A dramatic change in the antibody repertoire of isogeneic Xenopus is associated with the developmental transition from the larval to the adult form. To define this process in molecular genetic terms, VH genes will be identified in isogeneic """"""""clonal"""""""" animals using heterologous cross-hybridization, the extent of the VH gene family will be assessed and genomic organization will be established with segment specific probes (JH and constant region) derived from VH+ cDNAs as well as by DNA sequencing (D regions). The organization of the VH locus will be studied in lambda vectors and in newly developed cosmid vectors, which exhibit stable replication characteristics. Microscale mRNA isolation and high efficiency cDNA cloning in lambda will permit the identification of immunoglobulin mRNAs from antigen binding and polyclonally activated cells of both adult and developmentally blocked, larval animals. These will be characterized at the sequence level and their corresponding germline structures will be identified using synthetic probe techniques. Extended linkage maps will be constructed and the relationships between developmental expression, gene structure and chromosomal organization will be analyzed. Flanking region sequence comparisons will be used to determine whether specific DNA patterns can be correlated with developmental expression. Adult and larval genes will be injected into fertilized eggs and their regulation during development studied using gene specific probes. The ability to microinject large numbers of eggs with fusion gene constructs will greatly facilitate the study of essential regulatory sequences. Studies will be extended to include light chain genes. Overall we expect to characterize the developmental regulation of the antibody repertoire at the gene level and identify the control mechanisms that function in this system. Such findings have far reaching significance in terms of our understanding the developmental regulation of eukaryotic gene expression and the ontogenesis of adaptive immunity.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM038656-01
Application #
3295241
Study Section
Immunobiology Study Section (IMB)
Project Start
1987-07-01
Project End
1992-06-30
Budget Start
1987-07-01
Budget End
1988-06-30
Support Year
1
Fiscal Year
1987
Total Cost
Indirect Cost
Name
Tampa Bay Research Institute
Department
Type
DUNS #
City
Saint Petersburg
State
FL
Country
United States
Zip Code
33716
Litman, G W; Rast, J P; Shamblott, M J et al. (1993) Phylogenetic diversification of immunoglobulin genes and the antibody repertoire. Mol Biol Evol 10:60-72
Haire, R N; Buell, R D; Litman, R T et al. (1993) Diversification, not use, of the immunoglobulin VH gene repertoire is restricted in DiGeorge syndrome. J Exp Med 178:825-34
Amemiya, C T; Ohta, Y; Litman, R T et al. (1993) VH gene organization in a relict species, the coelacanth Latimeria chalumnae: evolutionary implications. Proc Natl Acad Sci U S A 90:6661-5
Litman, G W; Haire, R N; Hinds, K R et al. (1992) Evolutionary development of the B-cell repertoire. Ann N Y Acad Sci 651:360-8
Haire, R N; Ohta, Y; Litman, R T et al. (1991) The genomic organization of immunoglobulin VH genes in Xenopus laevis shows evidence for interspersion of families. Nucleic Acids Res 19:3061-6
Haire, R N; Amemiya, C T; Suzuki, D et al. (1990) Eleven distinct VH gene families and additional patterns of sequence variation suggest a high degree of immunoglobulin gene complexity in a lower vertebrate, Xenopus laevis. J Exp Med 171:1721-37
Amemiya, C T; Haire, R N; Litman, G W (1989) Nucleotide sequence of a cDNA encoding a third distinct Xenopus immunoglobulin heavy chain isotype. Nucleic Acids Res 17:5388
Reinisch, C L; Litman, G W (1989) Evolutionary immunobiology. Immunol Today 10:278-81