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R J Planta

Publications and source records attributed to R J Planta.

At least 55 records · Page 3Linked to original sources
PubMed1994

A G-protein beta subunit that is expressed in the central nervous system of the mollusc Lymnaea stagnalis identified through cDNA cloning.

We have cloned cDNA encoding a G-protein beta subunit from the central nervous system (CNS) of the mollusc Lymnaea stagnalis. The deduced protein is very homologous to other metazoan beta subunits. Thus, the Lymnaea CNS can be used as a model system to study beta gamma subunits in their native setting since its large neurons can be manipulated and studied relatively easily in vivo.

Amino Acid Sequence↗
PubMed1994

A G protein-coupled receptor with low density lipoprotein-binding motifs suggests a role for lipoproteins in G-linked signal transduction.

We have isolated and analyzed a cDNA from the central nervous system of the mollusc Lymnaea stagnalis encoding a putative receptor, which might be a natural hybrid between two different classes of receptor proteins. Preceded by a signal peptide, two types of repeated sequences are present in the N-terminal part of the protein. The first repeat displays a high sequence similarity to the extracellular binding domains of the low density lipoprotein receptor, which binds and internalizes cholesterol-containing apolipoproteins. The second repeat and the C-terminal part of the Lymnaea receptor are very similar to regions of a specific class of guanine nucleotide-binding protein-coupled receptors, the mammalian glycoprotein hormone receptors. The mRNA encoding the receptor is predominantly expressed in a small number of neurons within the central nervous system and to a lesser extent in the heart.

Amino Acid Sequence↗
PubMed1994

Separate structural elements within internal transcribed spacer 1 of Saccharomyces cerevisiae precursor ribosomal RNA direct the formation of 17S and 26S rRNA.

Structural features of Internal Transcribed Spacer 1 (ITS1) that direct its removal from Saccharomyces cerevisiae pre-rRNA during processing were identified by an initial phylogenetic approach followed by in vivo mutational analysis of specific structural elements. We found that S. cerevisiae ITS1 can functionally be replaced by the corresponding regions from the yeasts Torulaspora delbrueckii, Kluyveromyces lactis and Hansenula wingei, indicating that structural elements required in cis for processing are evolutionarily conserved. Despite large differences in size, all ITS1 regions conform to the secondary structure proposed by Yeh et al. [Biochemistry 29 (1990) 5911-5918], showing five domains (I-V; 5'-->3') of which three harbour an evolutionarily highly conserved element. Removal of most of domain II, including its highly conserved element, did not affect processing. In contrast, highly conserved nucleotides directly downstream of processing site A2 in domain III play a major role in production of 17S, but not 26S rRNA. Domain IV and V are dispensable for 17S rRNA formation although an alternative, albeit inefficient, processing route to mature 17S rRNA may be mediated by a conserved region in domain IV. Each of these two domains is individually sufficient for efficient production of 26S rRNA, suggesting two independent processing pathways. We conclude that ITS1 is organized into two functionally and structurally distinct halves.

Base Sequence↗
PubMed1994

Transcription regulation of ribosomal protein genes at different growth rates in continuous cultures of Kluyveromyces yeasts.

We have investigated the relationship between the growth rate of two Kluyveromyces strains that differ in their maximum growth rate, namely K. lactis (mumax = 0.5 h-1) and K. marxianus (mumax = 1.1 h-1), and the transcription rate of ribosomal protein (rp) genes in these strains. The growth rate of either strain was varied by culturing the cells in a chemostat under conditions of glucose limitation at different dilution rates. Although the steady-state levels of transcription of the rp-genes of both Kluyveromyces strains were tightly coupled to the cellular growth rate, no clear relationship between the level of rp-gene transcription and the amount of in vitro binding of the RAP1- and ABF1-like proteins to the promoters of these rp-genes was observed. Upon a sudden increase in the growth rate of a steady-state culture, the transcription of rp-genes of K. lactis showed a different response from that in K. marxianus. Whereas a substantial overexpression of the K. lactis rp-genes was found during at least 4-5 h, the level of expression of the K. marxianus rp-genes was almost immediately adjusted to the new growth rate.

Blotting, Northern↗
PubMed1994

Specific identification of Candida albicans by hybridization with oligonucleotides derived from ribosomal DNA internal spacers.

In order to develop DNA probes for rapid, sensitive and specific detection of the pathogenic yeast species Candida albicans, we carried out comparative sequence analysis of the two internal transcribed spacer regions (ITS1 and ITS2) of the ribosomal DNA (rDNA) units of C. albicans and the closely related pathogenic species C. tropicalis. While overall sequence similarity between the two species was considerable (65-75%), both ITS1 and ITS2 were found to contain distinct regions with sufficient sequence divergence to make them suitable as specific target sites for the identification of C. albicans. On the basis of these results one ITS1-derived (ANAB1) and two ITS2-derived (ANAB2 and ANAB3) oligonucleotides were selected, chemically synthesized, and used as hybridization probes. Their specificity and reliability were evaluated in dot-blot hybridization experiments with total genomic DNA from 13 strains of medically important Candida species, six strains of other yeast genera associated with man and animals, and ten strains previously identified as C. albicans by phenotypic criteria. Under well-defined hybridization conditions the three probes hybridized exclusively with DNA derived from strains belonging to the species C. albicans, thus demonstrating their potential clinical usefulness. The failure of four of the (presumed) C. albicans strains to show hybridization to the ITS probes sheds doubt upon their taxonomic classification, which is reinforced by other phenotypic aspects of these strains.

Base Sequence↗
PubMed1993

Cytoplasmic dynein is required for normal nuclear segregation in yeast.

We have identified the gene DYN1, which encodes the heavy chain of cytoplasmic dynein in the yeast Saccharomyces cerevisiae. The predicted amino acid sequence (M(r) 471,305) reveals the presence of four P-loop motifs, as in all dyneins known so far, and has 28% overall identity to the dynein heavy chain of Dictyostelium [Koonce, M. P., Grissom, P. M. & McIntosh, J. R. (1992) J. Cell Biol. 119, 1597-1604] with 40% identity in the putative motor domain. Disruption of DYN1 causes misalignment of the spindle relative to the bud neck during cell division and results in abnormal distribution of the dividing nuclei between the mother cell and the bud. Cytoplasmic dynein, by generating force along cytoplasmic microtubules, may play an important role in the proper alignment of the mitotic spindle in yeast.

Amino Acid Sequence↗
PubMed1993

Transcriptional regulation of the gene encoding cholesterol 7 alpha-hydroxylase in the rat.

The cytochrome P450 enzyme, cholesterol 7 alpha-hydroxylase (CYP7A), catalyses the first and rate-limiting step in the conversion of cholesterol to bile acids. Expression of the CYP7A gene is under complex physiological control, encompassing amongst others a feedback down-regulation by bile acids. Using the CYP7A cDNA of the rat as a probe, we isolated a rat genomic clone containing the 5' part of the gene, including approximately 3.6 kb of upstream sequences. Sequence analysis revealed the presence of several putative regulatory elements. Transient expression analyses of transfected primary hepatocytes demonstrated that the major transcription-activating region is located in the proximal 145 nucleotide (nt). Upon addition of taurocholate to the culture, a significant reduction of the transcriptional activity was observed, suggesting the presence of a bile acid-responsive element in the proximal region of the CYP7A promoter. In addition, evidence was obtained for the presence of a thyroxine-responsive site further upstream. After addition of taurocholate, steady-state CYP7A mRNA levels, as judged by Northern analysis of hepatocyte RNA, are eightfold reduced. On the other hand, the transcriptional activity of CYP7A, as shown both in CAT assays and run-on experiments, revealed only a threefold decrease. These experiments suggest that both transcriptional control and regulation of CYP7A mRNA stability play an important part in the feedback regulation of CYP7A activity in the rat.

Animals↗
PubMed1993

Structure of the ABF1-homologue from Kluyveromyces marxianus.

By transformation of a Saccharomyces cerevisiae mutant strain conditionally expressing the ABF1-gene, a Kluyveromyces marxianus DNA fragment carrying the gene encoding the ABF1-homologue of this yeast strain (KmABF1) was selected. Comparison of the sequence of the KmABF1 gene with that encoding Saccharomyces cerevisiae ABF1 and the previously isolated ABF1-gene from Kluyveromyces lactis (KlABF1) revealed distinct regions displaying considerable homology and therefore most likely representing sequences encoding essential domains. In addition to the domains putatively involved in DNA binding of the protein factor, two short conserved amino acid sequence elements at the C-termini of the homologous proteins were identified, which are proposed to play a part in their trans-acting functions. This is the first report on the structure of a regulatory protein factor from K. marxianus.

Amino Acid Sequence↗
PubMed1993

Gastric chief cell-specific transcription of the pepsinogen A gene.

The molecular mechanisms underlying the regulation of pepsinogen A (PGA) gene expression in mammalian cells are poorly understood. In this paper we describe the structural and functional analysis of the pepsinogen A gene promoter in the pig. By genomic Southern analyses we demonstrate that, in contrast with human PGA genes which are amplified and organized in haplotypes, only a single PGA gene is present per haploid porcine genome. With the aim of identifying promoter elements mediating the gastric mucosa cell-specific transcription of the PGA gene in pig, we isolated a PGA gene from a porcine genomic library. The nucleotide sequence of the first exon and 1.7 kb of the upstream DNA region were determined and compared with the corresponding regions of the human PGA gene encoding isozymogen Pg5. In order to study the promoter activity of the PGA gene a functional assay was developed: we succeeded in obtaining primary monolayer cultures of porcine gastric mucosal chief cells, suitable for transfection. Fragments of 5'-flanking and noncoding first exon sequences of the porcine and human PGA genes were linked to the chloramphenicol acetyltransferase (CAT) gene. The transcriptional activity of these hybrid genes was assessed in transient expression assays upon transfection (lipofection) of gastric and nongastric cells. Whereas PGA 5'-flanking sequences showed no promoter activity in nongastric cell types, the DNA region from -205 to +21 was found to be sufficient to direct expression of the porcine PGA constructs in a cell-specific manner. Further deletion analysis of the proximal promoter fragment identified several regions (-205 to -167, -127 to -67 and +2 to +21) acting synergistically in the transcriptional regulation of the PGA gene. In contrast, all human PGA-CAT constructs used failed to show promoter activity in porcine gastric chief cells, indicating species-specific control of PGA gene expression. In addition, the transcriptional activity of the porcine PGA promoter in chief cells from pig was completely abolished by in vitro CpG methylation. Footprint analyses of the proximal promoter fragment using nuclear extracts from either porcine gastric mucosal chief cells or liver revealed some notable differences between both extracts, which might reflect the interaction with (a) cell-specific factor(s).

Animals↗
PubMed1993

Known heat-shock proteins are not responsible for stress-induced rapid degradation of ribosomal protein mRNAs in yeast.

We have previously shown that the heat-induced enhanced decay of yeast mRNAs encoding ribosomal proteins (rp-mRNAs) requires ongoing transcription during the heat treatment [Herruer et al. (1988) Nucl. Acids Res. 16, 7917]. In order to determine whether this requirement reflects the need for heat-shock protein (hsp), we analysed the effect of heat shock on rp-mRNA levels in several yeast strains in which each of the heat-shock genes encoding hsp26, hsp35 or hsp83 had been individually disrupted. In all three strains we still observed increased degradation of rp-mRNAs immediately after the temperature shift, demonstrating that hsp26, hsp35 and hsp83 are not required for this effect. Accelerated turnover of rp-mRNA was also found to occur upon raising the growth temperature of a mutant strain that contains a disruption of the gene specifying the heat-shock transcription factor and in wild-type yeast cells treated with canavanine, an arginine analogue that will be incorporated into all known hsps and that is known to cause misfolding of the polypeptide chain. Latter observation suggests that enhanced rp-mRNA decay is a more general stress-related phenomenon. Taken together, these data strongly indicate that the trans-acting factor required for the increase in the rate of degradation of rp-mRNAs upon stress is not one of the known yeast hsps.

Canavanine↗
PubMed1993

Cloning and sequencing of the URA3 gene of Kluyveromyces marxianus CBS 6556.

The URA3 gene, coding for orotidine-5'-phosphate decarboxylase, from Kluyveromyces marxianus CBS 6556, was isolated from a genomic DNA library. The K. marxianus URA3 gene encodes a protein of 267 amino acids with a calculated molecular weight of 29.3 kDa. Comparison of the K. marxianus protein with the corresponding enzymes of Saccharomyces cerevisiae and Kluyveromyces lactis showed amino acid sequence identities of 81% and 88%, respectively. Using contour-clamped homogenous electric field gel electrophoresis, the genomic copy was found to be located on chromosome VI. We have used the cloned gene for the construction of a K. marxianus leu2 mutant. This mutant contains no heterologous sequences, which is essential to make it acceptable for application in the food industry.

Amino Acid Sequence↗
PubMed1993

The RHO4a and NUD1 genes on Saccharomyces cerevisiae chromosome XI.

Sequence analysis of a 4 kb fragment from the right arm of Saccharomyces cerevisiae chromosome XI, in combination with Northern hybridization experiments revealed the presence of two genes, designated RHO4a and NUD1. The first gene encodes a 32 kDa protein showing significant sequence similarity with members of the ras family. Its 3'-terminal sequence is virtually identical to a sequence published previously as the RHO4 gene [Matsui and Toh-e, Gene 114 (1992), 43-49], which, however, appears to start at an internal ATG codon. The RHO4a sequence also overlaps the 5'-terminal sequence of the RNC1 gene [Chow et al., Nucl. Acids Res. 20 (1992), 5215-5221] proposed to encode the yeast yNucR endo/exonuclease. The remainder of this RNC1 gene overlaps with the 5'-end of the NUD1 gene. However, the RNC1 sequence lacks a portion of 276 bp that in our fragment is part of the intergenic region separating RHO4a and NUD1. From these results we conclude that the proposed RNC1 gene is the result of a cloning artefact and that the yNucR protein is instead encoded by the NUD1 gene.

Amino Acid Sequence↗
PubMed1993

Expression of an alpha-galactosidase gene under control of the homologous inulinase promoter in Kluyveromyces marxianus.

For expression of the alpha-galactosidase gene from Cyamopsis tetragonoloba in Kluyveromyces marxianus CBS 6556 we have used the promoter of the homologous inulinase-encoding gene (INU1). The INU1 gene has been cloned and sequenced and the coding region shows an identity of 59% with the Saccharomyces cerevisiae invertase gene (SUC2). In the 5'-flanking region of INU1 we found a sequence (TAAATCCGGGG) that perfectly matches to the MIG1 binding consensus sequence (WWWWTSYGGGG) of the S. cerevisiae GAL1, GAL4 and SUC2 genes. Using the K. marxianus INU1 promoter and prepro-signal sequence, we obtained a high alpha-galactosidase production level (153 mg/l) and a secretion efficiency of 99%. Both the production level and the secretion efficiency were significantly reduced when the INU1 pro-peptide was deleted. With either the S. cerevisiae PGK or GAL7 promoter we could obtain only low alpha-galactosidase production levels (2 mg/l).

Amino Acid Sequence↗
PubMed1993

Growth-related expression of ribosomal protein genes in Saccharomyces cerevisiae.

The rate of ribosomal protein gene (rp-gene) transcription in yeast is accurately adjusted to the cellular requirement for ribosomes under various growth conditions. However, the molecular mechanisms underlying this co-ordinated transcriptional control have not yet been elucidated. Transcriptional activation of rp-genes is mediated through two different multifunctional transacting factors, ABF1 and RAP1. In this report, we demonstrate that changes in cellular rp-mRNA levels during varying growth conditions are not parallelled by changes in the in vitro binding capacity of ABF1 or RAP1 for their cognate sequences. In addition, the nutritional upshift response of rp-genes observed after addition of glucose to a culture growing on a non-fermentative carbon source turns out not to be the result of increased expression of the ABF1 and RAP1 genes or of elevated DNA-binding activity of these factors. Therefore, growth rate-dependent transcription regulation of rp-genes is most probably not mediated by changes in the efficiency of binding of ABF1 and RAP1 to the upstream activation sites of these genes, but rather through other alterations in the efficiency of transcription activation. Furthermore, we tested the possibility that cAMP may play a role in elevating rp-gene expression during a nutritional shift-up. We found that the nutritional upshift response occurs normally in several mutants defective in cAMP metabolism.

Base Sequence↗
PubMed1992

Molecular cloning of G protein alpha subunits from the central nervous system of the mollusc Lymnaea stagnalis.

The central nervous system of the pond snail, Lymnaea stagnalis, contains many large, identified neurons which can be easily manipulated making it an advantageous model system to elucidate in vivo the architecture of neuronal signal transduction pathways. We have isolated three cDNA clones encoding G protein alpha subunits that are expressed in the Lymnaea CNS, i.e. G alpha o, G alpha s and G alpha i. The deduced proteins exhibit a very high degree of sequence identity to their vertebrate and invertebrate counterparts. The strong conservation of G protein alpha subunits suggests that functional insights into G protein-mediated signalling routes obtained through the experimental amenability of the Lymnaea CNS will have relevance for similar pathways in the mammalian brain.

Amino Acid Sequence↗
PubMed1992

Kluyveromyces contains a functional ABF1-homologue.

ABF1 is a multifunctional protein present in Saccharomyces cerevisiae, involved in transcription-activation and -repression as well as in DNA-replication. Several lines of evidence indicate the occurrence in the related species Kluyveromyces lactis of a protein having similar properties to those of ABF1 in S. cerevisiae. In order to identify conserved functional domains in ABF1, we have cloned and sequenced the gene encoding the ABF1-homologue from K. lactis. KIABF1 is much smaller than ScABF1 (54.6 vs. 81.7 kD). It exhibits extensive homology with its S. cerevisiae counterpart in the N-terminal region. The C-terminal domain however, is divergent, with the striking exception of a stretch of 20 amino acids, which is virtually identical in the two proteins. KIABF1 can substitute ABF1 in S. cerevisiae, emphasizing the conservation of the multiple functions of this protein.

Amino Acid Sequence↗
PubMed1992

Functional analysis of internal transcribed spacer 2 of Saccharomyces cerevisiae ribosomal DNA.

Using the previously described "tagged ribosome" (pORCS) system for in vivo mutational analysis of yeast rDNA, we show that small deletions in the 5'-terminal portion of ITS2 completely block maturation of 26 S rRNA at the level of the 29 SB precursor (5.8 S rRNA-ITS2-26 S rRNA). Various deletions in the 3'-terminal part, although severely reducing the efficiency of processing, still allow some mature 26 S rRNA to be formed. On the other hand, none of the ITS2 deletions affect the production of mature 17 S rRNA. Since all of the deletions severely disturb the recently proposed secondary structure of ITS2, these findings suggest an important role for higher order structure of ITS2 in processing. Analysis of the effect of complete or partial replacement of S. cerevisiae ITS2 with its counterpart sequences from Saccharomyces rosei or Hansenula wingei, points to helix V of the secondary structure model as an important element for correct and efficient processing. Direct mutational analysis shows that disruption of base-pairing in the middle of helix V does not detectably affect 26 S rRNA formation. In contrast, introduction of clustered point mutations at the apical end of helix V that both disrupt base-pairing and change the sequence of the loop, severely reduces processing. Since a mutant containing only point mutations in the sequence of the loop produces normal amounts of mature 26 S rRNA, we conclude that the precise (secondary and/or primary) structure at the lower end of helix V, but excluding the loop, is of crucial importance for efficient removal of ITS2.

Base Sequence↗