Structure and expression of the Saccharomyces cerevisiae CRY1 gene: a highly conserved ribosomal protein gene.

The Saccharomyces cerevisiae CRY1 gene encodes ribosomal protein rp59, a component of the 40S ribosomal subunit. Mutations in CRY1 can confer resistance to the alkaloid cryptopleurine, an inhibitor of the elongation step of translation. The nucleotide sequence of the cloned CRY1 gene was determined. The predicted amino acid sequence shows that CRY1 encodes a 14,561-dalton polypeptide that has 88% amino acid sequence homology to the hamster or human S14 ribosomal protein responsible for emetine resistance and 45% homology to Escherichia coli ribosomal protein S11. Analysis of the DNA sequences upstream from CRY1 revealed the presence of three sequences, HOMOL1 (consensus, A/TACATCC/TG/ATA/GCA), RPG (consensus, ACCCA/GTACATT/CT/A), and a thymine-rich sequence, found upstream of more than 20 other cloned yeast genes encoding components of the translational apparatus. We exploited the ability to assay the expression of CRY1 in vivo by using the cryptopleurine resistance phenotype to demonstrate that these three consensus sequences are necessary for the transcription of CRY1. We previously showed that the upstream promoter element of the yeast RP39A gene consists of these identical sequence motifs. Therefore, we suggest that these three sequences define a consensus promoter element for the genes encoding the yeast translational apparatus. CRY1 is one of several hundred yeast genes, including ribosomal protein genes, whose expression is transiently decreased 10-fold upon heat shock. We found that the HOMOL1 and RPG consensus sequences are not necessary for the heat shock response of CRY1.

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Saccharomyces cerevisiae contains a complex multigene family related to the major heat shock-inducible gene of Drosophila.

Molecular and Cellular Biology ◽

10.1128/mcb.2.11.1388 ◽

1982 ◽

Vol 2 (11) ◽

pp. 1388-1398 ◽

Cited By ~ 83

Author(s):

T D Ingolia ◽

M R Slater ◽

E A Craig

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acid ◽

Heat Shock ◽

Dna Sequences ◽

Multigene Family ◽

Amino Acid Sequences ◽

Hsp 70 ◽

Rna Sequences ◽

Yeast Genes ◽

Differential Control

Saccharomyces cerevisiae contains a family of genes related to the major heat shock-induced gene of Drosophila (hsp 70). Two members of the multigene family (YG100 and YG101) were isolated. The primary DNA sequences of more than one-half of the protein-encoding regions of YG100 and YG101 were determined and compared with the Drosophila hsp 70 gene sequence; the predicted amino acid sequences were 72 and 64% homologous to the sequence of the Drosophila hsp 70 protein, respectively. The predicted amino acid sequences of the yeast genes were 65% homologous. Our results demonstrate a striking sequence conservation of hsp 70-related sequences in evolution. Hybridization of the S. cerevisiae genes to total S. cerevisiae DNA indicated that the multigene family consists of approximately 10 members. Hybridization of labeled RNAs from heat-shocked and control cells suggested that, like transcription of the Drosophila hsp 70 gene, transcription of YG100 or a closely related gene is enhanced after heat shock. However, the amount of RNA sequences homologous to YG101 was reduced after heat shock. A multigene family related to the hsp 70 gene exists in Drosophila; transcription of some members is induced by heat shock, whereas transcription of others is not. Our results suggest that S. cerevisiae, like Drosophila, contains a multigene family of hsp 70-related sequences under complex transcriptional regulation and that the differential control, as well as the nucleotide sequence, has been highly conserved in evolution.

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SSB, Encoding a Ribosome-Associated Chaperone, Is Coordinately Regulated with Ribosomal Protein Genes

Journal of Bacteriology ◽

10.1128/jb.181.10.3136-3143.1999 ◽

1999 ◽

Vol 181 (10) ◽

pp. 3136-3143 ◽

Cited By ~ 31

Author(s):

Nelson Lopez ◽

John Halladay ◽

William Walter ◽

Elizabeth A. Craig

Keyword(s):

Heat Shock ◽

Ribosomal Protein ◽

Ribosomal Proteins ◽

Chain Complex ◽

Mrna Levels ◽

Ribosomal Protein Genes ◽

Genes Encoding ◽

Synthetic Capacity ◽

Translational Apparatus ◽

Temperature Upshift

ABSTRACT Genes encoding ribosomal proteins and other components of the translational apparatus are coregulated to efficiently adjust the protein synthetic capacity of the cell. Ssb, a Saccharomyces cerevisiae Hsp70 cytosolic molecular chaperone, is associated with the ribosome-nascent chain complex. To determine whether this chaperone is coregulated with ribosomal proteins, we studied the mRNA regulation of SSB under several environmental conditions. Ssb and the ribosomal protein rpL5 mRNAs were up-regulated upon carbon upshift and down-regulated upon amino acid limitation, unlike the mRNA of another cytosolic Hsp70, Ssa. Ribosomal protein and Ssb mRNAs, like many mRNAs, are down-regulated upon a rapid temperature upshift. The mRNA reduction of several ribosomal protein genes and Ssb was delayed by the presence of an allele, EXA3-1, of the gene encoding the heat shock factor (HSF). However, upon a heat shock theEXA3-1 mutation did not significantly alter the reduction in the mRNA levels of two genes encoding proteins unrelated to the translational apparatus. Analysis of gene fusions indicated that the transcribed region, but not the promoter of SSB, is sufficient for this HSF-dependent regulation. Our studies suggest that Ssb is regulated like a core component of the ribosome and that HSF is required for proper regulation of SSB and ribosomal mRNA after a temperature upshift.

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Ribosomal protein S14 is encoded by a pair of highly conserved, adjacent genes on the X chromosome of Drosophila melanogaster

Molecular and Cellular Biology ◽

10.1128/mcb.8.10.4314-4321.1988 ◽

1988 ◽

Vol 8 (10) ◽

pp. 4314-4321

Author(s):

S J Brown ◽

D D Rhoads ◽

M J Stewart ◽

B Van Slyke ◽

I T Chen ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Drosophila Melanogaster ◽

Amino Acid ◽

Amino Acid Sequence ◽

Ribosomal Protein ◽

X Chromosome ◽

Duplicated Genes ◽

Ribosomal Protein Genes ◽

Yeast Saccharomyces Cerevisiae ◽

Genes Encoding

We describe a Drosophila DNA clone of tandemly duplicated genes encoding an amino acid sequence nearly identical to human ribosomal protein S14 and yeast rp59. Despite their remarkably similar exons, the locations and sizes of introns differ radically among the Drosophila, human, and yeast (Saccharomyces cerevisiae) ribosomal protein genes. Transcripts of both Drosophila RPS14 genes were detected in embryonic and adult tissues and are the same length as mammalian S14 message. Drosophila RPS14 was mapped to region 7C5-9 on the X chromosome. This interval also encodes a previously characterized Minute locus, M(1)7C.

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Ribosomal protein S14 is encoded by a pair of highly conserved, adjacent genes on the X chromosome of Drosophila melanogaster.

Molecular and Cellular Biology ◽

10.1128/mcb.8.10.4314 ◽

1988 ◽

Vol 8 (10) ◽

pp. 4314-4321 ◽

Cited By ~ 21

Author(s):

S J Brown ◽

D D Rhoads ◽

M J Stewart ◽

B Van Slyke ◽

I T Chen ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Drosophila Melanogaster ◽

Amino Acid ◽

Amino Acid Sequence ◽

Ribosomal Protein ◽

X Chromosome ◽

Duplicated Genes ◽

Ribosomal Protein Genes ◽

Yeast Saccharomyces Cerevisiae ◽

Genes Encoding

We describe a Drosophila DNA clone of tandemly duplicated genes encoding an amino acid sequence nearly identical to human ribosomal protein S14 and yeast rp59. Despite their remarkably similar exons, the locations and sizes of introns differ radically among the Drosophila, human, and yeast (Saccharomyces cerevisiae) ribosomal protein genes. Transcripts of both Drosophila RPS14 genes were detected in embryonic and adult tissues and are the same length as mammalian S14 message. Drosophila RPS14 was mapped to region 7C5-9 on the X chromosome. This interval also encodes a previously characterized Minute locus, M(1)7C.

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Saccharomyces cerevisiae contains a complex multigene family related to the major heat shock-inducible gene of Drosophila

Molecular and Cellular Biology ◽

10.1128/mcb.2.11.1388-1398.1982 ◽

1982 ◽

Vol 2 (11) ◽

pp. 1388-1398

Author(s):

T D Ingolia ◽

M R Slater ◽

E A Craig

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acid ◽

Heat Shock ◽

Dna Sequences ◽

Multigene Family ◽

Amino Acid Sequences ◽

Hsp 70 ◽

Rna Sequences ◽

Yeast Genes ◽

Differential Control

Saccharomyces cerevisiae contains a family of genes related to the major heat shock-induced gene of Drosophila (hsp 70). Two members of the multigene family (YG100 and YG101) were isolated. The primary DNA sequences of more than one-half of the protein-encoding regions of YG100 and YG101 were determined and compared with the Drosophila hsp 70 gene sequence; the predicted amino acid sequences were 72 and 64% homologous to the sequence of the Drosophila hsp 70 protein, respectively. The predicted amino acid sequences of the yeast genes were 65% homologous. Our results demonstrate a striking sequence conservation of hsp 70-related sequences in evolution. Hybridization of the S. cerevisiae genes to total S. cerevisiae DNA indicated that the multigene family consists of approximately 10 members. Hybridization of labeled RNAs from heat-shocked and control cells suggested that, like transcription of the Drosophila hsp 70 gene, transcription of YG100 or a closely related gene is enhanced after heat shock. However, the amount of RNA sequences homologous to YG101 was reduced after heat shock. A multigene family related to the hsp 70 gene exists in Drosophila; transcription of some members is induced by heat shock, whereas transcription of others is not. Our results suggest that S. cerevisiae, like Drosophila, contains a multigene family of hsp 70-related sequences under complex transcriptional regulation and that the differential control, as well as the nucleotide sequence, has been highly conserved in evolution.

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Molecular genetics of cryptopleurine resistance in Saccharomyces cerevisiae: expression of a ribosomal protein gene family.

Genetics ◽

10.1093/genetics/135.3.719 ◽

1993 ◽

Vol 135 (3) ◽

pp. 719-730

Author(s):

A G Paulovich ◽

J R Thompson ◽

J C Larkin ◽

Z Li ◽

J L Woolford

Keyword(s):

Saccharomyces Cerevisiae ◽

Ribosomal Protein ◽

Gene Fusion ◽

Null Allele ◽

Ribosomal Subunit ◽

Null Alleles ◽

Protein Synthesis Inhibitor ◽

Lacz Gene ◽

Ribosomal Subunits ◽

Cry1 Gene

Abstract The Saccharomyces cerevisiae CRY1 gene encodes the 40S ribosomal subunit protein rp59 and confers sensitivity to the protein synthesis inhibitor cryptopleurine. A yeast strain containing the cry1-delta 1::URA3 null allele is viable, cryptopleurine sensitive (CryS), and expresses rp59 mRNA, suggesting that there is a second functional CRY gene. The CRY2 gene has been isolated from a yeast genomic library cloned in bacteriophage lambda, using a CRY1 DNA probe. The DNA sequence of the CRY2 gene contains an open reading frame encoding ribosomal protein 59 that differs at five residues from rp59 encoded by the CRY1 gene. The CRY2 gene was mapped to the left arm of chromosome X, centromere-proximal to cdc6 and immediately adjacent to ribosomal protein genes RPS24A and RPL46. Ribosomal protein 59 is an essential protein; upon sporulation of a diploid doubly heterozygous for cry1-delta 2::TRP1 cry2-delta 1::LEU2 null alleles, no spore clones containing both null alleles were recovered. Several results indicate that CRY2 is expressed, but at lower levels than CRY1: (1) Introduction of CRY2 on high copy plasmids into CryR yeast of genotype cry1 CRY2 confers a CryS phenotype. Transformation of these CryR yeast with CRY2 on a low copy CEN plasmid does not confer a CryS phenotype. (2) Haploids containing the cry1-delta 2::TRP1 null allele have a deficit of 40S ribosomal subunits, but cry2-delta 1::LEU2 strains have wild-type amounts of 40S ribosomal subunits. (3) CRY2 mRNA is present at lower levels than CRY1 mRNA. (4) Higher levels of beta-galactosidase are expressed from a CRY1-lacZ gene fusion than from a CRY2-lacZ gene fusion. Mutations that alter or eliminate the last amino acid of rp59 encoded by either CRY1 or CRY2 result in resistance to cryptopleurine. Because CRY2 (and cry2) is expressed at lower levels than CRY1 (and cry1), the CryR phenotype of cry2 mutants is only expressed in strains containing a cry1-delta null allele.

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Association of RAP1 binding sites with stringent control of ribosomal protein gene transcription in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.11.5.2723-2735.1991 ◽

1991 ◽

Vol 11 (5) ◽

pp. 2723-2735 ◽

Cited By ~ 1

Author(s):

C M Moehle ◽

A G Hinnebusch

Keyword(s):

Gene Expression ◽

Saccharomyces Cerevisiae ◽

Amino Acid ◽

Ribosomal Protein ◽

Binding Sites ◽

Protein Gene ◽

Ribosomal Protein Gene ◽

Ribosomal Protein Genes ◽

Biosynthetic Genes ◽

Stringent Control

An amino acid limitation in bacteria elicits a global response, called stringent control, that leads to reduced synthesis of rRNA and ribosomal proteins and increased expression of amino acid biosynthetic operons. We have used the antimetabolite 3-amino-1,2,4-triazole to cause histidine limitation as a means to elicit the stringent response in the yeast Saccharomyces cerevisiae. Fusions of the yeast ribosomal protein genes RPL16A, CRY1, RPS16A, and RPL25 with the Escherichia coli lacZ gene were used to show that the expression of these genes is reduced by a factor of 2 to 5 during histidine-limited exponential growth and that this regulation occurs at the level of transcription. Stringent regulation of the four yeast ribosomal protein genes was shown to be associated with a nucleotide sequence, known as the UASrpg (upstream activating sequence for ribosomal protein genes), that binds the transcriptional regulatory protein RAP1. The RAP1 binding sites also appeared to mediate the greater ribosomal protein gene expression observed in cells growing exponentially than in cells in stationary phase. Although expression of the ribosomal protein genes was reduced in response to histidine limitation, the level of RAP1 DNA-binding activity in cell extracts was unaffected. Yeast strains bearing a mutation in any one of the genes GCN1 to GCN4 are defective in derepression of amino acid biosynthetic genes in 10 different pathways under conditions of histidine limitation. These Gcn- mutants showed wild-type regulation of ribosomal protein gene expression, which suggests that separate regulatory pathways exist in S. cerevisiae for the derepression of amino acid biosynthetic genes and the repression of ribosomal protein genes in response to amino acid starvation.

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Multiple GCD genes required for repression of GCN4, a transcriptional activator of amino acid biosynthetic genes in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.6.11.3990-3998.1986 ◽

1986 ◽

Vol 6 (11) ◽

pp. 3990-3998

Author(s):

S Harashima ◽

A G Hinnebusch

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acid ◽

Lacz Gene ◽

Gene Products ◽

Wild Type ◽

Double Mutation ◽

Double Stranded Rna ◽

Amino Acid Availability ◽

Genes Encoding ◽

New Alleles

GCN4 encodes a positive regulator of multiple unlinked genes encoding amino acid biosynthetic enzymes in Saccharomyces cerevisiae. Expression of GCN4 is coupled to amino acid availability by a control mechanism involving GCD1 as a negative effector and GCN1, GCN2, and GCN3 as positive effectors of GCN4 expression. We used reversion of a gcn2 gcn3 double mutation to isolate new alleles of GCD1 and mutations in four additional GCD genes which we designate GCD10, GCD11, GCD12, and GCD13. All of the mutations lead to constitutive derepression of HIS4 transcription in the absence of the GCN2+ and GCN3+ alleles. By contrast, the gcd mutations require the wild-type GCN4 allele for their derepressing effect, suggesting that each acts by influencing the level of GCN4 activity in the cell. Consistent with this interpretation, mutations in each GCD gene lead to constitutive derepression of a GCN4::lacZ gene fusion. Thus, at least five gene products are required to maintain the normal repressed level of GCN4 expression in nonstarvation conditions. Interestingly, the gcd mutations are pleiotropic and also affect growth rate in nonstarvation conditions. In addition, certain alleles lead to a loss of M double-stranded RNA required for the killer phenotype. This pleiotropy suggests that the GCD gene products contribute to an essential cellular function, in addition to, or in conjunction with, their role in GCN4 regulation.

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Genes for low-molecular-weight heat shock proteins of soybeans: sequence analysis of a multigene family.

Molecular and Cellular Biology ◽

10.1128/mcb.5.12.3417 ◽

1985 ◽

Vol 5 (12) ◽

pp. 3417-3428 ◽

Cited By ~ 65

Author(s):

R T Nagao ◽

E Czarnecka ◽

W B Gurley ◽

F Schöffl ◽

J L Key

Keyword(s):

Molecular Weight ◽

Amino Acid ◽

Heat Shock ◽

Dna Sequences ◽

Regulatory Element ◽

Amino Acid Sequences ◽

Striking Similarity ◽

Low Molecular Weight ◽

Genes Encoding ◽

Flanking Regions

Soybeans, Glycine max, synthesize a family of low-molecular-weight heat shock (HS) proteins in response to HS. The DNA sequences of two genes encoding 17.5- and 17.6-kilodalton HS proteins were determined. Nuclease S1 mapping of the corresponding mRNA indicated multiple start termini at the 5' end and multiple stop termini at the 3' end. These two genes were compared with two other soybean HS genes of similar size. A comparison among the 5' flanking regions encompassing the presumptive HS promoter of the soybean HS-protein genes demonstrated this region to be extremely homologous. Analysis of the DNA sequences in the 5' flanking regions of the soybean genes with the corresponding regions of Drosophila melanogaster HS-protein genes revealed striking similarity between plants and animals in the presumptive promoter structure of thermoinducible genes. Sequences related to the Drosophila HS consensus regulatory element were found 57 to 62 base pairs 5' to the start of transcription in addition to secondary HS consensus elements located further upstream. Comparative analysis of the deduced amino acid sequences of four soybean HS proteins illustrated that these proteins were greater than 90% homologous. Comparison of the amino acid sequence for soybean HS proteins with other organisms showed much lower homology (less than 20%). Hydropathy profiles for Drosophila, Xenopus, Caenorhabditis elegans, and G. max HS proteins showed a similarity of major hydrophilic and hydrophobic regions, which suggests conservation of functional domains for these proteins among widely dispersed organisms.

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