Genetic analysis of alpha 2-adrenergic receptors and blood pressure using Dahl salt-sensitive rats.

OBJECTIVE: To evaluate the role of alpha 2-adrenergic receptors in genetic hypertension by cosegregation analysis using Dahl rats. DESIGN: Inbred Dahl salt-sensitive (SS/Jr) rats were crossed with inbred Dahl salt-resistant (SR/Jr) rats; also, SS/Jr rats were crossed with several control strains, and large F2 populations were subsequently produced from each cross. All F2 populations were raised on a high-salt diet. The rats were genotyped, where possible, at the loci for three different subtypes of alpha 2-adrenergic receptors designated as classes I, II and III. The blood pressures of the rats classified by genotype at each alpha 2-adrenergic receptor subtype locus were compared using analysis of variance. METHODS: Genomic clones of three classes of alpha 2-adrenergic receptors were isolated from genomic lambda-phage libraries of SS/Jr or SR/Jr rat strains, or both, by screening with complementary DNA for human alpha 2-adrenergic receptors. Fragments of the rat genomic clones obtained were used for genotyping by restriction fragment length polymorphism. Also, cloned genomic DNA flanking the alpha 2-adrenergic receptors and containing microsatellites was sequenced; genotyping at informative microsatellite markers was performed using the polymerase chain reaction. Two of the three classes of rat alpha 2-adrenergic receptors were localized to rat chromosomes by linkage analysis or using a panel of mouse-rat hybrid somatic cell lines. RESULTS: Rat alpha 2-adrenergic receptor classes I and III genes were assigned to rat chromosomes 14 and 3, respectively. These correspond to alpha 2-adrenergic receptor genes on human chromosomes 4 and 2, respectively. Extensive cosegregation analysis, involving five alleles in six segregating populations for class I alpha 2-adrenergic receptors, yielded no evidence of an effect of these loci on blood pressure. Classes II and III alpha 2-adrenergic receptors could each be tested in only one population and there was no evidence for an effect of either receptor gene on genetic differences in blood pressure. The dopamine-1B receptor was closely linked to the class I alpha 2-adrenergic receptor on rat chromosome 14. Thus, the negative cosegregation of the class I receptor with blood pressure applies equally to the dopamine-1B receptor. CONCLUSIONS: Genetic analysis in segregating populations involving crosses of inbred Dahl salt-sensitive rats with five other strains provides no evidence for a genetic effect of class I alpha 2-adrenergic receptors, or of the dopamine-1B receptor, on blood pressure. Classes II and III alpha 2-adrenergic receptors also failed to cosegregate with blood pressure but, because only limited testing was possible with the classes II and III receptors, this negative result is not definitive.

Estimate of the genetic divergence between inbred Dahl salt-sensitive and salt-resistant rats.

OBJECTIVE: The genetic divergence of inbred Dahl salt-sensitive (SS/Jr) rats from inbred Dahl salt-resistant (SR/Jr) rats and various other inbred strains was measured. DESIGN: Structural differences in DNA between strains were evaluated. METHODS: Genetic variants were sought (1) by restriction fragment length polymorphism (RFLP) analysis, using 19 DNA probes, (2) by the polymerase chain reaction around microsatellites and (3) by DNA sequencing. RESULTS: It was estimated that 1 in 1532 bases of DNA were different between the SS/Jr and SR/Jr strains. In comparing SS/Jr and SR/Jr rats, it was also observed that one DNA probe in 10 will yield multiple RFLP, presumably as the result of large insertion/deletion events. A comparison of SS/Jr rats with seven other inbred strains showed that the percentage of loci that carry alleles different from SS/Jr rats varies from about 23% for Albino Surgery rats to 71% for Brown Norway rats. CONCLUSIONS: Although the SR/Jr strain is an appropriate contrasting strain for the genetic analysis of hypertension in SS/Jr rats, a genetic analysis involving crosses of SS/Jr rats and unrelated inbred strains is also likely to be useful in identifying genes that cosegregate with blood pressure because more informative genetic markers will be available than in a cross of SS/Jr with SR/Jr rats.

Inhibition of rotavirus in vitro transcription by optimal concentrations of monoclonal antibodies specific for rotavirus VP6.

Three monoclonal antibodies (MAbs) obtained from inoculation of mice with either a serotype 1 human rotavirus or rotavirus SA11 (serotype 3) inhibited the in vitro transcription of rotavirus SA11. Two of the MAbs exhibited a biphasic inhibitory response. Removal of antibody from MAb preparations by adsorption with Sepharose-Protein G reduced the inhibitory activity completely for all three MAb preparations. Analysis by radioimmunoprecipitation and Western blotting indicated that all three MAbs reacted with VP6. All MAbs also reacted with four group A rotavirus serotypes by ELISA, but did not cross-react with reovirus type 1, poliovirus type 2 or MA-104 cell lysates. Transcription of four rotavirus serotypes as well as epizootic diarrhoea of infant mice rotavirus was inhibited when tested with two of the MAbs. Transcription of both purified single-shelled virus and purified heat-activated double-shelled SA11 rotavirus was inhibited by purified MAb. Our results indicate that these MAbs can be used effectively to study the events associated with rotavirus transcription.