Lyb-2 is a molecular system involved in the function and regulation of B cells. Evidence is given that this 45,000 dalton cell surface glycoprotein is critical to immune responses to T-dependent antigen, that Lyb-2 is confined to B cells and is absent from terminal antibody-secreting cells, that the Lyb-2 locus is on chromosome 4 and includes the Lyb-2 protein structural gene, and that Lyb-2 exhibits a degree of serological polymorphism unusual in comparable systems, other than H-2:T1a and Ig.
In Aim 1, serological and structural studies should clarify presently equivocal relations of Lyb-2 to the Lyb-4 and Lyb-6 loci linked to Lyb-2, which may compose a family of allied genes. It will be ascertained whether Lyb-6 is truly a separate system distinct from Lyb-2. Unusual reports on the genetics of the Lyb-2 and Lyb-4 systms, which might be trivial or important, are the subject of Aim 2.
In Aim 3, since structural polymorphism can be significant in understanding molecular function, our finding that 2D chymotryptic peptide maps of Lyb-2 allotypes are strikingly different will be extended to mouse strains of the same serological Lyb-2 allotype; further biochemical methods will be applied to the peptide constitution of Lyb-2 of mice of similar and different Lyb-2 allotypes, and to new putative Lyb-2 specificities. Cloning and DNA sequencing of Lyb-2 are addressed in Aim 4, which includes the construction of cDNA probes enriched for Lyb-2 by subtractive hybridization for library screening, and the use of bacterial expression and mammalian expression vectors. This work will be aided by Lyb-2 monoclonal antibodies already made and others projected, by new Lyb-2 antibodies, by Lyb-2 congenic mouse strains which include an Lyb-2/Mup-1 recombinant congenic strain, and by the I.29 early B cell line which has five times more Lyb-2 than splenic B cells. Among the methods to be employed, a description is given of the projected use of Lyb-2 congenic mouse strains to identify restriction fragment polymorphisms representing Lyb-2 or flanking genes within the congenic allele donor segments, as has proved successful in our hands with other systems named.
Aim 5 concerns Lyb-2 biosynthesis, particularly in regard to intermediary products reactive with Lyb-2 antibody, carbohydrate composition, and kinetics. These data will bear also on the obtaining of amino acid sequence information to serve, among other purposes, for making synthetic oligodeoxynucleotide probes, which is one of the approaches to gene cloning projected in Aim 4.
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| Saga, Y; Tung, J S; Shen, F W et al. (1988) Organization of the Ly-5 gene. Mol Cell Biol 8:4889-95 |
| Tsuge, I; Shen, F W; Steinmetz, M et al. (1987) A gene in the H-2S:H-2D interval of the major histocompatibility complex which is transcribed in B cells and macrophages. Immunogenetics 26:378-80 |
| Saga, Y; Tung, J S; Shen, F W et al. (1987) Alternative use of 5' exons in the specification of Ly-5 isoforms distinguishing hematopoietic cell lineages. Proc Natl Acad Sci U S A 84:5364-8 |
| Tung, J S; Shen, F W; LaRegina, V et al. (1986) Antigenic complexity and protein-structural polymorphism in the Lyb-2 system. Immunogenetics 23:208-10 |
