Chapter 5 Isolation, Characterization, and Translation of mRNA from Yeast

Sripati, Conjeevaram E.; Warner, Jonathan R.

doi:10.1016/s0091-679x(08)62009-9

Cited by 51 publications

(13 citation statements)

References 31 publications

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“…Polysomes were prepared essentially according to the method described earlier (Sripati and Warner, 1978). Briefly, for harvesting cells, cultures were treated with cycloheximide, 100 pg/ml for 10 sec, and cooled rapidly by pouring on crushed frozen culture medium.…”

Section: Preparation Of Polysomesmentioning

confidence: 99%

The specific phosphorylation of a 40S ribosomal protein in growth‐arrested tetrahymena is induced by sodium

Cuny

Sripati

Hayes

1985

Journal Cellular Physiology

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In the past few years, in vivo phosphorylation of ribosomal proteins has been the subject of extensive studies and the results have shown that reversible phosphorylation of small subunit ribosomal protein S6, ubiquitous in eukaryotic cells, is apparently related to regulation of protein synthesis initiation. Thus the level of protein synthesis under various conditions is correlated with the level of S6 phosphorylation. In exponentially growing Tetrahymena, however, such phosphorylation does not occur, but when these cells are transferred to starvation buffers, the rate of protein synthesis is drastically reduced and a 40S ribosomal protein analogous to S6 of higher eukaryotic cells is fully and rapidly phosphorylated in all the ribosomes. We have studied the conditions which lead to this phosphorylation in growth-arrested Tetrahymena, in order to understand the physiological significance of this process. Our results show that there is no obvious correlation between this phosphorylation and starvation. Moreover, it is not a developmentally regulated process related to the conjugation cycle, but a modification induced by the presence of sodium ions or high concentration of Tris in the starvation buffer. The physiological significance of this process is discussed in terms of accumulation of negative charge density probably required for initiation of protein synthesis in the growth-arrested cells starving in Na+-containing buffers.

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Section: Preparation Of Polysomesmentioning

confidence: 99%

The specific phosphorylation of a 40S ribosomal protein in growth‐arrested tetrahymena is induced by sodium

Cuny

Sripati

Hayes

1985

Journal Cellular Physiology

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“…Total RNA was isolated from logarithmically growing yeasts by the method of Sripati and Warner (39). Poly(A)+ RNA was selected from the total RNA fraction with oligo(dT)-cellulose.…”

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confidence: 99%

Duplicate upstream activating sequences in the promoter region of the Saccharomyces cerevisiae GAL7 gene.

Tajima¹,

Nogi²,

Fukasawa³

1986

Mol. Cell. Biol.

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We constructed a series of deletions in the 5' noncoding region of the Saccharomyces cerevisiae GAL7 gene, fused them to the Escherichia coli gene lacZ, and introduced them into yeasts by using a multicopy vector. We then studied the effect of the deletions on P-galactosidase synthesis directed by the gene fusions in media with various carbon sources. This analysis identified a TATA box and two upstream activating sequences as necessary elements for galactose-controlled GAL7 transcription. Two upstream activating sequences exhibiting 71% homology with each other were located 255 and 168 base pairs, respectively, upstream of the GAL7 transcription start point. Each sequence consists of 21 base pairs, displaying an approximate rotational symmetry with a core consensus sequence of GAA--GCT CT -{CCG. At least one of the two sequences is required for galactose induction and also for glucose repression of the GAL7'-lac'Z gene. Analysis with host regulatory mutants Agal4 and Agal80 suggests that these sequences are the site at which the GAL4 product exerts its action to activate the GAL7 gene. We also observed that a deletion lacking both upstreamn activation sequences allowed the gene fusion to be expressed in the absence of galactose at about 10% of the fully induced level of the intact fusion. This constitutive expression depended on the presence of the TATA box of GAL7 in cis but not on a functional GAL4 gene. The level of the uncontrolled expression was decreased by increasing the distance between the TATA box and the pBR322 sequence in the vector plasmid.

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“…Care was taken that all cultures were of similar cell density at the time of harvest. RNA was extracted by the procedure described previously (24). For the kinetic RNA experiments, the RNA was prepared as described previously (18).…”

Section: Methodsmentioning

confidence: 99%

New positive and negative regulators for general control of amino acid biosynthesis in Saccharomyces cerevisiae.

Greenberg¹,

Myers²,

Skvirsky³

et al. 1986

Mol. Cell. Biol.

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The biosynthesis of most amino acids in Saccharomyces cerevisiae is coregulated. Starvation for a single amino acid results in the derepression of amino acid biosynthetic enzymes in many unrelated pathways. This phenomenon, known as general control, is mediated by both positive (GCN) and negative (GCD) regulatory genes. In this paper we describe the identification and characterization of several new regulatory genes for this system, GCN6, GCN7, GCN8, GCN9, and GCDS. A mutation in the negative regulator GCDS was isolated on the basis of its suppression of a gcn2 mutation. The effect of gedS is a posttranscriptional increase in histidine biosynthetic enzyme activity. Suppressors of ged5 which are deficient in derepression were in turn isolated. Eight such mutations, defining four new positive regulatory genes (GCN6 through GCN9), were obtained. These mutations are recessive, confer sensitivity to multiple amino acid analogs, and result in decreased mRNA levels for genes under general control. The GCN6 and GCN7 gene products were shown to be positive regulators for transcription of the GCN4 gene, the most direct-acting positive regulator thus far identified. The interaction of GCN6 and GCN7 with GCN4 is fundamentally different from that of previously isolated GCN genes. It should also be noted that these gen selections gave a completely different nonoverlapping set of mutations from earlier selections which relied on analog sensitivity. Thus, we may have identified a new class of GCN genes which are functionally distinct from GCNI through GCNS.

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Chapter 5 Isolation, Characterization, and Translation of mRNA from Yeast

Cited by 51 publications

References 31 publications

The specific phosphorylation of a 40S ribosomal protein in growth‐arrested tetrahymena is induced by sodium

The specific phosphorylation of a 40S ribosomal protein in growth‐arrested tetrahymena is induced by sodium

Duplicate upstream activating sequences in the promoter region of the Saccharomyces cerevisiae GAL7 gene.

New positive and negative regulators for general control of amino acid biosynthesis in Saccharomyces cerevisiae.

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