Integrating the porcine physical and linkage map using cosmid-derived markers

An essential part in the development of informative linkage maps is to include genetic markers that have been anchored by physical mapping. Here a set of 18 porcine cosmid-derived genetic markers are reported that have been mapped by linkge analysis, and that also have been physically localized by fluorescence in situ hybridization (FISH). Three different strategies were used to establish polymorphic markers from the cosmid clones. Firstly, dinucleotide microsatellite loci were derived by sequencing cosmid subclones containing (CA), repeats. Secondly, variable SINE 3′ poly(A) tracts (SINEVA) were identified by direct SINE-PCR amplification of cosmid clones. Thirdly, the cosmids were used in Southern blot hybridization to detect restriction fragment length polymorphisms (RFLPs). Compared with the most recent consensus compilation of the porcine gene map, the present assignment of markers to chromosomes Zp, 3, 4, 10, 12q, and 16 represents the first loci mapped to these chromosomes, for which linkage as well as in situ data are now available.     

A linkage group on pig chromosome 4 comprising the loci for blood group L, GBA, ATP1B1 and three microsatellites

Restriction fragment length polymorphisms (RFLPs) were described for the porcine loci for β-glucosidase (GBA) and the β-polypeptide 1 of the Na⁺, K⁺-transporting ATPase (ATP1B1). Linkage analyses using a three-generation pedigree provided evidence for the assignment of ATP1B1, GBA and two microsatellite loci (S0001 and S0067) to a previously described linkage group comprising the loci for blood group L (EAL) and an anonymous microsatellite (S0097). The linear order of the six markers was determined with confidence by multipoint analyses and the length of the linkage group was estimated at 88 CM. This linkage group was assigned to pig chromosome 4 on the basis of a previous physical localization of the ATP1B1 gene. In situ hybridization data for S0001 presented in this study were consistent with a localization on chromosome 4 and suggested a regional localization to 4pl2-pl3. The present study reveals conflicting data concerning the genetic localization of the K88 loci controlling the expression of the receptors for the E. coli pilus antigens. One group has reported data suggesting a loose linkage between K88 and EAL, now mapped to chromosome 4, whereas two other groups have found linkage between K88 and the transferrin locus (TF), mapped to chromosome 13 by in situ hybridization.     

Establishing Causality between Exposure to Metals and Effects on Fish

This study evaluates causal relationships between chronic exposure of fish to metals and effects at different levels of biological organization based on a weight-of-evidence approach. Criteria for evaluation of causality were strength, consistency, and specificity of the association, as well as biological gradient and plausibility. Field sampling was conducted three times between 1998 and 2000, in Furnas Stream, impacted by an abandoned lead mine, and in three other locations, including two reference and one impacted sites. Levels of Pb, Zn, Cd, and Ag in sediments from the Furnas Stream exceeded background levels, and their concentrations were above sediment quality guidelines. Residual levels of metals in fish tissue were high enough to indicate reduced growth, reproduction and/or survival according to toxicological benchmarks. Lead-induced biochemical changes (ALA-D activity depletion) were observed in two species of siluriform catfish. The condition factor of a predatory catfish was reduced, and the percentage of prey generalists was higher in Furnas than at the noncontaminated sites. Reduction in fish community diversity and density was observed. Integration of data provided supporting evidence that observed effects on fish from the Furnas Stream resulted from long-term exposure to metals, however influences from other stressors cannot be ruled out.     

Periodate-methylamine degradation of ribonucleotides in rat liver extracts in comparison to extracts of cultured cells

Periodate-methylamine degradation of ribonucleotides, which enables separation of deoxyribonucleotides in cell extracts by high-performance liquid chromatography, was tested on the acid-soluble fraction of L1210 cells, Ehrlich ascites tumor (ELD) cells, liver tissue, and liver cell nuclei. It was shown that dTTP, dCTP, and dATP could be clearly separated in L1210 and ELD extracts. In samples from liver tissue, however, dTTP coeluted with another compound, the dCTP peak often eluted very close to another peak, and only dATP separated quite satisfactorily. Furthermore, extracts from liver cell nuclei, isolated in glycerol, were not directly susceptible to periodate oxidation. This problem can be avoided by use of a different procedure for the isolation of cell nuclei.     

Letter: Crystallization and preliminary x-ray investigation of bovine erythrocyte carbonic anhydrase

The major form of bovine erythrocyte carbonic anhydrase has been prepared by a new method in which the conventional chloroform-ethanol treatment is replaced by chromatography on DEAE-Sephadex. Single crystals, suitable for high resolution X-ray diffraction studies, have been obtained from enzyme prepared by this method. The space group is P6₁22. The unit cell contains 12 enzyme molecules and has the dimensions $\text{a = b = 68}\text{A}\text{?}$, $\text{c = 244}\text{A}\text{?}$.     

DLA Class II Alleles Are Associated with Risk for Canine Symmetrical Lupoid Onychodystropy (SLO)

Symmetrical lupoid onychodystrophy (SLO) is an immune-mediated disease in dogs affecting the claws with a suggested autoimmune aethiology. Sequence-based genotyping of the polymorphic exon 2 from DLA-DRB1, -DQA1, and -DQB1 class II loci were performed in a total of 98 SLO Gordon setter cases and 98 healthy controls. A risk haplotype (DRB1*01801/DQA1*00101/DQB1*00802) was present in 53% of cases and 34% of controls and conferred an elevated risk of developing SLO with an odds ratio (OR) of 2.1. When dogs homozygous for the risk haplotype were compared to all dogs not carrying the haplotype the OR was 5.4. However, a stronger protective haplotype (DRB1*02001/DQA1*00401/DQB1*01303, OR = 0.03, 1/OR = 33) was present in 16.8% of controls, but only in a single case (0.5%). The effect of the protective haplotype was clearly stronger than the risk haplotype, since 11.2% of the controls were heterozygous for the risk and protective haplotypes, whereas this combination was absent from cases. When the dogs with the protective haplotype were excluded, an OR of 2.5 was obtained when dogs homozygous for the risk haplotype were compared to those heterozygous for the risk haplotype, suggesting a co-dominant effect of the risk haplotype. In smaller sample sizes of the bearded collie and giant schnauzer breeds we found the same or similar haplotypes, sharing the same DQA1 allele, over-represented among the cases suggesting that the risk is associated primarily with DLA-DQ. We obtained conclusive results that DLA class II is significantly associated with risk of developing SLO in Gordon setters, thus supporting that SLO is an immune-mediated disease. Further studies of SLO in dogs may provide important insight into immune privilege of the nail apparatus and also knowledge about a number of inflammatory disorders of the nail apparatus like lichen planus, psoriasis, alopecia areata and onycholysis.     

Endotoxin and staphylococcus aureus enterotoxin a induce different patterns of cytokines

The effects of Staphylococcus aureus enterotoxin A (SEA) and lipopolysaccharide (LPS) in cytokine production were assessed at the single cell level in cells obtained from healthy blood donors. Cytokine production was studied with UV-microscopy of fixed and permeabilized cells stained with cytokine specific monoclonal antibodies. The cytokines evaluated included tumour necrosis factor (TNF)-α, interleukin (IL)-1α, IL-1β, IL-6, IL-8, IL-10, IL-2, IL-4, interferon (IFN)-γ and TNF-β. LPS exhibited marked production of IL-1α, IL-1β, TNF-α, IL-6 and IL-8. After LPS stimulation IL-1α, IL-1β, TNF-α and IL-8 were the dominating products, all peaking at or before 4 hours after cell stimulation. In addition, IL-10 production was evident after 12 hours of cell stimulation. The T-lymphocyte-derived cytokines TNF-β, IL-2, IFN-γ and IL-4 were never detected in the cultures. All cytokine production, except IL-8, was downregulated at 96 hours.In contrast, peak production of IL-1α, IL-1β and IL-8, which were the dominant products, occurred after 12 hours in the SEA-stimulated cultures. Further, a significant T-lymphocyte production of TNF-β, TNF-α, IFN-γ and IL-2 was found with peak production 12–48 hours after initiation. Only low amounts of IL-6 were evident.The two types of cytokine pattern and kinetics found may correspond to the different clinical conditions after invasive Gram-negative Escherichia coli vs Gram-positive Staphylococcus aureus infections in humans, with a much more rapid onset of disease after E. coli infections. The data may also imply that different immunomodulating approaches should be considered in life-threatening diseases with the two microbacterial agents.