Extensive intragenic recombination and patterns of linkage disequilibrium at the CSN3 locus in European rabbit

Kappa-casein (CSN3) plays an important role in stabilising the Ca-sensitive caseins in the micelle. The European rabbit (Oryctolagus cuniculus) CSN3 has previously been shown to possess two alleles (A and B), which differ deeply in their intronic regions (indels of 100 and 1550 nucleotides in introns 1 and 4, respectively). Furthermore, a correlation between several reproductive performance traits and the different alleles was described. However, all these data were exclusively collected in rabbit domestic breeds, preventing a deeper understanding of the extensive polymorphism observed in the CSN3 gene. Additionally, the techniques available for the typing of both indel polymorphisms were until now not suitable for large-scale studies. In this report, we describe a simple, PCR-based typing method to distinguish rabbit CSN3 alleles. We analyse both ancient wild rabbit populations from the Iberian Peninsula and France, and the more recently derived English wild rabbits and domestic stocks. A new allele (C) showing another major indel (250 bp) in intron 1 was found, but exclusively detected in Iberian wild rabbits. In addition, our survey revealed the occurrence of new haplotypes in wild populations, suggesting that intragenic recombination is important in creating genetic diversity at this locus. This easy and low cost single-step PCR-based method results in an improvement over previous described techniques, can be easily set up in a routine molecular laboratory and would probably be a valuable tool in the management of rabbit domestic breeds.     

Mechanisms for intragenic complementation at the human argininosuccinate lyase locus.

Argininosuccinate lyase (ASL) is a homotetrameric enzyme that catalyzes the reversible cleavage of argininosuccinate to arginine and fumarate. Deficiencies in the enzyme result in the autosomal, recessive disorder argininosuccinic aciduria. Considerable clinical and genetic heterogeneity is associated with this disorder, which is thought to be a consequence of the extensive intragenic complementation identified in patient strains. Our ability to predict genotype-phenotype relationships is hampered by the current lack of understanding of the mechanisms by which complementation can occur. The 3-dimensional structure of wild-type ASL has enabled us to propose that the complementation between two ASL active site mutant subunits, Q286R and D87G, occurs through a regeneration of functional active sites in the heteromutant protein. We have reconstructed this complementation event, both in vivo and in vitro, using recombinant proteins and have confirmed this hypothesis. The complementation events between Q286R and two nonactive site mutants, M360T and A398D, have also been characterized. The M360T and A398D substitutions have adverse effects on the thermodynamic stability of the protein. Complementation between either the M360T or the A398D mutant and the stable Q286R mutant occurs through the formation of a more stable heteromeric protein with partial recovery of catalytic activity. The detection and characterization of a novel complementation event between the A398D and D87G mutants has shown how complementation in patients with argininosuccinic aciduria may correlate with the clinical phenotype.     

Molecular analysis of intragenic recombination at the tryptophan synthetase locus in Neurospora crassa

Fifteen different classically generated and mapped mutations at the tryptophan synthetase locus in Neurospora crassa have been characterized to the level of the primary sequence of the gene. This sequence analysis has demonstrated that intragenic recombination is accurate to order mutations within one open reading frame. While classic genetic analysis correctly ordered the mutations, the position of mutations characterized by gene sequence analysis was more accurate. A leaky mutation was found to have a wild-type primary sequence. The presence of unique polymorphisms in the primary sequence of the trp-3 gene from strain 861 confirms that it has a unique history relative to the other strains studied. Most strains that were previously shown to be immunologically nonreactive with antibody preparations raised against tryptophan synthetase protein were shown to have nonsense mutations. This work defines 14 alleles of the N. crassa trp-3 gene.     

Intrahaplotype polymorphism at the Brassica S locus

The S locus receptor kinase and the S locus glycoproteins are encoded by genes located at the S locus, which controls the self-incompatibility response in Brassica. In class II self-incompatibility haplotypes, S locus glycoproteins can be encoded by two different genes, SLGA and SLGB. In this study, we analyzed the sequences of these genes in several independently isolated plants, all of which carry the same S haplotype (S(2)). Two groups of S(2) haplotypes could be distinguished depending on whether SRK was associated with SLGA or SLGB. Surprisingly, SRK alleles from the two groups could be distinguished at the sequence level, suggesting that recombination rarely occurs between haplotypes of the two groups. An analysis of the distribution of polymorphisms along the S domain of SRK showed that hypervariable domains I and II tend to be conserved within haplotypes but to be highly variable between haplotypes. This is consistent with these domains playing a role in the determination of haplotype specificity.     

Restriction fragment length polymorphism at the HLA-C locus

We have used the HLA-C-specific DNA probe pC250 to investigate restriction fragment length polymorphism (RFLP) at the HLA-C locus. Genomic Southern blot hybridization included DNA prepared from a panel of homozygous typing cells representing serological specificities Cw1 to Cw8 and also from samples representing Cw blanks. Although many restriction nucleases failed to reveal any polymorphism, RFLPs were evident with Taq I, Pvu II, Bst XI, Nde 1, and Nci I in addition to the previously reported Eco RI. In the case of Bst XI, a unique RFLP defined a subset of serologically defined Cw blanks. Comparison of RFLP sizes with restriction fragment lengths obtained from the known HLA-Cw3 gene sequence permitted the localization of intragenic C locus RFLLs and the identification of a variable Taq I site in the second intron, a variable Nci I site near the end of the fourth exon, and a variable Pvu lI site in the fifth intron.