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R J Planta

Publications and source records attributed to R J Planta.

At least 73 records · Page 4Linked to original sources
PubMed1992

A system to study transcription by yeast RNA polymerase I within the chromosomal context: functional analysis of the ribosomal DNA enhancer and the RBP1/REB1 binding sites.

We have developed a novel system to study transcription by yeast RNA polymerase I (Pol I) of mutated rDNA units within the chromosomal context. For this, complete rDNA units carrying specific oligonucleotide tags in both the 17S and 26S rRNA genes were integrated into the chromosomal rDNA locus. Using this novel system, we analysed the action of the rDNA enhancer in stimulating transcription within the chromosomal context. We found that the enhancer acts as a stimulatory element in both directions, mainly on its two most proximal rRNA operons. Deletion of the sequences between the enhancer and the Pol I promoter in the tagged, integrated unit indicated that this part of the intergenic spacer contains no other transcriptional regulatory elements for Pol I. We also applied the system to study the function of the rDNA binding protein RBP1/REB1. For this purpose, we analysed tagged units in which either one or both of the binding sites for this protein have been inactivated. We found that mutations of both binding sites strongly diminish the transcription of the adjacent operon. The protein is hypothesized to play a crucial role in keeping the chromosomal rDNA units in an optimal spatial configuration by anchoring consecutive enhancers and promoters to the nucle(ol)ar matrix.

Base Sequence↗
PubMed1992

Molecular cloning and physical analysis of an 8.2 kb segment of chromosome XI of Saccharomyces cerevisiae reveals five tightly linked genes.

The nucleotide sequence of 6472 base pairs of an 8.2 kb segment of Saccharomyces cerevisiae chromosome XI has been determined. The sequence contains a cluster of four long open reading frames (ORF) designated YKL2, YKL3, YKL4 and TGL1 in the same orientation, flanked at the 5'-end by a divergent incomplete ORF (YKL1). Transcription and Southern analysis of the four complete ORFs showed that all are expressed and are present in single copy on the haploid genome. The average codon adaptation index of the coding regions is approximately 0.2, suggesting that these genes are lowly expressed. The upstream regions of all four genes as well as the YKL1 ORF contain putative promoter elements previously found to be characteristic of nuclear genes encoding mitochondrial proteins. Significant sequence similarities were found between the YKL3 protein and Escherichia coli ribosomal protein S2 as well as between the TGL1 protein and triglyceride lipases from rat salivary gland and human gastric tissue. The 3'-end of the 6472 bp nucleotide sequence overlaps with the upstream region of the previously identified CTK1 gene, encoding the largest subunit of CTD kinase (Lee, J.M. and Greenleaf, A.L., 1991, Gene Expression 2, 149-167), thereby increasing the number of genes on the 8.2 kb fragment to at least five. The transcripts of these genes represent approximately 83% of the DNA fragment, making it one of the most highly transcribed regions of the yeast chromosome analysed to date.

Amino Acid Sequence↗
PubMed1992

Structural and putative regulatory sequences of Kluyveromyces ribosomal protein genes.

The transcription of the majority of the ribosomal protein (rp) genes of Saccharomyces cerevisiae is activated by cis-acting elements, designated RPG boxes, which specifically bind the multifunctional protein RAP1 in vitro. To investigate to what extent this global system of transcription regulation has been conserved, we have isolated a number of rp genes of the related yeast species Kluyveromyces lactis and Kluyveromyces marxianus, whose counterparts in Saccharomyces are controlled by RAP1. The coding regions of these genes showed a sequence similarity of about 90% when compared to their Saccharomyces counterparts. In contrast, little or no sequence similarity was found between the upstream regions and the intervening sequences of Kluyveromyces and Saccharomyces homologs. However, the occurrence and the position of the introns is conserved. The sequence data also show that the physical linkage that exists in S. cerevisiae between the rp genes encoding RP59 (CRY1), S24 and L46 is conserved in Kluyveromyces. Northern analysis demonstrated that each of the isolated Kluyveromyces genes is transcriptionally active. By sequence comparison we identified a number of conserved sequences in the upstream region of each of the Kluyveromyces rp genes, which we designated the X, Z and RPGK boxes. The last one is highly similar, though not identical, to the S. cerevisiae RPG box. Functional analysis of the intergenic region between the genes encoding Kluyveromyces ribosomal proteins S24 and L46 showed that the RPGK box (+Z box) functions as a transcriptional activator, while the X box acts as a transcriptional repressor. Band-shift assays confirmed the existence of a RAP1-like protein in Kluyveromyces that binds to the RPGK box but not to the S. cerevisiae RPG box. In contrast, S. cerevisiae RAP1 did recognize the RPGK box.

Amino Acid Sequence↗
PubMed1992

Structure and expression of the ABF1-regulated ribosomal protein S33 gene in Kluyveromyces.

The abundant multifunctional protein ABF1 of Saccharomyces cerevisiae binds to the upstream region of several genes, including some ribosomal protein genes like the one encoding protein S33. Deletion of the ABF1-binding sequence lowers the transcription of these genes three- to more than ten-fold. We have isolated the S33 genes of two related yeast species, Kluyveromyces lactis and Kluyveromyces marxianus. Comparison of the nucleotide sequences of these S33 genes with their counterpart from S. cerevisiae shows a strong sequence similarity covering the whole of the coding regions. In contrast, little or no sequence similarity is found in the 5'-flanking regions of the three genes. Also the trailer regions differ considerably in both length and sequence from one species to another. An ABF1-binding site is present in the upstream region of the S33 gene of K. marxianus. Retardation analyses showed that this sequence is able to bind a protein present in Kluyveromyces cells with a molecular mass somewhat lower than that of S. cerevisiae ABF1. Functional analyses, using a beta-glucuronidase reporter system, showed that the ABF1-binding site is indeed involved in transcription activation of the K. marxianus S33 gene in Kluyveromyces cells. A S. cerevisiae ABF1-gene-specific probe showed only weak hybridization with Kluyveromyces DNA and Northern blots did not show a signal. These results indicate that S. cerevisiae and Kluyveromyces contain functionally related but structurally dissimilar ABF1-type proteins.

Amino Acid Sequence↗
PubMed1992

Multiple-copy integration of the alpha-galactosidase gene from Cyamopsis tetragonoloba into the ribosomal DNA of Kluyveromyces lactis.

We have developed a vector system for high-copy-number integration into the ribosomal DNA of the yeast Kluyveromyces lactis. This system is analogous to the pMIRY-system developed for Saccharomyces cerevisiae. Plasmids containing a portion of K. lactis rDNA for targeted homologous recombination, as well as the S. cerevisiae TRP1 gene with various promoter deletions, were constructed and, after transformation to K. lactis, analyzed for both copy number and stability. These plasmids were found to be present in about 60 copies per cell and were stably maintained during growth under non-selective conditions. Using this vector system, we expressed a fusion construct containing the S. cerevisiae GAL7 promoter, the SUC2 (invertase) signal sequence and the gene coding for alpha-galactosidase from the plant Cyamopsis tetragonoloba. Although the maximum copy number of these integrated plasmids was only about 15, we nevertheless obtained a high level of alpha-galactosidase production (250 mg/l) with a secretion efficiency of about 95%. When compared to extrachromosomal K. lactis vectors containing the same fusion construct, the multicopy integrants showed a much higher alpha-galactosidase production level and a considerably higher stability under non-selective conditions.

DNA Transposable Elements↗
PubMed1992

Molecular cloning of a cDNA encoding a member of a novel cytochrome P450 family in the mollusc Lymnaea stagnalis.

We isolated a cDNA encoding a cytochrome P450 from the mollusc Lymnaea stagnalis. The mRNA is 2.1 nucleotides long and contains an open reading frame encoding a protein of 545 amino acids. A conserved home-binding domain, characteristic of cytochrome P450 proteins, is present in the deduced amino acid sequence. The Lymnaea cytochrome P450 protein shares less than 40% positional identity with any known member of the cytochrome P450 superfamily, and therefore, represents a separate family, which we propose to name CYP10. The CYP10 mRNA is shown to be uniquely and abundantly expressed in the female gonadotropic hormone producing dorsal bodies of L. stagnalis.

Amino Acid Sequence↗
PubMed1992

Osmostress-induced changes in yeast gene expression.

When Saccharomyces cerevisiae cells are exposed to high concentration of NaCl, they show reduced viability, methionine uptake and protein biosynthesis. Cells can acquire tolerance against a severe salt shock (up to 1.4 M NaCl) by a previous treatment with 0.7 M NaCl, but not by a previous heat shock. Two-dimensional analysis of [3H]-leucine-labelled proteins from salt-shocked cells (0.7 M NaCl) revealed the elevated rate of synthesis of nine proteins, among which were the heat-shock proteins hsp12 and hsp26. Northern analysis using gene-specific probes confirmed the identity of the latter proteins and, in addition, demonstrated the induction of glycerol-3-phosphate dehydrogenase gene expression. The synthesis of the same set of proteins is induced or enhanced upon exposure of cells to 0.8 M sucrose, although not as dramatically as in an iso-osmolar NaCl concentration (0.7 M).

Adaptation, Physiological↗
PubMed1992

Analysis of the promoter of the cytochrome P-450 2B2 gene in the rat.

About 3 kb of the promoter region of the gene encoding cytochrome P-450 2B2 (CYP2B2) in the rat were sequenced and searched for potential cis-acting elements. Apart from putative binding sites for (liver-specific) protein factors, a region showing homology with the LINE 1 retrotransposon element was also found. Three proximal promoter fragments, encompassing nucleotides -579 to -372, -372 to -211, and -211 to +1, respectively, were shown to contain binding sites for multiple protein factors by bandshift analyses. The strongest protein-binding element, designated BRE (basic regulatory element), occurs between -103 to -66. Its structure is very similar to a negative control element in the murine cmyc promoter and displays a composite feature having a tandemly repeated sequence homology with the BTE (basic transcription element; Yanagida et al., 1990) separated by a CCAAA-box. The use of a deletion series of this template in in vitro transcription assays, provided evidence that the BRE serves as a major cis-acting element in the (regulated) transcription activation of the CYP2B2 gene.

Animals↗
PubMed1991

The yeast RNA polymerase I promoter: ribosomal DNA sequences involved in transcription initiation and complex formation in vitro.

Using an in vitro transcription system for Saccharomyces cerevisiae RNA polymerase I, we have analyzed Pol I promoter deletion mutants and mapped the boundaries of the promoter between positions -155 and +27. The 5'-boundary of the minimal core promoter capable of transcription initiation, however, was found to lie between -38 and -26. The 3'-deletion extending to -2 and -5 still allowed some transcription, suggesting that the positioning of Pol I is directed by upstream sequences. The results of in vitro analysis of linker scanning mutants (LSMs) combined with the deletion analysis showed that the promoter consists of three domains: two essential core domains (I: -28 to +8 and II: -76 to -51) and a transcription modulating upstream domain (III: -146 to -91). These results are in general agreement with those obtained in vivo (1). Using a template competition assay we also analyzed these mutant promoters for their ability to form a stable preinitiation complex. We found that the ability of 5'-deletion mutants to sequester an essential factor(s) correlates with their transcriptional activity. In contrast, several 3'-deletions and some LSMs in domain I and II decrease transcription activity greatly without significantly decreasing competition ability. The results indicate that the stimulatory function of domain III is achieved through its interaction with an essential transcription factor(s), although the other domains also participate in this interaction, perhaps directly or through another protein factor.

Base Sequence↗
PubMed1991

Functional analysis of the promoter of the gene encoding the acidic ribosomal protein L45 in yeast.

The gene encoding the acidic ribosomal protein L45 in yeast is expressed coordinately with other rp-genes. The promoter region of this gene harbours binding sites for CP1 and ABF1. We demonstrate that the CP1-site is not involved in the transcription activation of the L45-gene. Rather, the ABF1-site, through deviating from the consensus sequence (RTARY3N3ACG), appears to be essential for efficient transcription. Replacement of this site by a consensus RAP1-binding site (an RPG box) did not alter the transcriptional yield of the L45-gene. An additional transcription activating region is present downstream of the ABF1-site. The relevant nucleotide sequence, which is repeated in the L45-gene promoter, gives rise to complex formation with a yeast protein extract in a bandshift assay. The results indicate that the L45-gene promoter has a complex architecture.

Base Sequence↗
PubMed1991

Mechanism of high-copy-number integration of pMIRY-type vectors into the ribosomal DNA of Saccharomyces cerevisiae.

Targeted integration of the yeast plasmid pMIRY2 into the ribosomal DNA (rDNA) of Saccharomyces cerevisiae by homologous recombination results in transformants carrying 100-200 copies of the plasmid per cell which are stably maintained over a large number of generations [Lopes et al., Gene 79 (1989) 199-206]. These properties make pMIRY2 an attractive vector for high-level production of (heterologous) proteins by yeast cells. We have investigated the mechanism underlying high-copy-number (hcn) integration of pMIRY-type plasmids and show that either targeting to a location outside the rDNA locus or use of the wild-type LEU2, instead of the deficient LEU2d gene, as selection marker reduces the copy number to the low value characteristic of standard integrating (YIp-type) yeast plasmids. Further experiments demonstrate that the hcn of pMIRY-type plasmids is achieved by amplification of a small number of copies initially integrated into the rDNA locus. Amplification depends upon the strong selection pressure created by the extremely low expression of the deficient LEU2d gene, but not on the presence of this gene per se. The hcn integration also occurs when either the TRP1 or URA3 gene is used as the selection marker, provided expression of the marker gene is severely curtailed, e.g., by removal of most of its 5'-flanking region.

DNA, Fungal↗
PubMed1991

The conserved GTPase center and variable region V9 from Saccharomyces cerevisiae 26S rRNA can be replaced by their equivalents from other prokaryotes or eukaryotes without detectable loss of ribosomal function.

Using the "tagged" rRNA gene system, which allows in vivo mutational analysis of Saccharomyces cerevisiae rRNA, we studied the role of two distinct structural elements of 26S rRNA in ribosome biogenesis and function--namely, the evolutionarily highly conserved "GTPase center" located in domain II and the eukaroyote-specific variable region V9 in domain III. Replacement of the S. cerevisiae GTPase center with its counterpart from Escherichia coli did not affect the assembly of the mutant 26S rRNA into functional (as judged by their polysomal distribution) 60S subunits, indicating that the E. coli GTPase center functions efficiently in the context of the heterologous rRNA. Removal of most of the S. cerevisiae V9 region or replacement of this segment by the equivalent segment from mouse 28S rRNA also did not affect the formation of functional 60S subunits carrying the mutant 26S rRNA. Therefore, the V9 region does not seem to play a role in the biological functioning of the yeast 60S subunits, and these subunits appear to be able to accommodate V9 regions of various size and secondary structure without apparent loss of function.

Escherichia coli↗
PubMed1991

Cloning and disruption of the LEU2 gene of Kluyveromyces marxianus CBS 6556.

The LEU2 gene, coding for beta-isopropylmalate dehydrogenase, of the yeast Kluyveromyces marxianus was isolated and sequenced. An open reading frame, coding for a protein with a molecular weight of 38 kDa was found. Comparison of the deduced amino acid sequence of the LEU2 gene with the corresponding enzymes of three other yeasts and two thermophilic bacteria, revealed extensive sequence similarities. The cloned gene could complement a leuB mutation of Escherichia coli and a leu2 mutation of Saccharomyces cerevisiae. Using orthogonal field alternation gel electrophoresis, the genomic copy of the gene was found to be located at chromosome VI or VII. Analysis of the 5'-untranslated region indicated the presence of a putative binding site for the LEU3 protein, which is involved in the leucine-specific regulation of transcription. We show that the cloned gene can be used for the construction of a non-reverting K. marxianus leu2 mutant.

3-Isopropylmalate Dehydrogenase↗
PubMed1991

Turnover rate of yeast PGK mRNA can be changed by specific alterations in its trailer structure.

The effect of insertions in the 5'- and 3'-untranslated regions (UTR) of the Saccharomyces cerevisiae mRNA encoding phosphoglycerate kinase (PGK) on the stability of the transcript in vivo was determined. None of the structural alterations in the 5'-UTR affected mRNA turnover significantly, despite the strong negative effect on translational efficiency of some of these alterations previously observed. We conclude that the structure of the 5'-UTR is not important for the relatively high affinity of PGK mRNA in yeast cells. Moreover, translation cannot be a major factor in determining the rate of turnover of this mRNA. Insertion of either a polyG or polyU, but not a polyA or polyC, tract into the 3'-UTR of PGK mRNA increased its half-life by a factor of about two. Introduction of a hairpin structure containing 18 G.C base pairs had only a slight stabilizing effect. We argue that the stabilization by the structural changes in the 3'-UTR is due to altered folding of the mutant mRNA which retards a rate-limiting endonucleolytic cleavage step in the normal turnover pathway of PGK mRNA. The stabilizing effect of local structural alterations in the 3'-UTR opens the possibility for further increasing the product yield of a (heterologous) gene cloned in yeast cells.

Base Sequence↗