C site, that we found at a subset of meiotic genes. In agreement with prior studies, URS1 mutations lead to elevated IME2 UAS activity in the absence of IME1. However, the URS1 mutations prevent any further stimulation of UAS activity by IME1. Repression through URS1 has been shown to require the UME6 gene product. We find that activation of the IME2 UAS by IME1 also requires the UME6 gene product. Thus, UME6 and the URS1 site both have dual negative and positive roles at the IME2 UAS. We propose that IME1 modifies UME6 to convert it from a negulator to a positive Regulor.     

Selection for Early Meiotic Mutants in Yeast

In the yeast Saccharomyces cerevisiae, only a/alpha cells can enter meiosis; a and alpha cells cannot. Because a/alpha cells are typically diploid and a and alpha cells are typically haploid, this cell type restriction ensures that only diploid cells enter meiosis. Entry into meiosis is accompanied by an increase in expression of the IME1 gene; the IME1 product (IME1) then activates IME2 and other meiotic genes. We have found that IME1 expression is toxic to starved haploid cells, presumably because IME1 directs them into meiosis. IME1 toxicity is greater in rad52 mutants, in which meiotic recombination causes lethal damage. Suppressors of IME1 toxicity include recessive mutations in two genes, RIM11 and RIM16 (Regulator of Inducer of Meiosis), that are required for IME1 to activate IME2 expression. RIM11 maps near CIN4 on chromosome XIII.     

Identification of essential intron sequences that enhance gene expression independently of splicing in the yeast Saccharomyces cerevisiae

Gene expression in eukaryotes is enhanced by the presence of introns in a process known as intron-mediated enhancement (IME), but its mechanism remains unclear. In Saccharomyces cerevisiae, sequences at the 5′-splice sites (SS) and branch point sites (BPS) are highly conserved compared with other higher eukaryotes. Here, the minimum intron sequence essential for IME was investigated using various short introns and a yeast codon-optimized luciferase gene as an IME model. Mutations at the 5′-SS conserved sequence and branch point in the QCR10 intron caused splicing deficiency with either a complete loss or a marked decrease in IME. By contrast, however, the 3′-AG to tG mutant was spliced and retained IME function. Moreover, heterologous introns, which did not show IME in S. cerevisiae, gained splicing competency and IME ability by substitutions to the S. cerevisiae-type 5′-SS and BPS sequences. Intriguingly, several deletion mutants between the 5′-SS and BPS in introns exhibited high levels of IME despite a loss in splicing competency. In most cases, further deletions or substitutions did not recover splicing competency and were found to decrease IME. However, a 16-nt variant consisting of the conserved 5′-SS and BPS sequences and 3′-CAG showed an IME level comparable with that of the wild-type intron. These results indicate that IME can be independent of splicing in S. cerevisiae while intron sequences at the 5′-SS and BPS play an essential role in IME.     

RIM4 encodes a meiotic activator required for early events of meiosis in Saccharomyces cerevisiae

RIM4 was previously found to be required for both the IME1- and IME2-dependent pathways of meiotic gene expression in Saccharomyces cerevisiae. We now demonstrate that RIM4 is also required for meiotic division and recombination. Furthermore, rim4Delta mutants show defects in premeiotic DNA synthesis, which can be suppressed by deletion of the SIC1 gene, which encodes a Cdk inhibitor. Expression of RIM4 is induced early in meiosis, and is dependent on IME1 but not IME2. Indeed, RIM4 itself is essential for the meiotic expression of IME2. These results suggest that RIM4 is epistatic to IME2, and is required for multiple steps during sporulation. In agreement with this interpretation, overexpression of RIM4 induces low levels of sporulation in rich medium.     

Helicobacter pylori vaccine development and use: a cost-effectiveness analysis using the Institute of Medicine Methodology

Background. Prophylactic vaccination has been suggested as a better strategy than antibiotics to control Helicobacter pylori infection. We evaluated the cost-effectiveness (CE) of H. pylori vaccine development and use in the United States and developing countries, using a method developed by the Institute of Medicine (IOM).
Methods. The IOM model includes costs of vaccine development, vaccination program, and averted medical treatments; morbidity and mortality prevented; expected efficacy and use; and proportion of disease that is vaccine-preventable. The model employs infant mortality equivalence (IME) to estimate disease burden; with IME, the societal cost of infection-related morbidity is expressed as equivalent to a specific rate of infant deaths. We tested model assumptions by univariate sensitivity analyses.
Results. In the United States, H. pylori vaccine would save 1,176 IME and would cost $58.71 million (1997 dollars) annually, yielding a CE ratio of $49,932 per IME; the health benefits would exceed all IOM-studied vaccines, even when efficacy dropped to 55%. H. pylori vaccine could be cost-saving if priced at less than $60 per course. In developing countries, H. pylori vaccine would rank unfavorably both in terms of health benefits (33,518 IME) and costs ($5,254 million). None of the changes in assumptions improved significantly the H. pylori vaccine's ranking relative to other IOM-studied vaccines.
Conclusions. Compared to other vaccines evaluated in the IOM study, H. pylori vaccine warrants public resource allocation for accelerated development and use in the United States but not for use in developing countries.     

Status-dependent selection in the dimorphic beetle Onthophagus taurus.

The occurrence of alternative reproductive phenotypes is widespread in most animal taxa. The majority of known examples best fit the notion of alternative tactics within a conditional strategy where the fitness pay-offs depend on an individual's competitive ability or status. Individuals are proposed as "choosing" the tactic that maximizes their fitness, given their status relative to others in the population. Theoretically, status-dependent selection should determine when an animal should switch between alternative tactics. While a number of studies have demonstrated unequal fitness pay-offs associated with alternative tactics, none, to our knowledge, have examined the fitness functions necessary for predicting when individuals should switch between tactics. Here, we use a dimorphic male beetle in order to provide the first empirically derived fitness function across alternative reproductive phenotypes. Our data provide empirical support for a game-theoretic conditional strategy that has evolved under status-dependent selection.     

Genomic footprinting of the yeast zinc finger protein Rme1p and its roles in repression of the meiotic activator IME1.

The zinc finger protein Rme1p is a negative regulator of the meiotic activator IME1 in Saccharomyces cerevisiae . Prior studies have shown that Rme1p binds in vitro to a site near nt -2030 in the IME1 upstream region, but a genomic mutation in that site has little effect on repression of IME1 . To identify Rme1p binding sites in vivo , we have examined the binding of Rme1p to genomic sites through in vivo footprinting. We show that Rme1p binds to two sites in the IME1 upstream region, near nt -1950 and -2030. Mutations in both binding sites abolish repression of chromosomal IME1 by Rme1p, whereas a mutation in either single site causes partial derepression. Therefore, both Rme1p binding sites are essential for repression of IME1 . Prior studies have shown that repression by Rme1p depends upon RGR1 and SIN4 , which specify RNA polymerase II mediator subunits that are required for normal nucleosome density. We find that RGR1 and SIN4 are not simply required for Rme1p to bind to DNA in vivo . These results suggest that Rme1p functions directly as a repressor of IME1 and that Rgr1p and Sin4p are required for DNA-bound Rme1p to exert repression.