Biomedical subjects
A Jeffreys
Publications and source records attributed to A Jeffreys.
Instability of long inverted repeats within mouse transgenes.
Various sequences in the mammalian genomes are unstable. One class of sequence arrangement is long inverted repeats, which are known to be unstable in bacteria and yeast. While in mammals some evidence suggests that short inverted repeats (<10 bp long) may show instability, nothing is known about the stability of long inverted repeats. Here we describe two unrelated multicopy transgenes in the mouse (loci 109 and OX1-5), each of which contains a long inverted repeat that shows substantial mitotic instability. This instability also occurs in the germline so that mutant transgenes appear within pedigrees at a high frequency. The mutation processes acting at these two inverted repeats are complex and can involve insertion or deletion, and can result in stabilization of the transgene. At transgene 109 mutational events range from very small rearrangements at the centre of the inverted repeat to complete transgene deletion. In addition we show that the rates of mutation at the inverted repeat of transgene OX1-5 can vary between the male and female germlines and between inbred strains of mice, suggesting the possibility of a genetic analysis to identify loci that modulate inverted repeat instability.
A somatic BRCA1 mutation in an ovarian tumour.
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Increased rate of spontaneous mitotic recombination in T lymphocytes from a Bloom's syndrome patient using a flow-cytometric assay at HLA-A locus.
Bloom's syndrome (BS) is an autosomal recessive disorder conferring high propensity for cancer and displaying a high degree of genetic instability; the frequency of sister chromatid exchange is characteristically 10 times above background. The symmetrical four-armed chromatid interchanges are much more readily detected in peripheral blood lymphocytes of BS patients, suggesting that the frequency of somatic recombination is also increased. In the present study, the rate of spontaneous loss of HLA-A allele expression was estimated following fluctuation analysis in cultured T lymphocytes using a flow-cytometric assay. It was found to be 10 times or more higher than normal in lymphocytes from a BS patient. Molecular and chromosome analyses showed that all 13 independent variants from the patient were most likely derived from somatic recombinations. Further tests for loss of heterozygosity at a closely linked proximal locus, HLA-DQA1, showed that as many as half of the recombinants retained heterozygosity irrespective of the donor. The results suggest that the HLA region is hyperrecombinogenic in somatic cells and that the elevated recombination rate in BS cells results from the general increase at ordinary sites and not from random creation of unusual sites for recombination.
Development of a flow-cytometric HLA-A locus mutation assay for human peripheral blood lymphocytes.
A flow-cytometric technique was developed to measure the frequency of variant lymphocytes lacking expression of HLA-A2 or A24 allele products among donors heterozygous for HLA-A2 or A24. It was found that the variant frequency of lymphocytes in peripheral blood was of the order of 10(-4) and increased with donor age. Molecular analyses of mutant clones revealed that about one-third were derived from somatic recombinations and that the remaining two-thirds did not show any alterations after Southern blotting analysis. In contrast, mutants obtained after in vitro X-ray mutagenesis study were found to be mostly derived from large chromosomal deletions. A small-scale study on atomic bomb survivors did not show a significant dose effect.
Application of DNA "fingerprints" to paternity determinations.
26 cases of disputed paternity were tested by the methods routinely used in Scandinavian countries and by the DNA "fingerprinting" technique. In all the studied cases the results of DNA analyses were similar to those obtained with the routine examinations based on protein polymorphisms; and, in the cases where even HLA typing did not distinguish between tentative fathers, the results obtained with DNA analyses were unambiguous.
Use of minisatellite DNA probes for recognition and characterization of relapse after allogeneic bone marrow transplantation.
Restriction fragment length polymorphisms can be used to distinguish blood and marrow cells from close relatives. We used two probes that recognize a series of dispersed and highly polymorphic tandem-repetitive minisatellite regions in the human genome that can be detected via a shared 10-15 base pair core sequence similar to the generalized recombination sequence (chi) of E. coli. We have studied the resulting individual-specific DNA fingerprints in 15 patients before and after allogeneic bone marrow transplantation performed for chronic myeloid leukaemia and in two patients transplanted for acute leukaemia. Early engraftment could be demonstrated at 3 weeks post-transplant based on the recognition of cells of donor origin. One patient who failed to engraft had only recipient type marrow cells 3 months post-transplant. Nine patients who relapsed after transplantation had only cells of recipient origin. In one patient who relapsed after transplantation with T-cell depleted donor marrow, fractionation studies showed that his T-cells at relapse were of recipient origin. We conclude that these minisatellite probes are valuable for characterizing the origin of different cell populations after marrow transplantation and could be useful for characterizing relapse when donor and recipient are of the same sex.
The 5' flanking region of human epsilon-globin gene.
The structural analysis of the 2.0 kb region upstream from the epsilon-globin gene has been carried out. A genomic DNA map around the gene was worked out in some detail to ensure that the cloned DNA was representative of the actual chromosomal arrangement. Furthermore, a new technique was developed to precisely map a reiterated DNA sequence present 1.5 kb to the 5' side of the gene. The complete nucleotide sequence of the 2.0 kb 5' flanking region was then determined and overlapped with the gene. The sequence included the reiterated DNA sequence which is homologous to the so-called AluI family of repeats. Unusual stretches of sequence 50 nucleotides long, where A + T represent about 90% of the bases, are present at both the 5' and 3' sides of the repeat.
Proteins made in mitochondria of cultured animal cells.
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Differences in the products of mitochondrial protein synthesis in vivo in Human and mouse cells and their potential use as markers for the mitochondrial genome in human--mouse somatic cell hybrids.
The proteins synthesized in the mitochondria of mouse and human cells grown in tissue culture were examined by electrophoresis in polyacrylamide gels. The proteins were labelled by incubating the cells in the presence of [(35)S]methionine and an inhibitor of cytoplasmic protein synthesis (emetine or cycloheximide). A detailed comparison between the labelled products of mouse and human mitochondrial protein synthesis was made possible by developing radioautograms after exposure to slab-electrophoresis gels. Patterns obtained for different cell types of the same species were extremely similar, whereas reproducible differences were observed on comparison of the profiles obtained for mouse and human cells. Four human-mouse somatic cell hybrids were examined, and in each one only components corresponding to mouse mitochondrially synthesized proteins were detected.