Biomedical subjects
Publications and source records attributed to R Williamson.
Direct gene analysis was used in the prenatal exclusion of ornithine transcarbamylase (OTC) deficiency; karyotyping the family's previously affected child had already identified the defect as a small deletion. A cloned OTC gene and two other X chromosome-specific probes were mixed, and hybridised to fetal DNA obtained from cultured amniocytes; the fetus was shown to be a normal male.
Cloned coding sequences for rat and human ornithine transcarbamylase (OTC) were obtained by screening a rat and a human cDNA library respectively with a synthetic oligonucleotide corresponding to 27 bases of the rat sequence. These clones, 1100 bp long for the rat clone and 1300 bp for the human, contain approximately 80% of the human OTC coding sequence. The OTC mRNA length determined by Northern blot analysis is 1700bp. The human OTC sequence was shown to be localised Xp11.4-Xp21 using somatic cell hybrids. There is a frequent RFLP revealed with the restriction enzyme MspI. OTC is located more closely to the Duchenne muscular dystrophy mutation than previously reported markers such as RC8 and L1.28, and therefore should prove useful in carrier detection and haplotype analysis of families carrying the mutation causing the disease.
We have used a cDNA clone for Chinese hamster 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase to isolate a genomic recombinant for human HMG-CoA reductase. The identity of the gene was confirmed by partial sequence analysis. Several unique fragments that will be useful for restriction fragment length polymorphism (RFLP) studies were identified. In situ hybridisation of a 2.6 kb unique fragment of the gene to human metaphase chromosomes localised the human HMGCoA reductase gene to human chromosome 5q12.
We have synthesised a mixed oligonucleotide 17 bases long and used it to isolate cDNA clones for apolipoprotein CI (apo CI) from an adult liver cDNA library. The partial sequence of one of these clones confirms its identity. We have used this probe and Southern blotting techniques to identify the human apo CI gene in DNA from a series of rodent X human somatic cell hybrids. Our results provide evidence for the assignment of this gene to human chromosome 19.
It has been suggested that a protein factor causing ciliary dyskinesis is a marker for the basic defect causing cystic fibrosis (CF), and that the structural gene for this protein may be (amongst others) on human chromosome 4. We have isolated two DNA sequences mapping to chromosome 4 which show restriction fragment length polymorphisms (RFLPs), and have followed their segregation in families in which cystic fibrosis occurs. Eleven families with a total of 30 children with CF and ten unaffected sibs were studied. We have also followed the inheritance of RFLPs revealed by two probes mapping to chromosome 4 and obtained from another laboratory, polymorphisms revealed by cloned coding sequences for albumin and fibrinogen, and the inheritance of the MNS blood group. Although the level of albumin is altered in children with CF, the gene does not segregate with CF, and therefore albumin can be excluded as the site of the basic defect. Tight linkage with CF was not found with any of the seven markers investigated, and therefore, assuming that the markers (excepting MNS and fibrinogen) are unlinked to one another, approximately half of the total genetic length of chromosome 4 may be excluded.
DNA samples from 60 unrelated UK patients with familial hypercholesterolaemia (FH) were screened by Southern blot hybridisation to detect gross alterations in the low density lipoprotein (LDL) receptor gene. One patient was found to have a 2kb deletion in the 3' part of the gene. The deletion cosegregates with the FH phenotype in his family. This finding is compatible with the deletion being the cause of FH in this case and makes a presymptomatic test based on DNA analysis available for this family. The defects in most of the other patients are likely to be due to point mutations.
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We studied in vitro mutants of Klebsiella, Enterobacter, and Serratia cross-resistant to nalidixic acid, trimethoprim, and chloramphenicol that were similar to mutants found in vivo. The sole mechanism for this type of resistance appeared to be a reduction in permeability of the cell envelope. The mutants had significantly lower rates of uptake of glucose and chloramphenicol, but binding of chloramphenicol to ribosomes was normal. In addition, the amounts of dihydrofolate reductase were similar in both wild-type and cross-resistant mutants of Klebsiella. Examination of the bacterial outer membrane revealed that the amount of at least one major protein, with a molecular size of approximately 40 kilodaltons, was decreased in the mutants. Therefore the resistance seemed likely to be due to the reduction in quantity of these outer membrane proteins, possibly porins, in the mutant bacteria.
Three benzylpenicillin-resistant, clinical isolates of Enterococcus faecium (MIC values 16-64 micrograms ml-1) contained six penicillin-binding proteins (PBPs), of which PBP5 was the most abundant and had the lowest affinity for the antibiotic. Four benzylpenicillin-susceptible strains (MIC values 0.031-0.5 microgram ml-1) were obtained as spontaneous derivatives from these above organisms. There were significant decreases in the amounts of PBP5 in each of the derivatives, with the concomitant appearance of a new, higher affinity PBP (5*) in three strains. Increased amounts of PBP5, with no changes in PBP5*, were found in several mutants with intermediate-level benzylpenicillin-resistance (MIC values 1-8 micrograms ml-1) selected from two of the susceptible strains. Examination of 18 other clinical isolates, with a wide range of susceptibilities to benzylpenicillin (MIC values 0.062-128 micrograms ml-1), showed that PBP5* was present in 13 strains, and PBP5 in all of them, but in differing amounts. The results concerning the relative amounts and relative affinities of PBPs 5* and 5 allowed the categorization of the various strains into six groups, within which organisms had somewhat similar susceptibilities to benzylpenicillin.
The linkage relationships of the serum arylesterase paraoxonase (PON) was examined in our Danish material of normal families and in Danish and English cystic fibrosis families. Highest lod scores were found between PON and cystic fibrosis. The combined lod score for this relationship was z = 2.69 at theta = 0.07 in males and theta = 0.00 in females. When scored in accordance with a tentative three allele model for PON, the score was z = 3.70 at the same theta values. Linkage studies for PON against 64 other polymorphic marker systems did not give any lod score above +1.3 and PON still remains chromosomally unassigned. By the present screening about 2/3 of the genome could tentatively be excluded as the region of PON and cystic fibrosis.
A comparative study of 60 strains of nutritionally variant streptococci (NVS) with 34 strains of Streptococcus mitis and 37 strains of Streptococcus sanguis II showed the presence of a red chromophore which was absent in the other streptococcal species. By using the conventional microbiological tests, only small differences were found between the NVS and the two other related species. In contrast a clear-cut delineation was found by the API 20 Strep system of identification. All NVS contained pyrrolidonylarylamidase, an enzyme which was absent in S. mitis and S. sanguis II strains, and lacked the alkaline phosphatase enzyme which was present in 56% of S. mitis strains and 62% of S. sanguis II strains. According to the additional enzymatic and biochemical tests of the API 20 Strep system, there were three biotypes among NVS. The major biotype included 33 of 60 strains which were characterized by the presence of both alpha- and beta-galactosidases and the capacity to hydrolyze trehalose. This biotype also showed a specific pattern of penicillin-binding proteins. These results show that NVS are recognized as a separate variety distinct from S. mitis and S. sanguis II species, despite some common biochemical properties. Moreover, the delineation of 33 strains with a specific biotype and a specific penicillin-binding protein pattern strongly suggests that a large part of NVS strains belong to an individual species.
The gene coding for coagulation factor X was studied in a family segregating chromosomal abnormalities involving chromosomes 13 and 6. An individual monosomic for 13q34 was deficient in levels of clotting factors VII and X, while her brother, who is trisomic for 13q34, had elevated levels. DNA dosage studies with a cloned human factor X gene demonstrated that the low levels of factor X expression in the individual with the chromosome 13q34 deletion were due to the absence of one copy of the factor X structural gene. This confirms the assignment of the human gene coding for factor X to 13q34.
We describe the basic principles of recombinant DNA technology and how this methodology has been used to isolate and characterise cloned genes coding for alpha 1-antitrypsin. We show how these probes can be used for diagnosis and to study molecular variants of alpha 1-antitrypsin which may predispose individuals to develop lung disease.
A somatic cell hybrid has been constructed and characterized using fibroblasts from a phenotypically normal woman who possesses an X chromosome with an interstitial deletion of the short arm. High-resolution banding indicates that the deleted segment is either Xp22.13-p11.4 or Xp22.11-p11.23. Southern blot hybridization to previously mapped DNA sequences confirms that the missing segment of the X chromosome is a deletion and not an interstitial translocation and supports the cytogenetic interpretation that the deletion extends proximal of Xp11.3 and therefore probably comprises Xp22.11-p11.23. Three further DNA sequences have been localized to the region of the deleted segment. The following order has been assigned to the seven probes used: Xpter-RC8-pXUT22-(OA1,C7,M2C)-L1.28-RD6 -Xcen.
Total messenger RNA (mRNA) was isolated from adult human liver and copied to give complementary DNA (cDNA) with reverse transcriptase. Double stranded cDNA was cloned by GC tailing into the PstI site of the plasmid pAT153; approximately 2000 recombinant clones were isolated. The sensitivity of the bacterial cells to ampicillin was used as a marker to study the stability of the library during consecutive overnight growth in mixed liquid culture. A significant change in the composition of the library was observed after three overnight cultures. A heterologous rat albumin cDNA recombinant and two specific oligonucleotides were used to screen the library, and clones containing sequences of the human mitochondrial rRNA- and the alpha-1 antitrypsin-genes were isolated.
The cytological location of six cloned DNA sequences on the human X chromosome has been determined to a high resolution by direct hybridisation 'in situ' to metaphase chromosomes. Each locus has been identified using clones which also detect restriction fragment length polymorphisms by Southern hybridisation. The six loci identified are spaced along the chromosome from Xp22 to Xq28. By combining data obtained using this powerful sequence localisation technique with that from hybrid cell panels and from family studies, it is possible to compare physical and genetic distances, and to demonstrate that the frequency of reciprocal genetic exchange is not uniform along the chromosome length.
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