Robin Holliday

Altered recombination frequencies in radiation sensitive strains of Ustilago

Abstract Current models of genetic recombination predict that one or more of the enzymic mechanics responsible for the repair of damaged DNA would also play some part in the process of gene conversion and possibly also in that of crossing over. This prediction has been tested in Ustilago maydis by examining the effect of radiation-sensitive mutants on these processes. Three mutants, uvs -1, uvs -2 and uvs -3, which are recessive, unlinked and phenotypically distinct, have been used in recombination studies. Diploids have been synthesised which are heterozygous for 3 biochemical markers; heteroallelic for mutants in the structural gene for nitrat reductase, and either heterozygous or homozygous for each of the 3 radiation sensitive mutants, or for both uvs -1 and uvs -2. With these diploids the frequencies of spontaneous and UV-induced mitotic gene conversion and crossing over have been measured. In addition, the effect of the mutants on crossing over and conversion at meiosis has been examined. Of the 3 mutants, uvs -1 and uvs -2 have a strong influence on recombination, particularly in preventing UV-induced mitotic gene conversion; and in combination they also abolish spontaneous mitotic conversion. uvs -1 diploids have a high rate of spontaneous and induced mitotic segration which appears to be the result of terminal deletions following chromosome breakage. uvs -2 reduces mitotic crossing over and also has the striking effect of completely blocking meiotic division. uvs -3 has much weaker effects on recombination. The results demonstrate that there is a direct relationship between radiation sensitivity and ability to recombine, and it is to some extent possible to explain the data on the basis of particular deficiencies in the repair processes.

Errors in Protein Synthesis and Clonal Senescence in Fungi

Irreversible senescence in fungi—a cytoplasmic condition—may be the result of a breakdown in the maintenance of the accuracy of protein synthesis. Premature senescence in Podospora can be induced by treatments which may reduce the accuracy of amino-acid incorporation. A predicted interrelationship between phenotypic reversion of a base substitution mutation, suppression and ageing is shown in Neurospora.

Strong effects of 5-azacytidine on the in vitro lifespan of human diploid fibroblasts

Azacytidine (5-aza-CR) and azadeoxycytidine (5-aza-CdR) are known to inhibit the methylation of cytosine (5-mC) in DNA, and their effects on the long-term growth of human fibroblasts, strain MRC-5, have been examined. A single treatment with either analogue initially inhibits growth, but the cells recover to normal morphology, growth rate and cell density at confluence. However, a memory of the treatment is retained, since the cells subsequent lifespan is considerably reduced in comparison with controls, and the terminal stages of growth are indistinguishable from senescent cultures of untreated cells. The effect of 5-aza-CR or 5-aza-CdR does not appear to be closely related to the concentration used, or to the length of treatment up to about half-way through the total lifespan. Sequential doses have cumulative effects on longevity. There is evidence that the pattern of 5-mC in mammalian DNA is inherited via cell division; therefore, a reduction in 5-mC induced by a pulse treatment of 5-aza-CR or 5-aza-CdR will be transmitted to all descendants. The results are consistent with independent observations that the level of 5-mC declines continually during the serial subculture of human diploid cells. The analogues would be expected to precipitate this decline and thereby advance the physiological age of the culture. The results provide support for the view that the random loss of methyl groups in DNA may eventually have deleterious consequences, such as aberrant epigenetic changes in gene expression.

Commitment to senescence: A model for the finite and infinite growth of diploid and transformed human fibroblasts in culture

As selection may be expected to act against sensescent cells, the observed finite lifespan of human diploid fibroblast cultures suggests that cells become committed to senescence while still outwardly healthy. On this basis a mathematical model is developed for the growth and development of human diploid fibroblast cultures. The division of an uncommitted cell is assumed to produce committed daughter cells with a fixed probability, P , the “probability of commitment”. An incubation period of M cell divisions must then elapse between commitment and the death of the resultant clone. This model is compatible with at least three possible ageing mechanisms. The model predicts that an initially healthy population will double in size with each successive cell division (Stage 1 growth) until, after M divisions, the first deaths occur. For P > 0·5 the population then becomes rapidly extinct. For P M is sufficiently small, or the population size sufficiently large, the experimental routine of subculturing will lead to loss by dilution of the uncommitted cells and the population is then mortal. The final stage of growth (Stage 3) occurs as the last live cells become senescent and die. The predicted growth is found to agree well with some experimental data for human fibroblasts. The model suggests a possible explanation for both the observed finite lifespan of human diploid fibroblast cultures and the apparent immortality of lines transformed by an oncogenic virus or derived from malignant tissue, since reducing the incubation period or the probability of commitment can convert a population with limited growth potential to one which grows indefinitely.

Mechanism for RNA splicing of gene transcripts

It has been demonstrated that structural genes in eukaryotes contain intervening sequences (introns) which are absent from expressed sequences (exons) [l-6]. There is evidence that intervening sequences are transcribed [7], but subsequently the introns are removed by a process which is now known as RNA splicing [ 8,9]. RNA splicing must be a very precise process since it results in the joining of successive coding sequences to produce an unambiguous message. We wish to propose a mechanism for RNA splicing which depends on intermolecular hybridisation and which is closely analogous to the genetic recombination of DNA molecules. In both processes the point at which the breakage and reunion of polynucleotide genes occurs is determined by the base pairing of complementary sequences (hybrid DNA or hybrid RNA). In this way the addition of extraneous nucleotides or the deletion of essential nucleotides is prevented.

Chromosome changes during the in vitro ageing of MRC-5 human fibroblasts.

Summary The frequency of polyploidy, aneuploidy and chromosome abnormalities was recorded at intervals throughout the lifespan of fetal lung fibroblasts strain MRC-5. The frequency of polyploidy rose significantly at passage 50, which is close to the beginning of the senescent phase, and a further increase was seen at passage 60, which is near the end of the lifespan. Aneuploidy and chromosome abnormalities increase significantly only at passage 60. The effect of incubation at 32, 34 and 40°C was also examined. Senescence is accelerated at 40°C, and this is accompanied by a dramatic rise in chromosome abnormalities and polyploidy. No similar effect is seen at the lower temperatures. The overall results suggest that chromosome changes are a secondary consequence of the cellular deterioration which leads to ageing, rather than being a major cause of ageing.

A rapid rise in cell variants during the senescence of populations of human fibroblasts

An attempt has been made to measure the frequency of mutations throughout the lifespan of human fibroblast strain MRC-5. A novel procedure has been used which involves staining individual cells for high levels of glucose-6-phosphate dehydrogenase. Evidence is presented that this phenotype is due to mutation. The frequency of variants was scored from passage 16 until the final phase of senescence (passage 60). There is an exponential increase of stained cells throughout this period. The results are in agreement with the general error theory, which proposes that aging is due to a breakdown in the fidelity of information transfer between macromolecules.

DNA methylation and epigenetic mechanisms

Genes are essential for the transmission of genetic information from generation to generation, and this mechanism of inheritance is fully understood. Genes are also essential for unfolding the genetic program for development, but the rules governing this process are obscure. Epigenetics comprises the study of the switching on and off of genes during development, the segregation of gene activities following somatic cell division, and the stable inheritance of a given spectrum of gene activities in specific cells. Some of these processes may be explained by DNA modification, particularly changes in the pattern of DNA methylation and the heritability of that pattern. There is strong evidence that DNA methylation plays an important role in the control of gene activity in cultured mammalian cells, and the properties of a CHO mutant strain affected in DNA methylation are described. Human diploid cells progressively lose cytosine methylation during serial subculture, and this may be related to their in vitro senescence. There is also evidence that DNA modifications can be inherited through the germ line. Classical genetics is based on the study of all types of change in DNA base sequence, but the rules governing the activity of genes by epigenetic mechanisms are necessarily different. Their elucidation will depend both on a theoretical framework for development and on experimental studies at the molecular, chromosomal, and cellular levels.

Increased error frequency of DNA polymerases from senescent human fibroblasts

Abstract The error catastrophe theory of ageing predicts that the fidelity of DNA polymerase should be reduced in extracts from senescent cells. This prediction has been experimentally verified with MRC-5 human diploid fibroblasts. Using “cytoplasmic” DNA polymerase α the old/young ratio of error frequencies was 3.4 with the poly[d(A-T)]/dGTP/Mg 2+ system, 1.9 with poly[d(A-T)]/dGTP/Mn 2+ and 2.0 with poly[d(I-C)]/dTTP/Mg 2+ . With DNA polymerase γ the old/young ratio was 3.8. The fidelity of DNA polymerase was examined at seven points during the life span of MRC-5 fibroblasts and the increase in error frequency with cell age was found to be statistically highly significant ( P By means of control experiments, artifactual explanations of the results can be eliminated. The close correlation between homologous and non-homologous DNA synthesis was demonstrated by following the time-course of the reaction, varying the enzyme concentration and by other means. The non-homologous dNMP incorporation was sensitive to DNase, but not RNase or protease treatment. No terminal transferase activity could be detected. The discrimination of the DNA polymerase from young and old extracts was constant over a wide range of homologous and non-homologous dNTP concentrations. Degradation of the products of the misincorporation assay to 3′-dNMPs revealed that a guanine-thymine base mispairing was the main method of non-homologous synthesis in young and old cells while degradation to 5′-dNMPs revealed that dGTP was not contaminated by other homologous dNTPs. By use of DNA polymerase with a lower error frequency than MRC-5 polymerase, the contamination of poly[d(A-T)] by cytosine could be ruled out. Mixing experiments could not detect a diffusible agent in young or old extracts which was capable of modifying the error frequency of a DNA polymerase from another extract. Since the DNA polymerase extract was not pure, an alternative explanation is possible for these results. Nevertheless, these control experiments do strongly suggest that the misincorporation assay measures the frequency of DNA polymerase-directed errors which are present as single base substitutions.

Genetic characterization of rec -1, a mutant of Ustilago maydis defective in repair and recombination

Detailed physiological and genetic studies of haploid and diploid strains have revealed a complex phenotype for the rec -1 mutation in Ustilago maydis . The mutant is defective in the repair of damage by UV light, ionizing radiation and nitrosoguanidine. Four alleles are all recessive and have the same sensitivity to UV, suggesting the loss of a single cellular function. A significant fraction of non-viable cells is formed during growth, and in diploid strains considerable variation in colony size and morphology is seen. The spontaneous frequency of mutation is greater than in wild-type cells, but there is little, if any, enhancement by irradiation. rec -1 also has pleiotropic effects on genetic recombination. The spontaneous level of mitotic allelic or non-allelic recombination is abnormally high, but the relative increase after irradiation is much lower than in control diploids. Allelic recombination is strongly associated with the expression of a hetozygous recessive distal marker, and it is shown that this is often due to hemizygosity rather than to homozygosity of this marker. The results indicate that allelic recombination is due to crossing over rather than gene conversion, but that the cross over is often associated with a chromatid break. rec -1 interacts with other radiation sensitive mutants, such as rec -2. Diploids homozygous for both are totally deficient in allelic recombination. In crosses between rec -1 strains meiosis is defective, with a low viability of meiotic products and frequent production of aneuploids or diploids among the survivors. The overall phenotype of rec -1 strains can best be explained in terms of the loss of a regulatory function, which leads to uncontrolled recombination during mitosis and meiosis, and the loss of a recombination repair pathway which is normally induced by agents which damage DNA.