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H T Jacobs

Publications and source records attributed to H T Jacobs.

At least 37 records · Page 2Linked to original sources
PubMed1999

Heteroplasmic segregation associated with trisomy-9 in cultured human cells.

In cybrid cells carrying the mitochondrial A3243G MELAS mutation, which were also heteroplasmic for the G12300A suppressor mutation, we observed a transient episode of heteroplasmic instability, resulting in a wide diversification in G12300A heteroplasmy levels and a shift in the average heteroplasmy level from 11 to 29%. These cells were found to be trisomic for chromosome 9, whereas a minority of cells that retained disomy-9 showed no instability. Coculture experiments implied that trisomy-9 cells exhibited a significant growth advantage, but neither heteroplasmy levels, respiratory phenotype nor trisomy-9 itself had direct selective value under standard culture conditions. Mitochondrial nucleoid number was the same (50-100) in cells that had or had not experienced transient heteroplasmic instability, but 1-2 orders of magnitude less than the segregation number in such cells. These findings support the idea that mtDNA partition is under nuclear genetic control, and implicate a locus on chromosome 9 in this regulation.

Cells, Cultured↗
PubMed1999

Modelling in Escherichia coli of mutations in mitoribosomal protein S12: novel mutant phenotypes of rpsL.

The rpsL gene of Escherichia coli encodes the highly conserved rps12 protein of the ribosomal accuracy centre. We have used the E. coli gene to model the phenotypic effects of specific substitutions found in the mitochondrial gene for rps12. Variants created by in vitro mutagenesis were tested in two different plasmid vector systems, in both streptomycin-sensitive and streptomycin-resistant hosts. A substitution with respect to eubacterial rps12 (K87-->Q), found in all metazoan and fungal mitochondrial orthologues thus far studied, is associated with low-level resistance to streptomycin and a modest (15%) drop in translational elongation rate, but without significant effects on translational accuracy. An amino-acid replacement at a highly conserved leucine residue (L56-->H), associated with the phenotype of sensitivity to mechanical vibration and hemizygous female lethality in Drosophila, creates a functionally inactive but structurally stable protein that is not assembled into ribosomes. The presence in the cell of the mutant, but not wild-type, rpsL greatly downregulates the level of a prominent polypeptide of approximately 50 kDa. These results indicate novel structure-function relationships in rps12 genes affecting translational function, ribosome assembly and drug sensitivity, and indicate a novel regulatory pathway that may influence ribosome biogenesis.

Cell Division↗
PubMed1999

Molecular phenotype of the np 7472 deafness-associated mitochondrial mutation in osteosarcoma cell cybrids.

The nucleotide pair (np) 7472 insC mitochondrial DNA mutation in the tRNA(Ser)(UCN) gene is associated with sensorineural deafness, combined in some individuals with a wider syndrome including ataxia and myo-clonus. Previous studies in osteosarcoma cell cybrids revealed only a mild respiratory defect linked to the mutation. We have investigated the biochemical and molecular consequences of the mutation, using a panel of seven osteosarcoma cell cybrids containing 100% mutant mtDNA, plus two cybrids carrying 100% wild-type mtDNA from the same patient. The mutation is associated with a mild growth deficit in selective (galactose) medium that is only significant in combination with a reduced mtDNA copy number, suggesting a mechanism that might modulate clinical phenotype. The mutation results in a 65% drop in the steady-state level of tRNA(Ser)(UCN), but causes at most only a very mild and quantitative abnormality of mitochondrial protein synthesis, associated with modest hypersensitivity to doxycyclin. No evidence for a specific defect in aminoacylation was obtained, and unlike the case with the np 7445 mutation, the pattern of RNA processing of light strand transcripts of the ND6 region was not systematically altered. Comparing the np 7472 and np 7445 mutant phenotypes in cultured cells suggests that sensorineural deafness can result from a functional insufficiency of mitochondrial tRNA(Ser)(UCN), to which some cells of the auditory system are especially vulnerable.

Base Sequence↗
PubMed1998

Chromosomal locations of three human nuclear genes (RPSM12, TUFM, and AFG3L1) specifying putative components of the mitochondrial gene expression apparatus.

We have mapped the chromosomal locations of three human nuclear genes for putative components of the apparatus of mitochondrial gene expression, using a combination of in situ hybridization and interspecies hybrid mapping. The genes RPMS12 (mitoribosomal protein S12, a conserved protein component of the mitoribosomal accuracy center), TUFM (mitochondrial elongation factor EF-Tu), and AFG3L1 (similar to the yeast genes Afg3 and Rca1 involved in the turnover of mistranslated or misfolded mtDNA-encoded polypeptides) were initially characterized by a combination of database sequence analysis, PCR, cloning, and DNA sequencing. RPMS12 maps to chromosome 19q13.1, close to the previously mapped gene for autosomal dominant hearing loss DFNA4. The TUFM gene is located on chromosome 16p11.2, with a putative pseudogene or variant (TUFML) located very close to the centromere of chromosome 17. AFG3L1 is located on chromosome 16q24, very close to the telomere. By virtue of their inferred functions in mitochondria, these genes should be regarded as candidates of disorders sharing features with mitochondrial disease syndromes, such as sensorineural deafness, diabetes, and retinopathy.

ATPases Associated with Diverse Cellular Activitie↗
PubMed1998

Familial mitochondrial DNA depletion in liver: haplotype analysis of candidate genes.

Two sons and one daughter of healthy consanguineous parents presented with fatal hepatic failure in association with severe depletion of mitochondrial (mt)DNA in liver; a third son is healthy. Other published cases of mtDNA depletion concern single members of a family, which excludes the use of haplotype analysis. In the family presented here, the inheritance of the genes for mitochondrial transcription factor A (mtTFA), nuclear respiratory factor 1 (NRF-1), mitochondrial single-stranded DNA-binding protein (mtSSBP), and endonuclease G (EndoG) was studied using microsatellite markers linked to these genes. The inheritance of the gene for mtDNA polymerase (pol gamma) was studied using a polymorphic CAG repeat present within the coding region of the gene. EndoG and mtSSBP were excluded, but mtTFA remains a candidate. Pol gamma or NRF-1 involvement would be compatible only with autosomal dominant inheritance. Coding sequence analysis of NRF-1 and mtTFA revealed no novel mutations in affected individuals.

Chromosomes, Human↗
PubMed1998

Expression of the nuclear gene encoding mitochondrial ATP synthase subunit alpha in early development of Drosophila and sea urchin.

Complementary DNAs encoding nuclear-coded mitochondrial ATP synthase subunit alpha of Drosophila melanogaster and Strongylocentrotus purpuratus were obtained by a combination of library screening and redundant PCR. The entire coding sequence of the precursor polypeptide was inferred for both species. Southern blots to genomic DNA indicated that the gene is almost certainly single-copy in both organisms. Northern blots to RNA from staged developmental series showed that ATP synthase subunit alpha mRNA is represented in the egg, declines in abundance during cleavage, and is replenished by zygotic transcription in both species. However, the extent and timing of these changes differ significantly in the two species studied. Nuclear-coded and mitochondrially encoded ATP synthase genes appear to be temporally co-regulated in Drosophila, but not sea urchin development.

Amino Acid Sequence↗
PubMed1998

A tRNA suppressor mutation in human mitochondria.

Mitochondrial mutations are associated with a wide spectrum of human diseases. A common class of point mutations affects tRNA genes, and mutations in the tRNA-leu(UUR) gene (MTTL1) are the most frequently detected. In earlier studies, we showed that lung carcinoma cybrid cells containing high levels (greater than 95%) of mutated mtDNA from a patient with the pathological nucleotide pair (np) 3243 tRNA-leu(UUR) mutation can remain genotypically stable over time, and exhibit severe defects in mitochondrial respiratory metabolism. From such a cybrid containing 99% mutated mtDNA, we have isolated a spontaneous derivative that retains mutant mtDNA at this level but which has nevertheless reverted to the wild-type phenotype, based on studies of respiration, growth in selective media, mitochondrial protein synthesis and biogenesis of mitochondrial membrane complexes. The cells are heteroplasmic for a novel anticodon mutation in tRNA-leu(CUN) at np 12300, predicted to generate a suppressor tRNA capable of decoding UUR leucine codons. The suppressor mutation represents approximately 10% of the total mtDNA, but was undetectable in a muscle biopsy sample taken from the original patient or in the parental cybrid. These results indicate that the primary biochemical defect in cells with high levels of np 3243 mutated mtDNA is the inability to translate UUR leucine codons.

Anticodon↗
PubMed1998

Mitochondrial tRNALeu isoforms in lung carcinoma cybrid cells containing the np 3243 mtDNA mutation.

We have investigated the representation of structural isoforms of the two mitochondrial leucyl tRNAs in lung carcinoma cybrid cell lines containing the np 3243 (MELAS) mtDNA mutation, alone or in combination with the np 12300 suppressor mutation. The mutant tRNALeu(UUR) is aminoacylated very poorly or not at all, whereas the suppressor tRNALeu(CUN) is efficiently aminoacylated. Deacylated mitochondrial tRNALeu(CUN) is present, in all human cells tested, in two structural isoforms that are separable on denaturing gels, indicating a difference in primary structure. The ratio of the two isoforms differs between cell types and is strongly biased towards one isoform in lung carcinoma cybrids containing high levels of the np 3243 mutation, compared with control cybrids. We propose that structural modification of tRNALeu(CUN) could be a natural suppression mechanism for the np 3243 and other mitochondrial tRNALeu(UUR) mutations and could underlie some of the phenotypic variability of np 3243 disease.

Acylation↗
PubMed1997

Metazoan nuclear genes for mitoribosomal protein S12.

We have characterized nuclear genes for mitoribosomal protein S12 (mt-rps12) a major component of the ribosomal accuracy centre, in human, mouse and Drosophila melanogaster. In human and Drosophila, and probably also in mouse, there is a single intron within the coding region, located in the mitochondrial targeting pre-sequence. In humans, the mRNA structure is highly suggestive of translational regulation. In all three species, there is an amino-acid substitution with respect to eubacterial homologues in a residue implicated in aminoglycoside resistance. The only viable mutant allele of the Drosophila gene, associated with a bang-sensitive phenotype (paralysis upon mechanical vibration, arising from a mechanoreceptor cell defect) also has a novel substitution in a conserved region implicated in translational fidelity. Given the involvement of the mitoribosomal accuracy centre in human sensorineural deafness by virtue of rRNA mutations, our results indicate that this fly mutant may be a useful animal model of this disorder, and earmark the gene for mt-rps12 as a candidate in human hearing impairment.

3T3 Cells↗
PubMed1997

Immunochemical characterization of a novel mitochondrially located protein encoded by a nuclear gene within the DFNB8/10 critical region on 21q22.3.

A novel protein encoded by the C210RF2 gene in chromosomal locus 21q22.3 was characterized by immunochemistry. This chromosomal region is known to contain genes for human diseases such as non-syndromic autosomal recessive deafness (DFNB8/10) and autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED). Polyclonal murine antisera were produced against the multivalent peptides deduced from the amino acid sequence of the polypeptide. Immunological reactivity of the obtained antisera was tested with primary cells or established cell lines. On western blotting, the polyclonal sera recognized a single protein product of 25 Kd expressed in cell lines of epithelial and lymphoid origin. Subsequent immunochemistry of several human tissues indicated the ubiquitous expression of the protein. Immunofluorescence studies and co-staining with a mitochondrial-specific dye suggest the subcellular localization of the protein to mitochondria. Mitochondrial localization is also predicted by computer analysis of the polypeptide sequence. As deafness is known to be caused in some instances by defects in mitochondrial function, C210RF2 is a plausible candidate gene for DFNB8/10.

Amino Acid Sequence↗
PubMed1997

The study of cell-death proteins in the outer mitochondrial membrane by chemical cross-linking.

Chemical cross-linking was used to study the interactions of the anti-cell-death protein Bcl2 with other proteins in the outer mitochondrial membrane. Cross-linking of mitochondrial surface proteins produced a large Bcl2-containing complex (>200 kDa), and a Bcl2-derived peptide was shown to cross-link specifically with a mitochondrial protein identified by immunoblotting as Raf-1 kinase.

Amino Acid Sequence↗
PubMed1997

Molecular phenotype of a human lymphoblastoid cell-line homoplasmic for the np 7445 deafness-associated mitochondrial mutation.

We have studied mitochondrial gene expression and metabolic function in a human lymphoblastoid cell-line homoplasmic for the np 7445, deafness-associated mitochondrial DNA mutation. The mutation maps to the 3' termini of the oppositely oriented genes encoding cytochrome oxidase subunit I (COI) and tRNA-ser(UCN). In comparison with control lymphoblastoid cells, we detected a marked depletion (> 60%) of tRNA-ser(UCN). There was, however, no significant impairment of respiratory function, no alteration to the structure or abundance of COI mRNA or its precursors, and no detectable abnormality of mitochondrial protein synthesis. We also found considerable tissue-variation in the abundance of tRNA-ser(UCN). We propose that the tissue-specific phenotype associated with this mutation results from an inherent deficiency in the processing of the mutant pre-tRNA, that becomes limiting for protein synthesis only in a restricted set of cells of the auditory system in which the tRNA is, for other reasons, already at a critically low level.

Blotting, Northern↗
PubMed1997

Behaviour of a population of partially duplicated mitochondrial DNA molecules in cell culture: segregation, maintenance and recombination dependent upon nuclear background.

We have studied the dynamics of mitochondrial DNA maintenance and segregation in human cells using serial cybrid transfer of partially duplicated mitochondrial DNA, from a mitochondrial myopathy patient, to two distinct recipient cell types. The results indicate two radically different outcomes dependent upon nuclear background. In one case (lung carcinoma) there is systematic loss of the partial duplication by an implied recombinational mechanism. In another nuclear background (osteosarcoma) the duplicated molecules can survive, having only a marginal effect on mitochondrial respiratory function. Moreover, in the osteosarcoma nuclear background further disturbances of mtDNA maintenance frequently follow from cybrid transfer. These are progressive, catastrophic loss of mtDNA and further rearrangement to generate partially triplicated molecules. The results imply differential expression of nuclear genes regulating mtDNA copy number, replication and recombination in different human cell types.

Cell Nucleus↗
PubMed1997

Mitochondrial deafness.

Hearing impairment is a common disorder, largely genetic in origin, and showing classical features of a heterogeneous genetic disease. Up to 100 independently acting nuclear genes are involved in the disorder, of which around 30 have been mapped, but only a handful identified. Mutations in mitochondrial DNA also play a significant role in both syndromic and nonsyndromic sensorineural hearing impairment. Environmental agents such as aminoglycoside antibiotics and as yet unidentified nuclear genes interact with mitochondrial mutations in the expression of auditory phenotypes. The spectrum of different mitochondrial mutations associated with hearing impairment, taken together with mechanistic studies at the molecular level, suggests that the pathogenic process involves the accumulation of abnormal translation products inside mitochondria, in sensitive cells of the auditory system. This leads to a prediction of the involvement of a novel class of nuclear genes in hearing impairment, namely those with roles in 'mitochondrial protein quality control'.

Adenosine Triphosphatases↗
PubMed1995

The relationship between mRNA half-life and gene function in the yeast Saccharomyces cerevisiae.

Saccharomyces cerevisiae (Sc) mRNAs have been described as falling into two major classes with respect to mRNA half-life [Santiago et al., Nucleic Acids Res. 14 (1986) 8347-8360]. We have used DNA sequence analysis to address the functional roles of eleven of the thirteen cDNAs upon which Santiago et al. based their conclusions. Eight had been described as copies of short half-life and five as copies of long-half-life mRNAs. We show here that five members of the short-half-life class encode known Sc cytosolic ribosomal proteins (rp). One further short-half-life cDNA appears to encode a new Sc rp related to higher eukaryotic rp S12. Among the long-half-life cDNAs, one encodes the glucose-inducible glycolytic enzyme enolase, while another is related to the mouse housekeeping gene MER5.

Animals↗
PubMed1995

Different cellular backgrounds confer a marked advantage to either mutant or wild-type mitochondrial genomes.

After the introduction of mitochondria with a mixture of mutant and wild-type mitochondrial DNA (mtDNA) into a human rho degree cell line (143B.206), Yoneda et al. [Yoneda, M., Chomyn, A., Martinuzzi, A., Hurko, O. & Attardi, G. (1992) Proc. Natl. Acad. Sci. USA 89, 11164-11168] observed a shift in the proportion of the two mitochondrial genotypes in a number of cybrid clones. In every case where a shift was observed, there was an increase in the proportion of mutant mtDNA. By using the same cell line (143B.206 rho degree), we also generated cybrids that were either stable in their mitochondrial genotype or showed an increase in the proportion of mutant mtDNA. However, temporal analysis of the same mutant mtDNA type in another rho degree cell line revealed a quite distinct outcome. Those clones that showed a change shifted toward higher levels of wild-type rather than mutant mtDNA. These results indicate that the nuclear genetic background of the recipient (rho degree) cell can influence the segregation of mutant and wild-type mitochondrial genomes in cell cybrids.

Base Sequence↗
PubMed1995

Characterisation of Saccharomyces cerevisiae genes encoding ribosomal protein YL6.

We have characterised a Saccharomyces cerevisiae cDNA (cDNA13), originally isolated on the basis of the short half-life of the corresponding mRNA. We show here that its sequence is closely related to that of the genes encoding ribosomal proteins K37, KD4 and K5 of Schizosaccharomyces pombe. 'mRNA13' also behaves like other mRNAs encoding ribosomal proteins, in that its abundance increases sharply when glucose is added to cells grown on ethanol (nutrient-upshift), and declines when cells are subjected to a mild heat-shock. Unspliced mRNA13 accumulates when cells bearing a temperature-sensitive splicing mutation are grown at the restrictive temperature. The gene(s) corresponding to cDNA13, like other ribosomal protein genes of S. cerevisiae, thus contain an intron. Southern blot analysis indicates the presence of two separate loci related to cDNA13 in the S. cerevisiae genome. From the sequence of one of these, a complete polypeptide sequence was deduced. The first 40 amino acids are identical to those of YL6, a S. cerevisiae ribosomal protein characterised only by N-terminal protein sequence analysis. There is clear evidence within the genomic sequence for the predicted intron, and for elements similar to those that regulate expression of other S. cerevisiae ribosomal protein genes.

Amino Acid Sequence↗