Determination of haemophilia A carrier status from hair samples using polymerase chain reaction technique.

The usefulness of intragenic restriction fragment length polymorphisms (RFLPs) for BclI, HindIII and XbaI, adapted for polymerase chain reaction (PCR), was tested for the detection of haemophilia A carrier status in the consultant of a family in which only haematological information was available on the inheritance of the trait. Hair follicles were used as the non-invasive source of DNA. The mother was found to be homozygous for BclI and heterozygous for HindIII sites, whereas her status as regards informativeness could not be established for XbaI. On the basis of HindIII RFLP, the daughter was found to be a carrier of the haemophilia trait. This was confirmed by sequencing the amplified intron 19 of the mother and the daughter. The RFLP for XbaI did not appear to be suitable for carrier detection using PCR due to the difficulty of establishing homozygosity or heterozygosity from the results of digestion of the amplified product.

Structural and functional conservation of the human homolog of the Schizosaccharomyces pombe rad2 gene, which is required for chromosome segregation and recovery from DNA damage.

The rad2 mutant of Schizosaccharomyces pombe is sensitive to UV irradiation and deficient in the repair of UV damage. In addition, it has a very high degree of chromosome loss and/or nondisjunction. We have cloned the rad2 gene and have shown it to be a member of the Saccharomyces cerevisiae RAD2/S. pombe rad13/human XPG family. Using degenerate PCR, we have cloned the human homolog of the rad2 gene. Human cDNA has 55% amino acid sequence identity to the rad2 gene and is able to complement the UV sensitivity of the rad2 null mutant. We have thus isolated a novel human gene which is likely to be involved both in controlling the fidelity of chromosome segregation and in the repair of UV-induced DNA damage. Its involvement in two fundamental processes for maintaining chromosomal integrity suggests that it is likely to be an important component of cancer avoidance mechanisms.

Evolutionary conservation of excision repair in Schizosaccharomyces pombe: evidence for a family of sequences related to the Saccharomyces cerevisiae RAD2 gene.

Cells mutated at the rad13 locus in the fission yeast, Schizosaccharomyces pombe are deficient in excision-repair of UV damage. We have cloned the S.pombe rad13 gene by its ability to complement the UV sensitivity of a rad13 mutant. The gene is not essential for cell proliferation. Sequence analysis of the cloned gene revealed an open reading-frame of 1113 amino acids with structural homology to the RAD2 gene of the distantly related Saccharomyces cerevisiae. The sequence similarity is confined to three domains, two close to the N-terminus of the encoded protein, the third being close to the C-terminus. The central region of about 500 amino acids shows little similarity between the two organisms. The first and third domains are also found in a related yet distinct pair of homologous S.pombe/S.cerevisiae DNA repair genes (rad2/YKL510), which have only a very short region between these two conserved domains. Using the polymerase chain reaction with degenerate primers, we have isolated fragments from a gene homologous to rad13/RAD2 from Aspergillus nidulans. These findings define new functional domains involved in excision-repair, as well as identifying a conserved family of genes related to RAD2.

Non-transmissible and self-transmissible plasmids conferring drug resistance in clinical isolates of Serratia marcescens from hospitals in Riyadh, Saudi Arabia.

Sixty Serratia marcescens isolates were obtained from patient specimens from three different hospitals in the city of Riyadh. These were tested for their antibiotic resistance factors using eleven different antibiotics. Their ability to transfer antibiotic resistance plasmids to a sensitive E. coli strain (RR1) was tested by transformation and conjugation experiments. Agarose gel electrophoresis was used to determine the size and number of the R-plasmids. Southern blotting was used to assess homologies between antibiotic resistance plasmids from different isolates. Among the isolates tested, 36.7% contained plasmids, and all these were from strains isolated from two hospitals. No R-plasmids could be detected among the multiple resistant strains isolated from the third hospital. Among the strains that contained plasmids, approximately 63.6% transferred multiple antibiotic resistance to E. coli and the rest transferred only one antibiotic resistance marker. The majority of strains carrying out the plasmids showed similarities in band number and size. In view of the similarities this group was denoted the predominant group, and selected for further molecular investigations. Restriction endonuclease digests of plasmids from this group gave the same restriction pattern which confirmed that they were closely related. Hybridization experiments using these plasmids and nick-translated 32p-labelled pBR-322 DNA probe, showed that all the large bands (36 kb) are related and exhibit homology with pBR-322.

Incidence and transfer of R-plasmids at a hospital in Saudi Arabia.

One hundred and fifty Gram-negative bacteria isolated from patient specimens at King Faisal Specialist Hospital were examined for their ability to transfer antibiotic resistance plasmids to a sensitive Escherichia coli recipient in conjugation and transformation experiments. Agarose gel electrophoresis was used to enumerate and size the R-plasmids found, and Southern DNA hybridization was used to assess similarities between antibiotic resistance plasmids from different bacteria and sources. Of the bacterial isolates tested 65% contained plasmids, 70% of these transferred antibiotic resistance to E. coli, and 40% transferred multiple, linked resistances on R-plasmids. DNA hybridization of these R-plasmids demonstrated widespread similarities between plasmids from different bacterial genera and from different hospital locations. In particular, a gene encoding ampicillin resistance appeared especially widespread, indicating that a transposon may be mediating transmission of this resistance.

Gamma processing of Arabic bread for immune system-compromised cancer patients.

Arabic bread prepared from local Saudi flour contained a total of up to 10(4) organisms per g. Most of these were bacterial spores that survived the baking process (1.3 X 10(2) to 3.5 X 10(3] and a small number of yeasts and molds (10 to 40 cells per g). The organisms in Arabic bread appear to be harmless to healthy individuals. However, for immune system-compromised cancer patients and bone marrow transplant recipients, it is prudent to irradiate the bread to reduce microbial contamination. The decimal reduction doses (10% survival) for the most radiation-resistant organisms (spore formers) in bread were 0.11 to 0.15 Mrad. Accordingly, 0.6 Mrad was sufficient to reduce the number of spores in Arabic bread by a factor of 10,000, i.e., to less than 1/g. This treatment constitutes radiation pasteurization (radicidation), and to this extent, provides a margin of microbiological safety. Sensory evaluation by the nine-point hedonic scale showed no detectable loss of organoleptic quality of bread up to 0.6 Mrad, while irradiation to 2.5 Mrad induced unacceptable organoleptic changes.