Biomedical subjects

P Sloof

Publications and source records attributed to P Sloof.

35 records · Page 2Linked to original sources

PubMed1987

Evolution of the mitochondrial protein synthetic machinery.

Comparative analysis of the components of the mitochondrial translational apparatus reveals a remarkable variability. For example the mitochondrial ribosomal rRNAs, display a three-fold difference in size in different organisms as a result of insertions or deletions, which affect specific areas of the rRNA molecule. This suggests that such areas are either not essential for mitoribosome function or that they can be replaced by proteins. Also mitochondrial tRNAs and mitoribosomal proteins are much less conserved than their cytoplasmic counterparts. Not only do the mitochondrial translational molecules vary in properties, also the location of the genes from which they are derived is not the same in all cases: mitochondrial tRNA genes which usually are found in the mtDNA, may have a nuclear location in protozoa and, conversely, only in fungi one finds a mitoribosomal protein gene in the organellar genome. The high rate of change of the components of the mitochondrial protein synthesizing machinery is accompanied by a number of unique features of the translation process: (i) the mitochondrial genetic code differs substantially from the standard code in a species-specific manner; (ii) special codon-anticodon recognition rules are followed; (iii) unusual mechanisms of translational initiation may exist. These observations suggest that the evolutionary pressures that have shaped the present day mitochondrial translational apparatus have been different in different organisms and also distinct from those acting on the cytoplasmic machinery. In spite of the interspecies variability, however, many features of the mitochondrial and bacterial protein synthetic apparatus show a clear resemblance, providing support for the hypothesis of a prokaryotic endosymbiont ancestry of mitochondria.

Animals↗

PubMed1986

Major transcript of the frameshifted coxII gene from trypanosome mitochondria contains four nucleotides that are not encoded in the DNA.

The mitochondrial cytochrome oxidase (cox) subunit II gene from trypanosomes contains a frameshift at amino acid 170. This gene is highly conserved in different trypanosome species, suggesting that it is functional. Sequence determination of coxII transcripts of T. brucei and C. fasciculata reveals four extra, reading frame-restoring nucleotides at the frameshift position that are not encoded in the DNA. Southern blot analysis of DNA of both trypanosome species failed to show the existence of a second version of the coxII gene. We conclude, therefore, that the extra nucleotides are added during or after transcription of the frameshift gene by an RNA-editing process.

Animals↗

PubMed1985

Further characterization of the extremely small mitochondrial ribosomal RNAs from trypanosomes: a detailed comparison of the 9S and 12S RNAs from Crithidia fasciculata and Trypanosoma brucei with rRNAs from other organisms.

We have determined the nucleotide sequence of a maxi-circle segment from the insect trypanosome Crithidia fasciculata mitochondrial DNA, on which the genes for the major maxicircle transcripts of 9S and 12S are localized. The 5'-terminal sequences of these RNAs were determined by wandering spot analysis. The map coordinates of the 9S and 12S RNAs from Trypanosoma brucei were adjusted with respect to a previous report with the aid of primer extension analysis with reverse transcriptase. This approach allowed us to align the corresponding genes from both organisms which show an overall sequence homology of 77%. The 9S and 12S RNA genes from the two trypanosome species contain sequences, closely related to some of the regions that are universally conserved among ribosomal RNAs from members of the three primary kingdoms and their organelles, even though the overall level of sequence homology is extremely low. These universal sequences occur at positions in the 9S and 12S RNAs that are analogous to those occupied by their counterparts in authentic ribosomal RNAs. The characteristic secondary structure elements flanking these universal sequences in genuine ribosomal RNAs can also be formed in the trypanosomal 9S and 12S RNAs. These results provide unequivocal evidence for a ribosomal function of the 9S and 12S RNAs of trypanosomal mitochondria, notwithstanding their extremely small size (estimated to be 612 and 1141 nucleotides in C. fasciculata, 611 and 1150 nucleotides in T. brucei) and their unusual base composition (83% A+U).

Animals↗

PubMed1984

The sequence of the gene for cytochrome c oxidase subunit I, a frameshift containing gene for cytochrome c oxidase subunit II and seven unassigned reading frames in Trypanosoma brucei mitochondrial maxi-circle DNA.

A 9.2 kb segment of the maxi-circle of Trypanosoma brucei mitochondrial DNA contains the genes for cytochrome c oxidase subunits I and II (coxI and coxII) and seven Unassigned Reading Frames ("URFs"). The genes for coxI and coxII display considerable homology at the aminoacid level (38 and 25%, respectively) to the corresponding genes in fungal and mammalian mtDNA, the only striking point of divergence being an unusually high cysteine content (about 4.5%). The reading frame coding for cytochrome c oxidase subunit II is discontinuous: the C-terminal portion of about 40 aminoacids, is present in the DNA-sequence in a -1 reading frame with respect to the N-terminal moiety. URF5, 8 and 10, show a low but distinct homology (about 20%) to mammalian mitochondrial URF-1, 4 and 5, respectively. In URF5, the first AUG is found at codon 145, whereas extensive homology to mammalian URF-1 sequences occurs upstream of this position. The possibility exists that UUG can serve as an initiator codon. URF7 and URF9 have a highly unusual aminoacid composition and do not possess AUG or UUG initiator codons. These URFs probably do not have a protein-coding function. The segment does not contain conventional tRNA genes.

Amino Acid Sequence↗

PubMed1983

Size fractionation of Trypanosoma brucei DNA: localization of the 177-bp repeat satellite DNA and a variant surface glycoprotein gene in a mini-chromosomal DNA fraction.

We have size-fractionated intact DNA from Trypanosoma brucei into a major large DNA fraction (greater than 350S) and minor middle-sized (60-250S) and small (less than 60S) DNA fractions. Large DNA contains the rRNA genes, the basic copy genes for several variant surface glycoproteins (VSGs), including one which lies near a telomer, and the expression-linked copies of the two VSG genes. The middle-sized DNA contains at least one VSG gene, but the hybridization of this fraction with probes for the conserved repetitive sequences that mark the edges of the transposed segments of VSG genes, suggests that it may contain many VSG genes. The 177-bp repeat satellite DNA is also exclusively found in this fraction.

Animals↗

PubMed1983

Characterization of satellite DNA in Trypanosoma brucei and Trypanosoma cruzi.

We have determined the properties of the simple-sequence satellite DNAs from two protozoa, Trypanosoma brucei and Trypanosoma cruzi. The T. brucei satellite DNA contains 29 mol% guanine plus cytosine and is made up of long tandem arrays of a 177 base-pair repeat. Sequence heterogeneity in these repeats is limited and restricted to certain positions as shown by sequence analysis, restriction enzyme digestion and two-dimensional analysis of nucleotides bordering the AluI and HhaI recognition sites in the repeat. The repeat contains two copies of a 19 base-pair sequence differing by a single base-pair substitution and several additional copies of part of this sequence. Sequence variants of the repeat are clustered in the DNA. Satellite DNA is not detectably linked to other DNA and no transcripts of this DNA are found in T. brucei. The T. cruzi satellite DNA repeat is 196 base-pairs long and contains 53 mol% guanine plus cytosine. Direct repetitions longer than eight base-pairs were not observed in the nucleotide sequence of this repeat. The nucleotide sequences of the satellites of T. brucei and T. cruzi are not related. In cell fractionation experiments, the T. brucei and T. cruzi satellite DNAs were recovered from the nuclear fraction. Micrococcal nuclease digestion of nuclear fractions yielded 193 and 197 base-pair nucleosomal oligomers in T. brucei and T. cruzi, respectively; these oligomers contained satellite DNA but not the extranuclear kinetoplast DNA. The 193 base-pair nucleosomal repeat of T. brucei is significantly different from the 177 base-pair satellite repeat. Satellite and nucleosomal repeats are, therefore, not in phase in T. brucei. These satellite DNAs are the first to be observed in protozoa, and we conclude that their properties are similar to those of satellites from animals or plants.

Animals↗

PubMed1983

Activation of the genes for variant surface glycoproteins 117 and 118 in Trypanosoma brucei.

We have studied the activation of genes for VSGs (variant surface glycoproteins) in Trypanosoma brucei (strain 427) in six independently isolated trypanosome clones; four expressing the gene for VSG 118 and two the gene for VSG 117. In all cases, gene activation is brought about by a duplicative transposition of the gene to an expression site located close to the end of a chromosome. The DNA segments flanking the expression-linked extra gene copy are nearly devoid of restriction enzyme recognition sites and their lengths vary by more than 10,000 base-pairs among different variants. From the correspondence of five upstream restriction sites, we conclude that the same expression site is used in each case. The transposition event does not lead to detectable alterations in the sequence coding for the mature protein. All restriction enzyme recognition sites detected in the basic copy gene are present also in each of the expression-linked copies. This argues against the introduction of mutations by an error-prone polymerase during the synthesis of the expression-linked copy. In five of the six variants, the 3' end of the VSG messenger RNA differs from that of the corresponding basic copy gene by multiple point mutations, insertions and deletions, starting at positions varying from 16 nucleotides upstream to 113 downstream of the last codon of the mature protein. We attribute this end alteration to the recombination process that introduces the gene into the expression site. We confirm that the expression-linked gene copy is more sensitive to DNase I than the corresponding basic copy gene. This appears to be due to its activated state and not to its location near the end of a chromosome, because another basic copy VSG gene permanently located near a chromosome end is not hypersensitive to DNase I. The mature transcripts of the 117 and 118 genes all possess the same 35 nucleotides at their 5' ends and these are not encoded contiguously in the basic gene copies with the remainder of the mRNAs. This extends our previous conclusion, that mature VSG mRNAs are formed by a splicing process in which the 35-nucleotide sequence encoded in the expression site is fused onto the body of the mRNA contributed by the transposed gene.

Animals↗

PubMed1983

Folding of prokaryotic DNA. Isolation and characterization of nucleoids from Bacillus licheniformis.

Intact and fast-sedimenting nucleoids of Bacillus licheniformis were isolated under low-salt conditions and without addition of detergents, polyamines or Mg2+. These nucleoids were partially unfolded by treatment with RNase and completely unfolded by treatments that disrupt protein-DNA interactions, like incubation with proteinase K, 0.1% sodium dodecyl sulphate and high ionic strength. Ethidium bromide intercalation studies on RNase-treated, proteinase-K-treated and non-treated nucleoids in combination with sedimentation analysis of DNase-I-treated nucleoids revealed that DNA is organized in independent, negatively supertwisted domains. In contrast to the DNA organization in bacterial nucleoids, isolated under high-salt conditions and in the presence of detergents (Stonington & Pettijohn, 1971; Worcel & Burgi, 1972), the domains of supertwisted DNA in the low-salt-isolated nucleoids studied here are restrained by protein-DNA interactions. A major role for nascent RNA in restraining supertwisted DNA was not observed. The superhelix density of B. licheniformis nucleoids calculated from the change of the sedimentation coefficient upon ethidium bromide intercalation, was of the same order of magnitude as that of other bacterial nucleoids and eukaryotic chromosomes, isolated under high-salt conditions: namely, -0.150 (corrected to standard conditions: 0.2 M-NaCl, 37 degrees C; Bauer, 1978). Electron microscopy of spread nucleoids showed relaxed DNA and regions of condensed DNA. Spreading in the presence of 100 micrograms ethidium bromide per ml revealed only condensed structures, indicating that nucleoids are intact. From spreadings of proteinase-K-treated nucleoids we infer that supertwisted DNA and the protein-DNA interactions, responsible for restraining the superhelical DNA conformation, are localized in the regions of condensed DNA.

Bacillus↗

PubMed1978

RNA-RNA interactions in the binding site of protein L24 on 23S ribosomal RNA of Escherichia coli: 1. Evidence for their occurrence between widely separated sequence regions.

Protein L24, from the Escherichia coli ribosome, protects a large region of 23S RNA against ribonuclease digestion. This protected RNA consists of a series of non-contiguous subfragments encompassing about 480 nucleotides at the 5' -end of 23S RNA (1). The present work demonstrates that this RNA moiety remains intact, after removal of protein L24, indicating that the subfragments are maintained together by RNA-RNA interactions. Using a urea washing procedure, the weakly bound RNA subfragments were selectively removed leaving more strongly interacting subfragments that were identified, by gel electrophoresis and oligonucleotide fingerprinting, and shown to derive from widely separated sequence regions.

Base Sequence↗

PubMed1978

The binding site of ribosomal protein L1 from Escherichia coli on the 23-S ribosomal RNA from Bacillus stearothermophilus. A possible base-pairing scheme differing from that proposed for Escherichia coli.

The region of Bacillus stearothermophilus strain NCA 1503 23-S ribosomal RNA protected from T1 ribonuclease digestion by the 50-S ribosomal subunit protein L1 from Escherichia coli has been established. The sequence of 115 nucleotides is compared to the analogous region in E. coli. The similar behaviour of the RNA towards the recognition of protein L1 may be explained in terms of secondary base-pairing, even though there exists almost 40% difference between the primary nucleotide sequences.

Base Sequence↗

PubMed1976

The binding site of protein L1 on 23-S ribosomal RNA of Escherichia coli. 1. Isolation and characterization.

Ribonucleoproteins were prepared by ribonuclease digestion of a reconstitued complex of ribosomal protein L 1 and 23-S RNA from Escherichia coli. Three main ribonucleoproteins were identified. The largest was only obtained in an impure state at low ribonuclease concentration, whereas the two smaller ones, which were difficult to separate from one another electrophoretically, were stable over a range of enzyme concentrations. The two smaller ribonucleoproteins yielded a total of 13 RNA subfragments that were judged to be homogeneous electrophoretically. The latter were characterized for molecular weight and the subfragment composition of each of these ribonucleoproteins was established. Furthermore, the subfragments were shown to be maintained together in each ribonucleoprotein by RNA-RNA interactions. The primary and specific binding site of protein L1 was localized on one continuous RNA subfragment of about 110 nucleotides in length by two newly developed binding methods.

Binding Sites↗

PubMed1976

The binding site of protein L1 ON 23-S ribosomal RNA of Escherichia coli. 2. Identification of the rna region contained in the L1 ribonucleoproteins and determination of the order of the RNA subfragments within this region.

Ribonucleoproteins were obtained by T1 ribonuclease digestion of reconstitued complexes of ribosomal protein L1 AND 23-S RNA from Escherichia coli. The RNA region of the main ribonucleoprotein 2 was totally digested with T1 ribonuclease. The oligonucleotide products were characterised and they showed that this region comprises 148 nucleotides located between the 550th and 1000th necleotides from the 3' end of the 23-S RNA. Of the other two ribonucleoproteins, the largest ribonucleoprotein 1 contained an extra RNA sequence, of at least 15 nucleotides, that was located at the 5' end of the RNA region. The smallest ribonucleoprotein 3 lacked an RNA section towards the 3' end of the region. The order of the RNA subfragments and the enzymic cutting positions in the whole RNA region are given for the ribonucleoproteins. It is shown that protein L1 most strongly protects a continuous section of 115 nucleotides at the 5' end of the main RNA region. Finally, evidence is presented for a methylated base, and for two sequence heterogeneities, in this region of the 23-S RNA.

Alkaline Phosphatase↗

PubMed1976

An investigation of the binding sites of proteins S8, L23 and L24 on the ribosomal RNAs of Escherichia coli by electron microscopy.

An electron microscopic method was used to investigate the binding regions of proteins S8 on 16S RNA, and proteins L23 and L24 on 23S RNA. Regions of the RNA that were not stabilised by the protein were completely denatured in 80% dimethyl-sulfoxide. The lengths of these denatured RNA regions were compared with that of the whole denatured RNA. Conclusions are drawn concerning the approximate location of the three proteins and these results are correlated with both RNA structural data and RNA sequence data on the RNA binding regions of the proteins.

Bacterial Proteins↗

PubMed1975