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J Klootwijk

Publications and source records attributed to J Klootwijk.

At least 37 records · Page 2Linked to original sources
PubMed1983

Molecular cloning of the rDNA of Saccharomyces rosei and comparison of its transcription initiation region with that of Saccharomyces carlsbergensis.

We have cloned one complete repeating unit of rDNA from Saccharomyces rosei and determined its physical and genetic organization. Heteroduplex analysis of the rDNA units from S. rosei and S. carlsbergensis shows that the nontranscribed spacers are largely nonhomologous in sequence, whereas the transcribed regions are essentially homologous. We also determined the transcription initiation site for the 37S precursor RNA on S. rosei rDNA. Sequence comparison of the region surrounding the site of transcription initiation for the 37S RNA with the corresponding region of S. carlsbergensis revealed extensive homology from position -9 downstream into the external transcribed spacer. Very little homology was observed between position -9 and -55, but some homologous tracts are present upstream from position -55.

Base Sequence↗
PubMed1981

The primary and secondary structure of yeast 26S rRNA.

We present the sequence of the 26S rRNA of the yeast Saccharomyces carlsbergensis as inferred from the gene sequence. The molecule is 3393 nucleotides long and consists of 48% G+C; 30 of the 43 methyl groups can be located in the sequence. Starting from the recently proposed structure of E. coli 23S rRNA (see ref. 25) we constructed a secondary structure model for yeast 26S rRNA. This structure is composed of 7 domains closed by long-range base pairings as n the bacterial counterpart. Most domains show considerable conservation of the overall structure; unpaired regions show extended sequence homology and the base-paired regions contain many compensating base pair changes. The extra length of the yeast molecule is due to a number of insertions in most of the domains, particularly in domain II. Domain VI, which is extremely conserved, is probably part of the ribosomal A site. alpha-Sarcin, which apparently inhibits the EF-1 dependent binding of aminoacyl-tRNA, causes a cleavage between position 3025 and 3026 in a conserved loop structure, just outside domain VI. Nearly all of the located methyl groups, like in E. coli, are present in domain II, V and VI and clustered to a certain extent mainly in regions with a strongly conserved primary structure. The only three methyl groups of 26S rRNA which are introduced relatively late during the processing are found in single stranded loops in domain VI very close to positions which have been shown in E. coli 23S rRNA to be at the interface of the ribosome.

Base Sequence↗
PubMed1981

The nucleotide sequence of the intergenic region between the 5.8S and 26S rRNA genes of the yeast ribosomal RNA operon. Possible implications for the interaction between 5.8S and 26S rRNA and the processing of the primary transcript.

We have determined the nucleotide sequence of part of a cloned yeast ribosomal RNA operon extending from the 5.8S RNA gene downstream into the 5' -terminal region of the 26S RNA gene. We mapped the pertinent processing sites, viz. the 5' end of 26S rRNA and the 3'ends of 5.8S rRNA and its immediate precursor, 7S RNA. At the 3' end of 7S RNA we find the sequence UCGUUU which is very similar to the type I consensus sequence UCAUUA/U present at the 3' ends of 17S, 5.8S and 26S rRNA as well as 18S precursor rRNA in yeast. At the 5' end of the 26S RNA gene we find a sequence of thirteen nucleotides which is homologous to the type II sequence present at the 5' termini of both the 17S and the 5.8S RNA gene. These findings further support the suggestion put forward earlier (G.M. Veldman et al. (1980) Nucl. Acids Res. 8, 2907-2920) that both consensus sequences are involved in the recognition of precursor rRNA by the processing nuclease(s). We discuss a model for the processing of yeast rRNA in which a processing enzyme sequentially recognizes several combinations of a type I and a type II consensus sequence. We also describe the existence of a significant base complementarity between sequences in the 5' -terminal region of 26S rRNA and the 3' -terminal region of 5.8S rRNA. We suggest that base pairing between these sequences contributes to the binding between 5.8S and 26S rRNA.

Base Sequence↗
PubMed1980

The transcription termination site of the ribosomal RNA operon in yeast.

The site at which transcription of the ribosomal RNA operon in yeast is terminated was precisely localized. First, the exact position of the 3' end of the 26S rRNA gene was mapped on the rDNA on the basis of RNA- and DNA sequence data. Next, the 3' terminus of the primary transcript, 37S precursor rRNA, was established by hybridization experiments and sequence analysis. 37S pre-rRNA appears to be just 7 nucleotides longer at its 3' end than 26S rRNA. The non-coding strand around the termination site is extremely T-rich: 15 out of 18 nucleotides are T-residues. An extensive dyad symmetry is present in the sequence downstream from the termination site; a possible role of this structure in the regulation of transcription termination is discussed. The 3'-terminal 110 nucleotides of yeast 26S rRNA have approx. 50% and 60% homology with the corresponding regions of E. coli 23S rRNA and Xenopus laevis 28S rRNA, respectively.

Animals↗
PubMed1980

Some characteristics of processing sites in ribosomal precursor RNA of yeast.

The DNA sequences of the intergenic region between the 17S and 5.8S rRNA genes of the ribosomal RNA operon in yeast has been determined. In this region the 37S ribosomal precursor RNA is specifically cleaved at a number of sites in the course of the maturation process. The exact position of these processing sites has been established by sequence analysis of the terminal fragments of the respective RNA species. There appears to be no significant complementarity between the sequences surrounding the two termini of the 18S secondary precursor RNA nor between those surrounding the two termini of 17S mature rRNA. This finding implies that the processing of yeast 37S ribosomal precursor RNA is not directed by a double-strand specific ribonuclease previously shown to be involved in the processing of E. coli ribosomal precursor RNA [see Refs 1,2]. The processing sites of yeast ribosomal precursor RNA described in the present paper are all flanked at one side by a very [A+T]-rich sequence. In addition, sequence repeats are found around the processing sites in this precursor RNA. Finally, sequence homologies are present at the 3'-termini [6 nucleotides] and the 5'-termini [13 nucleotides] of a number of mature rRNA products and intermediate ribosomal RNA precursors. These structural features are discussed in terms of possible recognition sites for the processing enzymes.

Base Sequence↗
PubMed1979

Pseudouridylation of yeast ribosomal precursor RNA.

The pseudouridylation of ribosomal RNA of Saccharomyces carlsbergensis was investigated with respect to its timing during the maturation of rRNA and its sequence specificity. Analysis of 37-S RNA, the common precursor to 17-S, 5.8-S and 26-S rRNA and most probably the primary ribosomal transcript, shows that this RNA molecule contains already most if not all of the 36-37 pseudouridine residues found in the mature rRNAs. Thus pseudouridylation is, like 2'-0-ribosemethylation, an early event in the maturation of rRNA, taking place immediately after, or even during, transcription. The data presented show that the non-conserved sequences of 37-S precursor rRNA contain very few pseudouridine residues if any. The pseudouridine residues within the rRNA sequences are apparently clustered to a certain degree as can inferred from the occurrence of a single oligonucleotide containing 3 pseudouridines, which was obtained by digestion of 26-S rRNA with ribonuclease T1.

Base Sequence↗
PubMed1979

Extrachromosomal circular ribosomal DNA in the yeast Saccharomyces carlsbergensis.

Purified ribosomal DNA from Saccharomyces carlsbergensis contains a small proportion of circular DNA molecules with a contour length of 3 micron or integral multiples thereof. Hybridization of yeast ribosomal DNA with 26 S rRNA, using the R-loop technique, reveals that these circular molecules contain sequences complementary to yeast ribosomal RNA. We suggest that these extrachromosomal rRNA genes may be intermediates in the amplification of rRNA genes in yeast.

DNA, Circular↗
PubMed1979

The primary transcript of the ribosomal repeating unit in yeast.

Endgroup analysis of 37S ribosomal precursor RNA from Saccharomyces carlsbergensis has revealed that the major 5' endgroup is ppA-Up, with a molar yield of 0.8. This shows that most, if not all, 37S RNA molecules have preserved a transcriptional initiation sequence. Analysis of the 3' terminus of 37S RNA has shown the presence of a uridine rich oligonucleotide, tentatively identified as U6-8-A-NOH. This long stretch of uridines at the 3' end of 37S RNA may represent a transcriptional termination site. The two sets of data on the terminal sequences suggest that 37S ribosomal precursor RNA, if not already spliced, is a primary transcription product. Since the 3' terminus of 26S rRNA, U-U-U-G-UOH., appears to be clearly different from the 3' end of 37S RNA, we conclude that 37S ribosomal precursor RNA contains additional nucleotides 3'-distal to the 26S rRNA sequence.

Base Sequence↗
PubMed1978

Biosynthesis of a hypermodified nucleotide in Saccharomyces carlsbergensis 17S and HeLa-cell 18S ribosomal ribonucleic acid.

The biosynthesis of a hypermodified nucleotide, similar to or identical with 3-(3-amino-3-carboxypropyl)-1-methylpseudouridine monophosphate, present in Saccharomyces carlsbergensis 17S and HeLa-cell 18S rRNA, was investigated with respect to the sequence of reactions required for synthesis and their timing in ribosome maturation. In both yeast and HeLa cells methylation precedes attachment of the 3-amino-3-carboxypropyl group. In yeast the methylated precursor nucleotide was tentatively characterized as 1-methylpseudouridine. This precursor nucleotide was demonstrated in both 37S and most of the cytoplasmic 18S pre-rRNA (rRNA precursor) molecules. The synthesis of the hypermodified nucleotide is completed just before the final cleavage of 18S pre-rRNA to give 17S rRNA, so that the final addition of the 3-amino-3-carboxypropyl group is a cytoplasmic event. Comparable experiments with HeLa cells indicated that formation of 1-methylpseudouridine occurs at the level of 45S RNA and addition of the 3-amino-3-carboxypropyl group occurs in the cytoplasm on newly synthesized 18S RNA.

Base Sequence↗
PubMed1977

Secondary methylation of yeast ribosomal precursor RNA.

The timing of methylation of the ribosomal sequences of ribosomal precursor RNA (pre-rRNA) from the yeast Saccharomyces carlsbergensis was investigated by fingerprint analysis of the methylated oligonucleotides derived from the various precursors. From the total of 37 ribose and 6 base-methyl groups found in 26-S rRNA, the two copies of the base-methylated nucleoside m3U as well as the doubly methylated sequence Um-Gm psi are not yet present in 37-S RNA, the predominant common precursor of 26-S and 17-S rRNA. Introduction of these methyl groups into the ribosomal sequences appears to take place at the level of 29-S pre-rRNA, the immediate precursor to 26-S rRNA. From the total of 18 ribose-methylated and 6 base-methylated nucleosides found in 17-S rRNA, the latter group (one copy of m7G, the m62A-m62A- sequence and the hypermodified methylated nucleoside "mX") is completely missing in 37-S pre-rRNA. The methyl group of m7G is introduced into 18-S pre-rRNA, the direct precursor of 17-S rRNA, in the nucleus. The -m62A-m62A- sequence is methylated after transport of the 18-S pre-rRNA to the cytoplasm prior to the final maturation into 17-S rRNA.

Base Sequence↗
PubMed1977

Cowpea mosaic virus RNAs have neither m7GpppN ... nor mono-, di- or triphosphates at their 5' ends.

32P-labeled cowpea mosaic virus (CPMV) RNAs were analyzed for their 5' termini by three different fingerprinting standard procedures. These analyses showed unequivocally that CPMV RNAs lack a structure like m7GpppN ... at their 5' termini. In addition, neither mono-, di- or triphosphorylated [(p)(p)pN ....] termini were found in the respective fingerprints. The inability to label the 5' termini treatment with gamma-32P-ATP and polynucleotide kinase, either per se or after pretreatment with alkaline phosphatase, strongly suggests the absence of a free 5'-OH and a free 5'-phosphate at the 5' terminal nucleotide in CPMV RNAs. The various analyses also showed that no 2' -O-methylated nucleotides, N6-methyladenosines or pseudo-uridines are present in CPMV RNAs.

Adenosine Triphosphate↗
PubMed1977

Sequence of the 3'-terminal 21 nucleotides of yeast 17S ribosomal RNA.

The sequence of the 3'-terminal 21 nucleotides of 17S ribosomal RNA from the yeast Saccharomyces carlsbergensis has been determined to be (Y)G-m62A-m62A-C-U-C-G-C-G-G-A-A-G-G-A-U-C-A-U-U-AOH. This sequence shows extensive homology with the 3'-terminal sequence of 16S rRNA from Escherichia coli including the presence of the two adjacent N6-,N6-dimethyladenosines observed in the small subunit rRNA of eukaryotes as well as of many prokaryotes.

Base Sequence↗
PubMed1977

Terminal nucleotide sequences of 17-S ribosomal RNA and its immediate precursor 18-S RNA in yeast.

The 5' and 3'-terminal nucleotide sequences of 17-S rRNA and its immediate precursor 18-S RNA from the yeast Saccharomyces carlsbergensis have been analysed. Identification of the terminal oligonucleotides, as present in Ti ribonuclease digests, was performed by diagonal procedures. The major (molar yield 0.9) 5'-terminal oligonucleotide (molar yield 0.15) with the overall composition pU (U2,C2)G was observed. 18-S precursor RNA was found to contain the same 5'-terminal sequences as 17-S rRNA. However, the 3'-terminal sequences of the two types of RNA appeared to be different. The 17-S rRNA yields the oligonucleotide A-U-C-A-U-U-AOH while at least half of the 18-S RNA molecules contain the sequence U-U-U-C-A-A-U-AOH. In addition 18-S RNA yields several minor 3'-terminal oligonucleotides which appear to be structurally related to the major 3'-terminal sequence. These results demonstrate that the extra nucleotides in 18-S RNA relative to 17-S RNA are located exclusively at the 3'-terminus of the 18-S RNA molecule. The possibility that the 3'-terminal nucleotide sequence of 18-S RNA plays a role in the maturation process is discussed.

Base Sequence↗