Selection of a human chromosome 21 enriched YAC sub-library using a chromosome-specific composite probe.

The subdivision of total genomic human yeast artificial chromosome (YAC) libraries into specific chromosome clone collections will greatly facilitate the construction of an integrated genetic, physical and transcriptional map of the genome. We report the isolation of 388 YAC clones from a human library with an average insert size of 620 kilobases (kb) by the hybridization of a composite chromosome 21 probe to a high-density array of YAC clones. Roughly half of these clones hybridize to chromosome 21 by fluorescence in situ hybridization. These clones represent a twofold coverage of the chromosome. The technique offers the potential of sub-dividing whole genomic YAC libraries into their chromosomal elements to produce high-resolution tools for genome mapping.

A radiation hybrid map of the rat genome containing 5,255 markers.

A whole-genome radiation hybrid (RH) panel was used to construct a high-resolution map of the rat genome based on microsatellite and gene markers. These include 3,019 new microsatellite markers described here for the first time and 1,714 microsatellite markers with known genetic locations, allowing comparison and integration of maps from different sources. A robust RH framework map containing 1,030 positions ordered with odds of at least 1,000:1 has been defined as a tool for mapping these markers, and for future RH mapping in the rat. More than 500 genes which have been mapped in mouse and/or human were localized with respect to the rat RH framework, allowing the construction of detailed rat-mouse and rat-human comparative maps and illustrating the power of the RH approach for comparative mapping.

Current British veterinary attitudes to the use of perioperative antimicrobials in small animal surgery.

A questionnaire was sent to 2951 mixed and small animal veterinary practices to examine the use of perioperative antimicrobials in cats and dogs in the UK. The percentage of respondents who always used antimicrobials in two surgical procedures classified according to NRC criteria as 'clean' was 25.3 per cent for removal of a 1 cm cutaneous mass and 32.1 per cent for routine prescrotal castration. Factors considered important in decision-making about when to use antimicrobial agents included immunosuppression, presence of a drain, degree of wound contamination, potential for spillage of visceral contents and implantation of prosthesis. The most common antimicrobial agents mentioned were potentiated amoxicillin (98.0 per cent), amoxicillin (60.5 per cent), clindamycin (21.8 per cent), enrofloxacin (21.7 per cent), cephalexin (18.6 per cent) and metronidazole (12.7 per cent). Forty-three per cent of all responding veterinarians listed a long-acting preparation for perioperative use. The routes used were subcutaneous (76.1 per cent), intravenous (25.8 per cent), intramuscular (19.8 per cent), oral (13.5 per cent) and topical (7.7 per cent). Antimicrobials were given before surgery (66.6 per cent), during surgery (30.2 per cent), immediately after surgery (12.0 per cent) and after surgery (6.3 per cent). This survey has identified the suboptimal use of perioperative antimicrobials in small animal surgery with improvements needed with respect to timing, duration, choice of antimicrobial and a more prudent selection of surgical cases requiring prophylaxis.

Hamster cells with increased rates of DNA amplification, a new phenotype.

Baby hamster kidney (BHK) cells selected simultaneously with N-phosphonacetyl-L-aspartate (PALA) and methotrexate (MTX) gave rise to doubly resistant colonies at frequencies 20 to 260 times greater than the product of the independent frequencies found with PALA or MTX alone. Double resistance was due to amplification of both target genes, CAD and DHFR. Four independent doubly resistant "MP" lines were selected and characterized. Cells resistant to coformycin, pyrazofurin, or ouabain were generated from all four MP lines at rates up to 25 times greater than the rates for BHK cells. These three drugs select cells that have amplified the genes for their target enzymes. Therefore, we conclude that the four MP lines have an amplificator phenotype. All four grew much more slowly than BHK cells, indicating that the amplificator phenotype may be linked to significant defects in metabolism or cell division.

Neutralization and lipoprotein binding of lipopolysaccharides in tolerant rabbit serum.

We examined the ability of sera drawn from rabbits made tolerant to lipopolysaccharide (LPS) from Escherichia coli O18 to inhibit heterologous LPS-induced gelation of Limulus lysate. Compared with a pool of pretolerant sera, a pool of tolerant sera from these rabbits neutralized 5.5-fold more LPS from Salmonella typhimurium, 4.9-fold more LPS from Pseudomonas aeruginosa, and 10.0-fold more LPS from Klebsiella pneumoniae. Because previous studies have found that LPS bound to lipoprotein is less toxic than unbound LPS, we also studied the binding of LPS lipoprotein fractions in the serum pools by using fractionation with a cesium chloride-equilibrium density gradient. Radiolabeled LPS from E. coli O113 bound much more rapidly to lipoprotein fractions in tolerant serum than in pretolerant serum (after 11 min of incubation, 66.1% vs. 16.5% binding, respectively).

Construction of a mouse whole-genome radiation hybrid panel and application to MMU11.

Whole-genome radiation hybrids have been used to construct human genome maps that integrate different types of markers. To investigate this methodology in mammalian species other than humans, a panel of 164 mouse x hamster whole-genome radiation hybrids was constructed. This set of hybrids was used to produce a high-resolution map of a region on MMU11 that included microsatellite markers and cDNA sequences. The mouse homologue of the human SRY-related gene SOX9 was mapped to an interval of approximately 1.1 cM flanked by the microsatellite markers D11Mit11 and D11Mit291. This interval includes the region containing the mouse Tail-short mutation, a possible homologue of the human syndrome campomelic dysplasia, which is caused by mutations in SOX9. Our results suggest that whole-genome radiation hybrid technology will be a useful adjunct to mapping the genomes of nonhuman mammalian species.

A first-generation whole genome-radiation hybrid map spanning the mouse genome.

We have assembled a first-generation anchor map of the mouse genome using a panel of 94 whole-genome-radiation hybrids (WG-RHs) and 271 sequence-tagged sites (STSs). This is the first genome-wide RH anchor map of a model organism. All of the STSs have been previously localized on the genetic map and are located 8.8 Mb apart on average. This mouse WG-RH panel, known as T31, has an average retention frequency of 27.6% and an estimated potential resolution of 145 kb, making it a powerful resource for efficient large-scale expressed sequence tag mapping. [All of the mapping data for the maps presented here have been deposited at the Research Genetics, Inc., web site and can be freely accessed and downloaded at http://www.resgen.com/.]

An integrated YAC map of the human X chromosome.

The human X chromosome is associated with a large number of disease phenotypes, principally because of its unique mode of inheritance that tends to reveal all recessive disorders in males. With the longer term goal of identifying and characterizing most of these genes, we have adopted a chromosome-wide strategy to establish a YAC contig map. We have performed > 3250 inter Alu-PCR product hybridizations to identify overlaps between YAC clones. Positional information associated with many of these YAC clones has been derived from our Reference Library Database and a variety of other public sources. We have constructed a YAC contig map of the X chromosome covering 125 Mb of DNA in 25 contigs and containing 906 YAC clones. These contigs have been verified extensively by FISH and by gel and hybridization fingerprinting techniques. This independently derived map exceeds the coverage of recently reported X chromosome maps built as part of whole-genome YAC maps.