Evidence for complex multigenic inheritance of radiation AML susceptibility in mice revealed using a surrogate phenotypic assay.

The mapping of genes which affect individual cancer risk is an important but complex challenge. A surrogate assay of susceptibility to radiation-induced acute myeloid leukaemia (AML) in the mouse based on chromosomal radiosensitivity has been developed and validated. This assay was applied to the mapping of radiation-induced AML risk modifier loci by association with microsatellite markers. A region on chromosome (chr) 18 with strong association is identified and confirmed by backcross analysis. Additional loci on chrs 8 and 13 show significant association. A key candidate gene Rbbp8 on chr18 is identified. Rbbp8 is shown to be upregulated in response to X-irradiation in the AML sensitive CBA strain but not AML resistant C57BL/6 strain. This study demonstrates the strength of utilizing surrogate endpoints of cancer susceptibility in the mapping of mouse loci and identifies additional loci that may affect radiation cancer risk.

Microsatellite analysis of recurrent chromosome 2 deletions in acute myeloid leukaemia induced by radiation in F1 hybrid mice.

Deletions and/or rearrangements involving one copy of chromosome 2 are consistent and early events in the development of murine acute myeloid leukaemia (AML) by radiation. More than 90% of AMLs induced in the CBA strain of mice express such cytogenetic alterations, with chromosome 2 breakpoints clustering in the C and F regions of the chromosome. In inbred mouse strains, the molecular resolution of these breakpoints is problematic. However, by using x-ray-induced AMLs in FI progeny of genetically divergent CBA/H x C57BI, it has been possible to show region-specific loss of heterozygosity (LOH) in genetically linked sets of chromosome 2 microsatellite alleles from one of the two parental chromosomes. In the majority of cases, an acceptable concordance was shown for AML chromosome 2 deletion, as defined by microsatellites and as revealed by G-band cytogenetics. A degree of breakpoint clustering was found, but the identification of a number of deletion types, based on the position of proximal and distal breakpoints as defined by microsatellite analysis, strongly supports a leukaemogenic mechanism involving gene deletion. No bias towards loss of CBA or C57BI alleles was observed, and the gender of AML-presenting animals did not appear to influence the parental origin of the deletions. A molecular map of chromosome 2 breakpoints has now been established in FI AMLs as a first step towards the molecular cloning of breakpoint sequences.

Development of an assay method for purine catabolic enzymes in the mouse and its adaptation for use on an autoanalyzer.

An assay method has been developed for the purine catabolic enzymes adenosylhomocysteinase, adenosine deaminase (ADA), purine-nucleoside phosphorylase (PNP), and urate oxidase in mice. The assay links H2O2 produced during purine catabolism to the production of a dye complex. The assay method has been developed for ADA and PNP in erythrocytes and for all four enzymes in liver. The assay is cheap, sensitive, and easy to perform. The dye complex absorbs in the visible range, negating the need for an expensive ultraviolet spectrophotometer and allowing the use of an autoanalyzer.

Confirmation of assignment of the bovine K-casein locus by PCR.

The provision of a bovine gene map will allow the ready identification of genetic disease in cattle and will lead to the identification of the genetic loci responsible for quantitative traits of economic importance. An extension of the polymerase chain reaction to the identification of linkage in bovine-Chinese hamster cell hybrids has improved the speed and facility of the assignment of genes to linkage groups and thus makes it easier to achieve a bovine linkage map.

Adenosine deaminase, Ada, is in mouse chromosome 2H3, and is not allelic with wasted, wst.

The adenosine deaminase locus (Ada) in the mouse has been localized by in situ hybridization to band 2H3. Linkage analysis of backcross data has shown that Ada is 13.8 +/- 2.7 cM from the coat texture mutant, ragged, Ra. From the results of earlier work (Abbott, C. M., et al., Proc. Natl. Acad. Sci. USA 83:693, 1986), it had been suggested that wst was a low-activity allele of Ada, but this cannot be so because Ada and wst have been found to be nonallelic.

Analysis of polymorphism in the bovine casein genes by use of the polymerase chain reaction.

Methods have been devised for detecting polymorphisms in the bovine beta- and kappa-casein genes using the polymerase chain reaction (PCR) followed either by restriction enzyme digestion (to reveal a restriction fragment length polymorphism (RFLP] or by hybridization of an allele-specific oligonucleotide. These methods, as well as being faster and more sensitive than traditional RFLP methods, are of more general applicability since they can detect any change in DNA sequence. They require only a small sample of blood or semen and are applicable to animals of any age or sex. These methods make possible large-scale screening and thus selection for alleles at these loci. Typing of blood DNA can give erroneous results when the animal concerned is a twin; however, this can be overcome by retesting using milk or semen. Analysis of the kappa-casein genotype of Holstein-Friesian bulls gives frequencies for the A and B alleles of 0.80 and 0.20 respectively. Selection in favour of the B allele, which is superior for cheese production, could thus have a large effect. The A3 and B alleles at the beta-casein locus have been shown to be rare in the Holstein-Friesian population. Linkage disequilibrium exists between beta-casein B and kappa-casein B.

Benzamides can stimulate as well as inhibit the activity of nuclear ADP-ribosyltransferase.

Benzamides are potent inhibitors of nuclear ADP-ribosyltransferase and have been extensively used to demonstrate the involvement of ADP-ribosylation in cellular function. When permeabilized L1210 cells are treated with 50 microM 3-acetylamidobenzamide (3-aab) the enzyme is inhibited. However, when 50 nM 3-aab is used a two-fold stimulation of enzyme activity is produced. This anomalous stimulation is obtained with benzamides and nicotinamides and is correlated with their activity as inhibitors. Strikingly the steady-state level of poly(ADP-ribose) in intact cells is increased by these low levels of inhibitors. The mechanisms of this effect and its consequences for the experimental use of benzamides are discussed.

Deficiency of adenosine deaminase in the wasted mouse.

Wasted (wst) is a spontaneous mutation with autosomal recessive inheritance. Abnormally low levels of adenosine deaminase have been found in erythrocytes from the wasted mouse. Enzyme activity in wst/wst mice is reduced to 38% of that found in the erythrocytes from control mice, and the apparent Km for adenosine is reduced to 51% of control. These changes imply an alteration in the catalytic properties of the enzyme arising from a change in the primary structure of the protein. We postulate that wasted is a mutation in the structural gene for adenosine deaminase. In man, the autosomal recessive form of severe combined immunodeficiency is associated, in about one-third of cases, with a deficiency of adenosine deaminase. Wasted mice are immunodeficient, develop neurological abnormalities, and die soon after weaning. These features are shared with the human syndrome. We therefore further suggest that the wasted mouse is an animal model for this form of severe combined immunodeficiency that will have potential use in gene-therapy studies.

Nuclear ADP-ribosylation in the chick lens during embryonic development.

Nuclear ADP-ribosyltransferase is present in cells from the chick lens throughout embryonic development. The activity does not decrease when the cells become post-mitotic and commence terminal differentiation but declines slowly in both epithelia and fibre cells. At all stages studied the enzyme retains its ability to be activated by DNA strand breaks induced either by X-irradiation or by the action of an endogenous endonuclease. There is no correlation between the enzyme activity or the levels of its substrate NAD+ and the changes in DNA repair capacity which have been observed during the development of the lens.

The enhancement of cytotoxicity of N-methyl-N-nitrosourea and of gamma-radiation by inhibitors of poly(ADP-ribose) polymerase.

Inhibitors of poly(ADP-ribose) polymerase show a synergistic potentiation of cytotoxicity with certain DNA-damaging agents. Non-toxic concentrations of 5-methylnicotinamide dramatically potentiate the cytotoxicity of N-methyl-N-nitrosourea as tested by the cloning ability of mouse leukaemia (L1210) cells. A dose-enhancement factor of about 10 is observed. This potentiation is dependent on the concentration of 5-methylnicotinamide. The methylxanthines theobromine, theophylline and caffeine also increase the cytotoxicity of methylnitrosourea. Thymidine, in the presence of sufficient deoxycytidine to overcome the perturbation of deoxynucleotide metabolism, also potentiates the cytotoxicity of methylnitrosourea. Nicotinate, which is not an inhibitor of poly-(ADP-ribose) polymerase, has no effect on methylnitrosourea toxicity. A very small, but consistent, enhancement of the toxicity of gamma-radiation by the same inhibitors has been observed. We suggest that this potentiation of cytotoxicity is mediated by inhibition of (ADP-ribose)n biosynthesis; and that the biosynthesis is stimulated by DNA damage. We therefore propose that (ADP-ribose)n takes part in cellular repair mechanisms, either by modifying chromatin structure or by a specific participation in DNA repair.

The effect of gamma radiation and neocarzinostatin on NAD and ATP levels in mouse leukaemia cells.

When mouse leukemia cells are treated with gamma-radiation or neocarzinostatin the intracellular NAD and ATP levels fall rapidly. We have shown that the ATP response is a consequence of the decreased NAD level. We suggest that this low NAD level results in decreased glycolytic activity and that there is a subsequent accumulation of phosphorylated sugars associated with the fall in ATP. Under these extreme conditions, therefore, the NAD level probably regulates the rate of glycolysis in cells which are utilising a rapidly metabolisable sugar as their energy source.

The interaction of core histones with DNA: equilibrium binding studies.

The binding of core histone proteins to DNA, measured as a function of [NaCl[ is a reversible process. Dissociation and reassociation occurs in two stages. Between 0.7 and 1.2 M NaCl H2a H2b bind non-cooperatively as an equimolar complex with deltaGo = 1.6 Kcals/mole at 4 degree C and 1.0 M NaCl. Between 1.2 and 2.0 M NaCl H3 and H4 bind cooperatively as an equimolar complex with delta Go = 7.4 Kcal/mole at 4 degree C and 1.0 M NaCl. The proper binding of H2a and H2b requires the presence of bound H3 and H4. Nuclease digestion of the H3-H4 DNA produces a tetramer of H3-H4 bound to fragments of DNA 145, 125 and 104 base pairs long. Thus an H3-H4 tetramer can protect fragments of DNA as long as those found in complete core particles and must therefore span the nucleosome core particle.