An experimental approach to enumerate the genes coding for immunoglobulin variable-regions

Abstract

Critical to our understanding of the immune system diversity is the determination of the number of germ line V genes. The total number of V genes is given by the product: number of subgroups x number of germ line genes per subgroup. Studies of kappa chains and of embryonic DNA indicate 5-10 V genes per subgroup. Statistical analysis of the limited sequence data of mouse kappa chains suggest about 50 V kappa subgroups. We report here a general approach for direct estimation of the number of VL and VH subgroups expressed in normal spleen, and present data for V kappa. The kappa mRNA of the spleen is a heterogeneous population where different V kappa are linked to the same C kappa, i.e. C kappa equals total V kappa. The ratio C kappa/distinct V kappa approximates the number of subgroups since V kappa of the same subgroup cross hybridize while V kappa of different subgroups do not. This ratio was determined by molecular hybridization of cloned C kappa and V kappa DNA probes with spleen mRNA. The results indicate the expression of 280 V kappa subgroups in mouse. Assuming an average of 7 genes per subgroup, we estimate about 2000 V kappa germ line genes.

A cloned immunoglobulin cDNA fragment enhances transposition of IS elements into recombinant plasmids

“…The enhanced transposition is due to the particular combination of the cloned DNA and its position in the cloning vector. Recombinant plasmids containing longer stretches or the entire length of L-321 cDNA cloned by dc:dG "tailing" into the Pst I site of pBR322 did not acquire IS insertions (17,38), Likewise, scoring of a large number of individual transformant clones with pBR313 did not reveal any loss of the TcR marker as would result from IS insertions into the Tc region. It thus appears that the junctions of VBRI with the flanking plasmid sequences (pBR313 or pBR322), or another type of coupling between the cloned DNA and the cloning vector sequences are effective in the enhancement of transposition frequency.…”

Analyses of chicken immunoglobulin light chain cDNA clones indicate a few germline V lambda genes and allotypes of the C lambda locus.

“…If many different subgroups were expressed, then the V cDNA isolates should have revealed different subgroups in genomic DNA. This was tested in the mouse system which expresses about 200 different Vx subgroups (Zeelon et al, 1981). A mouse spleen cDNA library was screened with a mouse Cx probe.…”

“…The germline element refers to the inherited repertoire of the V, J and D gene segments which encode the variable regions of Ig polypeptide chains. In the case of x-chains, the number of germline V genes ranges from several hundred in mouse (Zeelon et al, 1981;Cory et al, 1981) to about 20-50 in human (Bently, 1984;Klobeck et al, 1984), with 4-5 J genes. The somatic element operates during B cell ontogenesis, involving combinatorial joining of V (D) and J in a flexible manner and somatic mutations of the rearranged genes (Hieter et al, 1982;Tonegawa, 1983).…”

Immunoglobulin Variable Region Genes

“…In general, major differences between VK myeloma sequences and serum immunoglobulin have not been found, but some bias in the myeloma pool is not ruled out since it comes largely from IgA myeloma proteins and no sequences are available from k chains with blocked N-termini [51]. Indeed, a preliminary estimate from the proportion of total spleen k mRNA molecules represented by a single VK group has suggested there might be 280 non crosshybridising subgroups of VK genes [59], If substantiated by similar studies with more probes, the germline repertoire would be some 10-fold higher than the values quoted above.…”