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I J Holt

Publications and source records attributed to I J Holt.

At least 19 recordsLinked to original sources
PubMed2001

Prominent mitochondrial DNA recombination intermediates in human heart muscle.

Recombination intermediates containing four-way (Holliday) junctions are generated during DNA repair and replication in many systems, including yeast mitochondrial DNA (mtDNA). In contrast, convincing evidence for recombination in mammalian mtDNA is lacking. We have used two-dimensional agarose-gel electrophoresis to analyse non-linear forms of mtDNA in human heart muscle. Replication intermediates from both the coupled and strand-asynchronous mtDNA replication pathways were detected. An additional class of non-linear molecules, with the electrophoretic properties of four-way junctions, was also prominent. These molecules were insensitive to topoisomerase I or RNase H, but were diminished by branch migration or RuvC treatment. Junctional molecules were detected in all regions of the mitochondrial genome, were found in myocardial DNA from young and old adults, but were present at lower levels in skeletal muscle and placenta. We suggest that they could represent intermediates of mtDNA repair, given their prevalence in the oxyradical-rich environment of heart muscle mitochondria.

Blotting, Southern↗
PubMed2000

Human mtDNA sublimons resemble rearranged mitochondrial genoms found in pathological states.

Sublimons, originally identified in plant mitochondria, are defined as rearranged mtDNA molecules present at very low levels. We have analysed the primary structures of sublimons found in human cells and tissues and estimated their abundance. Each tissue of a given individual contains a wide range of different sublimons and the most abundant species differ between tissues in a substantially systematic manner. Sublimons are undetectable in rho(0) cells, indicating that they are bona fide derivatives of mtDNA. They are most prominent in post-mitotic tissue subject to oxidative stress. Rearrangement break-points, often defined by short direct repeats, are scattered, but hotspot regions are clearly identifiable, notably near the end of the D-loop. The region between the replication origins is therefore frequently eliminated. One other hotspot region is located adjacent to a known site of protein binding, suggesting that recombination may be facilitated by protein-protein interactions. For a given primary rearrangement, both deleted and partially duplicated species can be detected. Although each sublimon is typically present at a low level, at most a few copies per cell, sublimon abundance in a given tissue can vary over three orders of magnitude between healthy individuals. Collectively, therefore, they can represent a non-negligible fraction of total mtDNA. Their structures are very similar to those of the rearranged molecules found in pathological states, such as adPEO and MNGIE; therefore, we propose that, as in plants, human mtDNA sublimons represent a pool of variant molecules that can become amplified under pathological conditions, thus contributing to cellular dysfunction.

Adult↗
PubMed2000

Impaired ATP synthase assembly associated with a mutation in the human ATP synthase subunit 6 gene.

Mutations in human mitochondrial DNA are a well recognized cause of disease. A mutation at nucleotide position 8993 of human mitochondrial DNA, located within the gene for ATP synthase subunit 6, is associated with the neurological muscle weakness, ataxia, and retinitis pigmentosa (NARP) syndrome. To enable analysis of this mutation in control nuclear backgrounds, two different cell lines were transformed with mitochondria carrying NARP mutant mitochondrial DNA. Transformant cell lines had decreased ATP synthesis capacity, and many also had abnormally high levels of two ATP synthase sub-complexes, one of which was F(1)-ATPase. A combination of metabolic labeling and immunoblotting experiments indicated that assembly of ATP synthase was slowed and that the assembled holoenzyme was unstable in cells carrying NARP mutant mitochondrial DNA compared with control cells. These findings indicate that altered assembly and stability of ATP synthase are underlying molecular defects associated with the NARP mutation in subunit 6 of ATP synthase, yet intrinsic enzyme activity is also compromised.

Adenosine Triphosphate↗
PubMed2000

Coupled leading- and lagging-strand synthesis of mammalian mitochondrial DNA.

Analysis of mammalian mtDNA by two-dimensional agarose gel electrophoresis revealed two classes of replication intermediate. One was resistant to single-strand nuclease digestion and displayed the mobility properties of coupled leading- and lagging- strand replication products. Intermediates of coupled, unidirectional mtDNA replication were found in mouse liver and human placenta and were the predominant species in cultured cells recovering from transient mtDNA replication. Replication intermediates sensitive to single-strand nuclease were most abundant in untreated cultured cells. These are presumed to derive from the orthodox, strand-asynchronous mode of mtDNA replication. These findings indicate that two modes of mtDNA replication operate in mammalian cells and that changes in mtDNA copy number involve an alteration in the mode of mtDNA replication.

Animals↗
PubMed2000

The np 3243 MELAS mutation: damned if you aminoacylate, damned if you don't.

The np 3243 MELAS mtDNA mutation in tRNA(leu(UUR))has been variously proposed as a loss-of-function or as a gain-of-function mutation, based on apparently contradictory studies in cultured cell lines. A new report describing the molecular effects of the mutation in vivo now mirrors this variability. This should prompt a more systematic re-investigation of cells carrying the mutation, in order to separate primary from secondary and pathogenic from compensatory effects, all of which may contribute to disease phenotype. Nuclear genetic and developmental background, mitochondrial haplotype, and epigenetic effects may all influence the pathological outcome. Defects in both base-modification and aminoacylation of the mutant tRNA could play critical roles.

Acylation↗
PubMed2000

Separation of intact pyruvate dehydrogenase complex using blue native agarose gel electrophoresis.

We show that the blue native gel polyacrylamide electrophoresis system (BN-PAGE) can be applied to pyruvate dehydrogenase complex (PDC). BN-PAGE has been used extensively to study the multisubunit enzymes of oxidative phosphorylation, as nondenaturing separation in the first dimension maintains holoenzyme integrity. However, the standard protocol was inappropriate for PDC as, at 10 MDa, it is approximately ten times larger than the largest respiratory chain enzyme complex. Therefore, agarose was substituted for polyacrylamide. Moreover, a substantial decrease in salt concentration was necessary to prevent dissociation of PDC. As with standard BN-PAGE, immunoblots of second-dimensional sodium dodecyl sulfate-PAGE (SDS-PAGE) provided more detailed information on specific subunits and subcomplexes. The method was applied to human heart mitochondrial fragments, control cultured human cells, rho0 cells that lack mitochondrial DNA, and two cell lines derived from patients with PDC deficiency. The PDC deficient cell lines showed a clear correlation between amount of PDC holoenzyme and disease severity. In cells lacking mitochondrial DNA, synthesis and assembly of all PDC subunits (all nuclearly encoded) appeared normal, suggesting that respiratory function has no regulatory role in PDC biogenesis. Blue native agarose gel electrophoresis coupled with standard second-dimensional SDS-PAGE provides a new tool to be used in conjunction with biochemical assays and immunoblots of one-dimensional SDS-PAGE to further elucidate the nature of PDC in normal and disease states. Furthermore, other cellular protein complexes of 1 MDa or more can be analysed by this method.

Electrophoresis, Agar Gel↗
PubMed2000

Rhabdomyosarcoma rho(0) cells: isolation and characterization of a mitochondrial DNA depleted cell line with 'muscle-like' properties.

Mutations of mitochondrial DNA are a significant cause of neuromuscular disease. Pathological mutant mitochondrial DNA has been studied in control nuclear backgrounds. These experiments entailed transfer of patient-derived mitochondria to rho(0) cells that lack mtDNA. A limitation of these studies has been the fact that the control nuclear backgrounds were unrelated to the affected tissues of patients. Therefore a rhabdomyosarcoma cell line that has 'muscle-like' properties was tested to determine whether it could be depleted of mtDNA. A human rhabdomyosarcoma cell line was treated with the DNA intercalating dye ethidium bromide (3, 8-diamino-5-ethyl-6-phenylphenanthridinium bromide) for 45 days. The treatment induced complete and permanent loss of mitochondrial DNA (rho(0)) in the rhabdomyosarcoma cells, as mtDNA remained undetectable after 8 months of growth in medium without drug. Crucially, the rhabdomyosarcoma rho(0) cells retained the ability to differentiate into myotubes with expression of muscle specific isoenzymes. The rhabdomyosarcoma rho(0) cell line provides a model system for studying pathological mutant mtDNA in cells that more closely resemble human muscle than the hitherto available human rho(0) cell lines.

Antigens, Differentiation↗
PubMed2000

Genotypic stability, segregation and selection in heteroplasmic human cell lines containing np 3243 mutant mtDNA.

The mitochondrial genotype of heteroplasmic human cell lines containing the pathological np 3243 mtDNA mutation, plus or minus its suppressor at np 12300, has been followed over long periods in culture. Cell lines containing various different proportions of mutant mtDNA remained generally at a consistent, average heteroplasmy value over at least 30 wk of culture in nonselective media and exhibited minimal mitotic segregation, with a segregation number comparable with mtDNA copy number (>/=1000). Growth in selective medium of cells at 99% np 3243 mutant mtDNA did, however, allow the isolation of clones with lower levels of the mutation, against a background of massive cell death. As a rare event, cell lines exhibited a sudden and dramatic diversification of heteroplasmy levels, accompanied by a shift in the average heteroplasmy level over a short period (<8 wk), indicating selection. One such episode was associated with a gain of chromosome 9. Analysis of respiratory phenotype and mitochondrial genotype of cell clones from such cultures revealed that stable heteroplasmy values were generally reestablished within a few weeks, in a reproducible but clone-specific fashion. This occurred independently of any straightforward phenotypic selection at the individual cell-clone level. Our findings are consistent with several alternate views of mtDNA organization in mammalian cells. One model that is supported by our data is that mtDNA is found in nucleoids containing many copies of the genome, which can themselves be heteroplasmic, and which are faithfully replicated. We interpret diversification and shifts of heteroplasmy level as resulting from a reorganization of such nucleoids, under nuclear genetic control. Abrupt remodeling of nucleoids in vivo would have major implications for understanding the developmental consequences of heteroplasmy, including mitochondrial disease phenotype and progression.

Base Sequence↗
PubMed1999

Introduction of heteroplasmic mitochondrial DNA (mtDNA) from a patient with NARP into two human rho degrees cell lines is associated either with selection and maintenance of NARP mutant mtDNA or failure to maintain mtDNA.

Mitochondria from a patient heteroplasmic at nucleo-tide position 8993 of mitochondrial DNA (mtDNA) were introduced into two human tumour cell lines lacking mtDNA. The donor mitochondria contained between 85 and 95% 8993G:C mtDNA. All detectable mtDNA in the mitochondrially transformed cells contained the pathological 8993G:C mutation 3 months after transformation. These results suggest that 8993G:C mtDNA had a selective advantage over 8993T:A mtDNA in both lung carcinoma and osteo-sarcoma cell backgrounds. In contrast, two other presumed pathological mtDNA variants were lost in favour of 'wild-type' mtDNA molecules in the same lung carcinoma cell background. Taken together, these findings suggest that the transmission bias of mtDNA variants is dependent upon a combination of nuclear background and mtDNA genotype. A second phenomenon observed was a marked decrease in the growth rate of many putative transformed cell lines after 6 weeks of culturing in selective medium, and in these cell lines mtDNA was not readily detectable by Southern blotting. Restriction endonuclease analysis and sequencing of amplified mtDNA demonstrated that the slow growing cells contained little or no mtDNA. It is concluded that these cells represented transient mitochondrial transformants.

Ataxia↗
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

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↗
PubMed1996

Complex I deficiency is associated with 3243G:C mitochondrial DNA in osteosarcoma cell cybrids.

143B.206 rho degrees cells were repopulated with mitochondria from a MELAS patient harbouring a mixture of 3243G:C and 3243A:T mitochondrial DNA. A number of biochemical assays were performed on selected cybrids with various proportions of the two types of mitochondrial DNA. These assays revealed a marked decrease in oxygen consumption with pyruvate, a complex I substrate, in cybrids containing 60% to 90% 3243G:C mitochondrial DNA. Moreover, these cybrids showed decreased synthesis of a number of polypeptides in a mitochondrial in vitro translation assay. A cybrid line with a very high level of 3243G:C mitochondrial DNA (95%) had additional deficiencies in complexes III and IV and there was a marked generalised decrease in mitochondrial translation in this cybrid. The observation of complex I deficiency is consistent with previously reported enzymatic measurements of muscle homogenates from MELAS patients with the 3243G:C mutation.

Cell Fusion↗
PubMed1996

Do sequence variants in the major non-coding region of the mitochondrial genome influence mitochondrial mutations associated with disease?

Several different mutations in human mitochondrial DNA (mtDNA) have been associated with disease, but their origins and the basis of the wide phenotypic variability remain to be elucidated. We initially investigated three patients with heteroplasmic disease associated mutations of mtDNA for the presence of cis mutations in the major non-coding region that might influence their origins or pathology. A T --> C transition at nt 16 189 previously identified in one patient with the 3243 G:C mutation was associated with heteroplasmic length variation. Identical length variation was found in patient-derived cybrid lines containing 0-97.5% 3243 G:C. Similarly, heteroplasmic length variation was demonstrated in 2/6 other probands with both the 3243 mutation and the 16,189 polymorphism. The distribution of length variants in probands and in asymptomatic family members was identical in all cases. Thus length variation appears to be independent of the level of 3243 mutant mtDNA and hence probably arose within both 3243 G:C and 3243 A:T mtDNAs. We suggest that the 16,189 polymorphism reflects a predisposition to the formation or fixation of several different mutations in mitochondrial tRNA-LeuUUR.

Base Sequence↗
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↗
PubMed1994

Comprehensive care of the child with a chronic condition. Part 1. Understanding chronic conditions in childhood.

Understanding the care of children with chronic illnesses and disabilities is an important part of the practice of pediatrics. Children with special health care needs and their families benefit from the support which comprehensive, coordinated, accessible, and responsive services provide. Although the primary care pediatrician is often most appropriate to serve as the overall coordinator of such services, many challenges to providing such care exist. Part 2, "Primary Care Management," will suggest guiding principles and management structures that allow the pediatrician to respond most effectively over time to the needs of children with chronic conditions and their families.

Child↗