Biomedical subjects

R J Planta

Publications and source records attributed to R J Planta.

At least 127 records · Page 7Linked to original sources

PubMed1985

Yeast contains two functional genes coding for ribosomal protein S10.

The DNA sequence of the second copy of the gene coding for yeast ribosomal protein S10 was determined and compared with the sequence of the first gene-copy. In addition, the sites at which the transcription of these genes start and terminate are identified. The amino acid coding regions of the two gene copies are virtually identical. The leader and in particular the trailer sequences, however, are significantly different, while the intervening sequences have hardly any homology. Taking advantage of the sequence differences we could establish that both genes are expressed in the vegetatively growing yeast cell; the respective transcripts, however, differ in their relative amounts.

Amino Acid Sequence↗

PubMed1985

Identification of yeast 60 S ribosomal proteins crosslinked to rRNA by 2-iminothiolane.

Saccharomyces carlsbergensis 60 S ribosomal subunits were treated with the hetero-bifunctional crosslinking agent 2-iminothiolane and then subjected to mild UV irradiation to introduce protein-rRNA crosslinks. The major crosslinked products were identified as proteins L2, L3, L5, L19 and L23 of which L5 was found to be crosslinked at a 3-5-fold higher efficiency than the other four. Several additional proteins were cross-linked to a detectable but much lower extent.

Cross-Linking Reagents↗

PubMed1985

The genes for yeast ribosomal proteins S24 and L46 are adjacent and divergently transcribed.

Unlike most yeast ribosomal protein genes studied so far the genes coding for S24 and L46 are adjacent on the genome. Sequence analysis showed that the two genes are transcribed divergently, their initiation codons being 630 bp apart. Taking the respective ATG translation start sites as reference points, the 5'- end of L46 mRNA was mapped at position -26, while the S24 mRNA showed two major 5'-ends mapping at positions -13 and -16 respectively. Unlike most other yeast ribosomal protein genes, the gene for S24 does not contain an intron. Its coding region encompasses 390 nucleotides encoding a protein of 14762 D. The gene for L46 on the other hand is split by an intron of 386 nucleotides starting after its second codon. This gene encodes a small, very basic protein having a molecular weight of 6334 D. Yeast ribosomal proteins S24 and L46 show striking homologies with ribosomal proteins from other organisms. In particular, yeast L46 is clearly the evolutionary counterpart of rat liver L39. A search of the intergenic region for sequence elements previously identified as common to most yeast ribosomal protein genes, revealed the presence of a single conserved box (RPG-box) roughly equidistant from the transcription initiation sites of both genes. We suggest that this box acts as a regulatory signal in either orientation and thus influences the expression of both genes simultaneously.

Amino Acid Sequence↗

PubMed1985

Yeast ribosomal protein L25 binds to an evolutionary conserved site on yeast 26S and E. coli 23S rRNA.

The binding site of the yeast 60S ribosomal subunit protein L25 on 26S rRNA was determined by RNase protection experiments. The fragments protected by L25 originate from a distinct substructure within domain IV of the rRNA, encompassing nucleotides 1465-1632 and 1811-1861. The protected fragments are able to rebind to L25 showing that they constitute the complete protein binding site. This binding site is remarkably conserved in all 23/26/28S rRNAs sequenced to date including Escherichia coli 23S rRNA. In fact heterologous complexes between L25 and E. coli 23S rRNA could be formed and RNase protection studies on these complexes demonstrated that L25 indeed recognizes the conserved structure. Strikingly the L25 binding site on 23S rRNA is virtually identical to the previously identified binding site of E. coli ribosomal protein EL23. Therefore EL23 is likely to be the prokaryotic counterpart of L25 in spite of the limited homology displayed by the amino acid sequences of the two proteins.

Amino Acid Sequence↗

PubMed1985

Assignment of human pepsinogen A locus to the q12-pter region of chromosome 11.

A 0.9kb cDNA fragment, corresponding to a large part of Rhesus monkey pepsinogen A mRNA, was used as probe for the chromosomal localization of the human pepsinogen A gene(s) using human-rodent somatic cell hybrids. Southern blot analysis of 14 human-Chinese hamster and three human-mouse cell hybrids, strongly indicates that the human PGA locus is on chromosome 11. The human-mouse hybrids, containing a translocation involving chromosome 11, allow sublocalization to the region q12-pter.

Animals↗

PubMed1985

Deletion mapping of the yeast Pol I promoter.

Deletions in the promoter region of the 37S pre-rRNA operon in yeast were constructed and analysed in vivo using an artificial ribosomal minigene present on an extrachromosomal yeast vector. Sequences required for correct transcription initiation were found to be located between positions -192 and +15 relative to the start; a 5'-deletion down to position -133 reduces the transcription yield of the minigene at least five-fold. To allow detection of transcription of the minigene in isolated nuclei of yeast transformed with a minigene-bearing plasmid we attempted to increase the minigene copy number. The transcription yield in vivo appeared not to be proportional to the copy number but was found to be greatly enhanced when two or three minigenes are present in tandem. alpha-Amanitin sensitivity of transcription of these minigenes in isolated nuclei proved that RNA polymerase I is responsible for their transcription.

Base Sequence↗

PubMed1985

The gene for yeast ribosomal protein S31 contains an intron in the leader sequence.

Analysis of the primary structure of the gene for yeast ribosomal protein S31 revealed two unusual features. First, an intron of 312 nucleotides is located within the 5'-untranslated region. Second, the coding sequence for the known amino-terminal peptide of the protein starts 13 codons downstream of the ATG initiation codon, suggesting that S31 is synthesized as a precursor which undergoes post-translational processing to the mature protein. Primer extension analysis showed that transcription of the S31 gene starts at multiple sites. The 5'-flanking region of the gene contains several, previously described, conserved sequence elements that may play a role in the coordinate expression of yeast ribosomal protein genes.

DNA Restriction Enzymes↗

PubMed1985

Conserved sequences upstream of yeast ribosomal protein genes.

Computer analysis has previously revealed the presence of a 12-nucleotide common sequence element (AACATCTGCATGACA; HOMOL1) in the upstream regions of several yeast ribosomal protein genes. By extending the sequence analysis of the 5'-flanking regions of a number of other ribosomal protein genes (including those encoding S10-1, S10-2, S33 and L16-2) we could establish that HOMOL1 occurs upstream of most but not all yeast ribosomal protein genes. Apart from HOMOL1 an additional conserved sequence (ACCCATACATTTA; RPG-box) was detected in front of nearly all yeast ribosomal protein genes, although in some cases it is present in the opposite orientation in the other strand. There seems to be no correlation between the occurrence of one box and that of the other. However when both boxes are present the RPG-box is always located 3' to the HOMOL1-sequence mostly at a distance of only a few nucleotides. A further one-to-one comparison of the upstream regions of several yeast ribosomal protein genes revealed extensive additional sequence homologies that are suggested to be involved in the coordinate control of ribosomal protein gene expression in yeast.

Base Sequence↗

PubMed1984

Stepwise dissociation of yeast 60S ribosomal subunits by LiCl and identification of L25 as a primary 26S rRNA binding protein.

Treatment of yeast 60S ribosomal subunits with 0.5 M LiCl was found to remove all but six of the ribosomal proteins. The proteins remaining associated with the (26S + 5.8S) rRNA complex were identified as L4, L8, L10, L12, L16 and L25. These core proteins were split off sequentially in the order (L16 + L12), L10, (L4 + L8), L25 by further increasing the LiCl concentration. At 1.0 M LiCl only ribosomal protein L25 remains bound to the rRNA. Upon lowering the LiCl concentration the core proteins reassociate with the rRNA in the reverse order of their removal. The susceptibility of the ribosomal proteins to removal by LiCl corresponds quite well with their order of assembly into the 60S subunit in vivo as determined earlier [Kruiswijk et al. (1978) Biochim. Biophys. Acta 517, 378-389]. Binding studies in vitro using partially purified L25 showed that this protein binds specifically to 26S rRNA. Therefore our experiments for the first time directly identify a eukaryotic ribosomal protein capable of binding to high-molecular-mass rRNA. Binding studies in vitro using a blot technique demonstrated that core proteins L8 and L16 as well as protein L21, though not present in any of the core particles, are also capable of binding to 26S rRNA to approximately the same extent as L25. About nine additional 60S proteins appeared to interact with the 26S rRNA, though to a lesser extent.

Carrier Proteins↗

PubMed1984

Structure and organization of two linked ribosomal protein genes in yeast.

The genes encoding yeast ribosomal proteins rp28 and S16A are linked and occur duplicated in the yeast genome. In both gene pairs the genes are approximately 600 bp apart and are both transcribed in the same direction. Both ribosomal protein genes resemble other ribosomal protein genes studied so far in many structural aspects. The genes are interrupted by an intron near the 5'-end of their coding sequence. In addition the flanking regions contain several conserved sequence elements, which may function in transcription initiation and termination. In agreement with findings concerning other cloned yeast ribosomal protein genes, upstream homology blocks occur that may be involved in coordinate control of ribosomal protein gene transcription. The complete pattern of conserved and diverged sequences between the two duplicate gene pairs is presented.

Amino Acid Sequence↗

PubMed1984

The primary structure of the gene encoding yeast ribosomal protein L16.

As part of our studies on the molecular basis for the coordinate expression of ribosomal protein genes in yeast we analyzed the primary structure of the gene encoding protein L16 of the large ribosomal subunit including the flanking sequences. L16 turned out to be a ribosomal protein with a molecular mass of 22662 Da and a net charge of +12. Both the 5'- and the 3'-end of the L16 mRNA were mapped by primer extension and S1 nuclease analysis. In the DNA regions flanking the coding sequence several conserved elements are present that may be involved in transcription initiation or termination.

Amino Acid Sequence↗

PubMed1984

Structural comparison of yeast ribosomal protein genes.

The primary structure of the genes encoding the yeast ribosomal proteins L17a and L25 was determined, as well as the positions of the 5'- and 3'-termini of the corresponding mRNAs. Comparison of the gene sequences to those obtained for various other yeast ribosomal protein genes revealed several similarities. In all split genes the intron is located near the 5'-side of the amino acid coding region. Among the introns a clear pattern of sequence conservation can be observed. In particular the intron-exon boundaries and a region close to the 3'-splice site show sequence homology. Conserved sequences were also found in the leader and trailer regions of the ribosomal protein mRNAs. The 5'-flanking regions of the yeast ribosomal protein genes appeared to contain sequence elements that many but not all ribosomal protein genes have in common, and therefore may be implicated in the coordinate expression of these genes. The amino acid coding sequences of the ribosomal protein genes show a biased codon usage. Like most yeast ribosomal protein molecules, L17a and L25 are particularly basic at their N-terminus.

Amino Acid Sequence↗

PubMed1984

Structure and function of the nontranscribed spacer regions of yeast rDNA.

The sequences of the nontranscribed spacers (NTS) of cloned ribosomal DNA (rDNA) units from both Saccharomyces cerevisiae and Saccharomyces carlsbergensis were determined. The NTS sequences of both species were found to be 93% homologous. The major disparities comprise different frequencies of reiteration of short tracts of six to sixteen basepairs. Most of these reiterations are found within the 1100 basepairs long NTS between the 3'-ends of 26S and 5S rRNA (NTS1). The NTS between the starts of 5S rRNA and 37S pre-rRNA (NTS2) comprises about 1250 basepairs. The first 800 basepairs of NTS NTS2 (adjacent to the 5S rRNA gene) are virtually identical in both strains whereas a variable region is present at about 250 basepairs upstream of the RNA polymerase A transcription start. In contrast to the situation in Drosophila and Xenopus no reiterations of the putative RNA polymerase A promoter are present within the yeast NTS. The strands of the yeast NTS reveal a remarkable bias of G and C-residues. Yeast rDNA was previously shown to contain a sequence capable of autonomous replication (ARS) (Szostak, J.W. and Wu, R (1979), Plasmid 2, 536-554). This ARS, which may correspond to a chromosomal origin of replication, was located on a fragment of 570 basepairs within NTS2.

Animals↗

PubMed1984

The in vivo and in vitro initiation site for transcription of the rRNA operon of Saccharomyces carlsbergensis.

We have performed a detailed analysis of the transcription initiation of the rRNA operon in the yeast Saccharomyces carlsbergensis. Electron microscopic analysis of R-looped pre-rRNA molecules together with a very sensitive S1-nuclease mapping showed the use of only a single transcription start at about 700 bp upstream of the 17S rRNA gene and not of the minor start sites proposed for the very closely related species S. cerevisiae by others [Bayev et al. (5), Swanson and Holland (6)]. The sequence of 730 bp of the initiating region is presented. In vitro transcription in concentrated lysates of yeast spheroplasts in the presence of (gamma-SH)ATP or (gamma-SH)GTP, followed by purification of the in vitro initiated RNA via Hg-agarose, revealed that on the endogenous template exactly the same site is used for transcription initiation as in vivo.

Base Sequence↗

PubMed1984

A conserved sequence element is present around the transcription initiation site for RNA polymerase A in Saccharomycetoideae.

To identify DNA elements involved in the initiation of rRNA transcription in yeast we located the start site of the rRNA operon of Kluyveromyces lactis and Hansenula wingei, both members of the Saccharomycetoideae, by S1 nuclease analysis and determined the surrounding nucleotide sequences. Comparison of these sequences with those of Saccharomyces carlsbergensis, S. cerevisiae and S. rosei (all belonging to the same yeast subfamily) reveals an identical sequence at the site of transcription initiation from position +1 to +7 which is part of a larger conserved region extending from position -9 to +23; the conserved heptanucleotide sequence is supposed to constitute an important part of the promoter for yeast RNA polymerase A. The non-transcribed spacers (NTS) upstream of position -9 have diverged strongly with the exception of two short elements around positions -75 and -135. The external transcribed spacer (ETS) downstream of position +23 is largely conserved between K. lactis, S. rosei and S. carlsbergensis except for a divergent region around position +75. On the other hand, the ETS of H. wingei has diverged significantly.

Ascomycota↗

PubMed1984

Transcription of an artificial ribosomal RNA gene in yeast.

We constructed an artificial yeast rRNA gene and studied its transcription after introduction into a recipient yeast strain. The artificial gene comprised a fragment containing the sequence from position -207 to +128 relative to the site of initiation of Saccharomyces carlsbergensis 37S pre-rRNA, followed by a marker fragment from Spirodela oligorhiza chloroplast DNA and finally a fragment containing the sequence from position -36 to +101 relative to the 3' end of the 26S rRNA gene. The resulting construct was cloned into the yeast-Escherichia coli shuttle vector pJDB207. Both Northern blot hybridization and R-loop analysis of RNA from transformed Saccharomyces cerevisiae cells revealed a discrete transcript of the expected length. S1 nuclease mapping as well as primer extension analysis showed that the major proportion of the transcripts was initiated at exactly the same site as 37S pre-rRNA. These results show that the respective rDNA fragments contain the information for correct initiation of transcription and formation of the 3' end. A minor proportion of the transcripts was initiated at a number of sites between positions -1 and -100 upstream of the predominant start. The proportion and the pattern of these upstream starts is affected by the vector context of the artificial rRNA gene.

Base Sequence↗

PubMed1984

Evolution of yeast ribosomal DNA: molecular cloning of the rDNA units of Kluyveromyces lactis and Hansenula wingei and their comparison with the rDNA units of other Saccharomycetoideae.

We have studied the evolution of the yeast ribosomal DNA unit to search for regions outside the rRNA genes that exhibit evolutionary constraints and therefore might be involved in control of ribosome biosynthesis. We have cloned one complete rDNA unit of Kluyveromyces lactis and Hansenula wingei and established the physical and genetic organisation of both units. Both species belong to the subfamily of the Saccharomycetoideae. The lengths of the rDNA units of K. lactis and H. wingei are 8.6 and 11.1 kb respectively, and both comprise the 5S rRNA gene in addition to the large rRNA operon. Sequence conservation was monitored by restriction enzyme mapping as well as heteroduplex analysis of the two cloned rDNA units with S. carlsbergensis rDNA. These analyses showed that, phylogenetically, K. lactis is closer to S. carlsbergensis than H. wingei. The non-transcribed spacers (NTS) of both K. lactis and H. wingei have diverged completely from S. carlsbergensis; moreover in H. wingei the NTS are about double the length of these in the other two species. The transcribed spacers of both K. lactis and H. wingei contain conserved tracts. A homologous sequence of about 60 bp was found in the middle of the external transcribed spacer of H. wingei upon heteroduplexing with S. carlsbergensis rDNA, whereas the sequence at the transcription initiation site itself was insufficiently homologous to form a duplex. The sequence of the homologous region was determined both in H. wingei and K. lactis and compared with that of S. carlsbergensis. The function of this conserved element within the external transcribed spacer is discussed.

Ascomycota↗

PubMed1983

Yeast ribosomal protein S33 is encoded by an unsplit gene.

The structure of the gene coding for ribosomal protein S33, - a protein which escapes the coordinate control of ribosomal protein synthesis in rna 2 mutant cells -, was determined by sequence analysis. The gene comprises an uninterrupted coding region of 204 nucleotides encoding a protein of 8.9 kD. Like for other yeast ribosomal protein genes that have been sequenced so far, a relatively strong codon bias was observed. By S1 nuclease mapping the 5' end of the S33 mRNA was shown to be located at 11 to 15 nucleotides upstream from the initiation codon.

Amino Acid Sequence↗