Isolation of cloned DNA sequences containing ribosomal protein genes from Saccharomyces cerevisiae.

Yeast mRNA enriched for ribosomal protein mRNA was obtained by isolating poly(A)+ small mRNA from small polysomes. A comparison of cell-free translation of this small mRNA and total mRNA, and electrophoresis of the products on two-dimensional gels which resolve most yeast ribosomal proteins, demonstrated that a 5-10 fold enrichment for ribosomal protein mRNA was obtained. One hundred different recombinant DNA molecules possibly containing ribosomal protein genes were selected by differential colony hybridization of this enriched mRNA and unfractionated mRNA to a bank of yeast pMB9 hybrid plasmids. After screening twenty-five of these candidates, five different clones were found which contain yeast ribosomal protein gene sequences. The yeast mRNAs complementary to these five plasmids code for 35S-methionine-labeled polypeptides which co-migrate on two-dimensional gels with yeast ribosomal proteins. Consistent with previous studies on ribosomal protein mRNAs, the amounts of mRNA complementary to three of these cloned genes are controlled by the RNA2 locus. Although two of the five clones contain more than one yeast gene, none contain more than one identifiable ribosomal protein gene. Thus there is no evidence for "tight" linkage of yeast ribosomal protein genes. Two of the cloned ribosomal protein genes are single-copy genes, whereas two other cloned sequences contain two different copies of the same ribosomal protein gene. The fifth plasmid contains sequences which are repeated in the yeast genome, but it is not known whether any or all of the ribosomal protein gene on this clone contains repetitive DNA.     

Newly identified genes contribute to vanillin tolerance in Saccharomyces cerevisiae

Exploring the mechanisms of tolerance in microorganisms to vanillin, which is derived from lignin, will benefit the design of robust cell factories that produce biofuels and chemicals using lignocellulosic materials. Our objective was to identify the genes related to vanillin tolerance in Saccharomyces cerevisiae. We investigated the effects on vanillin tolerance of several genes that have site mutations in the highly vanillin-tolerant strain EMV-8 compared to its parental line NAN-27. The results showed that overexpression of GCY1, a gene that encodes an aldo-keto reductase that also has mRNA-binding activity, YPR1, a paralog of GCY1 that encodes an aldo-keto reductase, PEX5, a gene that encodes a peroxisomal membrane signal receptor and MBF1, a gene that encodes a multiprotein bridging factor increase the specific growth rates (μ) by 49%, 41%, 44% and 48 %, respectively, in medium containing 6 mmol l⁻¹ vanillin. Among these gene products, Gcy1p and Ypr1p showed NADPH-dependent and NAD(P)H-dependent vanillin reductase activity, respectively. The reductase-inactive mutant Gcy1p^Y56F also increased vanillin tolerance in S. cerevisiae, suggesting that other mechanisms exist. Although TRS85 and PEX5, genes for which the mRNAs are binding targets of Gcy1p, were shown to be related to vanillin tolerance, both the mRNA and protein levels of these genes were not changed by overexpression of GCY1. The relationship between the mRNA-binding activity of Gcy1p and its positive effect on vanillin tolerance is still not clear. Finally, we found that the point mutation D112A in Mbf1p, which disrupts the binding of Mbf1p and the TATA element-binding protein (TBP), did not decrease the positive effect of Mbf1p on vanillin tolerance. This indicates that the binding of Mbf1p and TBP is not necessary for the positive effect on vanillin tolerance mediated by Mbf1p. We have successfully identified new genes related to vanillin tolerance and provided novel targets that can be used to improve the vanillin tolerance of S. cerevisiae. Moreover, we have extended our understanding of the proteins encoded by these genes.     

Functional analysis of a duplicated linked pair of ribosomal protein genes in Saccharomyces cerevisiae.

Ribosomal protein genes RP28 and S16A (RP55) are closely linked. Another set of this pair of genes exists in the genome (copy 2), genetically unlinked to copy 1. By using gene replacement techniques, we have shown that RP28 from copy 1 is required for vegetative growth and that the cells need S16A from copy 2 to achieve maximum growth rate.     

An acidic protein associated to ribosomes of Saccharomyces cerevisiae changes during cell cycle.

A large proportion of protein Ax, one of the three acidic proteins detected in S.cervisiae is found in the cell cytoplasm. The fraction of Ax associated with the ribosomes is partially released by treatment of these particles in conditions that remove the bound initiation factors but a residual amount remains tightly bound to the ribosomes.Protein Ax is associated with the large ribosomal subunit and the amount of protein in the particles duplicates as the celle enters the stationary phase.The amino acid composition of protein Ax is similar to those of other acidic proteins from the ribosomes of yeast and other species being very rich in alanine (21.3%), glycine (12.2%), aspartic acid (14.7%) and glumatic acid (14.3%).     

Acidic proteins from Saccharomyces cerevisiae ribosomes.

Two dimensional gel electrophoresis of ribosomal proteins from $\text{Saccharomyces}\text{cerevisiae}$ reveals the presence of three spots in the region corresponding to proteins of high acidic character. Washing the ribosomes with 0.4 M NH₄Cl and 50% ethanol, followed by chromatography of the extracted proteins on DEAE-cellulose, indicated the presence of two fractions of acidic proteins; (A and Ax), having very similar molecular weights (12.000–13.000), but phosphorylated to different extents. Fractions A and Ax are immunologically distinct and their immunologic properties differ from acidic proteins found in $\text{Escherichia}\text{coli}$, rat liver, and $\text{Artemia}\text{salina}$ ribosomes.Protein A can be resolved into two bands by electrofocusing, and two dimensional gel electrophoresis. The two components correspond to proteins L44 and L45 according to the standard nomenclature. Proteins Ax seems to correspond to the spot that moves above and to the left of L44 and L45 and is at least three times more phosphorylated than these two proteins.     

Disruption of single-copy genes encoding acidic ribosomal proteins in Saccharomyces cerevisiae.

Using the cloned genes coding for the ribosomal acidic proteins L44 and L45, constructions were made which deleted part of the coding sequence and inserted a DNA fragment at that site carrying either the URA3 or HIS3 gene. By gene disruption techniques with linearized DNA from these constructions, strains of Saccharomyces cerevisiae were obtained which lacked a functional gene for either protein L44 or protein L45. The disrupted genes in the transformants were characterized by Southern blots. The absence of the proteins was verified by electrofocusing and immunological techniques, but a compensating increase of the other acidic ribosomal proteins was not detected. The mutant lacking L44 grew at a rate identical to the parental strain in complex as well as in minimal medium. The L45-disrupted strain also grew well in both media but at a slower rate than the parental culture. A diploid strain was obtained by crossing both transformants, and by tetrad analysis it was shown that the double transformant lacking both genes is not viable. These results indicated that proteins L44 and L45 are independently dispensable for cell growth and that the ribosome is functional in the absence of either of them.     

Cloning and characterization of nuclear genes for two mitochondrial ribosomal proteins in Saccharomyces cerevisiae.

The genes for two large subunit proteins, YmL8 and YmL20, of the mitochondrial ribosome of Saccharomyces cerevisiae were cloned by hybridization with synthetic oligonucleotide mixtures corresponding to their N-terminal amino acid sequences. They were termed MRP-L8 and MRP-L20, respectively, and their nucleotide sequences were determined using a DNA sequencer. The MRP-L8 gene was found to encode a 26.8-kDa protein whose deduced amino acid sequence has a high degree of similarity to ribosomal protein L17 of Escherichia coli. The gene MRP-L20 was found to encode a 22.3-kDa protein with a presequence consisting of 18 amino acid residues. By Southern blot hybridization to the yeast chromosomes separated by field-inversion gel electrophoresis, the MRP-L8 and MRP-L20 genes were located on chromosomes X and XI, respectively. Gene disruption experiments indicate that their products, YmL8 and YmL20 proteins, are essential for the mitochondrial function and the absence of these proteins causes instability of the mitochondrial DNA.     

Ribosomal protein L2 in Saccharomyces cerevisiae is homologous to ribosomal protein L1 in Xenopus laevis. Isolation and characterization of the genes

By cross-hybridization with a cDNA probe for the Xenopus laevis ribosomal protein L1 we have been able to isolate the homologous genes from a Saccharomyces cerevisiae genomic library. We have shown that these genes code for a ribosomal protein which was previously named L2. In yeast, like in X. laevis, these genes are present in two copies per haploid genome and, unlike the vertebrate counterpart, they do not contain introns. Amino acid comparison of the X. laevis L1 and S. cerevisiae L2 proteins has shown the presence of a highly conserved protein domain embedded in very divergent sequences. Although these sequences are very poorly homologous, they confer an overall secondary structure and folding highly conserved in the two species.     

Tandemly arranged variant 5S ribosomal RNA genes in the yeast Saccharomyces cerevisiae.

Most of the ribosomal RNA genes of the yeast Saccharomyces cerevisiae are about 9 kilobases (kb) in size and encode both the 35S rRNA (processed to produce the 25S, 18S, and 5.8S species) and 5S rRNA. These genes are arranged in a single tandem array of 100 repeats. Below, we present evidence that at the centromere-distal end of this array is a tandem arrangement of a different type of rRNA gene. Each of these repeats is 3.6 kb in length and encodes a single 5S rRNA. The coding sequence of this gene is different from that of the "normal" 5S gene in three positions located at the 3' end of the gene.