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W H Mager

Publications and source records attributed to W H Mager.

90 records · Page 5Linked to original sources
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↗
PubMed1983

The genes coding for histone H3 and H4 in Neurospora crassa are unique and contain intervening sequences.

Sequences coding for histone H3 and H4 of Neurospora crassa could be identified in genomic digests with the use of the corresponding genes from sea urchin and X. laevis as hybridization probes. A 2.6 kb HindIII-generated N. crassa DNA fragment, showing homology with the heterologous histone H3-gene probes was cloned in a charon 21A vector. Using DNA from this clone as a homologous hybridization probe a 6.9 kb SalI-generated DNA fragment was isolated which in addition to the histone H3-gene also contains the gene coding for histone H4. Several lines of evidence demonstrate the presence of only a single histone H3- as well as a single histone H4-gene in N. crassa. The two genes are physically linked on the genome. DNA sequencing of the N. crassa histone H3- and H4-genes confirmed their identity and, in addition, revealed the presence of one short intron (67 bp) within the coding sequence of the H3-gene and even two introns (68 and 69 bp) within the H4-gene. The amino acid sequences of the N. crassa histones H3 and H4, as deduced from the DNA sequences, and those of the corresponding yeast histones differ only at a few positions. Much larger sequence differences, however, are observed at the DNA level, reflecting a diverging codon usage in the two lower eukaryotes.

Amino Acid Sequence↗
PubMed1982

The structure of the gene coding for the phosphorylated ribosomal protein S10 in yeast.

From previous studies on cloned yeast ribosomal protein genes we obtained evidence that a large number of them contain an intron [Bollen et al. (1982) Gene 18, 29-38]. In the temperature-sensitive rna2-mutant transcription of these genes leads to the accumulation of precursor RNAs at the restrictive temperature. These precursor mRNAs are several hundreds of nucleotides longer than the respective mature mRNAs. The split character of one of these ribosomal protein genes, viz. the gene coding for the major phosphorylated small-subunit protein S10, was further established by sequence analysis. The intervening sequence interrupts the coding sequence after the second codon and has a length of 352 nucleotides. Genomic Southern hybridizations with a DNA fragment carrying part of the S10-gene revealed that this gene is duplicated on the yeast genome. The molecular weight of S10 as deduced from the sequence analysis was estimated to be 31462 dal. Comparison of the N-terminal aminoacid sequence of the yeast ribosomal protein S10 with that of ribosomal protein S6 from rat liver revealed a striking homology between both proteins. Moreover, at the C-terminal end of the yeast ribosomal protein the sequence Arg-Ala-Ser-Ser-Leu-Lys is present which is very similar to the phosphorylation site of the rat liver protein S6.

Amino Acid Sequence↗
PubMed1982

Ribosomal protein genes of yeast contain intervening sequences.

From a colony bank of HindIII-generated yeast DNA fragments we have isolated a number of recombinant DNAs carrying genes for ribosomal proteins (e.g., S10, S16A, S20, S24, S31, S33, L16, L25 and L34) of the yeast Saccharomyces carlsbergensis. By electron microscopic analysis of the R-loops formed between various DNA fragments and yeast mRNA, we could locate the ribosomal protein genes on the physical maps of the cloned DNA fragments. The R-loop structures observed indicate that a number of the ribosomal protein genes contain an intervening sequence.

Base Sequence↗
PubMed1981

Isolation of cloned ribosomal protein genes from the yeast Saccharomyces carlsbergensis.

A colony bank of yeast dna obtained by cloning HindIII-generated fragments of total yeast nuclear DNA in Escherichia coli K-12 with the vector pBR322, was screened with a radioactive RNA probe enriched for a subset of ribosomal protein mRNAs. The selected recombinant DNA molecules were hybridized with poly(A)-containing mRNA under R-loop conditions. From the DNA-RNA hybrids the respective mRNAs were melted off and translated in vitro in a rabbit reticulocyte cell-free system. The translational products were analyzed by immunoprecipitation with antibodies raised against ribosomal proteins. The identity of the ribosomal protein gene products was further established by electrophoresis on two-dimensional gels. At least 15 recombinant DNA molecules were shown to contain ribosomal protein genes. Four of them, i.e. Y65, Y89, Y113 and Y138, have been characterized preliminarily.

DNA, Fungal↗
PubMed1980

Small-size mRNAs code for ribosomal proteins in yeast.

In order to identify and to study the ribosomal protein genes in yeast we have tried to purify the mRNA coding for ribosomal proteins. Poly(A)-containing RNA from the yeast Saccharomyces carlsbergensis was fractionated according to size using preparative sucrose gradient centrifugation. The various (size) fractions were translated in vitro in a wheat germ cell-free system. The products were analysed by sodium dodecylsulfate/polyacrylamide gradient gel electrophoresis as well as by acetic acid/urea gel electrophoresis. It was found that an mRNA fraction of about 9 S directs the synthesis in vitro of proteins that have properties characteristic of ribosomal proteins, i.e. they are both small and basic. The ribosomal nature of these proteins was further established by two-dimensional gel electrophoresis. This small-size mRNA fraction can be used as a probe for the identification of ribosomal protein genes in recombinant DNA molecules.

Electrophoresis, Polyacrylamide Gel↗
PubMed1979

Analysis of histones from the yeast Saccharomyces carlsbergensis.

Basic chromosomal proteins were isolated from the chromatin of the yeast Saccharomyces carlsbergensis by extraction with H2SO4 and were purified by ion-exchange chromatography. Electrophoresis of the purified fraction on acetic acid/urea gels revealed the presence of four main components. These four proteins were identified as histones H2A, H2B, H3 and H4 on the basis of their amino acid composition, molecular weight and solubility properties, all of which are very similar to the corresponding properties of the various histone proteins from other eukaryotic organisms. A fifth basic protein could be isolated from yeast chromatin by extraction with HClO4. The available evidence indicates this protein to be an H1-type histone. Yeast thus appears to contain a complete set of histone proteins which are strongly homologous to the histones occurring in higher eukaryotes.

Amino Acids↗
PubMed1978

Modification of yeast ribosomal proteins. Methylation.

Two-dimensional polyacrylamide-gel electrophoretic analysis of yeast ribosomal proteins uniformly labelled in vivo with [methyl-3H]methionine and [1-14C]methionine revealed that four ribosomal proteins are methylated, i.e. proteins S31, S32, L15 and L41. Lysine and arginine appear to be the predominant acceptors of the methyl groups. The degree of methylation ranges from 0.09 to 0.20 methyl group per modified ribosomal protein species.

Amino Acids↗
PubMed1978

Quantitative analysis of the protein composition of yeast ribosomes.

The molecular weights of the individual yeast ribosomal proteins were determined. The ribosomal proteins from the 40-S subunit have molecular weights ranging from 11 800 to 31 000 (average molecular weight = 21 300). The molecular weights of the 60-S subunit proteins range from 10 000 to 48 400 (average molecular weight = 21 800). Stoichiometric measurements, performed by densitometric scanning on ribosomal proteins extracted from high-salt dissociated subunits revealed that isolated ribosomal subunits contain, besides some protein species occurring in submolar amounts, a number of protein species which are present in multiple copies: S13, S27, L22, L31, L33, L34 and L39. The mass fractions of the ribosomal proteins which were found to be present on isolated ribosomes in non-unimolar amounts, were re-examined by using an isotope dilution technique. Applying this method to proteins extracted from mildely isolated 80-S ribosomes, we found that some protein species such as S32, S34 and L43 still are present in submolar amounts. On the other hand, however, we conclude that some other ribosomal proteins, in particular the strongly acidic proteins L44 and L45 get partially lost during ribosome dissociation. Proteins L44/L45 appears to be present on 80-S ribosomes in three copies.

Electrophoresis, Polyacrylamide Gel↗
PubMed1977

Transcriptional units for ribosomal proteins in yeast.

The effects of ultraviolet irradiation on the rates of synthesis of individual ribosomal proteins in yeast were examined and compared with the ultraviolet sensitivities of the synthesis of other yeast proteins. It was found that the synthesis of yeast ribosomal proteins is much more sensitive to ultraviolet irradiation than that of other yeast cellular proteins. Taking into account the half-life of yeast mRNA, the results obtained indicate that the genes coding for ribosomal proteins form part of long transcriptional units, which are much longer than the DNA stretch needed to code for a ribosomal protein of average molecular weight. Saturation hybridization of total poly(A)-containing mRNA with yeast nuclear DNA revealed that as much as 30% of DNA is complementary to yeast mRNA. Thus, the primary transcript of a protein gene on the average is about 1.7 times the length of the actual messenger. On the basis of the various experimental data we suggest a clustering of the yeast ribosomal protein genes in a number of common transcriptional units.

DNA↗
PubMed1975

Cellular site of synthesis of ribosomal proteins in yeast.

Looking for messenger RNA coding for yeast ribosomal protein, we devised a method to identify polysomes involved in ribosomal protein synthesis. Analysis of nascent protein elongated in vitro demonstrated that ribosomal proteins are synthesized both on membrane-associated and free polysomes.

Cell Fractionation↗
PubMedDate not supplied

Coordinate expression of ribosomal protein genes in yeast as a function of cellular growth rate.

The rate of ribosome formation in yeast is precisely adjusted to the physiological demands of the cell. During all growth conditions a balance is maintained in the production of all ribosomal constituents. Coordinate expression of the ribosomal protein (rp) genes is primarily accomplished at the transcriptional level. Transcription activation of the majority of the rp-genes is mediated through common upstream activating sequences, so-called RPG boxes, which occur usually in a tandem at a distance of 200-500 bp from the start codon. These RPG-boxes represent binding sites for a transcriptional activator, called TUF or RAP. The concentration of TUF parallels the cellular growth rate and evidence exists that the response of rp-genes upon nutritional changes is mediated by this factor. Recent findings indicate that TUF/RAP also activates other gene families involved in cellular growth rate. Furthermore, this multifunctional protein also binds to the mating-type silencer and telomeres in yeast. Some other rp-genes (e.g. those encoding S33 and L45) do not contain an RPG-box. They appear to be activated by another multifunctional protein, called ABF1 or SUF, by binding to another nucleotide motif. This multifunctional protein also activates other gene families, and in addition binds to the mating type silencer and ARS-elements.

Amino Acid Sequence↗
PubMedDate not supplied

Global regulators of ribosome biosynthesis in yeast.

Three abundant ubiquitous DNA-binding protein factors appear to play a major role in the control of ribosome biosynthesis in yeast. Two of these factors mediate the regulation of transcription of ribosomal protein genes (rp-genes) in yeasts. Most yeast rp-genes are under transcriptional control of Rap1p (repressor-activator protein), while a small subset of rp-genes is activated through Abf1p (ARS binding factor). The third protein, designated Reb1p (rRNA enhancer binding protein), which binds strongly to two sites located upstream of the enhancer and the promoter of the rRNA operon, respectively, appears to play a crucial role in the efficient transcription of the chromosomal rDNA. All three proteins, however, have many target sites on the yeast genome, in particular, in the upstream regions of several Pol II transcribed genes, suggesting that they play a much more general role than solely in the regulation of ribosome biosynthesis. Furthermore, some evidence has been obtained suggesting that these factors influence the chromatin structure and creat a nucleosome-free region surrounding their binding sites. Recent studies indicate that the proteins can functionally replace each other in various cases and that they act synergistically with adjacent additional DNA sequences. These data suggest that Abf1p, Rap1p, and Reb1p are primary DNA-binding proteins that serve to render adjacent cis-acting elements accessible to specific trans-acting factors.

Base Sequence↗