Germline genomic structure of the B10.A mouse Tcra-V2 gene subfamily.

The number of mouse Tcra-V gene segments varies from one individual to another and is estimated to be about 100. Southern blot analysis revealed that most of the Tcra-V are organized in clusters composed of copies of Tcra-V belonging to different subfamilies. We analyzed in detail a Tcra-V subfamily and looked for new Tcra-V in order to improve the knowledge of the mouse Tcra locus organization. A series of genomic clones derived from the B10.A mouse strain enclosing these clusters was used to determined the structure of all the Tcra-V2. We were able to identify ten Tcra-V2. This study showed that the Tcra-V2 can be organized into three structural subgroups. The distribution of the genes along the Tcra locus, plus their structural organization, indicates that successive duplications occurred during the processes of expansion and contraction of the Tcra-V gene subfamilies. Several Tcra-V2 are also identical, indicating recent duplications. The most divergent Tcra-V2 differ by 7.4% nucleotides, leading to 5.2% differences in amino acid contents.

T cell receptor V alpha gene segment with alternate splicing in the junctional region.

The locus encoding mouse TCR-alpha chain includes approximately 100 V alpha gene segments that can be organized in about 20 structural subfamilies. Southern blot analysis of a T cell line derived from the BALB/c strain, M5T, has indicated that both alpha loci were rearranged, as assessed by the deletion of the delta locus, and that the V alpha gene segment involved in one of the rearrangements did not belong to any of the V alpha subfamilies already described. Transcripts of TCR-alpha chains from the M5T line were cloned after cDNA synthesis and anchored-polymerase chain reaction, revealing a V alpha gene segment of an as yet unidentified subfamily, V alpha 5T. Molecular cloning of germ-line V alpha 5T gene segments has shown that this subfamily contained two members, one of them being a pseudogene. The two members were located to each extremity of the alpha locus associated with a member of the V alpha 13 and V alpha BWB subfamilies. Analysis of transcripts bearing the V alpha 5T gene segment in the M5T line as well as in thymocytes has revealed that J alpha are frequently absent. This is due to an alternate donor splice site generated at the V alpha 5T-J alpha junction that leads to a splicing from the end of V alpha 5T to C alpha instead of the J alpha to C alpha conventional splicing. The impact of J alpha spliced-out transcripts on the allelic exclusion process is discussed.

Restriction fragment length polymorphism and evolution of the mouse immunoglobulin constant region gamma loci.

Genomic DNA from twelve laboratory mouse strains, in addition to 21 wild-derived strains belonging to different taxa (Mus musculus domesticus, Mus musculus musculus, Mus spretus, Mus macedonicus, and Mus spicilegus) and four mouse strains that are evolutionarily more distant, were analyzed by Southern blot for polymorphism of the Ig heavy chain constant region gamma isotype (Igh-C gamma) and for the distribution of the duplicated Igh-1 (C gamma 2 alpha) haplotype. Distinct allelic forms of each Igh-C locus could be defined by restriction fragment length polymorphism (RFLP). In laboratory mouse strains RFLP proved to be more sensitive in the detection of Igh-4 (C gamma 1) alleles than serological methods. Taq I digestion allowed the definition of two alleles in the Igh-8 (C gamma 3) locus, which is absolutely conserved at the protein levels. More extensive RFLP could be found in wild strains belonging to the subgenus Mus and in the evolutionarily more distant Mus species belonging to other subgenera. In previous studies we have shown that the Igh-1 locus is duplicated in M. m. musculus subspecies. We now extend this observations to the wild mouse strains belonging to M. spicilegus and M. macedonicus species and to the evolutionarily more distant wild mouse strain Mus pahari (subgenus coelomys), which is thought to have diverged from domestic mice about 5 million years ago. In addition, we found a similar RFLP pattern in ten of 18 wild mice trapped in India, suggesting that the haplotype containing the two Igh-1-like genes, organized in tandem as distinct isotypes, is widely spread in natural populations. The evolution of murine Igh-C gamma-encoded isotypes is also discussed.

Further evidence that BALB/c and C57BL/6 gamma 2a genes originate from two distinct isotypes.

Gene conversion by the corresponding gamma 2b gene has been proposed to explain the multiple differences between the nucleic acid sequences of BALB/c (Igh-1a) and C57BL/6 (Igh-1b) gamma 2a immunoglobulin allelic genes. However, genetic analysis indicates that duplicated forms of gamma 2a genes are not only present in Eastern Asia, but also in European wild mouse populations which suggests a widespread phenomenon. In order to verify whether the gamma 2a-related isotypic genes, namely gamma 2c and gamma 2a, could correspond to those present as alleles in domestic mice (Igh-1b and Igh-1a), a genomic library from Mus m.musculus strain (MAI) was constructed. Extensive mapping of the recombinant phages and Southern blot analysis with several restriction enzymes gave the complete organization of these loci: gamma 2b (18 kb) gamma 2c (17 kb) gamma 2a (14 kb) epsilon. The homology in flanking, coding and intervening region sequences indicates that MAI gamma 2c and gamma 2a related genes correspond to C57BL/6 and BALB/c Igh-1 alleles respectively. Also, Southern blot analysis using several probes derived from exonic and intronic regions between gamma 2b and gamma 2a genes shows a 2.0- to 3.0-kb difference in the distance between gamma 2b and gamma 2a genes of BALB/c strain as compared to C57BL/6. Taken together, these results indicate that BALB/c and C57BL/6 gamma 2a genes could originate from different isotypes.

K2-tartarate gradient centrifugation in the isolation of purified influenza virus haemagglutinin after bromelain cleavage.

A method of haemagglutinin (HA) purification by means of K2-tartarate gradient centrifugation is described. Different influenza virus strains possessing the antigenic formulas H1N1, H2N2 and H3N2 yielded pure and immunologically active HA samples.