Biomedical subjects
Publications and source records attributed to D J Shaw.
In this paper we report the study of the segregation of three chromosome 19 markers known to be linked to myotonic dystrophy in nine families with von Recklinghausen neurofibromatosis. Clear evidence against linkage was found for all three markers excluding the von Recklinghausen neurofibromatosis gene from the myotonic dystrophy region of chromosome 19.
Chromosome 19 is currently the most fully mapped of the smaller chromosomes, with about 40 loci assigned to it (HGM8). Major inherited disorders on this chromosome include myotonic dystrophy and familial hypercholesterolaemia. Other loci include five blood groups, a cluster of apolipoprotein genes, and the receptors for insulin and polio virus. A number of cloned genes and random DNA sequences identify polymorphisms which, together with blood group and other protein polymorphisms, have been used to establish a framework for ordering the loci and estimating genetic distances. Hybrid cell lines allow loci to be assigned to one of eight different regions and a detailed genetic map of the chromosome will be possible in the near future.
Three chromosome 19 markers known to be linked to myotonic dystrophy have been studied in nine families with peripheral neurofibromatosis (Von Recklinghausen's disease). Clear evidence against linkage has been found for all three markers, excluding the peripheral neurofibromatosis gene from the myotonic dystrophy region of chromosome 19. Previous reports of co-inheritance of the two disorders in families cannot therefore be explained on the basis of close genetic linkage between the loci.
Recent advances in agarose gel electrophoresis of large DNA fragments raise the possibility of an entirely new approach to mapping mammalian genomes. In this article is discussed the potential of this technology for tackling problems such as construction of linkage maps, identifying chromosome translocation breakpoints, and moving from linked markers to genes causing diseases such as the muscular dystrophies and Huntington's chorea.
A series of mouse-human somatic cell hybrid lines (WILF) were derived from a hybrid that was originally thought to have chromosome 19 as its only human chromosome. In situ hybridization has been used to assess the amount of human material present in the different lines. All appear to contain different numbers of human chromosome fragments. A series of X-chromosome-specific DNA sequences hybridized against DNA from the lines revealed that material from the X long arm is present in several cases. Chromosome 19-specific DNA sequences used in a similar way show that fragmentation of this this chromosome has occurred with subsequent segregation of the fragments in different lines. The localization of these markers to various regions of chromosome 19, and their relation to the fragments observed in the WILF lines, is discussed.
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The human apolipoprotein CII gene probe detects a restriction fragment length polymorphism located on chromosome 19. We have investigated the linkage of this polymorphism to the myotonic dystrophy locus in families. The two loci are closely linked with a maximum Lod score of 7.877 at 4% recombination. The close linkage and informativeness of the APOC2 polymorphism suggest that this probe may be of use for presymptomatic diagnosis of the myotonic dystrophy gene. The APOC2 gene was localised to the region 19p13-19q13 using somatic cell hybrids, providing further evidence that the myotonic dystrophy locus is situated in the central region of chromosome 19.
The genes encoding apolipoproteins CI, CII, and E have been previously localized to chromosome 19. By use of rodent-human hybrid cell lines containing translocations of chromosome 19 we have now mapped these three genes to the region 19p13-19q13 and most probably 19p13-19cen. The clustering of APOC1, APOC2, and APOE must reflect their common evolutionary background and suggests that they may be coordinately regulated. Polymorphisms detected for any one gene will be useful for inheritance studies of all three.
Extracts of hamster-human and mouse-human hybrids, some with translocations involving chromosome 19, have been assayed for both human spleen ferritin (rich in L subunits) and human heart ferritin (rich in H subunits). Hybrid lines retaining part of the long arm of chromosome 19 including the region 19q13.3----19qter produced human "L" type ferritin. This confirms the previous assignment of the "ferritin gene" to chromosome 19 (Caskey et al. 1983). However, lines retaining chromosome 11 were found to contain human "H" type ferritin suggesting that the gene for the "H" subunit is on this chromosome. The presence of chromosome 6 was not necessary for the expression of either "H" or "L" type human ferritin. It thus seems unlikely that the gene for idiopathic haemochromatosis is a ferritin gene.
The inheritance of seven restriction fragment length polymorphisms detected by DNA probes has been studied in families with Duchenne and Becker muscular dystrophies (DMD and BMD). The probes used have all been mapped to the short arm of the X-chromosome, four being distal and three proximal to the disease loci located within the Xp21 region. Linkage analysis of the DNA polymorphisms in relation to the two disorders showed similar genetic distances. Data obtained from DMD and BMD families have been combined to give more precise values for the different recombination fractions. Combined use of these polymorphic DNA markers will be of practical value in the genetic counselling of women at risk for Duchenne and Becker muscular dystrophy.
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The inheritance of a restriction fragment length polymorphism (RFLP) detected by a cloned DNA sequence (p754) from the short arm of the X chromosome has been studied in 14 Duchenne muscular dystrophy kindreds and six Becker muscular dystrophy kindreds. The linkage data obtained suggest that both the DMD and BMD loci are located in the same region (p21) on the short arm of the X chromosome at a distance of about 15 to 20 cM from the 754 locus. Data from families informative for both the p754 and L1.28 polymorphisms suggest that p754 is closer to the disease loci than is L1.28.
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A study has been undertaken to determine the efficiency of current methods in providing an adequate amount of chorionic villus DNA for antenatal diagnosis using recombinant DNA techniques or enzyme assay. Chorionic biopsies were obtained from 40 women undergoing elective first trimester termination of pregnancy (8-12 weeks) under general anaesthesia. The villus tissue was isolated from maternal tissue under a dissection microscope and the presence of any remaining contamination was ascertained by conventional histology and immuno-cytochemical examinations. A high level of success was achieved in obtaining a pure fetal sample. In the first 20 samples the DNA yield obtained using the method of Williamson et al [1] was found to be 0.5 +/- 0.5 micrograms/mg wet weight of villus tissue (mean +/- 1 SD). In the subsequent 20 biopsies using a modified procedure, the yield was significantly improved to 1.0 +/- 0.65 (p less than 0.002). A normal range for the enzyme iduronate sulphatase, which is deficient in Hunter's syndrome (mucopolysaccharidosis II), is also reported. It is suggested that as little as 20 mg of chorionic villi may be used to provide sufficient material for a reliable study using recombinant DNA or biochemical methods.
A panel of human-rodent somatic cell hybrids containing translocation derivatives of human chromosome 19 has been used to assign the markers peptidase D, complement component 3, lysosomal mannosidase, lysosomal DNAase, chorionic gonadotropin beta-subunit, and a new polymorphic DNA sequence, to specific regions of chromosome 19. This has allowed the relative orientations of the genetic and physical maps to be established, and provides the framework for a search for the genes responsible for inherited disorders on chromosome 19, such as myotonic dystrophy and neurofibromatosis.
The fnr gene is essential for the expression of anaerobic respiratory metabolism in Escherichia coli. Genetic and biochemical studies support the view that its product. Fnr, is a transcriptional regulatory protein specific for genes encoding anaerobic respiratory functions (fumarate, nitrate and nitrite reductases, hydrogenase, etc.). In this respect Fnr may be considered analogous to the well-characterized catabolite gene activator protein (CAP), which mediates the control of catabolite-sensitive gene transcription. With a view to identifying its function, the fnr gene has recently been cloned and the primary structure of the Fnr protein deduced from the nucleotide sequence. This has revealed the presence of three regions of sequence homology with CAP. One corresponds to the DNA-binding site, a region of about 20 highly conserved amino acids that is believed to form a characteristic three-dimensional structure in several transcriptional regulators. The other regions of homology are in the nucleotide binding domain of CAP but the residues that interact with cAMP are not identical in Fnr. These homologies suggest that Fnr and CAP may have similar three-dimensional structures and that the regulation of anaerobic energy metabolism may involve interaction between Fnr and an unidentified effector molecule.
The nucleotide sequence of a 1.64 kb fragment of E. coli DNA containing the fnr gene (regulatory gene for fumarate and nitrate reduction) was determined using the dideoxy chain termination method. The fnr coding region (750 bp) was identified, and the initiation and termination points of fnr transcription were located by RNA:DNA hybridisation with single-stranded M13 probes. The DNA fragment also contained the 5' end of a separately transcribed gene of unknown function. The deduced molecular weight (27947) of the Fnr protein was in agreement with that of the protein identified by the maxicell procedure, and the primary structure contained regions of homology with several transcriptional regulator proteins.
The position of a gene (fnr) that is essential for growth of Escherichia coli with fumarate or nitrate as electron acceptor was located within an 11.5 kb HindIII fragment of bacterial DNA by deletion analysis with fnr transducing phages (lambda fnr) and by sub-cloning restriction fragments into multicopy plasmids. The functional gene was isolated in a 1.65 kb BamHI-HindIII fragment of a hybrid plasmid pGS24. The fnr gene product was identified as a protein of Mr 31 000, by post-infection labelling and by the 'maxicell' method. Organisms containing the multicopy plasmid (pGS24) overproduced fumarate reductase to the same extent as cultures containing a comparable fumarate reductase plasmid (pNU31) during anaerobic growth; the fnr plasmid also overcame the repression of fumarate reductase synthesis that is normally observed during aerobic growth. Similar effects on nitrate reductase synthesis were also observed. The results support the view that the fnr gene product functions as a positive regulator or a specific sigma factor for expression of anaerobic energy-generating systems.