Signals sufficient for 3'-end formation of yeast mRNA.

ABSTRACT The alcohol dehydrogenase II (ADH2) gene of the yeast, Saccharomyces cerevisiae, is not transcribed during growth on fermentable carbon sources such as glucose. Growth of yeast cells in a medium containing only nonfermentable carbon sources leads to a marked increase or derepression of ADH2 expression. The recessive mutation, adr6-1, leads to an inability to fully derepress ADH2 expression and to an inability to sporulate. The ADR6 gene product appears to act directly or indirectly on ADH2 sequences 3' to or including the presumptive TATAA box. The upstream activating sequence (UAS) located 5' to the TATAA box is not required for the Adr6- phenotype. Here, we describe the isolation of a recombinant plasmid containing the wild-type ADR6 gene. ADR6 codes for a 4.4-kb RNA which is present during growth both on glucose and on nonfermentable carbon sources. Disruption of the ADR6 transcription unit led to viable cells with decreased ADHII activity and an inability to sporulate. This indicates that both phenotypes result from mutations within a single gene and that the adr6-1 allele was representative of mutations at this locus. The ADR6 gene mapped to the left arm of chromosome XVI at a site 18 centimorgans from the centromere.

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Trans-acting regulatory mutations that alter transcription of Saccharomyces cerevisiae histone genes

Molecular and Cellular Biology ◽

10.1128/mcb.7.12.4204-4210.1987 ◽

1987 ◽

Vol 7 (12) ◽

pp. 4204-4210

Author(s):

M A Osley ◽

D Lycan

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Cycle ◽

Fusion Gene ◽

Chromosome Replication ◽

Lacz Fusion ◽

Histone Genes ◽

Promoter Elements ◽

Regulatory Mutations ◽

Cycle Dependent ◽

Beta Galactosidase

Using a Saccharomyces cerevisiae strain containing an integrated copy of an H2A-lacZ fusion gene, we screened for mutants which overexpressed beta-galactosidase as a way to identify genes which regulate transcription of the histone genes. Five recessive mutants with this phenotype were shown to contain altered regulatory genes because they had lost repression of HTA1 transcription which occurs upon inhibition of chromosome replication (D. E. Lycan, M. A. Osley, and L. Hereford, Mol. Cell. Biol. 7:614-621, 1987). Periodic transcription was affected in the mutants as well, since the HTA1 gene was transcribed during the G1 and G2 phases of the cell cycle, periods in the cell cycle when this gene is normally not expressed. A similar loss of cell cycle-dependent transcription was noted for two of the three remaining histone loci, while the HO and CDC9 genes continued to be expressed periodically. Using isolated promoter elements inserted into a heterologous cycl-lacZ fusion gene, we demonstrated that the mutations fell in genes which acted through a negative site in the TRT1 H2A-H2B promoter.

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3'-end-forming signals of yeast mRNA.

Molecular and Cellular Biology ◽

10.1128/mcb.15.11.5983 ◽

1995 ◽

Vol 15 (11) ◽

pp. 5983-5990 ◽

Cited By ~ 58

Author(s):

Z Guo ◽

F Sherman

Keyword(s):

Saccharomyces Cerevisiae ◽

Cleavage Sites ◽

Yeast Saccharomyces Cerevisiae ◽

Higher Eukaryotes ◽

A Site

It was previously shown that three distinct but interdependent elements are required for 3' end formation of mRNA in the yeast Saccharomyces cerevisiae: (i) the efficiency element TATATA and related sequences, which function by enhancing the efficiency of positioning elements; (ii) positioning elements, such as TTAAGAAC and AAGAA, which position the poly(A) site; and (iii) the actual site of polyadenylation. In this study, we have shown that several A-rich sequences, including the vertebrate poly(A) signal AATAAA, are also positioning elements. Saturated mutagenesis revealed that optimum sequences of the positioning element were AATAAA and AAAAAA and that this element can tolerate various extents of replacements. However, the GATAAA sequence was completely ineffective. The major cleavage sites determined in vitro corresponded to the major poly(A) sites observed in vivo. Our findings support the assumption that some components of the basic polyadenylation machinery could have been conserved among yeasts, plants, and mammals, although 3' end formation in yeasts is clearly distinct from that of higher eukaryotes.

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Genomic Considerations for the Modification of Saccharomyces cerevisiae for Biofuel and Metabolite Biosynthesis

Microorganisms ◽

10.3390/microorganisms8030321 ◽

2020 ◽

Vol 8 (3) ◽

pp. 321 ◽

Cited By ~ 1

Author(s):

James T. Arnone

Keyword(s):

Saccharomyces Cerevisiae ◽

Native Species ◽

Genetic Manipulation ◽

Scale Up ◽

Biosynthetic Pathways ◽

Yeast Saccharomyces Cerevisiae ◽

Position Effects ◽

Industrial Use ◽

A Site ◽

Global Population

The growing global population and developing world has put a strain on non-renewable natural resources, such as fuels. The shift to renewable sources will, thus, help meet demands, often through the modification of existing biosynthetic pathways or the introduction of novel pathways into non-native species. There are several useful biosynthetic pathways endogenous to organisms that are not conducive for the scale-up necessary for industrial use. The use of genetic and synthetic biological approaches to engineer these pathways in non-native organisms can help ameliorate these challenges. The budding yeast Saccharomyces cerevisiae offers several advantages for genetic engineering for this purpose due to its widespread use as a model system studied by many researchers. The focus of this review is to present a primer on understanding genomic considerations prior to genetic modification and manipulation of S. cerevisiae. The choice of a site for genetic manipulation can have broad implications on transcription throughout a region and this review will present the current understanding of position effects on transcription.

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Signals that produce 3' termini in CYC1 mRNA of the yeast Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.13.12.7836-7849.1993 ◽

1993 ◽

Vol 13 (12) ◽

pp. 7836-7849

Author(s):

P Russo ◽

W Z Li ◽

Z Guo ◽

F Sherman

Keyword(s):

Saccharomyces Cerevisiae ◽

Site Directed Mutagenesis ◽

Yeast Cells ◽

In Vitro Mutagenesis ◽

Yeast Saccharomyces Cerevisiae ◽

Upstream Element ◽

A Site ◽

Derivatives Of ◽

Acting In Concert

The cyc1-512 mutant was previously shown to contain a 38-bp deletion, 8 nucleotides upstream from the major wild-type poly(A) site, in the CYC1 gene, which encodes iso-1-cytochrome c of the yeast Saccharomyces cerevisiae. This 38-bp deletion caused a 90% reduction in the CYC1 transcripts, which were heterogeneous in size, aberrantly long, and presumably labile (K. S. Zaret and F. Sherman, Cell 28:563-573, 1982). Site-directed mutagenesis in and adjacent to the 38-bp region was used to identify signals involved in the formation and positioning of CYC1 mRNA 3' ends. In addition, combinations of various putative 3' end-forming signals were introduced by in vitro mutagenesis into the 3' region of the cyc1-512 mutant. The combined results from both studies suggest that 3' end formation in yeast cells involves signals having the following three distinct but integrated elements acting in concert: (i) the upstream element, including sequences TATATA, TAG ... TATGTA, and TTTTTATA, which function by enhancing the efficiency of downstream elements; (ii) downstream elements, such as TTAAGAAC and AAGAA, which position the poly(A) site; and (iii) the actual site of polyadenylation, which often occurs after cytidine residues that are 3' to the so-called downstream element. While the upstream element is required for efficient 3' end formation, alterations of the downstream element and poly(A) sites generally do not affect the efficiency of 3' end formation but appear to alter the positions of poly(A) sites. In addition, we have better defined the upstream elements by examining various derivatives of TATATA and TAG ... TATGTA, and we have examined the spatial requirements of the three elements by systematically introducing or deleting upstream and downstream elements and cytidine poly(A) sites.

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Mutational Analysis of the Structure and Localization of the Nucleolus in the Yeast Saccharomyces cerevisiae

The Journal of Cell Biology ◽

10.1083/jcb.143.1.23 ◽

1998 ◽

Vol 143 (1) ◽

pp. 23-34 ◽

Cited By ~ 80

Author(s):

M. Oakes ◽

J.P. Aris ◽

J.S. Brockenbrough ◽

H. Wai ◽

L. Vu ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Nuclear Envelope ◽

Mutational Analysis ◽

Fusion Gene ◽

Nuclear Periphery ◽

Rdna Locus ◽

Coding Region ◽

Yeast Saccharomyces Cerevisiae ◽

Pol I ◽

Rdna Expression

The nucleolus in Saccharomyces cerevisiae is a crescent-shaped structure that makes extensive contact with the nuclear envelope. In different chromosomal rDNA deletion mutants that we have analyzed, the nucleolus is not organized into a crescent structure, as determined by immunofluorescence microscopy, fluorescence in situ hybridization, and electron microscopy. A strain carrying a plasmid with a single rDNA repeat transcribed by RNA polymerase I (Pol I) contained a fragmented nucleolus distributed throughout the nucleus, primarily localized at the nuclear periphery. A strain carrying a plasmid with the 35S rRNA coding region fused to the GAL7 promoter and transcribed by Pol II contained a rounded nucleolus that often lacked extensive contact with the nuclear envelope. Ultrastructurally distinct domains were observed within the round nucleolus. A similar rounded nucleolar morphology was also observed in strains carrying the Pol I plasmid in combination with mutations that affect Pol I function. In a Pol I–defective mutant strain that carried copies of the GAL7-35S rDNA fusion gene integrated into the chromosomal rDNA locus, the nucleolus exhibited a round morphology, but was more closely associated with the nuclear envelope in the form of a bulge. Thus, both the organization of the rDNA genes and the type of polymerase involved in rDNA expression strongly influence the organization and localization of the nucleolus.

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Signals that produce 3' termini in CYC1 mRNA of the yeast Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.13.12.7836 ◽

1993 ◽

Vol 13 (12) ◽

pp. 7836-7849 ◽

Cited By ~ 32

Author(s):

P Russo ◽

W Z Li ◽

Z Guo ◽

F Sherman

Keyword(s):

Saccharomyces Cerevisiae ◽

Site Directed Mutagenesis ◽

Yeast Cells ◽

In Vitro Mutagenesis ◽

Yeast Saccharomyces Cerevisiae ◽

Upstream Element ◽

A Site ◽

Derivatives Of ◽

Acting In Concert

The cyc1-512 mutant was previously shown to contain a 38-bp deletion, 8 nucleotides upstream from the major wild-type poly(A) site, in the CYC1 gene, which encodes iso-1-cytochrome c of the yeast Saccharomyces cerevisiae. This 38-bp deletion caused a 90% reduction in the CYC1 transcripts, which were heterogeneous in size, aberrantly long, and presumably labile (K. S. Zaret and F. Sherman, Cell 28:563-573, 1982). Site-directed mutagenesis in and adjacent to the 38-bp region was used to identify signals involved in the formation and positioning of CYC1 mRNA 3' ends. In addition, combinations of various putative 3' end-forming signals were introduced by in vitro mutagenesis into the 3' region of the cyc1-512 mutant. The combined results from both studies suggest that 3' end formation in yeast cells involves signals having the following three distinct but integrated elements acting in concert: (i) the upstream element, including sequences TATATA, TAG ... TATGTA, and TTTTTATA, which function by enhancing the efficiency of downstream elements; (ii) downstream elements, such as TTAAGAAC and AAGAA, which position the poly(A) site; and (iii) the actual site of polyadenylation, which often occurs after cytidine residues that are 3' to the so-called downstream element. While the upstream element is required for efficient 3' end formation, alterations of the downstream element and poly(A) sites generally do not affect the efficiency of 3' end formation but appear to alter the positions of poly(A) sites. In addition, we have better defined the upstream elements by examining various derivatives of TATATA and TAG ... TATGTA, and we have examined the spatial requirements of the three elements by systematically introducing or deleting upstream and downstream elements and cytidine poly(A) sites.

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Different classes of polyadenylation sites in the yeast Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.11.6.3060 ◽

1991 ◽

Vol 11 (6) ◽

pp. 3060-3069 ◽

Cited By ~ 50

Author(s):

S Irniger ◽

C M Egli ◽

G H Braus

Keyword(s):

Saccharomyces Cerevisiae ◽

Fusion Gene ◽

Signal Sequence ◽

Test System ◽

Dependent Manner ◽

Yeast Saccharomyces Cerevisiae ◽

Sequence Elements ◽

Polyadenylation Sites ◽

Point Mutagenesis

This report provides an analysis of the function of polyadenylation sites from six different genes of the yeast Saccharomyces cerevisiae. These sites were tested for their ability to turn off read-through transcription into the URA3 gene in vivo when inserted into an ACT-URA3 fusion gene. The 3' ends of all polyadenylation sites inserted into the test system in their natural configuration are identical to the 3' ends of the chromosomal genes. We identified two classes of polyadenylation sites: (i) efficient sites (originating from the genes GCN4 and PHO5) that were functional in a strict orientation-dependent manner and (ii) bidirectional sites (derived from ARO4, TRP1, and TRP4) that had a distinctly reduced efficiency. The ADH1 polyadenylation site was efficient and bidirectional and was shown to be a combination of two polyadenylation sites of two convergently transcribed genes. Sequence comparison revealed that all efficient unidirectional polyadenylation sites contain the sequence TTTTTAT, whereas all bidirectional sites have the tripartite sequence TAG...TA (T)GT...TTT. Both sequence elements have previously been proposed to be involved in 3' end formation. Site-directed point mutagenesis of the TTTTTAT sequence had no effect, whereas mutations within the tripartite sequence caused a reduced efficiency for 3' end formation. The tripartite sequence alone, however, is not sufficient for 3' end formation, but it might be part of a signal sequence in the bidirectional class of yeast polyadenylation sites. Our findings support the assumption that there are at least two different mechanisms with different sequence elements directing 3' end formation in yeast.

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The PHO84 gene of Saccharomyces cerevisiae encodes an inorganic phosphate transporter

Molecular and Cellular Biology ◽

10.1128/mcb.11.6.3229-3238.1991 ◽

1991 ◽

Vol 11 (6) ◽

pp. 3229-3238

Author(s):

M Bun-Ya ◽

M Nishimura ◽

S Harashima ◽

Y Oshima

Keyword(s):

Saccharomyces Cerevisiae ◽

Fusion Gene ◽

Amino Acid Residues ◽

Sugar Transporters ◽

Pi Transport ◽

Constitutive Synthesis ◽

A Site ◽

Mutant Cells ◽

Hydropathy Analysis ◽

Beta Galactosidase

The PHO84 gene specifies Pi-transport in Saccharomyces cerevisiae. A DNA fragment bearing the PHO84 gene was cloned by its ability to complement constitutive synthesis of repressible acid phosphatase of pho84 mutant cells. Its nucleotide sequence predicted a protein of 596 amino acids with a sequence homologous to that of a superfamily of sugar transporters. Hydropathy analysis suggested that the secondary structure of the PHO84 protein consists of two blocks of six transmembrane domains separated by 74 amino acid residues. The cloned PH084 DNA restored the Pi transport activity of pho84 mutant cells. The PHO84 transcription was regulated by Pi like those of the PHO5, PHO8, and PHO81 genes. A PHO84-lacZ fusion gene produced beta-galactosidase activity under the regulation of Pi, and the activity was suggested to be bound to a membrane fraction. Gene disruption of PHO84 was not lethal. By comparison of nucleotide sequences and by tetrad analysis with GAL80 as a standard, the PHO84 locus was mapped at a site beside the TUB3 locus on the left arm of chromosome XIII.

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Characterization and mutational analysis of a cluster of three genes expressed preferentially during sporulation of Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.6.7.2443 ◽

1986 ◽

Vol 6 (7) ◽

pp. 2443-2451 ◽

Cited By ~ 40

Author(s):

A Percival-Smith ◽

J Segall

Keyword(s):

Saccharomyces Cerevisiae ◽

Intergenic Region ◽

Mutational Analysis ◽

Fusion Gene ◽

Lacz Fusion ◽

Regulatory Sequences ◽

Wild Type ◽

Base Pairs ◽

Differential Hybridization ◽

Dna Library

A differential hybridization screen of a genomic yeast DNA library previously identified 14 genes of Saccharomyces cerevisiae that are expressed preferentially during sporulation. Three of these sporulation-specific genes, SPS1, SPS2, and SPS3, have been shown to be closely linked. A mutational analysis has demonstrated that expression of the SPS1 gene, but not the SPS2 gene, is essential for the completion of sporulation. A diploid MATa/MAT alpha strain homozygous for a disruption of the SPS1 gene failed to form asci when subjected to sporulation conditions. The 3' end of the transcript encoded by the SPS1 gene was found to map only 185 base pairs from the 5' end of the SPS2 gene. The SPS1-SPS2 intergenic region was shown to contain all of the regulatory sequences necessary for the sporulation-specific activation of the SPS2 gene as assessed by expression of a translational SPS2-lacZ fusion gene present on a replicating, centromere-containing plasmid. The fusion gene was found to be expressed at the same time during sporulation as the chromosomal wild-type SPS2 gene.

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