Cell cycle-dependent transcription of CLN2 is conferred by multiple distinct cis-acting regulatory elements

1994 ◽  
Vol 14 (7) ◽  
pp. 4788-4801
Author(s):  
D Stuart ◽  
C Wittenberg
Keyword(s):  
Cell Cycle ◽  
Mutational Analysis ◽  
Regulatory Elements ◽  
Upstream Region ◽  
Yeast Saccharomyces Cerevisiae ◽  
Transcriptional Initiation ◽  
Cis Acting ◽  
Upstream Activating Sequence ◽  
Dependent Cell ◽  
Cycle Dependent

The budding yeast Saccharomyces cerevisiae CLN1, CLN2, and CLN3 genes encode functionally redundant G1 cyclins required for cell cycle initiation. CLN1 and CLN2 mRNAs accumulate periodically throughout the cell cycle, peaking in late G1. We show that cell cycle-dependent fluctuation in CLN2 mRNA is regulated at the level of transcriptional initiation. Mutational analysis of the CLN2 promoter revealed that the major cell cycle-dependent upstream activating sequence (UAS) resides within a 100-bp fragment. This UAS contains three putative SWI4-dependent cell cycle boxes (SCBs) and two putative MluI cell cycle boxes (MCBs). Mutational inactivation of these elements substantially decreased CLN2 promoter activity but failed to eliminate periodic transcription. Similarly, inactivation of SWI4 decreased CLN2 transcription without affecting its periodicity. We have identified a second UAS in the CLN2 upstream region that can promote cell cycle-dependent transcription with kinetics similar to that of the intact CLN2 promoter. Unlike the major CLN2 UAS, this newly identified UAS promotes transcription in cells arrested in G1 by inactivation of cdc28. This novel UAS is both necessary and sufficient for regulated transcription driven by a CLN2 promoter lacking functional SCBs and MCBs. Although this UAS itself contains no SCBs or MCBs, its activity is dependent upon SWI4 function. The characteristics of this novel UAS suggest that it might have a role in initiating CLN2 expression early in G1 to activate the positive feedback loop that drives maximal Cln accumulation.

scholarly journals Cell cycle-dependent transcription of CLN2 is conferred by multiple distinct cis-acting regulatory elements.

1994 ◽  
Vol 14 (7) ◽  
pp. 4788-4801 ◽  
Author(s):  
D Stuart ◽  
C Wittenberg
Keyword(s):  
Cell Cycle ◽  
Mutational Analysis ◽  
Regulatory Elements ◽  
Upstream Region ◽  
Yeast Saccharomyces Cerevisiae ◽  
Transcriptional Initiation ◽  
Cis Acting ◽  
Upstream Activating Sequence ◽  
Dependent Cell ◽  
Cycle Dependent

The budding yeast Saccharomyces cerevisiae CLN1, CLN2, and CLN3 genes encode functionally redundant G1 cyclins required for cell cycle initiation. CLN1 and CLN2 mRNAs accumulate periodically throughout the cell cycle, peaking in late G1. We show that cell cycle-dependent fluctuation in CLN2 mRNA is regulated at the level of transcriptional initiation. Mutational analysis of the CLN2 promoter revealed that the major cell cycle-dependent upstream activating sequence (UAS) resides within a 100-bp fragment. This UAS contains three putative SWI4-dependent cell cycle boxes (SCBs) and two putative MluI cell cycle boxes (MCBs). Mutational inactivation of these elements substantially decreased CLN2 promoter activity but failed to eliminate periodic transcription. Similarly, inactivation of SWI4 decreased CLN2 transcription without affecting its periodicity. We have identified a second UAS in the CLN2 upstream region that can promote cell cycle-dependent transcription with kinetics similar to that of the intact CLN2 promoter. Unlike the major CLN2 UAS, this newly identified UAS promotes transcription in cells arrested in G1 by inactivation of cdc28. This novel UAS is both necessary and sufficient for regulated transcription driven by a CLN2 promoter lacking functional SCBs and MCBs. Although this UAS itself contains no SCBs or MCBs, its activity is dependent upon SWI4 function. The characteristics of this novel UAS suggest that it might have a role in initiating CLN2 expression early in G1 to activate the positive feedback loop that drives maximal Cln accumulation.

Identification of an upstream activating sequence and an upstream repressible sequence of the pyruvate kinase gene of the yeast Saccharomyces cerevisiae

1989 ◽  
Vol 9 (2) ◽  
pp. 442-451
Author(s):  
M Nishizawa ◽  
R Araki ◽  
Y Teranishi
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Carbon Source ◽  
Pyruvate Kinase ◽  
Transcriptional Activation ◽  
Regulatory Elements ◽  
Noncoding Region ◽  
Yeast Cells ◽  
Kinase Gene ◽  
Yeast Saccharomyces Cerevisiae ◽  
Upstream Activating Sequence

To clarify carbon source-dependent control of the glycolytic pathway in the yeast Saccharomyces cerevisiae, we have initiated a study of transcriptional regulation of the pyruvate kinase gene (PYK). By deletion analysis of the 5'-noncoding region of the PYK gene, we have identified an upstream activating sequence (UASPYK1) located between 634 and 653 nucleotides upstream of the initiating ATG codon. The promoter activity of the PYK 5'-noncoding region was abolished when the sequence containing the UASPYK1 was deleted from the region. Synthetic UASPYK1 (26mer), in either orientation, was able to restore the transcriptional activity of UAS-depleted mutants when placed upstream of the TATA sequence located at -199 (ATG as +1). While the UASPYK1 was required for basal to intermediate levels of transcriptional activation, a sequence between -714 and -811 was found to be necessary for full activation. On the other hand, a sequence between -344 and -468 was found to be responsible for transcriptional repression of the PYK gene when yeast cells were grown on nonfermentable carbon sources. This upstream repressible sequence also repressed transcription, although to a lesser extent, when glucose was present in the medium. The possible mechanism for carbon source-dependent regulation of PYK expression through these cis-acting regulatory elements is discussed.

scholarly journals A presumptive helicase (MOT1 gene product) affects gene expression and is required for viability in the yeast Saccharomyces cerevisiae.

1992 ◽  
Vol 12 (4) ◽  
pp. 1879-1892 ◽  
Author(s):  
J L Davis ◽  
R Kunisawa ◽  
J Thorner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gene Product ◽  
Essential Gene ◽  
Single Gene ◽  
Temperature Sensitive ◽  
Yeast Saccharomyces Cerevisiae ◽  
Sequence Element ◽  
Cis Acting ◽  
Upstream Activating Sequence ◽  
Identical Manner

Exposure of a haploid yeast cell to mating pheromone induces transcription of a set of genes. Induction is mediated through a cis-acting DNA sequence found upstream of all pheromone-responsive genes. Although the STE12 gene product binds specifically to this sequence element and is required for maximum levels of both basal and induced transcription, not all pheromone-responsive genes are regulated in an identical manner. To investigate whether additional factors may play a role in transcription of these genes, a genetic screen was used to identify mutants able to express pheromone-responsive genes constitutively in the absence of Ste12. In this way, we identified a recessive, single gene mutation (mot1, for modifier of transcription) which increases the basal level of expression of several, but not all, pheromone-responsive genes. The mot1-1 allele also relaxes the requirement for at least one other class of upstream activating sequence and enhances the expression of another gene not previously thought to be involved in the mating pathway. Cells carrying mot1-1 grow slowly at 30 degrees C and are inviable at 38 degrees C. The MOT1 gene was cloned by complementation of this temperature-sensitive lethality. Construction of a null allele confirmed that MOT1 is an essential gene. MOT1 residues on chromosome XVI and encodes a large protein of 1,867 amino acids which contains all seven of the conserved domains found in known and putative helicases. The product of MOT1 is strikingly homologous to the Saccharomyces cerevisiae SNF2/SW12 and RAD54 gene products over the entire helicase region.

scholarly journals Spc98p Directs the Yeast γ-Tubulin Complex into the Nucleus and Is Subject to Cell Cycle-dependent Phosphorylation on the Nuclear Side of the Spindle Pole Body

1998 ◽  
Vol 9 (4) ◽  
pp. 775-793 ◽  
Author(s):  
Gislene Pereira ◽  
Michael Knop ◽  
Elmar Schiebel
Keyword(s):  
Cell Cycle ◽  
Spindle Pole Body ◽  
Mitotic Checkpoint ◽  
Spindle Pole ◽  
Nuclear Localization Sequence ◽  
Checkpoint Control ◽  
Yeast Saccharomyces Cerevisiae ◽  
Pole Body ◽  
Cycle Dependent ◽  
Cytoplasmic Side

In the yeast Saccharomyces cerevisiae, microtubules are organized by the spindle pole body (SPB), which is embedded in the nuclear envelope. Microtubule organization requires the γ-tubulin complex containing the γ-tubulin Tub4p, Spc98p, and Spc97p. The Tub4p complex is associated with cytoplasmic and nuclear substructures of the SPB, which organize the cytoplasmic and nuclear microtubules. Here we present evidence that the Tub4p complex assembles in the cytoplasm and then either binds to the cytoplasmic side of the SPB or is imported into the nucleus followed by binding to the nuclear side of the SPB. Nuclear import of the Tub4p complex is mediated by the essential nuclear localization sequence of Spc98p. Our studies also indicate that Spc98p in the Tub4p complex is phosphorylated at the nuclear, but not at the cytoplasmic, side of the SPB. This phosphorylation is cell cycle dependent and occurs after SPB duplication and nucleation of microtubules by the new SPB and therefore may have a role in mitotic spindle function. In addition, activation of the mitotic checkpoint stimulates Spc98p phosphorylation. The kinase Mps1p, which functions in SPB duplication and mitotic checkpoint control, seems to be involved in Spc98p phosphorylation. Our results also suggest that the nuclear and cytoplasmic Tub4p complexes are regulated differently.

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

1993 ◽  
Vol 13 (6) ◽  
pp. 3792-3801 ◽  
Author(s):  
R Foster ◽  
G E Mikesell ◽  
L Breeden
Keyword(s):  
Cell Cycle ◽  
Normal Cell ◽  
Cell Cycle Regulation ◽  
Cell Cycle Progression ◽  
Cis Acting ◽  
Upstream Activating Sequence ◽  
A Cell ◽  
Cis And Trans ◽  
Cycle Regulation ◽  
Normal 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.

Immunoglobulin molecular genetics: the prospects for mutational analysis of the chromosomal immunoglobulin genes

Genome ◽  
1989 ◽  
Vol 31 (1) ◽  
pp. 175-181 ◽  
Author(s):  
Marc J. Shulman ◽  
Lucine Bosnoyan ◽  
Catherine Collins ◽  
Nancy Pennell ◽  
Mark D. Baker
Keyword(s):  
Gene Expression ◽  
Homologous Recombination ◽  
Mutational Analysis ◽  
Regulatory Elements ◽  
Hybridoma Cells ◽  
Immunoglobulin Genes ◽  
Chromosomal Dna ◽  
Cis Acting ◽  
Chromosomal Genes ◽  
Recombination Gene

Homologous recombination between transferred and chromosomal DNA can be used to effect precise, predetermined modifications of the chromosomal genes. Ultimately this phenomenon should allow the assessment of genetic regulatory elements as they function in the normal chromosomal environment. We have previously described a system for isolating mutant hybridoma cells that are defective in immunoglobulin (Ig) production, with a view toward using these mutants to define cis-acting elements that influence Ig gene expression. Here we describe results that indicate that homologous recombination between transferred and chromosomal Ig genes can be used to map Ig mutations by marker rescue.Key words: homologous recombination, gene expression.

scholarly journals Characterization of a short, cis-acting DNA sequence which conveys cell cycle stage-dependent transcription in Saccharomyces cerevisiae.

1991 ◽  
Vol 11 (1) ◽  
pp. 329-337 ◽  
Author(s):  
E M McIntosh ◽  
T Atkinson ◽  
R K Storms ◽  
M Smith
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Dna Replication ◽  
Transcription Initiation ◽  
Site Directed Mutagenesis ◽  
Upstream Region ◽  
Sequence Element ◽  
Cell Cycle Stage ◽  
Transcription System ◽  
Cycle Dependent

Comparison of the 5'-flanking regions of several cell cycle-regulated DNA replication genes of Saccharomyces cerevisiae has revealed the presence of a common sequence, 5'-ACGCGT-3', which is upstream and proximal to mapped transcription initiation sites. This sequence, which is the cleavage site for the restriction endonuclease MluI, is present twice in the upstream region of the yeast thymidylate synthase gene TMP1. Previous studies have implicated these MluI sites as critical components in the cell cycle-dependent transcription of TMP1. In this study, we examined more closely the importance of the ACGCGT sequences for the transcription of this gene. Using site-directed mutagenesis in combination with deletion analysis and subcloning experiments, we found that (i) while both of the TMP1 MluI sites contribute to the total transcription of this gene, the distal site is predominant and (ii) the 9-bp sequence ACGCGTTAA encompassing the distal MluI site exhibits properties of a cell cycle-stage dependent upstream activation sequence element. The results of this study support the notion that the ACGCGT sequence is an integral component of a transcription system which coordinates the cell cycle-dependent expression of DNA replication genes in S. cerevisiae.

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

1993 ◽  
Vol 13 (6) ◽  
pp. 3792-3801
Author(s):  
R Foster ◽  
G E Mikesell ◽  
L Breeden
Keyword(s):  
Cell Cycle ◽  
Normal Cell ◽  
Cell Cycle Regulation ◽  
Cell Cycle Progression ◽  
Cis Acting ◽  
Upstream Activating Sequence ◽  
A Cell ◽  
Cis And Trans ◽  
Cycle Regulation ◽  
Normal 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.

scholarly journals The growth-related, translationally controlled protein P23 has properties of a tubulin binding protein and associates transiently with microtubules during the cell cycle

1999 ◽  
Vol 112 (8) ◽  
pp. 1257-1271 ◽  
Author(s):  
Y. Gachet ◽  
S. Tournier ◽  
M. Lee ◽  
A. Lazaris-Karatzas ◽  
T. Poulton ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Mutational Analysis ◽  
Binding Protein ◽  
Binding Domain ◽  
Dependent Manner ◽  
Cell Extracts ◽  
Tubulin Binding ◽  
Cycle Dependent ◽  
Almost All

The translationally controlled protein P23 was discovered by the early induction of its rate of synthesis after mitogenic stimulation of mouse fibroblasts. P23 is expressed in almost all mammalian tissues and it is highly conserved between animals, plants and yeast. Based on its amino acid sequence, P23 cannot be attributed to any known protein family, and its cellular function remains to be elucidated. Here, we present evidence that P23 has properties of a tubulin binding protein that associates with microtubules in a cell cycle-dependent manner. (1) P23 is a cytoplasmic protein that occurs in complexes of 100–150 kDa, and part of P23 can be immunoprecipitated from HeLa cell extracts with anti-tubulin antibodies. (2) In immunolocalisation experiments we find P23 associated with microtubules during G1, S, G2 and early M phase of the cell cycle. At metaphase, P23 is also bound to the mitotic spindle, and it is detached from the spindle during metaphase-anaphase transition. (3) A GST-P23 fusion protein interacts with alpha- and beta-tubulin, and recombinant P23 binds to taxol-stabilised microtubules in vitro. The tubulin binding domain of P23 was identified by mutational analysis; it shows similarity to part of the tubulin binding domain of the microtubule-associated protein MAP-1B. (4) Overexpression of P23 results in cell growth retardation and in alterations of cell morphology. Moreover, elevation of P23 levels leads to microtubule rearrangements and to an increase in microtubule mass and stability.

scholarly journals EGT2 gene transcription is induced predominantly by Swi5 in early G1.

1996 ◽  
Vol 16 (7) ◽  
pp. 3264-3274 ◽  
Author(s):  
B Kovacech ◽  
K Nasmyth ◽  
T Schuster
Keyword(s):  
Cell Cycle ◽  
Transcriptional Activation ◽  
Cell Separation ◽  
S Phase ◽  
Dependent Manner ◽  
Yeast Saccharomyces Cerevisiae ◽  
Concentration Dependent Manner ◽  
A Cell ◽  
Cycle Dependent ◽  
Ho Gene

In a screen for cell cycle-regulated genes in the yeast Saccharomyces cerevisiae, we have identified a gene, EGT2, which is involved in cell separation in the G1 stage of the cell cycle. Transcription of EGT2 is tightly regulated in a cell cycle-dependent manner. Transcriptional levels peak at the boundary of mitosis and early G1 The transcription factors responsible for EGT2 expression in early G1 are Swi5 and, to a lesser extent, Ace2. Swi5 is involved in the transcriptional activation of the HO gene during late G1 and early S phase, and Ace2 induces CTS1 transcription during early and late G1 We show that Swi5 activates EGT2 transcription as soon as it enters the nucleus at the end of mitosis in a concentration-dependent manner. Since Swi5 is unstable in the nucleus, its level drops rapidly, causing termination of EGT2 transcription before cells are committed to the next cell cycle. However, Swi5 is still able to activate transcription of HO in late G1 in conjunction with additional activators such as Swi4 and Swi6.

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