Biomedical subjects
R Holliday
Publications and source records attributed to R Holliday.
Recurrent mucormycosis of the paranasal sinuses in an immunologically competent host.
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Quantitative genetic variation and developmental clocks.
It is well-known that most genetic variation affects quantitative traits, and natural or artificial selection can act to change quantitative features of organisms more rapidly than qualitative ones. Surprisingly, variability is not confined to outbred species, but also occurs in inbred mice at a much higher rate than expected from known mutation rates. The size and shape of organisms and their constituent parts are, at least in part, controlled by the number of cell divisions, and there is published evidence for the existence of developmental clocks, which may count cell divisions. A molecular model for a developmental clock was previously proposed. It depends on the DNA methylation of repeated sequences of DNA, where the methylation of each additional sequence is tied to DNA synthesis and therefore cell division. The number of repeats specifies the number of divisions which will occur before a signal is produced which can activate or inactivate one or more genes. It is known that crossing over occurs between sister chromatids, and where tandemly repeated sequences occur unequal exchange can generate a larger or smaller number of repeats. An example of this is seen in the well-known variability of "minisatellite" sequences in human DNA. Unequal sister chromatid exchange can occur in mitotic and meiotic cells in the germ line, and in the case of developmental clock sequences could generate variation in clock length which in turn would directly affect quantitative traits. These events can be regarded as a special case of molecular drive during evolution.
Is DNA methylation of X chromosome genes stable during aging?
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Gene silencing in mammalian cells by uptake of 5-methyl deoxycytidine-5'-triphosphate.
Chinese hamster ovary (CHO) cells were subjected to electroporation in the presence of 5-methyl deoxycytidine-triphosphate. This treatment increases by 10 to 100-fold the frequency of cells lacking thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase, or adenine phosphoribosyltransferase. The inactivation of the genes coding for these enzymes is thought to occur following the direct incorporation of the methylated nucleotide triphosphate into DNA. The enzyme-deficient clones were stable, but almost all were reactivated at high frequency by the demethylating agent 5-azacytidine, to produce derivatives with enzyme activity. The results indicate that there is a direct relationship between DNA methylation and gene silencing.
Emergencies in AIDS patients: the otolaryngologic perspective.
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Molecular biology and the new paradigm.
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DNA methylation and epigenetic inheritance.
Classical genetics has revealed the mechanisms for the transmission of genes from generation to generation, but the strategy of the genes in unfolding the developmental programme remains obscure. Epigenetics comprises the study of the mechanisms that impart temporal and spatial control on the activities of all those genes required for the development of a complex organism from the zygote to the adult. Epigenetic changes in gene activity can be studied in relation to DNA methylation in cultured mammalian cells and it is also possible to isolate and characterize mutants with altered DNA methylase activity. Although this experimental system is quite far removed from the epigenetic controls acting during development it does provide the means to clarify the rules governing the silencing of genes by specific DNA methylation and their reactivation by demethylation. This in turn will facilitate studies on the control of gene expression in somatic cells of the developing organism or the adult. The general principles of epigenetic mechanisms can be defined. There are extreme contrasts between instability or switches in gene expression, such as those in stem-line cells, and the stable heritability of a specialized pattern of gene activities. In some situations cell lineages are known to be important, whereas in others coordinated changes in groups of cells have been demonstrated. Control of numbers of cell divisions and the size of organisms, or parts of organisms, is also essential. The epigenetic determination of gene expression can be reversed or reprogrammed in the germ line. The extent to which methylation or demethylation of specific DNA sequences can help explain these basic epigenetic mechanisms is briefly reviewed.
The history of the DNA heteroduplex.
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The limited proliferation of cultured human diploid cells: regulation or senescence?
It has been widely accepted that the limited life span of human diploid fibroblasts in culture provides a valid experimental model for the study of aging at the cellular level. In spite of innumerable investigations the underlying cause of cessation of growth is not known. Many approaches are being used to test the specific hypothesis that cells at the end of their life span produce an inhibitor of the initiation of DNA synthesis which irreversibly arrests cells in G1, rather than resulting in cell death. This implies that there is positive control of cell proliferation and that the final population of noncycling cells is in a stable state. There appears to be a basic contradiction between this interpretation of published data and the long-standing view that cells actually become progressively senescent during phase III of their growth in vitro. The DNA inhibitor theory can be criticized on a number of grounds, and there is considerable evidence that fibroblasts at the end of their life span are heterogeneous and have a very complex pleiotropic phenotype. One effect of these changes would be to prevent DNA synthesis and normal progression through the cell cycle. The conclusion is that phase III cells are indeed senescent, presumably as a result of a general failure to maintain the integrity of macromolecules and other cellular components.
Mechanisms for the control of gene activity during development.
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Paradoxes between genetics and development.
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Evidence for allelic exclusion in Chinese hamster ovary cells.
Earlier results suggested that the functional hemizygosity of genes in pseudodiploid Chinese hamster ovary (CHO) cells is due to the silencing of one allele by DNA methylation. From this one could make a strong prediction that we have now been able to confirm by genetic experiments, using thymidine kinase (TK) alleles. TK- mutants induced by ethylmethane sulphonate (EMS) were all revertible to TK+ at high frequency by the demethylating agent 5-azacytidine (5-aza-CR). This revertibility was due to reactivation of a silent nonmutant TK allele. Further mutagenesis by EMS yielded TK- derivatives that were no longer revertible by 5-aza-CR; these are assumed to have mutations in both alleles. TK- cells were also transfected with equine herpes virus TK+ DNA, and the TK+ derivatives were shown to be markedly less stable than cells with the normal TK+ gene. CHO cells lack metallothionein activity (sensitive to cadmium), and also require proline for growth, because genes have become silenced during the establishment of the cell line. In both cases 5-aza-CR reactivates these genes to give the cadmium resistant and proline independent phenotypes. Long-term experiments with reactivants in the absence of selection showed that the genes become silent, presumably as a result of de novo methylation. A strain resistant to cytosine arabinoside (araCR) was also resistant to 5-azadeoxycytidine (5-aza-CdR), but not to 5-aza-CR, which would be expected if the araCR strain lacked deoxycytidine kinase.(ABSTRACT TRUNCATED AT 250 WORDS)
Genomic imprinting and allelic exclusion.
In diploid cells, allelic exclusion reduces genes to functional haploidy, because only one of two alleles is active. It is best known in cells producing immunoglobulins, but other examples also exist. X-chromosome inactivation in female mammals is related to allelic exclusion, but in this case the dosage compensation mechanism extends to the whole chromosome. Functional hemizygosity in some mammalian cell lines is probably also due to allelic exclusion, where one autosomal allele is active and the other is methylated and inactive. In early development, it may be important to have only one functional copy of specific regulatory genes. If one considers the possible mechanisms whereby genes are switched from an active to an inactive form, or vice versa, complications arise if the same type of switch operates in two homologous chromosomes segregating independently at mitosis. This complication is avoided if one of the genes is totally inactive. It is therefore suggested that important regulatory gene are subject to allelic exclusion and that this provides a basis for genomic imprinting. Male or female gametes complement in the zygote, because they may have different inactive genes, and the active allele in each case is then functionally haploid in the zygote and developing embryo. These haploid genes would be those involved in critical switches of gene activity during the developmental process. Allelic exclusion imposed by imprinting might be based on the heritable DNA methylation of the regulatory regions of silent genes.
X-chromosome reactivation and ageing.
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Food, reproduction and longevity: is the extended lifespan of calorie-restricted animals an evolutionary adaptation?
Calorie restriction results in an increased lifespan and reduced fecundity of rodents. In a natural environment the availability of food will vary greatly. It is suggested that Darwinian fitness will be increased if animals cease breeding during periods of food deprivation and invest saved resources in maintenance of the adult body, or soma. This would increase the probability of producing viable offspring during an extended lifespan. The diversion of limited energy resources from breeding to maintenance of the soma is seen as an evolutionary adaptation, fully compatible with the 'disposable soma' theory of the evolution of ageing.
Maintenance of DNA methylation level in SV40-infected human fibroblasts during their in vitro limited proliferative life span.
Methylation level as expressed by the molar ratio of 5-methylcytosine content to the combined content of cytosine and 5-methylcytosine was determined by HPLC and uv adsorption of cellular DNA extracted from SV40-infected and pretransformed MRC-5 human diploid fibroblasts (HDFs) during their limited in vitro life span. The level decreased slightly during early passages, and then was maintained within a certain range in the subsequent pretransformed stage of serial passages. When HDFs were treated with 5-aza-2'-deoxycytidine (5-aza-CdR) at an effective concentration shortly after the SV40 infection, the level decreased and then increased or was maintained again within a certain range in the subsequent pretransformed state. The proliferative life span potential of SV40-infected HDFs was not significantly decreased by the 5-aza-CdR treatment. These results are in contrast to the established observations for uninfected HDFs, that methylation level decreases during serial passages, and that, after treatment with 5-aza-CdR, the level, as well as the proliferative life span, is decreased in comparison to untreated populations. These results show that SV40-infected pretransformed HDFs are in an intermediate state between normal finite growth and an established permanent line, in that they retain limited in vitro cell proliferation, while acquiring the ability to maintain methylation levels.