Search PubMed⌕ Search

Biomedical subjects

R J Planta

Publications and source records attributed to R J Planta.

At least 109 records · Page 6Linked to original sources
PubMed1988

Mild temperature shock affects transcription of yeast ribosomal protein genes as well as the stability of their mRNAs.

Shifting the temperature of a yeast culture from 23 degrees to 36 degrees C results in a sudden and severe (greater than 85%) decline in the cellular levels of ribosomal protein (rp-)mRNAs. Recovery during continued growth at 36 degrees C occurs within 1 h. The use of hybrid genes carrying different portions of the region upstream of the gene coding for ribosomal protein L25 revealed that this characteristic, coordinate temperature shock phenomenon does not depend on the presence of specific upstream DNA sequences. Analysis of a heterologous gene carrying a synthetic UASrpg (upstream activation site of yeast ribosomal protein genes) provided conclusive evidence that the rp-characteristic, transient heat shock response is not mediated through the UASrpg elements. The addition of the transcription inhibitor 1,10-phenantroline prior to a 23 degrees to 36 degrees C heat shock inhibited the severe decline of the rp-mRNA levels. The latter observation indicates that transcription is required for the rp-gene- specific response to heat shock. A milder temperature shift, from 23 degrees to 30 degrees C, gave rise to a two-fold decrease in mRNA levels for all genes studied, both ribosomal and non-ribosomal. Together, these results indicate that a temperature shift causes a temporary general transcriptional arrest in yeast cells, resulting in an over-all decrease in mRNA levels. In addition, an enhanced nucleolytic break-down of pre-existing rp-mRNAs accounts for the dramatic drop in the steady state amounts of these mRNAs observed upon a 23 degrees----36 degrees C shift. This enhanced breakdown is caused directly or indirectly by a factor whose synthesis is induced by the heat shock treatment.

DNA, Fungal↗
PubMed1988

Cloning and sequencing of rhesus monkey pepsinogen A cDNA.

The complete nucleotide sequence of Rhesus monkey (Macaca mulatta) pepsinogen A (PGA) cDNA was determined from two partially overlapping cDNA clones, covering the whole coding sequence and part of the flanking sequences. The nucleotide and deduced amino acid sequences were compared to known PGA sequences from other species. The degree of similarity with human PGA appeared to be 96% at the nucleotide sequence level and 94% at the amino acid sequence level. In the coding region the divergence was highest in the activation peptide. The amino acid sequence similarity between Japanese monkey (Macaca fuscata) PGA and Rhesus monkey PGA was shown to be 99%. Using the cDNA as probe in Southern hybridization of EcoRI-digested human and Rhesus monkey genomic DNAs, PGA patterns with inter-individual differences were observed. The hybridization patterns are compatible with the existence of a PGA multigene family in both species.

Amino Acid Sequence↗
PubMed1988

Genomic structure and evolution of the human pepsinogen A multigene family.

A human cosmid library was screened with a pepsinogen A (PGA) cDNA probe, yielding 18 clones with (parts of) one, two or three PGA genes. By aligning these cosmids a restriction map of a PGA gene quadruplet was obtained in which the four genes are arranged in a highly ordered fashion in a head-to-tail orientation. Using the length in kilobases of the large polymorphic EcoRI fragment of the PGA genes, this quadruplet can be described as 15.0-12.0-12.0-16.6. An AvaII polymorphism allowed us to identify the two PGA haplotypes of the individual whose DNA had been cloned in the cosmid library to be a gene triplet and a gene quadruplet. By comparing the restriction maps of the central 12.0 genes in these multiplets to those of the flanking 15.0 and 16.6 genes, we postulate that these central genes arose from unequal but homologous crossing over between two 15.0-16.6 gene pairs. This hypothesis provides for the creation of a variety of haplotypes by additional cross overs and mutations. Southern blots of family and population material supports the existance of at least five common PGA haplotypes, including a single-gene haplotype, giving rise to a large number of different EcoRI patterns. The single PGA gene is probably the reciprocal crossing over product. Comparison between the DNA and protein polymorphisms suggests further micro-heterogeneity in the different PGA haplotypes.

Biological Evolution↗
PubMed1987

Transcriptional control of yeast ribosomal protein synthesis during carbon-source upshift.

Shifting a yeast culture from an ethanol-based medium to a glucose-based medium causes a coordinate increase of the cellular levels of ribosomal protein mRNAs by about a factor 4 within 30 min. Making use of hybrid genes encompassing different portions of the 5'-flanking region of the L25-gene, we could show that the increase in mRNAs is a transcriptional event, mediated through DNA sequences upstream of the ribosomal protein (rp) genes. Further analysis revealed that sequence elements are involved that many rp-genes have in common and that previously were identified as transcription activation sites (RPG-boxes or UASrpg). Using appropriate deletion mutants of the fusion genes we could demonstrate that a single RPG-box is sufficient for the transcriptional upshift. In addition, both copy genes encoding rp28 which differ considerably in their extent of transcriptional activity, show the upshift effect in a proportional manner. Definite proof for the role of the UASrpg in nutritional regulation was obtained by examining the effect of a synthetic RPG-box on transcription.

Ethanol↗
PubMed1987

Analysis of upstream activation sites of yeast ribosomal protein genes.

Transcription of the gene encoding yeast ribosomal protein L25 was previously shown to be activated through tandemly arranged upstream sequence elements that most rp-genes in yeast have in common. A single copy of such a conserved element is now demonstrated to restore transcription of an inactivated heterologous gene, which confirms its role as a genuine UAS: UASrpg. Though a single box is sufficient to activate transcription, most rp-genes harbor two neighbouring elements. Northern analysis of mutants of the L25 upstream region lacking either the gene-distal (RPG1) or the gene-proximal (RPG2) box provided evidence that RPG2 is significantly more effective than RPG1 in vivo. Moreover the sum of the effects of the individual boxes as measured separately is significantly lower than their joint effect, supporting cooperative interaction between the two boxes in vivo. Making use of oligomer-insertion experiments several additional features of the UASrpg were elucidated. First of all we confirmed that the extent of transcription activation by the UASrpg depends upon the orientation of the element. Secondly we show that a certain minimal distance (greater than 100 n) between UASrpg and the transcription initiation site is required for transcription activation. Finally, internal deletion of the L25-upstream region as well as oligomer-insertion shed some light on the nucleotide requirements of the UASrpg.

Base Sequence↗
PubMed1987

Ribosomal proteins EL11 from Escherichia coli and L15 from Saccharomyces cerevisiae bind to the same site in both yeast 26 S and mouse 28 S rRNA.

The heterologous interaction of Escherichia coli ribosomal protein EL11 with yeast 26 S and mouse 28 S rRNA was studied by analysing the ability of this protein to form a specific complex with various synthetic rRNA fragments that span the structural equivalent of the EL11 binding site present in these eukaryotic rRNAs. The fragments were obtained by SP6 polymerase-directed in-vitro run-off transcription of parts of the yeast or mouse large rRNA gene cloned behind the SP6 promoter. EL11 was found to protect an oligonucleotide fragment of 63 nucleotides from both the yeast and mouse transcripts against digestion by RNase T1. In both cases, the position of this fragment in the L-rRNA sequence coincides almost exactly with that of the fragment previously found to be protected by EL11 in E. coli 23 S rRNA. Moreover, the protected yeast fragment was shown to be able to re-bind to EL11 by a nitrocellulose filter binding assay. A ribosomal protein preparation from Saccharomyces cerevisiae containing L15 (YL23) as well as the acidic proteins L44', L44 and L45 protects exactly the same oligonucleotide fragment as does EL11 in both the yeast and mouse transcripts. Evidence is provided that L15, which is known to be structurally and functionally equivalent to EL11, is the rRNA-binding protein in this preparation. Thus the structural equivalent of the EL11 binding site present in yeast 26 S rRNA constitutes the second example of functional conservation of a ribosomal protein-binding site on rRNA between prokaryotes and eukaryotes.

Animals↗
PubMed1987

Specific binding of TUF factor to upstream activation sites of yeast ribosomal protein genes.

Transcription activation of yeast ribosomal protein genes is mediated through homologous, 12-nucleotide-long and, in general, duplicated upstream promoter elements (HOMOL1 and RPG, referred to as UASrpg). As shown previously, a yeast protein factor, TUF, interacts specifically with these conserved boxes in the 5'-flanking sequences of the elongation factor genes TEF1 and TEF2 and the ribosomal protein gene RP51A. We have now extended our studies of TUF-UASrpg binding by analysing--using footprinting and gel electrophoretic retardation techniques--the genes encoding the ribosomal proteins L25, rp28 (both copy genes), S24 + L46 and S33. Most, but not all, conserved sequence elements occurring in front of these genes, turned out to represent binding sites for the same factor, TUF. The two functionally important boxes that are found in a tandem arrangement (a characteristic of many rp genes) upstream of the L25 gene are indistinguishable in their factor binding specificity. Large differences were shown to exist in the affinity of the TUF factor for the various individual boxes and in the half-life of the protein-DNA complexes. No binding cooperativity could be demonstrated on adjacent sites on L25 or RP51A promoters. Based on binding data, the UASrpg sequence ACACCCATACAT appears to be the one recognized most efficiently by the TUF factor. Previously, no conserved box was found in front of the gene encoding S33. Nevertheless, complex formation with the protein fraction used was observed in the upstream region of the S33 gene. Competition experiments disclosed the existence of an additional binding component, distinct from TUF. This component may possibly regulate a subset of genes for the translational apparatus.

Fungal Proteins↗
PubMed1987

Molecular cloning of a pair of human pepsinogen A genes which differ by a Glu----Lys mutation in the activation peptide.

Three human cosmid clones containing pepsinogen A (PGA) encoding sequences were isolated from a genomic bank derived from a single individual. One cosmid contains two PGA genes in tandem in a head-to-tail orientation, while the other two cosmids each contain a single PGA gene. The three cosmids were characterized by restriction mapping and sequence analysis (exons 1 and 2 and flanking regions). As judged from these data, three of the four PGA genes isolated appear to be nearly identical, but one of the tandem genes is clearly different from the other genes. The first exon of all four genes codes for the same amino acid sequence. However, in the second exon of one of the tandem genes we found a nucleotide substitution giving rise to a Glu----Lys substitution of the 43rd amino acid residue of the activation peptide, leading to a charge difference of the corresponding isozymogens. The presence of two distinct PGA genes in the isolated gene pair conclusively proves the multigene structure of the PGA system. These genes might be responsible for at least part of the electrophoretic polymorphism at the protein level.

Amino Acid Sequence↗
PubMed1987

Structural and putative regulatory sequences of the gene encoding ribosomal protein L25 in Candida utilis.

Using a heterologous probe containing a fragment of the L25-gene from Saccharomyces carlsbergensis we have isolated a DNA-fragment of Candida utilis carrying the gene encoding ribosomal protein L25. This gene is present in a single copy on the C. utilis genome, though as two distinguishable alleles. Both alleles have been isolated and sequenced including their flanking regions. The nucleotide sequence of the amino acid coding region of the C. utilis gene turned out to be highly homologous (83%) to the L25-gene of S. carlsbergensis. At the protein level the degree of homology is about 87%. Codon usage in both organisms appears to be somewhat different. Just like the Saccharomyces gene, the L25 gene in C. utilis appears to be split in its 5th codon, though the identity of this codon has changed. Intron as well as 5'- and 3'-flanking sequences have almost completely diverged, with some notable exceptions. Of the intervening sequences the 5'- and 3'-splice sites as well as the putative lariat branch site are conserved. In the 5'-flanking region, at a distance of about 330 n from the initiation codon, a conserved nucleotide element is present that is very similar to the upstream transcription activation site previously found in front of the ribosomal protein genes in Saccharomyces.

Amino Acid Sequence↗
PubMed1987

Interaction of ribosomal proteins L25 from yeast and EL23 from E. coli with yeast 26S and mouse 28S rRNA.

The interaction of ribosomal protein EL23 from E. coli and L25 from yeast with yeast 26S rRNA was analysed by nitrocellulose filter binding and RNase protection experiments using both intact rRNA and various fragments prepared by in vitro transcription of cloned yeast rDNA regions in the SP6 system. The results show that EL23 efficiently and specifically interacts with the region of 26S rRNA previously identified as the binding site for the yeast ribosomal protein L25. A comparison of the oligonucleotides resulting from limited RNase T1 digestion of the heterologous EL23/26S rRNA complex with those obtained by the same treatment of the homologous L25/26S rRNA complex showed that the molecular details of the two r-protein/rRNA interactions are highly similar if not identical. Using the synthetic 26S rRNA fragments we could demonstrate that all information for the formation of a biologically active binding site is located within the region of the rRNA delimited by the sequences protected by L25 against RNase T1 digestion. Part of the sequence at the 3' end of the 5'-distal protected region, however, was found not to be essential for r-protein binding although it does enhance the efficiency of this binding. Binding experiments using synthetic mouse 28S rRNA fragments showed that neither EL23 nor L25 interact with the structural equivalent of their respective cognate binding sites present in this mammalian rRNA. We argue that the structure of the expansion sequence present in this region of mouse 28S rRNA is a major cause of this failure.

Amino Acid Sequence↗
PubMed1986

Conserved sequence elements upstream of the gene encoding yeast ribosomal protein L25 are involved in transcription activation.

Previous studies have revealed the occurrence of two closely linked conserved sequence elements, designated as HOMOL 1 and RPG box, in front of most yeast ribosomal protein genes examined. To investigate whether these conserved nucleotide elements play a role in the regulation of ribosomal protein gene expression, we performed deletion analysis of the DNA region upstream of the gene encoding ribosomal protein L25. To that end we constructed a hybrid gene consisting of the pertinent 5'-flanking sequence and the Escherichia coli galK marker gene. The effects on the transcription of this fusion gene of Bal31-generated deletions were measured by Northern analysis of RNA isolated from the respective transformed yeast cells. The results demonstrate that removal of one box has a detrimental effect on the level of transcription, whereas after the deletion of both boxes hardly any transcription can be observed. Subsequently we inserted synthetic oligonucleotides in the upstream region of an L25 gene from which the original boxes had been removed. Expression of the inactivated hybrid gene turned out to be restored even by insertion of one RPG element. Moreover, the RPG box functions in both orientations, though not with equal efficiency.

Base Sequence↗
PubMed1986

3'-End formation of transcripts from the yeast rRNA operon.

Deletion analysis of artificial rRNA minigenes transformed into Saccharomyces cerevisiae revealed that a 110 bp long fragment corresponding to positions -36 to +74 relative to the 3'-end of the 26S rRNA gene, is both necessary and sufficient for obtaining transcripts whose 3'-termini are identical to those of 26S and 37S (pre-)rRNA. These termini are produced via processing of longer transcripts because in an rna 82.1 mutant the majority of the minigene transcripts extend further downstream. Since the rna 82.1 mutation inactivates an endonuclease involved in the 3'-processing of 5S pre-rRNA it is concluded that the maturation of 37S- and that of 5S pre-rRNA requires a common factor. Comparison of the spacer sequences between Saccharomyces carlsbergensis, Saccharomyces rosei and Hansenula wingei revealed several conserved sequence blocks within the region between +10 and +55. These conserved sequence tracts, which are part of a longer region showing dyad symmetry, are supposed to be involved in the interaction with the processing component(s). Deletion of the sequences required for the formation of the 3'-ends of 26S rRNA and 37S pre-rRNA revealed a putative terminator for transcription by RNA polymerase I situated at position +210. This site maps within a DNA fragment that also contains the enhancing element for rDNA transcription by RNA polymerase I.

Enhancer Elements, Genetic↗
PubMed1986

The cellular level of yeast ribosomal protein L25 is controlled principally by rapid degradation of excess protein.

When the gene dosage for the primary rRNA-binding ribosomal protein L25 in yeast cells was raised about 50-fold, the level of mature L25 transcripts was found to increase almost proportionally. The plasmid-derived L25 transcripts were structurally indistinguishable from their genomic counterparts, freely entered polysomes in vivo and were fully translatable in a heterologous in vitro system. Nevertheless, pulse-labelling for periods varying from 3-20 min did not reveal a significant elevation of the intracellular level of L25-protein. When pulse-times were decreased to 10-45 s, however, we did detect a substantial overproduction of L25. We conclude that, despite the strong RNA-binding capacity of the protein, accumulation of L25 is not controlled by an autogenous (pre-)mRNA-targeted mechanism similar to that operating in bacteria, but rather by extremely rapid degradation of excess protein produced.

Fungal Proteins↗
PubMed1986

Variant-specific differences in human unsaturated transcobalamin II.

Electrophoresis and subsequent autoradiography of 57Co-cobalamin (57 Co-Cbl)-labeled serum show intensity differences between the genetic variants of human transcobalamin II (TC2), suggesting differences in the unsaturated (apo-) TC2 concentration. In order to distinguish between variant-specific differences in the Cbl binding affinity and those in the total-TC2 concentration, techniques were developed to determine total, apo-, and holo-TC2. Prolonged incubation at 37 degrees C with a 20-fold excess of 57Co-Cbl resulted in an almost complete exchange of endogenously bound Cbl, which allowed determination of the total TC2. The holo-TC2 concentration of both gene products in TC2 heterozygotes could be estimated by comparison of the labeling levels of apo- and total TC2, using densitometric quantification of the autoradiographs. By means of ion-exchange chromatography, TC2 could be separated from other Cbl-binding proteins, permitting a simple quantitative assay of apo- and total TC2, the results of which correlate fairly well with those measured by an immunoadsorption assay. The results obtained in the present investigation indicate that the variant-specific variation in the apo-TC2 concentration is caused by differences in the total-TC2 concentration rather than in the Cbl binding affinity.

Apoproteins↗
PubMed1985

Correct removal by splicing of a Neurospora intron in yeast.

Processing of intron-containing nuclear messenger RNAs in yeast require an internal conserved sequence (ICS) element, UACUAAC. Similar elements (ugCUAGAC) have been identified in sequences interrupting nuclear genes of the related ascomycete Neurospora crassa. To examine the structural splicing requirements in yeast, we constructed hybrid genes containing the intron of the Neurospora histone H3 gene and cloned them into high copy number yeast vectors. Subsequently we analyzed the RNAs transcribed in yeast from the fusion genes by Northern analysis and primer extended sequencing. It turned out that the Neurospora intron, which contains the sequence element UGCUAAC, can be removed, though very inefficiently, provided that it is located near the 5'-end of the primary transcript. This proves that an A at the second position of the ICS is no absolute requirement for splicing in yeast. In addition, the results indicate that the yeast splicing machinery is intron-position dependent.

Amino Acid Sequence↗