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L Breeden

Publications and source records attributed to L Breeden.

At least 19 recordsLinked to original sources
PubMed2001

Statistical modeling of large microarray data sets to identify stimulus-response profiles.

A statistical modeling approach is proposed for use in searching large microarray data sets for genes that have a transcriptional response to a stimulus. The approach is unrestricted with respect to the timing, magnitude or duration of the response, or the overall abundance of the transcript. The statistical model makes an accommodation for systematic heterogeneity in expression levels. Corresponding data analyses provide gene-specific information, and the approach provides a means for evaluating the statistical significance of such information. To illustrate this strategy we have derived a model to depict the profile expected for a periodically transcribed gene and used it to look for budding yeast transcripts that adhere to this profile. Using objective criteria, this method identifies 81% of the known periodic transcripts and 1,088 genes, which show significant periodicity in at least one of the three data sets analyzed. However, only one-quarter of these genes show significant oscillations in at least two data sets and can be classified as periodic with high confidence. The method provides estimates of the mean activation and deactivation times, induced and basal expression levels, and statistical measures of the precision of these estimates for each periodic transcript.

CDC28 Protein Kinase, S cerevisiae↗
PubMed2000

CLN1 and its repression by Xbp1 are important for efficient sporulation in budding yeast.

Xbp1, a transcriptional repressor of Saccharomyces cerevisiae with homology to Swi4 and Mbp1, is induced by stress and starvation during the mitotic cycle. It is also induced late in the meiotic cycle. Using RNA differential display, we find that genes encoding three cyclins (CLN1, CLN3, and CLB2), CYS3, and SMF2 are downregulated when Xbp1 is overexpressed and that Xbp1 can bind to sequences in their promoters. During meiosis, XBP1 is highly induced and its mRNA appears at the same time as DIT1 mRNA, but its expression remains high for up to 24 h. As such, it represents a new class of meiosis-specific genes. Xbp1-deficient cells are capable of forming viable gametes, although ascus formation is delayed by several hours. Furthermore, Xbp1 target genes are normally repressed late in meiosis, and loss of XBP1 results in their derepression. Interestingly, we find that a deletion of CLN1 also reduces the efficiency of sporulation and delays the meiotic program but that sporulation in a Deltacln1 Deltaxbp1 strain is not further delayed. Thus, CLN1 may be Xbp1's primary target in meiotic cells. We hypothesize that CLN1 plays a role early in the meiotic program but must be repressed, by Xbp1, at later stages to promote efficient sporulation.

Base Sequence↗
PubMed1999

The MSN1 and NHP6A genes suppress SWI6 defects in Saccharomyces cerevisiae.

Ankyrin (ANK) repeats were first found in the Swi6 transcription factor of Saccharomyces cerevisiae and since then were identified in many proteins of eukaryotes and prokaryotes. These repeats are thought to serve as protein association domains. In Swi6, ANK repeats affect DNA binding of both the Swi4/Swi6 and Mbp1/Swi6 complexes. We have previously described generation of random mutations within the ANK repeats of Swi6 that render the protein temperature sensitive in its ability to activate HO transcription. Two of these SWI6 mutants were used in a screen for high copy suppressors of this phenotype. We found that MSN1, which encodes a transcriptional activator, and NHP6A, which encodes an HMG-like protein, are able to suppress defective Swi6 function. Both of these gene products are involved in HO transcription, and Nhp6A may also be involved in CLN1 transcription. Moreover, because overexpression of NHP6A can suppress caffeine sensitivity of one of the SWI6 ANK mutants, swi6-405, other SWI6-dependent genes may also be affected by Nhp6A. We hypothesize that Nhp6A and Msn1 modulate Swi6-dependent gene transcription indirectly, through effects on chromatin structure or other transcription factors, because we have not been able to demonstrate that either Msn1 or Nhp6A interact with the Swi4/Swi6 complex.

Ankyrins↗
PubMed1997

Xbp1, a stress-induced transcriptional repressor of the Saccharomyces cerevisiae Swi4/Mbp1 family.

We have identified Xbp1 (XhoI site-binding protein 1) as a new DNA-binding protein with homology to the DNA-binding domain of the Saccharomyces cerevisiae cell cycle regulating transcription factors Swi4 and Mbp1. The DNA recognition sequence was determined by random oligonucleotide selection and confirmed by gel retardation and footprint analyses. The consensus binding site of Xbp1, GcCTCGA(G/A)G(C/A)g(a/g), is a palindromic sequence, with an XhoI restriction enzyme recognition site at its center. This Xbpl binding site is similar to Swi4/Swi6 and Mbp1/Swi6 binding sites but shows a clear difference from these elements in one of the central core bases. There are binding sites for Xbp1 in the G1 cyclin promoter (CLN1), but they are distinct from the Swi4/Swi6 binding sites in CLN1, and Xbp1 will not bind to Swi4/Swi6 or Mbp1/Swi6 binding sites. The XBP1 promoter contains several stress-regulated elements, and its expression is induced by heat shock, high osmolarity, oxidative stress, DNA damage, and glucose starvation. When fused to the LexA DNA-binding domain, Xbp1 acts as transcriptional repressor, defining it as the first repressor in the Swi4/Mbp1 family and the first potential negative regulator of transcription induced by stress. Overexpression of XBP1 results in a slow-growth phenotype, lengthening of G1, an increase in cell volume, and a repression of G1 cyclin expression. These observations suggest that Xbp1 may contribute to the repression of specific transcripts and cause a transient cell cycle delay under stress conditions.

Amino Acid Sequence↗
PubMed1995

Acute exposure to morphine suppresses cytotoxic T-lymphocyte activity.

Chronic exposure (11 days) to morphine has previously been shown to suppress splenic and peritoneal cytotoxic T-lymphocyte activity through mu-opioid receptors. The present study was undertaken to assess the effects of varying the frequency of exposure to morphine on cytotoxic T-lymphocyte activity in C3H/HeN mice immunized with C57BL/6 splenocytes. Mice subchronically treated with morphine (50.0 mg/kg) showed no measurable suppression of splenic natural killer activity or splenic or peritoneal cytotoxic T-lymphocyte activity. However, mice treated acutely with 50.0 mg/kg of morphine exhibited a significant suppression of peritoneal but not splenic cytotoxic T-lymphocyte activity. Naltrexone pretreatment of mice receiving an acute dose of morphine blocked the suppression, implicating the involvement of opioid receptors. Using column depletion chromatography, peritoneal exudate cells mediating cytotoxic T-lymphocyte activity were both CD4+CD8- and CD4-CD8+. Collectively, the results suggest that the duration of opioid (morphine) exposure differentially affects peritoneal cytotoxic T-lymphocyte activity. These results may have important implications regarding immunity to viral infections in individuals who abuse drugs such as heroin.

Animals↗
PubMed1994

Three independent forms of regulation affect expression of HO, CLN1 and CLN2 during the cell cycle of Saccharomyces cerevisiae.

The G1 cyclins (CLNs) bind to and activate the CDC28 kinase during the G1 to S transition in Saccharomyces cerevisiae. Two G1 cyclins are regulated at the RNA level so that their RNAs peak at the G1/S boundary. In this report we show that the cell cycle regulation of CLN1 and CLN2 is partially determined by the restricted expression of SW14, a known trans-activator of SCB elements. When SWI4 is constitutively expressed or deleted, cell cycle regulation of CLN1/2 is reduced but not eliminated. In the absence of SwI6, another known regulator of both SCB and MCB elements, cell cycle regulation of the CLNs is also reduced, and the Start-dependence of HO transcription is eliminated. This indicates that SwI6 also plays an important role in the normal cell cycle regulation of all three promoters. When both SwI6 activity and the transcriptional regulation of SW14 are eliminated, cell cycle regulation is further reduced, indicating that these are two independent pathways of regulation. However, a twofold fluctuation in transcript levels still persists under these conditions. This reveals a third source of cell cycle control, which could affect SwI4 activity post-transcriptionally, or reflect the existence of another unidentified regulator of these promoters.

CDC28 Protein Kinase, S cerevisiae↗
PubMed1994

Differential effects of Cdc68 on cell cycle-regulated promoters in Saccharomyces cerevisiae.

Swi4 and Swi6 form a complex which is required for Start-dependent activation of HO and for high-level expression of G1 cyclin genes CLN1 and CLN2. To identify other regulators of this pathway, we screened for dominant, recessive, conditional, and allele-specific suppressors of swi4 mutants. We isolated 16 recessive suppressors that define three genes, SSF1, SSF5, and SSF9 (suppressor of swi four). Mutations in all three genes bypass the requirement for both Swi4 and Swi6 for HO transcription and activate transcription from reporter genes lacking upstream activating sequences (UASs). SSF5 is allelic with SIN4 (TSF3), a gene implicated in global repression of transcription and chromatin structure, and SSF9 is likely to be a new global repressor of transcription. SSF1 is allelic with CDC68 (SPT16). cdc68 mutations have been shown to increase expression from defective promoters, while preventing transcription from other intact promoters, including CLN1 and CLN2. We find that CDC68 is a required activator of both SWI4 and SWI6, suggesting that CDC68's role at the CLN promoters may be indirect. The target of CDC68 within the SWI4 promoter is complex in that known activating elements (MluI cell cycle boxes) in the SWI4 promoter are required for CDC68 dependence but only within the context of the full-length promoter. This result suggests that there may be both a chromatin structure and a UAS-specific component to Cdc68 function at SWI4. We suggest that Cdc68 functions both in the assembly of repressive complexes that form on many intact promoters in vivo and in the relief of this repression during gene activation.

Base Sequence↗
PubMed1993

Analysis of the SWI4/SWI6 protein complex, which directs G1/S-specific transcription in Saccharomyces cerevisiae.

SWI4 and SWI6 play a crucial role in START-specific transcription in Saccharomyces cerevisiae. SWI4 and SWI6 form a specific complex on the SCB (SWI4/6-dependent cell cycle box) sequences which have been found in the promoters of HO and G1 cyclin genes. Overproduction of SWI4 eliminates the SWI6 dependency of HO transcription in vivo and results in a new SWI6-independent, SCB-specific complex in vitro, which is heterogeneous and reacts with SWI4 antibodies. The C terminus of SWI4 is not required for SWI6-independent binding of SWI4 to SCB sequences, but it is necessary and sufficient for association with SWI6. Both SWI4 and SWI6 contain two copies of a 33-amino-acid TPLH repeat, which has been implicated in protein-protein interactions in other proteins. These repeats are not required for the SWI4-SWI6 association. Alanine substitutions in both TPLH repeats of SWI6 reduce its activity but do not affect the stability of the protein or its association with SWI4. However, these mutations reduce the ability of the SWI4/6 complex to bind DNA. Deletion of the lucine zipper motif in SWI6 also allows SWI4/6 complex formation, but it eliminates the DNA-binding ability of the SWI4/6 complex. This indicates that the integrity of two different regions of SWI6 is required for DNA binding by the SWI4/6 complex. From these data, we propose that the sequence-specific DNA-binding domain resides in SWI4 but that SWI6 controls the accessibility of this domain in the SWI4/6 complex.

Amino Acid Sequence↗
PubMed1993

Multiple SWI6-dependent cis-acting elements control SWI4 transcription through the cell cycle.

The Saccharomyces cerevisiae SWI4 gene encodes an essential transcription factor which controls gene expression at the G1/S transition of the cell cycle. SWI4 transcription itself is cell cycle regulated, and this periodicity is crucial for the normal cell cycle regulation of HO and at least two of the G1 cyclins. Since the regulation of SWI4 is required for normal cell cycle progression, we have characterized cis- and trans-acting regulators of SWI4 transcription. Deletion analysis of the SWI4 promoter has defined a 140-bp region which is absolutely required for transcription and can function as a cell cycle-regulated upstream activating sequence (UAS). The SWI4 UAS contains three potential MluI cell cycle boxes (MCBs), which are known cell cycle-regulated promoter elements. Deletion of all three MCBs in the SWI4 UAS decreases the level of SWI4 mRNA 10-fold in asynchronous cultures but does not abolish periodicity. These data suggest that MCBs are involved in SWI4 UAS activity, but at least one other periodically regulated element must be present. Since SWI6 is known to bind to MCBs and regulate their activity, the role of SWI6 in SWI4 expression was analyzed. Although the MCBs cannot account for the full cell cycle regulation of SWI4, mutations in SWI6 eliminate the normal periodicity of SWI4 transcription. This suggests that the novel cell cycle-regulated element within the SWI4 promoter is also SWI6 dependent. The constitutive transcription of SWI4 in SWI6 mutant cells occurs at an intermediate level, which indicates that SWI6 is required for the full activation and repression of SWI4 transcription through the cell cycle. It also suggests that there is another pathway which can activate SWI4 transcription in the absence of SWI6. The second activator may also target MCB elements, since SWI4 transcription drops dramatically when they are deleted.

Base Sequence↗
PubMed1992

SWI6 protein is required for transcription of the periodically expressed DNA synthesis genes in budding yeast.

In budding yeast many genes are expressed under cell-cycle control in late G1. These include a large group of DNA synthesis genes, the HO gene involved in mating-type switching, CTS1 (chitinase) and also CLN1 and CLN2 (ref. 4) encoding G1 cyclins. Two factors, encoded by the SWI4 and SWI6 genes, are required for HO (ref. 5), CLN (refs 6, 7) and CTS1 (ref. 3) gene expression and, at least in the HO promoter, bind to CACGA4 upstream sequences (CCBs). This motif is not found upstream of the DNA synthesis genes, which instead have a hexamer element, ACGCGT1 (MCB), an MluI restriction site, that is recognized by a cell-cycle regulated transcription complex DSC1 (ref. 1). This MluI-activation system consisting of the MCBs and DSC1 is conserved in fission yeast where a DSC1-like complex controls the cdc22+ ribonucleotide reductase gene. The Schizosaccharomyces pombe cdc10+ gene encodes a component of DSC1 (ref. 10) and, significantly, this has homology with both the Swi4 and Swi6 proteins. Here we show that Swi6 is an essential component of DSC1 and that deletion of SWI6 impairs the cell-cycle regulation of the DNA synthesis genes, as well as CLN1 and CLN2. Thus Swi6 is the common factor in regulation of all the above genes and may therefore be responsible for the timing of their expression in late G1.

Base Sequence↗
PubMed1991

Cell cycle-specific expression of the SWI4 transcription factor is required for the cell cycle regulation of HO transcription.

Expression of the HO endonuclease triggers mating-type switching in Saccharomyces cerevisiae. Transcription of the HO gene is start-dependent and restricted to the late G1/early S phase of haploid mother cells. The HO promoter contains 10 copies of a cell cycle-regulated upstream activation sequence, which is activated by SWI4 and SWI6. SWI4 mRNA levels vary at least 10-fold throughout the cell cycle and rise sharply just before the rise in HO mRNA levels. Constitutive synthesis of SWI4 mRNA leads to constitutive synthesis of HO mRNA. These data suggest that the cell cycle regulation of SWI4 mRNA is required for the tight cell cycle regulation of HO transcription. High-level constitutive synthesis of SWI4 also suppresses swi5 and swi6 mutations, suggesting that SWI4 is the predominant activator of HO transcription and that mutations in negative regulators of SWI4 could be isolated as suppressors of swi6 mutations. One recessive suppressor of swi6 (ssx1-1) that allowed high-level expression of SWI4 during alpha-factor arrest and constitutive expression of both SWI4 and HO after release from the arrest was isolated. This result suggests that SSX1 has a negative regulatory role in the cell-cycle regulation of SWI4 mRNA accumulation.

Cell Cycle↗
PubMed1987

The bases of the tRNA anticodon loop are independent by genetic criteria.

We employed two methods to study the translational role of interactions between anticodon loop nucleotides. Starting with a set of previously constructed weakly-suppressing anticodon loop mutants of Su7, we searched for second-site revertants that increase amber suppressor efficiency. Though hundreds of revertants were characterized, no second-site revertants were found in the anticodon loop. Second site reversion was detected in the D-stem, thereby demonstrating the efficacy of the search method. As a second method for detecting interactions, we used site-directed mutagenesis to construct multiple mutations in the anticodon loop. These multiple mutants are very weak suppressors and have translational activities that are equal to or lower than that predicted for the independent action of single mutations. We conclude that although the anticodon loop sequence of Su7 has an optimal structure for the translation of amber codons, we find no evidence that interactions between loop bases can enhance translational efficiency.

Anticodon↗
PubMed1987

Cell cycle control of the yeast HO gene: cis- and trans-acting regulators.

In this paper, we investigate the role of a short repeated sequence (CACGA4) in the cell-cycle regulation of HO. We show that this sequence activates transcription of a heterologous gene in a cell-cycle-dependent fashion indistinguishable from that of the wild-type HO promoter. We also show that, in addition to SWI1 through SWI5, at least five other genes (SWI6 through SWI10) are required for HO transcription. These genes fit into three distinct classes with respect to their targets within the HO promoter. SWI4 and SWI6 are specifically required for CACGA4-mediated activation of transcription. SWI1, SWI2, and SWI5 are required for transcription from sequences physically separate from and independent of the CACGA4 sequences. SWI3 may be required for both. Since all the SWI genes are required for HO transcription, the HO promoter must contain at least two essential upstream activation sequences, which are affected by different trans-acting factors and are subject to different types of control.

Cell Cycle↗
PubMed1986

Actions of the anticodon arm in translation on the phenotypes of RNA mutants.

In previous publications, we have shown that it is practical to study the translational activity of tRNAs by replacement and alteration of the anticodon arm sequence of the genus on a plasmid clone. Experiments in which the anticodon arm sequence is transplanted between tRNA genes suggest that the translational activity is determined by these sequences. We have therefore made every variant of the anticodon loop and the three base-pairs of the stem proximal to the loop, in order to resolve the relation between the structure of Su7Am tRNATrp, and its function. All derivatives conserved the normal secondary structure of the molecule, which was known to be essential for translational activity. The probability of translation of the amber codon by these suppressors is measured in this work. This translational activity in vivo is rationalized in terms of data on the copy numbers of the plasmid clones, the nucleotide modifications of the tRNAs, the steady-state level of the mature tRNA, and the aminoacylation of these molecules. Nucleotide modification levels vary among these tRNAs, giving information about the specificities of modification systems that make O-methylribose, pseudouridine, and modified A in the anticodon arm. However, for this series of tRNAs, none of these modifications has a strong effect on translational efficiency of the tRNAs. A few of the substitutions reduce aminoacylation of the tRNAs with glutamine, as determined by comparison of suppression in normal strains and related strains, which have 25-fold elevated levels of the glutaminyl-tRNA synthetase (GlnRS). The substitutions that have the largest effect on GlnRS action are, unexpectedly, purines for conserved pyrimidines on the 5' side of the anticodon loop. Data on the concentrations of tRNA in vivo suggest that the anticodon loop and helix contribute similarly to the determination of the steady-state level of the tRNAs. This level varies sevenfold, though all tRNAs are processed from a homologous precursor made from the same transcription unit. Effects on levels appear to be mediated by changes in anticodon arm structure. A robust equation that relates aminoacyl-tRNA levels to suppressor efficiency is developed in order to resolve effects on tRNA levels and on ribosomal steps: E = A/(K + A), where E is efficiency, A is aminoacyl-tRNA concentration, and K is the effective concentration, or cellular tRNA content required for an individual tRNA to have an efficiency of 0.50. The tRNAs vary in their intrinsic ability to function on the ribosome (represented by K), after other influences have been normalized.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acyl-tRNA Synthetases↗
PubMed1985

Characterization of a "silencer" in yeast: a DNA sequence with properties opposite to those of a transcriptional enhancer.

The mating type of yeast is determined by the allele, either a or alpha, at the MAT locus. Two other loci, HML and HMR, contain complete copies of the alpha and a genes, respectively, which are not expressed. The four SIR gene products are required in trans for repression of the silent loci, as are cis-acting sites on either side of HML and HMR, about 1000 bp from the mating-type promoters. We demonstrate that one of these cis-acting sequences, HMRE, is able to switch off at least two nonmating-type promoters. In common with enhancers, it is able to function in either orientation, relatively independently of its position with respect to the regulated promoter, and can act on promoters 2600 bp away. However since HMRE represses, rather than enhances, transcription we have called it a "silencer" sequence.

Base Sequence↗