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Biomedical subjects

R Williamson

Publications and source records attributed to R Williamson.

At least 325 records · Page 18Linked to original sources
PubMed1987

A rapid method to identify cosmids containing rare restriction sites.

A procedure for identifying specific cosmid clones containing recognition sites for "rare cutting" restriction enzymes has been developed. Cosmid clones containing human inserts were selected by hybridisation to human repetitive DNA. An oligonucleotide corresponding to the NotI recognition site, eight bases long, was labelled and used to probe DNA samples from one hundred cosmids. By optimising the difference in melting characteristics between eight-base perfect match and six-base match/two base mismatch hybrids, we were able to detect the cosmids containing either NotI (8 bp match) or XmaIII/EagI (6 bp match) sites. The generation of a map for rare cutter sites along a human chromosome, or a chromosome region, should be simplified using this approach, which will enable the identification of a set of "milestones" at intervals of several hundred kilobases (kb) along the DNA sequence.

Base Sequence↗
PubMed1987

Human gene mapping.

It is now possible to map the human genome completely with a set of closely linked markers. Over 500 coding genes have been cloned and localized, as have approximately 2000 anonymous DNA fragments, most of which recognize two-allele polymorphisms that are caused by single base changes which alter the recognition site for a restriction enzyme (restriction fragment length polymorphisms). Most human chromosomes have been mapped, with markers in defined order placed approximately 10 map units apart. Chromosomes X and 21 are particularly well mapped, with over 200 probes ordered on X. The strategy during the next few years will encompass moving from a linkage map to a set of overlapping cosmid or phage clones, and finally to a complete sequence of regions of chromosomes and entire chromosomes. A complete sequence of the human genome should transform our understanding of development, the control of gene expression, and the parameters of genetic disease.

Chromosome Mapping↗
PubMed1987

Two unusual cases of first trimester prenatal diagnosis of cystic fibrosis using DNA probes.

There are now several DNA probes which localize the cystic fibrosis mutation (CF) to chromosome 7q2.2-q3.1. The most tightly linked probes, pJ3.11 and met, are useful for first trimester prenatal diagnosis for many families provided that there is at least one living child affected by CF (Farrall et al., 1986). We describe here two families seeking prenatal diagnosis for CF which present unusual counselling problems. The first is an extended family in which there is no living affected member with CF; the second, a consanguinous marriage at risk both for cystic fibrosis and beta-thalassaemia. In both cases first trimester chorionic villus sampling and DNA haplotype analysis predicted that the fetus is a carrier for CF, and in the doubly affected family a carrier for beta-thalassaemia as well. Both pregnancies resulted in live births and subsequent immunoreactive trypsin estimations were both in the normal range.

Chorionic Villi↗
PubMed1987

Isolation of a polymorphic genomic clone from chromosome 7. Physical and genetic linkage studies to markers around the cystic fibrosis locus.

A peptide prepared from purified factor 13B (F13B) was sequenced, and a single, long oligonucleotide corresponding to its cognate DNA sequence was constructed and used to screen a chromosome 7 specific genomic library. The positive clone isolated, designated pKV13, was only related to F13B at the oligonucleotide region, but has proved to be a valuable chromosome 7 marker. pKV13 maps to 7pter-q22 in hybrid cell lines, and is present in a chromosome-mediated gene transfer (CMGT) cell line that also contains met and other 7q probes. pKV13 defines a common MspI restriction fragment length polymorphism (RFLP), and is genetically linked to two markers on the long arm of chromosome 7, B79a and COLIA2, both themselves linked to the cystic fibrosis locus. Multipoint linkage analysis demonstrates that KV13 maps centromeric to both B79a and COLIA2. pKV13 has been used to demonstrate the existence of rearrangements within CMGT hybrids, and will also prove valuable in multipoint linkage studies of other 7q markers. Finally, pKV13 provides a new polymorphic locus for the characterisation of 7q deletions in myeloid disorders such as myelodysplastic syndrome.

Amino Acid Sequence↗
PubMed1987

Crossovers in two German cystic fibrosis families determine probe order for MET, 7C22 and XV-2c/CS.7.

We have followed the segregation of the probes pJ3.11, 7C22, pB79a, and MET through cystic fibrosis families in the German Democratic Republic with two affected sibs. Two families with a crossover between MET and the CF phenotype were detected. In one of these families recombination was also observed between the DNA probe 7C22 and CF, and between the markers XV-2c and CF, which suggests that XV-2c, MET and 7C22 are all on the same side of CF. The other MET recombinant family is informative with XV-2c and does not recombine, which excludes the genetic order XV-2c--MET--CF if multiple recombinant events are disregarded. These two families together demonstrate that recombinations may occur in a very small genetic interval, which has important implications for prenatal diagnosis based on data from linked markers.

Crossing Over, Genetic↗
PubMed1987

Biochemical and genetic exclusion of calmodulin as the site of the basic defect in cystic fibrosis.

Recent physiological studies have shown a defective beta-adrenergic regulation of chloride transport and protein secretion in tissues affected by cystic fibrosis. The exact biochemical nature of this abnormality is unknown, but an intracellular second messenger may be involved. We have tested the hypothesis that calmodulin is the site of the basic defect in CF using biochemical and molecular genetic techniques. We report here that there is no gross structural abnormality in the calmodulin protein from CF submandibular glands, and that although there are at least three distinct sequences that cross-hybridise with a calmodulin cDNA probe in the human genome, none of these can be the locus of CF. A polymorphism at the locus of a calmodulin cross-hybridising sequence at human chromosome 7p2 is described.

Alleles↗
PubMed1987

Exclusion of the Friedreich ataxia gene from chromosome 19.

Friedreich ataxia, a progressive neurodegenerative disorder, is an autosomal recessive disease with a carrier frequency of 1/110 in the United Kingdom. The pathophysiological basis for the disease is not known and the chromosomal location of the mutation remains unidentified. As part of an attempt to map the mutation using linked DNA markers, we demonstrate that the Friedreich ataxia gene is excluded from human chromosome 19. This study also demonstrates that the insulin receptor, which maps to chromosome 19 and may be associated with abnormal biochemical features in some patients, is not the basic defect.

Alleles↗
PubMed1987

Patterns of polymorphism and linkage disequilibrium for cystic fibrosis.

Four polymorphic markers that map within 80 kb of an HTF island which is genetically very close to the cystic fibrosis locus have been identified. We have analyzed the linkage disequilibrium between each of these markers and the cystic fibrosis mutation in 89 families from four European countries, Denmark, Finland, Spain, and Great Britain. Strong linkage disequilibrium between three polymorphic sites and cystic fibrosis was observed. The markers on the J3.11 (D7S8) side of the HTF island show stronger disequilibrium than those on the met side. Linkage disequilibrium between markers and disease alters the probability that a person of a given haplotype is a carrier in some populations and helps to identify regions of a sequence that are most likely to contain the cystic fibrosis mutation.

Chromosome Mapping↗
PubMed1987

The structural gene for lecithin:cholesterol acyl transferase (LCAT) maps to 16q22.

We have used a cDNA clone for human lecithin:cholesterol acyl transferase (LCAT) and Southern blotting techniques to identify the human LCAT gene in DNA from a series of rodent X human somatic cell hybrids. Our results are compatible with the location of the gene on human chromosome 16, and this has been confirmed using in situ hybridization of the LCAT cDNA to human metaphase chromosomes. These results confirm the earlier studies on LCAT-deficient patients, indicating that the structural gene for LCAT is on human chromosome 16q22.

Animals↗
PubMed1987

Control of end-plate channel properties by neurotrophic effects and by muscle activity in rat.

1. The formation of ectopic neuromuscular synapses was induced in rat soleus muscle by implantation of the fibular nerve into the proximal part of the muscle and subsequent sectioning of the soleus nerve. The gating properties of acetylcholine (ACh) receptors at the newly formed end-plates were examined by analysis of acetylcholine-induced membrane current fluctuations. 2. In agreement with earlier studies, the apparent mean open time of end-plate channels decreased during synaptic development from about 4 ms to about 1 ms (-60 mV membrane potential, 22 degrees C) within 7-18 days after the soleus nerve had been cut. 3. When the fibular nerve was cut at an early stage of end-plate development, fast-gating channels with apparent mean open times of 1 ms characteristic of mature end-plates did not develop within the next 10-14 days. 4. When the fibular nerve was cut at an early stage of end-plate development and the soleus muscle was then stimulated chronically via implanted electrodes, fast-gating channels did develop in the absence of the nerve terminals within 4-6 days. 5. When impulse conduction in the transplanted fibular nerve was blocked chronically at the time of soleus nerve section such that ectopic end-plates formed in inactive muscle, fast-gating channels developed within 12-14 days. 6. The results show that motoneurones control the conversion from slow-gating fetal to fast-gating adult-type ACh receptor channels at ectopic end-plates in rat soleus muscles. The conversion occurs in the absence of impulse activity provided the nerve continues to be present. However, it also occurs in the absence of the nerve provided the muscle is active and had received an early priming influence from the nerve. Thus, nerve-evoked muscle activity and nerve-released trophic influences complement each other in controlling the gating properties of junctional ACh receptor channels.

Action Potentials↗
PubMed1987

The cystic fibrosis locus.

The identification of the cystic fibrosis locus (CF) provides a model for the study of single gene defects where the biochemical lesion is not known. Using families each of which has several affected siblings, it was possible to exclude a number of 'candidate genes' which had previously been proposed as possible sites of the CF mutation. Exclusion mapping of the genome using polymorphic protein and DNA markers showed that CF is on the long arm of human chromosome 7. The most closely linked flanking markers were identified, and human chromosome fragments containing them (and therefore the CF locus) were isolated in rodent cell lines by chromosome-mediated gene transfer. The transgenome was then analysed using cosmid contig mapping, pulse-field gel electrophoresis, HTF island identification and linkage disequilibrium. In this way, a candidate coding sequence has been identified which always segregates with CF.

Animals↗
PubMed1987

Linkage of DNA probe B79a (D7S13) to cystic fibrosis.

We have conducted, in 64 affected families, a study of linkage between the anonymous DNA segment pB79a (D7S13) and the locus for cystic fibrosis (CF) on chromosome 7q. The maximum lod score was 12.60 at theta = .08 (confidence bounds .045-.135). Although D7S13 is not sufficiently close to CF for routine use in DNA-based prenatal diagnosis, it will be helpful in certain families when other nearby markers are uninformative. D7S13 will also be useful for refining the linkage map of the CF region.

Cystic Fibrosis↗