Biomedical subjects
Publications and source records attributed to R Benne.
The chromosomal location of the human gene coding for peroxisomal 3-oxoacyl-CoA thiolase (ACAA) was determined with the aid of cDNA and genomic probes by screening of rodent x human somatic cell hybrids and in situ hybridization. The results localize the gene to chromosome region 3p22----p23.
Explore the source record for details and available documents.
The sequence of two fragments derived from the variable region of the kinetoplast maxicircle of Trypanosoma brucei has been determined. One fragment (1334 nucleotides, situated immediately upstream of the 12S and 9S ribosomal RNA genes) consists of non-repetitious DNA, which does not hybridize to other maxicircle regions. The other (844 nt, located between 1.7 and 2.55 kb downstream of the NADH-dehydrogenase subunit 5 gene) contains arrays of repetitive sequences which are also found outside this area. Hybridization analysis suggests that approximately 60% of the remaining part of the divergent region, which has not yet been fully analyzed, consists of similar sequences. Neither segment contains genes for mitochondrial proteins or tRNAs, as judged from computer analysis. This conclusion is supported by the fact that maxicircle DNA of trypanosome species other than T. brucei does not cross-hybridize to either fragment. Northern blot analysis and S1 nuclease experiments demonstrate, however, that both maxicircle regions are transcribed into RNAs of varying length (100-3000 nt), albeit at a low level. The function of these transcripts, that are derived from both DNA strands, and the likely absence of protein and tRNA genes from the variable region of the T. brucei maxicircle is discussed.
In trypanosome mitochondria an RNA editing process is operative, which co- or post-transcriptionally alters the nucleotide sequence of transcripts by insertion and/or deletion of U residues at specific sites. To increase our understanding of the mechanism of this process we have compared the nucleotide sequence of the frameshifted mitochondrial MURF3 gene from Crithidia fasciculata to that of a large number of MURF3 cDNAs. We found cDNAs derived from transcripts edited at two different sites in the protein coding sequence: (i) at the frameshift position five extra U residues connect the two reading frames and (ii) at the 5' terminus 22 inserted Us shift a putative initiator codon out of phase. The collection also contained cDNAs that were derived from non-edited transcripts. Partially edited sequences were not found, except in one cDNA, which contained an edited frameshift site in combination with a non-edited 5' terminus. The analysis further showed that MURF3 transcripts have a 3'-terminal poly(AU) extension, which varies in sequence. The implications of these results are discussed.
Explore the source record for details and available documents.
A 3.2 kb segment of the maxicircle of Crithidia fasciculata mitochondrial (mt) DNA contains the gene for cytochrome oxidase subunit III (coxIII), the N-terminal portion of the gene for apocytochrome b (cytb) and two partially overlapping Unassigned Reading Frames (C.URF2/1). Transcript analysis of the segment reveals that both the coxIII gene and the C.URF2/1 area are transcribed into a pair of RNA products. With the C. fasciculata gene version as a probe, a coxIII gene could not be detected in nuclear and mtDNA of Trypanosoma brucei, indicating that the cytochrome oxidases of these two closely related trypanosome species may differ. The nucleotide homology in the N-terminal region of the coxIII and cytb genes in T. brucei, Leishmania tarentolae and C. fasciculata starts at a UUA leucine codon, which adds further support to the hypothesis that apart from AUG, other initiator triplets are used in trypanosomal mitochondria: UUG, CUG and UUA, all triplets coding for leucine in the universal code. Finally, the possibility is discussed that the two overlapping URFs (C.URF2/1) in fact represent a single, frameshift containing, gene.
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.
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.
eIF-2 purified from neuroblastoma cells consists of three subunits, which appear to be of molecular weight identical to those of the subunits of rabbit reticulocyte eIF-2. A protein fraction has been isolated from neuroblastoma cells with characteristics similar to eRF from reticulocytes: stimulation of amino acid incorporation in a hemin-deprived reticulocyte lysate, the removal of GDP from eIF-2-GDP complexes, a 4-5-fold stimulatory effect in a two-step reaction measuring 40 S preinitiation complex formation and a 3-3.5-fold stimulation in the methionyl-puromycin synthesis. In the methionyl-puromycin-synthesizing system phosphorylated eIF-2 is not responsive to the addition of this fraction from neuroblastoma cells. The protein fraction contains eRF which seems to be similar to the eRF isolated from Ehrlich ascites tumor cells and somewhat distinct from the reticulocyte factor. Incubation of neuroblastoma cell lysate in the presence of [gamma-32P]ATP results in the phosphorylation of a protein of Mr 36 000, migrating on SDS-polyacrylamide gels to the position of eIF-2 alpha. This protein is also phosphorylated in vitro by HRI from reticulocytes. These results may reflect a common underlying principle for the quantitative regulation of protein synthesis in eukaryotic cells.
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).
The rate of initiation of protein synthesis appears to be controlled at the level of recycling of eIF-2. In this process a new factor, designated eRF, plays an important role. The factor has been purified from the post-ribosomal supernatant and has been called formerly anti-HRI and anti-inhibitor [Amesz, H., Goumans, H., Haubrich-Morree, Th., Voorma, H.O., and Benne, R. (1979) Eur. J. Biochem. 98, 513-520]. Its effect on the initiation of protein synthesis has been studied in several assays: a small but distinct effect is found in the assay for the formation of a ternary complex between eIF-2, GTP and Met-tRNA; a 4-5-fold stimulation is obtained in assays for 40S preinitiation complex formation and in the methionyl-puromycin reaction. In the latter assay a catalytic use of eIF-2 occurs provided that eRF is present. eRF forms a complex with eIF-2 which results in a decrease of the affinity of eIF-2 for GDP, giving it the properties of a GDP/GTP exchange factor. The model stresses the catalytic use of eIF-2 in initiation provided that conditions are met for GDP/GTP exchange by a transient complex formation between eIF-2 and eRF. On the other hand, it is shown that phosphorylation of eIF-2 by the hemin-regulated inhibitor (HRI) abolishes the recycling of eIF-2, by the formation of another stable complex comprising eIF-2 alpha P, GDP and eRF.
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.
From ribosomal washes of neuroblastoma cells infected with Semliki Forest virus (SFV) a protein of Mr 33000 was purified, which comigrated with the viral capsid protein on sodium dodecyl sulfate/polyacrylamide gels and was recognized by antibodies against the capsid protein of SFV. This protein selectively inhibits the translation of host and early viral 42S mRNA in vitro, but has no effect on late viral 26S and encephalomyocarditis virus mRNA translation. Eukaryotic initiation factor 4B and cap-binding protein restore the translation of host and 42S mRNA to control levels. The capsid protein specifically prevents the binding of host mRNA into 80S initiation complexes, but has no effect on that of late viral mRNA. We propose that the capsid protein is the component responsible for the shut-off of host protein synthesis in SFV-infected cells and for the decreased translational activity of the crude ribosomal washes from these cells.
The phosphoenolpyruvate:glucose phosphotransferase system (PTS) of Salmonella typhimurium is involved both in glucose transport and in the regulation and synthesis of adenylate cyclase and several transport systems. The crr gene has been implicated in this regulating mechanism. A 9.6-kb segment of the S. typhimurium chromosome containing the crr gene was cloned in pAT153. The cloned fragment also complemented cysA mutations but did not contain a functional pts operon which is closely linked to the crr gene and codes for two enzymes of the PTS. Although cysA and crr have been reported to be located on opposite sides of ptsHI, our results suggest that the correct gene order is cysK-ptsHI-crr-cysA. Expression of crr plasmids in a maxicell system yielded two proteins which reacted with specific anti-serum against IIIGlc. The apparent mol. wts. in SDS-polyacrylamide gels were 20 000 and 21 000, the former corresponding to the major band of purified IIIGlc. Both forms were also observed in bacterial extracts and purified IIIGlc. The crr gene was localized on a 1-kb EcoRI-EcoRV fragment of the 9.6-kb insert and sequenced. It codes for a single protein (18 556 D) containing 169 amino acid residues and identified as IIIGlc.
The nucleotide sequence of a 2.5-kb segment of the maxi-circle of Trypanosoma brucei mtDNA has been determined. The segment contains the gene for apocytochrome b, which displays about 25% homology at the amino acid level to the apocytochrome b gene from fungal and mammalian mtDNAs. Northern blot and S1 nuclease analyses have yielded accurate map positions of an RNA species in an area that coincides with the reading frame. The segment also contains two pairs of overlapping unassigned reading frames, which lack homology with any known mitochondrial gene or URF. The DNA sequence in these areas is AG-rich (70%), resulting in URFs with an unusually high level of glycine and charged amino acids (60%). They may not encode proteins, in spite of their size and the fact that abundant transcripts are mapped in these areas.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
Explore the source record for details and available documents.