Biomedical subjects
W Masson
Publications and source records attributed to W Masson.
Abnormalities of copper accumulation in cell lines established from nine different alleles of mottled are the same as those found in Menkes disease.
Menkes disease (MD) is caused by a defect in copper homeostasis and has a recognised mouse model, mottled (Atp7aMo). Copper uptake and retention assays performed on fibroblast cultures have been used successfully for pre- and postnatal diagnosis of Menkes disease. We report here the results of these assays applied to primary fibroblast cultures established from nine independent mottled alleles associated with phenotypes of varying severity maintained on identical genetic backgrounds. No significant differences were found between the different alleles, or between the mottled cultures and fibroblasts established from MD patients. Thus, in the mouse, the data obtained for copper retention/uptake at the cellular level do not correlate with the severity of the phenotype.
The use of compound heterozygotes and Hprt selection to analyze X-linked mottled alleles associated with prenatal lethality.
X-linked mutant alleles associated with prenatal male lethality are difficult to analyze because only heterozygous females are readily available for study. Genomic analysis of the mutant allele is facilitated by the construction of somatic cell hybrids because this enables the segregation of the X Chromosomes (Chrs) that carry the mutant and wild-type alleles. We describe here a method that ensures that the X Chr carrying the mutant allele is retained in somatic cell hybrids in an active selectable state. This is achieved by mating heterozygous females to males that carry a mutation at the hypoxanthine phosphoribosyl transferase (Hprt) locus. The resultant F1 females are compound heterozygotes, and when cells from these females are fused to HPRT- Chinese hamster cells and subjected to selection in HAT medium, the only survivors are those hybrid cells that retain an active X Chr carrying the mutant allele together with the wild-type Hprt allele. We use hybrids constructed by this method to demonstrate that there are no gross deletions or genomic rearrangements present in three mottled alleles associated with prenatal male lethality.
Analysis of large deletions in the HPRT gene of primary human fibroblasts using the polymerase chain reaction.
Spontaneous and X-ray-induced mutants of the HPRT gene were isolated from two primary human fibroblast lines. The limited life-span of the mutants restricted the use of methods requiring large quantities of DNA, and the polymerase chain reaction (PCR) was used in particular to check for the presence of multiple genomic sites in mutant analysis. Robust PCR primers were designed to amplify sites of up to 1 kb, mostly with 1-kb spacings between sites, over the entire 56-kb HPRT gene region. Using PCR, large deletions were found in 43% of independent X-ray-induced mutants, and their breakpoints were localized where these fell within the gene. Anonymous DNA sites in the Xq26 chromosomal region containing HPRT (covering > or = 1.5 Mb) were also amplified by PCR to assess codeletion with HPRT; sites up to 1 Mb distal to the gene (DXS86, DXS10) were codeleted in some mutants, but no mutant was found with loss of a proximal site (DXS79).
DNA methylation changes in the IL-1 (2F) chromosomal region of some radiation-induced acute myeloid leukaemias carrying chromosome 2 rearrangements.
Acute myeloid leukaemias (AML) arising in irradiated CBA/H mice frequently have breakpoints in the F region of chromosome 2. The closely linked cytokine genes interleukin (IL)-1 alpha and beta map to this region, and the beta gene is deregulated in some AMLs. Using pulsed-field gel electrophoresis techniques, we show here that an 800 kb 2F region encoding IL-1 alpha and beta is not obviously rearranged in six leukaemias carrying chromosome 2 abnormalities. However, changes in IL-1 region DNA methylation in three leukaemias may be consistent with loss of hypermethylated sequences from one chromosome copy. These possible 2F region losses are discussed in relation to genomic imprinting and its potential role in murine myeloid leukaemogenesis.
Interleukin-1 beta gene deregulation associated with chromosomal rearrangement: a candidate initiating event for murine radiation-myeloid leukemogenesis?
The incidence of acute myeloid leukemia (AML) in CBA/H mice following exposure to single acute doses of ionizing radiation has previously been determined. A high proportion of these AMLs are characterized by rearrangement of murine chromosome 2 in the C2 and/or E5-F regions, and there is evidence that these events are a direct consequence of radiation damage to multipotential hemopoietic cells. Using a combination of in situ chromosome hybridization and mRNA analyses, we show that the cytokine gene interleukin-1 beta (IL-1 beta) is encoded in the chromosome 2 F region and is translocated in a chromosome 2---2 rearrangement in an x-ray-induced AML (N36). Also, IL-1 beta is specifically deregulated in N36 and in two other chromosome 2-rearranged AMLs but not in a fourth, which has two cytogenetically normal chromosome 2 copies. We suggest that radiation-induced specific chromosome 2 rearrangement associated with IL-1 beta deregulation may initiate murine leukemogenesis through the uncoupling of normal proliferative control mechanisms in multipotential hemopoietic cells.