PubMed Health⌕ Search

Biomedical subjects

G S Groot

Publications and source records attributed to G S Groot.

At least 19 recordsLinked to original sources
PubMed1986

The gene for Spirodela oligorhiza chloroplast ribosomal protein homologous to E. coli ribosomal protein L16 is split by a large intron near its 5' end: structure and expression.

The nucleotide sequence of a Spirodela chloroplast DNA fragment, which directs the synthesis of a approximately 15 kD chloroplast ribosomal protein in an E. coli cell free system, has been determined. The deduced aminoacid sequence of the open reading frame shows extensive homology with E. coli ribosomal protein L16. Primer extension analysis, S1 nuclease mapping and nucleotide sequence analysis indicate that the chloroplast L16 gene (rpl16) is interrupted by a 1411 bp intron, which separates a short 5' exon from a large 3' exon. The shorter in vitro synthesized ribosomal protein results from an artificial initiation event at an internal ATG codon in the 3' exon. The sequences at the 5' and 3' splice sites of the intron are similar to consensus sequences described for other, class II intron containing, protein coding chloroplast genes. Northern hybridization experiments reveal 6 stable transcripts of rpl16 ranging from 500 b to greater than 4000 b. As determined by S1 nuclease mapping, the 3'-end of the smallest transcript maps exactly after the stem of a proposed termination signal. Finally, the implications of the finding of a cluster of several chloroplast ribosomal protein genes and possible polycistronic transcription of this chloroplast DNA region, are discussed in relation to the organization and expression of ribosomal protein genes found in the S10 operon of E. coli.

Amino Acid Sequence↗
PubMed1986

The genes encoding chloroplast ribosomal proteins S7 and S12 are located in the inverted repeat of Spirodela oligorhiza chloroplast DNA.

We have used a variety of methods to localize the genes for ribosomal proteins S7 and S12 on Spirodela chloroplast DNA. Heterologous hybridization with a rps12 gene specific probe from Euglena has revealed the presence of rps12 homologous sequences within the inverted repeat of Spirodela chloroplast DNA on the fragment BamHI-V. In the partial nucleotide sequence of this fragment, two regions of amino acid sequence homology to Euglena S12 can be identified, separated from each other by a 542 bp intron with conserved boundary sequences. As was found for Nicotiana S12, the Spirodela S12 coding regions are for 85 amino acids homologous (79%) to E. coli S12 (starting from residue 38 to the C-terminus). Likewise, we are unable to identify the 37 5' terminal codons of rps12 in Spirodela. The functionality of the Spirodela rps12 sequence is discussed. The rps7 gene is located adjacent to rps12. Chloroplast ribosomal protein C-S11 (homologous to S7) has been detected by immunoprecipitation with both a polyspecific anti 30S serum and an anti C-S11 serum, among the in vitro translation products of mRNAs selected by Spirodela chloroplast DNA fragments BamHI-V and BamHI-P. Since in a DNA dependent E. coli cell free system, only BamHI-V appears to be capable of synthesis of C-S11, it is concluded that rps7 is located entirely within BamHI-V and is transcribed into a mRNA which extends into BamHI-P. As determined by Northern hybridization experiments, rps7 is cotranscribed with rps12; a stable transcript of approx. 1100 b is detected in total cellular Spirodela RNA with either rps12 and rps7 gene specific probes. The rps12 probe additionally detects an approx. 600 b transcript, which presumably corresponds to the excised rps12 intron RNA. Finally we have examined the expression of both rps7 and rps12 during light induced chloroplast development by Northern blotting and by immunoblotting. It is shown, that the steady-state levels of neither chloroplast ribosomal protein transcripts, nor those of the chloroplast ribosomal proteins itself, change significantly during the greening process.

Amino Acid Sequence↗
PubMed1985

Induction of cytochrome P-450b,e-type isozymes by polychlorinated biphenyls in rat liver. Molecular cloning of induced mRNAs.

We have studied the induction of cytochrome P-450b,e-type antigen and mRNA by phenobarbital, 4,4'-dichlorobiphenyl and 2,4,5,2',4',5'-hexachlorobiphenyl in male Wistar rat liver. Chronic treatment of rats with 4,4'-dichlorobiphenyl leads to a relatively slow, 20-fold increase in the cytochrome P-450b,e-type antigen level and to an equivalent increase in the concentration of the corresponding mRNA. Treatment of rats with phenobarbital or 2,4,5,2',4',5'-hexachlorobiphenyl results in a faster and more pronounced increase of cytochrome P-450b,e-type antigen and mRNA levels. Analysis of clones from cDNA banks showed that two types of sequences are induced by phenobarbital corresponding to cytochrome P-450b and cytochrome P-450e respectively. 2,4,5,2',4',5'-Hexachlorobiphenyl appears to induce primarily a cytochrome P-450b-type sequence. The implications of these results for the study of the mechanism of induction are discussed.

Animals↗
PubMed1984

Protein synthesis during chloroplast development in Spirodela oligorhiza. Coordinated synthesis of chloroplast-encoded and nuclear-encoded subunits of ATPase and ribulose-1,5-bisphosphate carboxylase.

We have studied qualitative and quantitative changes of several parameters during chloroplast development in Spirodela oligorhiza (duckweed). On a dry weight basis, the amount of protein increases from 2.5% (w/w) in dark-grown to 7.8% (w/w) in light-grown fronds. At the same time the amount of starch drops from 50% to 27% (w/w). Using an immunochemical quantification method we have found that during greening of etiolated plants the amount of all subunits of the ATPase complex per frond increases 10-fold, whereas the level of the subunits of ribulose-1,5-biphosphate carboxylase increases 50-fold. Cytochrome f was found to be present in dark-grown Spirodela and the amount of this polypeptide per frond increases about 30-fold. The concentration of a polypeptide that possibly represents a cytochrome b6 subunit increases about 10-fold upon greening. The molar ratio of the CF1-beta and CF1-gamma subunits of the ATPase complex varies over 2-3, while in all stages of chloroplast development studied the molar ratio of the carboxylase subunits is about 1. As these values are in agreement with the stoichiometrical amounts in the native protein complexes, we conclude that the synthesis of CF1-beta and CF1-gamma, as well as the synthesis of the large and small carboxylase subunits, are strictly coordinated during chloroplast biogenesis in Spirodela oligorhiza.

Adenosine Triphosphatases↗
PubMed1983

The nucleotide sequences of the regions flanking the genes coding for 23S, 16S and 4.5S ribosomal RNA on chloroplast DNA from Spirodela oligorhiza.

The nucleotide sequences of the flanking regions of the genes coding for Spirodela oligorhiza chloroplast ribosomal RNA's have been determined. We have compared these sequences to the corresponding ones in chloroplast DNA of other plants and of E. coli and find a striking sequential or structural homology. The region 5'-proximal to the gene coding for 16S rRNA contains a gene coding for tRNAval, which is transcribed from the same strand. In this area three prokaryotic promoter motifs are found: one located in front of the tRNAval gene and two in the intergenic space between this gene and the 16S rRNA gene. The middle one is used for the start of the transcription of the large ribosomal RNA precursor.

Base Sequence↗
PubMed1981

RNA synthesis in isolated yeast mitochondria.

Isolated yeast mitochondria incorporate added UTP into RNA. Amongst the products formed are the two rRNAs, 4S RNA and several components presumed to be mRNAs. In omega+ strains (containing an intervening sequence in the 21S rRNA gene) besides mature 21S rRNA a transcript could be detected still containing nucleotide sequences transcribed from this intervening sequence. In omega- strains (not containing this intervening sequence) also a longer form of the 21S rRNA could be observed. These results suggest that isolated yeast mitochondria are capable of carrying out RNA synthesis and processing, including splicing.

DNA, Mitochondrial↗
PubMed1980

Mutations affecting RNA splicing and the interaction of gene expression of the yeast mitochondrial loci cob and oxi-3.

In Saccharomyces cerevisiae strains KL14-4A and 777-3A, four intervening sequences of 1900 (l alpha beta), 1400 (l beta gamma), 1300 (l gamma delta) and 650 bp (l delta epsilon) separate the five coding sequences (alpha-epsilon) of the structural gene (cob) for cytochrome b. Its major transcript is an 18S RNA (2200 nucleotides) which is likely to be the functional mRNA. The lengths of a series of larger transcripts and their hybridization with probes specific for different intervening sequences are consistent with their being intermediates in a splicing process which generates 18S RNA from a giant primary transcript (greater than or equal to 7.5 kb) covering the whole cob region. There is no absolute order of splicing. The intervening sequence l alpha beta is excised in two stages. The first generates a stable 10S RNA, coded for by sequences immediately downstream of the 18S RNA coding segment alpha. The function of this RNA is unknown. Its excision is an early step in processing, whereas excision of the remainder of l alpha beta is a late event. We have studied four cytochrome b-deficient mutants. These map in intervening sequences and are splicing-defective. They accumulate 22S-28S RNAs which contain one or more intervening sequences. The l alpha beta mutants synthesize long, novel polypeptides, antigenically related to cytochrome b, possibly as a result of read-through into the intervening sequences. Several cob mutants also display alterations in their transcripts of oxi-3, the locus which codes for cytochrome c oxidase subunit I. This indicates that interactions between cob and oxi-3 exist at the level of RNA processing.

Cytochromes↗
PubMed1979

Transcription maps of mtDNAs of two strains of saccharomyces: transcription of strain-specific insertions; Complex RNA maturation and splicing.

We have developed a two-dimensional method for simultaneously mapping on the yeast mtDNA genome all the transcripts representing more than 0.01% of mtRNA. In two yeast strains, Saccharomyces carlsbergensis NCYC-74 and Saccharomyces cerevisiae KL14-4A, about 25 discrete transcripts were found apart from tRNAs. The mtDNAs of these strains differ by the absence (NCYC-74) or presence (KL 14-4A) of various large insertions located within genetically active regions. The transcripts can all be related to known loci on the genetic map. In nearly all cases the RNAs are much longer than required to specify the known protein product of the locus concerned. The organization of the transcripts is similar in the two strains except at the positions of the large insertions (500-3300 bp) in the oxi-3 and cob loci. The sequences of these insertions are present in RNA species larger than 25S, but are absent from smaller transcripts of the same regions. This is probably due to splicing, since the coding sequences for most of these smaller transcripts are noncontiguous. The smaller transcripts of other loci also seem to arise from processing of larger RNA species. The oxi-3 locus, containing the structural gene for cytochrome c oxidase subunit l, is transcribed in a very complex fashion that suggests differential splicing into partially overlapping transcripts. This may indicate that oxi-3 has additional genetic functions, including possible control of the biosynthesis of cytochrome c oxidase holoenzyme or its assembly into the mitochondrial inner membrane. As in the case of the eucaryote nucleus, the regulation of mitochondrial gene expression seems to occur more at the level of RNA processing than has been recognized thus far.

Chromosome Mapping↗
PubMed1979

The identification of apocytochrome b as a mitochondrial gene product and immunological evidence for altered apocytochrome b in yeast strains having mutations in the COB region of mitochondrial DNA.

The yeast mitochondrial translation product of Mr 30 000 is identical with apocytochrome b. After labelling in vivo with [35S]sulphate in the presence of cycloheximide, the radioactivity in this product present in solubilized submitochondrial particles, was completely recovered in pure cytochrome bc1 complex as a single polypeptide. We show that this translation product is identical with apocytochrome b using peptide mapping by limited proteolysis according to Cleveland et al. [J. Biol. Chem. 250 (1977) 8236-8242] and by immunoprecipitation with a specific antiserum against apocytochrome b. New mitochondrial translation products in 36 strains of Saccharomyces cerevisiae having mutations in the COB region of the mitochondrial DNA, are precipitated by this antiserum. This is consistent with the assumption that many of the cob mutations are localized in the structural gene for apolcytochrome b on mitochondrial DNA. Mutations in two intervening sequences can give rise to products related to apocytochrome b that are considerably longer than normal apocytochrome b. We discuss the hypothesis that in these mutants splicing of the messenger RNA does not occur correctly and that, as a consequence of this, ribosomes read through in an intervening sequence.

Apoproteins↗
PubMed1978

Electrophoretic behaviour of yeast mitochondrial translation products.

We have studied the mobility of yeast mitochondrial translation products during electrophoresis on polyacrylamide gels of different composition and found that these polypeptides can be divided into two groups. One, to which subunit II of cytochrome c oxidase belongs, behaves normal as all water-soluble reference proteins. The other, to which cytochrome b and subunits I and III of cytochrome c oxidase belong, shows a free electrophoretic mobility about twice as fast as the first group. Conditions have been found to separate cytochrome c1 from cytochrome b.

Cytochromes↗
PubMed1977

Fine structure physical mapping of 4S RNA genes on mitochondrial DNA of Saccharomyces cerevisiae.

We have localized the genes for mitochondrial 4S RNA on the physical map of the mtDNA of several Saccharomyces cerevisiae strains by hybridization of iodinated 4S RNA to the restriction fragments obtained with endonucleases HindII + III, EcoRI and HapII. The data indicate that 5-8 of the 4S RNA genes are dispersed over a large area of the genome whereas the rest (about 18 genes) is located within an area of about 9000 bp in length (about 18 genes) is located within an area of about 9000 bp in length (about 12% of the genome) between the markers for chloramphenicol and paromomycin resistance (RIB 1 and PAR 1 loci). Within this region a cluster is present of 5 genes on a DNA fragment of 460 bp.

DNA↗
PubMed1976

The cytochrome bc) complex of yeast mitochondria. Site of translation of the polypeptides in vivo.

1. Yeast cells were labelled with radioactive amino acids in the presence of cycloheximide and the cytochrome bc1 complex was isolated from them as described in the preceding paper (Katan, M.B.., Pool, L. & Groot, G.S.P. (1976)Eur. J. Biochem, 65, 95-105). After analysis of this preparation by sodium dodecylsulphate polyacrylamide gel electrophoresis only one band, with an apparent Mr of 32000, was found to have incorporated radioactivity. The amount of label in the band was low, but could be increased approximately 5-fold by preincubating the cells in erythromycin before the labelling period. 2. Cells were labelled in the presence of chloramphenicol and the cytochrome bc1 complex was isolated by (NH4)2SO4 fractionation. Upon electrophoresis in the presence of sodium dodecylsulphate only four of the six bands that belong to the complex were found to have incorporated radioactivity; no radioactivity was found in the bands with an Mr of 40000 and 17000. The same result was obtained after labelling in the presence of acriflavin. If, however, the cytochrome bc1 complex was isolated by immunoprecipitation, all bands were found to have incorporated radioactivity in the presence of chloramphenicol. The amount of radioactivity in the Mr 32000 band was now clearly depressed. 3. It is concluded that of the seven polypeptides of the cytochrome bc1 complex of yeast only one is made on mitochondrial ribosomes. This polypeptide has an Mr of 32000 and is probably associated with cytochrome b.

Cycloheximide↗