Human T cell receptor V beta gene polymorphism.

Southern blot hybridizations with human T cell receptor V beta gene probes were used to determine the sizes of the various V beta gene subfamilies. An analysis of DNA samples from 100 unrelated individuals identified a single individual who lacked one V beta gene segment. A second individual had an apparently different repertoire of V beta gene segments in one subfamily, as assayed by hybridization, possibly due to a gene conversion event. An analysis with four restriction enzymes of DNA from 30 consanguineous donors detected restriction fragment length polymorphisms associated with 12 of 14 V beta gene segment subfamilies examined. In an analysis of DNAs from a large panel of unrelated individuals, some alleles at these loci were found to be in linkage disequilibrium, indicating a potentially close physical linkage. The segregation of three polymorphisms, two associated with V beta gene segment loci and one associated with the C beta genes, was compatible with Mendelian inheritance, and demonstrated that highly informative haplotypes could be generated. The high degree of polymorphism observed in the human T cell receptor beta chain complex should allow exploration of possible associations between T cell receptor genes and inherited diseases involving the immune system.

Analysis of a human V beta gene subfamily.

We have isolated and sequenced five germline V beta gene segments that are homologous to the V region of the YT35 cDNA encoding the beta chain of the T cell antigen receptor from the tumor MOLT-3. One of these gene segments is identical to the YT35 V segment, and therefore is the corresponding germline V beta gene segment encoding the YT35 cDNA. The other four V beta members exhibit 77-98% homology to the YT35 V gene segment. Two of these V beta gene segments are pseudogenes. Analyses of the coding region sequences reveal that, although the V beta segments are very diverse, they are mutating at a rate comparable to that observed in most eukaryotic genes. Analyses of the genomic clones show that the spacing distance between germline V beta gene segments ranges from 3 kb to greater than 30 kb, and the entire V beta 8 subfamily appears to be linked by a total of no more than 110 kb of DNA.

Characterization of the T cell receptor repertoire causing collagen arthritis in mice.

Collagen type II-induced arthritis (CIA) is generated in susceptible rodent strains by intradermal injections of homologous or heterologous native type II collagen in complete Freund's adjuvant. Symptoms of CIA are analogous to those of the human autoimmune disease, rheumatoid arthritis. CIA is a model system for T cell-mediated autoimmune disease. To study the T cell receptor (TCR) repertoire of bovine type II-specific T cells that may be involved in the pathogenesis of CIA in DBA/1Lac.J (H-2q) mice, 13 clonally distinct T cell hybridomas specific for bovine type II collagen have been established and the alpha and beta chains of their TCRs have been analyzed. These T cell hybridomas recognize epitopes that are shared by type II collagens from distinct species and not by type I collagens, and exhibit a highly restricted TCR-alpha/beta repertoire. The alpha chains of the TCRs employ three V alpha gene subfamilies (V alpha 11, V alpha 8, and V alpha 22) and four J alpha gene segments (J alpha 42, J alpha 24, J alpha 37, and J alpha 32). The V alpha 22 is a newly identified subfamily consisting of approximately four to six members, and exhibits a high degree of polymorphism among four mouse strains of distinct V alpha haplotypes. In addition, the beta chains of the TCRs employ three V beta gene subfamilies (V beta 8, V beta 1, and V beta 6), however the V beta 8.2 gene segment is preferentially utilized (58.3%). In contrast, the J beta gene segment usage is more heterogeneous. On the basis of the highly limited TCR-alpha/beta repertoire of the TCRs of the panel of bovine type II-specific T cell hybrid clones, a significant reduction (60%) of the incidence of arthritis in DBA/1Lac.J mice is accomplished by the use of anti-V beta 8.2 antibody therapy.

Helper and killer T cells do not express B cell immunoglobulin joining and constant region gene segments.

We have analyzed four kinds of T cells for rearrangement and expression of immunoglobulin genes. These cells include: (a) whole thymus; (b) WEHI-22, a T-cell lymphoma; (c) HT-1, an major histocompatibility complex-restricted T helper line; and (d) CTLLi6, an H-2 alloreactive killer cell line. None of the B-cell joining and constant gene segments are rearranged in the T cells. The monoclonal cells do not express any C kappa, C lambda, Cmu or C alpha RNA species. Small amounts of C kappa, C alpha, and Cmu sequences are present in RNA prepared from the thymus, although the significance of this RNA for T-cell antigen receptor synthesis is uncertain. The data support the hypothesis that expression of B-cell joining and C gene segments is unnecessary for T-cell helper and T-cell killer activity.

A low polymorphic mouse H-2 class I gene from the Tla complex is expressed in a broad variety of cell types.

We have previously described the isolation of pH-2d-37, a cDNA clone that encodes a so far unknown, poorly polymorphic, class I surface molecule. We report here the isolation of the corresponding gene, its nucleotide sequence, and its localization in the Tla region of the murine MHC. Using a RNase mapping assay, we have confirmed that the second domain coding region of the 37 gene displays very limited polymorphism, and that the gene is transcribed in a broad variety of cell types, in contrast to the genes encoding the known Qa and TL antigens. Possible functions are discussed.

T and B cells that recognize the same antigen do not transcribe similar heavy chain variable region gene segments.

We have attempted to determine whether T cells and B cells that have the same antigenic specificity and whose receptors share idiotypic determinants in fact express similar VH gene segments. To do this, we have obtained and characterized a cDNA clone containing the entire coding sequence for the VH gene from a glutamic acid60/alanine30/tyrosine10 (GAT)-binding immunoglobulin that carries the CGAT idiotype. The GAT-VH clone was hybridized to Northern blots of GAT-specific T cell RNAs; there was no evidence of a T cell transcript that hybridized to the GAT-VH probe. The T cells analyzed included: (a) 10 GAT-binding suppressor T cell hybridomas, 6 of which secreted factors with CGAT idiotypic determinants, (b) one GAT-specific helper T cell hybridoma, and (c) two GAT-specific helper T cell lines grown in the absence of feeder cells. The detection limit of the Northern blot analysis was 1-2 copies of a particular mRNA species per cell for the hybridomas and 5-10 copies per cell for the T cell lines. Therefore, we conclude that T and B lymphocytes responding to GAT do not utilize similar VH gene segments. Furthermore, the presence of idiotypic determinants on T lymphocytes does not necessarily imply close structural similarity between T and B cell antigen receptors.

Clonal variation in cell surface display of an H-2 protein lacking a cytoplasmic tail.

Truncated variants of the gene encoding H-2Ld, an integral membrane protein encoded by the major histocompatibility complex, were constructed by in vitro mutagenesis to elucidate the function of charged amino acids found on the cytoplasmic side of the transmembrane (TM) region. Analysis of cloned L cells transfected with these genes shows that the seven amino acids following the TM segment, four of which are basic, enhance the cell surface expression of H-2Ld protein but are not required for it. However, some clones do not express a tailless H-2Ld protein on the cell surface but express it intracellularly where it has a long half-life. Turnover measurements on cell surface H-2Ld proteins suggest that the basic residues following the TM segment are not a "stop transfer" sequence (Blobel, G., 1980, Proc. Natl. Acad. Sci. USA., 77:1496-1500) which anchors the H-2Ld protein in the membrane. Pulse-chase and endoglycosidase H sensitivity studies show that H-2Ld proteins lacking some or all of the basic residues and H-2Ld proteins which have a full-length cytoplasmic tail are processed with different kinetics. These results suggest an involvement of the membrane-proximal region of the cytoplasmic tail in the intracellular transport of H-2Ld. We further suggest that the L cell clones which do and do not express a tailless H-2Ld protein on the cell surface differ in the ability to transport a tailless integral membrane protein to the cell surface.

Immunoglobulin structure: amino terminal sequences of kappa chains from genetically similar mice (BALB/c).

The amino terminal portion of 20 kappa chains from the highly inbred BALB/c mouse has been examined on an automatic protein sequencer. These proteins can be divided into at least nine groups (subgroups) based on sequence patterns which are so distinct that each subgroup is probably encoded by at least one germ-line gene. The subgroups of mouse kappa chains are generally quite different from those of human kappa chains.

Protein sequence analysis: automated microsequencing.

The automated microsequencing of proteins can now be carried out at the 5- to 10-picomoles (submicrogram) level on polypeptides obtained directly from one- and two-dimensional gel electrophoresis. The techniques are applicable to polypeptides ranging in size from small peptides (less than 10 residues) to large proteins (more than 1000 residues).

DNA sequences mediating class switching in alpha-immunoglobulins.

Immunoglobulin class switching involves specific DNA rearrangements of the gene segments coding for heavy chain constant regions (CH) during B lymphocyte differentiation. In two different cases of C mu to C alpha switching examined here (T15 and M603) and one taken from the literature (MC101), three different sites on the 5' side of C mu and three different sites on the 5' side of C alpha are joined together in the process of CH switching. The sequences surrounding the three germ-line C alpha sites of recombination are highly conserved blocks of 30 nucleotides that may serve as recognition sequences for CH switching to the C alpha gene. This putative recognition sequence is repeated 17 times in approximately 1400 nucleotides of the germ-line Calpha 5' flanking sequence. The lack of homology between this C alpha sequence and sequences reported for the C gamma 1 and C gamma 2b switch sites suggests that heavy chain switching is mediated by class-specific recognition sequences and, presumably, class-specific regulatory mechanisms. In addition, it appears that in one example (MC101) CH switching progressed from C mu to C alpha to C gamma 1. This switching pathway may present difficulties for the simple deletional model of CH switching.

Rat transforming growth factor type 1: structure and relation to epidermal growth factor.

The complete amino acid sequence of rat transforming growth factor type 1 has been determined. This growth factor, obtained from retrovirus-transformed fibroblasts, is structurally and functionally related to mouse epidermal growth factor and human urogastrone. Production of this polypeptide by various neoplastic cells might contribute to the continued expression of the transformed phenotype.

Human fibroblast interferon: amino acid analysis and amino terminal amino acid sequence.

The purification of human fibroblast interferon has been simplified to a two-step procedure consisting of affinity chromatography on Blue Sepharose and sodium dodecyl sulfate polyacrlamide gel electrophoresis. A preliminary amino acid composition and the sequence of the 13 amino-terminal residues of homogeneous interferon prepared by this method is reported.

Acetylcholine receptor: complex of homologous subunits.

The acetylcholine receptor from the electric ray Torpedo californica is composed of five subunits; two are identical and the other three are structurally related to them. Microsequence analysis of the four polypeptides demonstrates amino acid homology among the subunits. Further sequence analysis of both membrane-bound and Triton-solubilized, chromatographically purified receptor gave the stoichiometry of the four subunits (40,000:50,000:60,000:65,000 daltons) as 2:1:1:1, indicating that this protein is a pentameric complex with a molecular weight of 255,000 daltons. Genealogical analysis suggests that divergence from a common ancestral gene occurred early in the evolution of the receptor. This shared ancestry argues that each of the four subunits plays a functional role in the receptor's physiological action.

Developmentally controlled expression of immunoglobulin VH genes.

Although antibody diversity arises mainly from apparently random combinatorial and somatic mutational mechanisms acting upon a limited number of germline antibody genes, the antibody repertoire develops in an ordered fashion during mammalian ontogeny. A series of early pre-B and B-lymphocyte cell lines were examined to determine whether an ordered rearrangement of gene families of the variable region of immunoglobulin heavy chains (VH) may be the basis for the programmed development of the antibody response. The results indicated that the VH repertoire of fetal B-lineage cells is largely restricted to the VH 7183 gene family and that subsequent recruitment of additional VH gene families occurs during neonatal development. These results have important implications in understanding the ontogeny of immune function.

Synthesis of a site-specific DNA-binding peptide.

The Hin recombinase binds to specific sites on DNA and mediates a recombination event that results in DNA inversion. In order to define the DNA-binding domain of the Hin protein two peptides 31 and 52 amino acids long were synthesized. Even though the 31mer encompassed the sequence encoding the putative helix-coil-helix-binding domain, it was not sufficient for binding to the 26-base pair DNA crossover site. However, the 52mer specifically interacted with the site and also effectively inhibited the Hin-mediated recombination reaction. The 52mer bound effectively to both the 26-base pair complete site and to a 14-base pair "half site." Nuclease and chemical protection studies with the 52mer helped to define the DNA base pairs that contributed to the specificity of binding. The synthetic peptide provides opportunities for new approaches to the study of the nature of protein-DNA interaction.

Amino terminal sequence of the major component of human lymphoblastoid interferon.

Homogeneous human lymphoblastoid interferon with an apparent molecular size of 18,500 daltons was characterized by its amino acid composition. Analysis of the amino terminal sequence by Edman degradation indicates that the sequence is unique.

Mouse interferons: amino terminal amino acid sequences of various species.

Mouse interferons of three size classes (A, 35,000 to 40,000 daltons; B, 26,000 to 33,000 daltons; and C, 20,000 daltons) were purified from Ehrlich ascites tumor cells infected with Newcastle disease virus. The sequences of the first 24 amino acids (No. 17 has not been identified) of interferons A and B are identical. The sequence of the first 20 amino acids of interferon C differs from that of A and B in 18 positions. There is partial homology in amino terminal sequence between mouse interferons A (or B) and a human fibroblast interferon and between mouse interferon C and a human lymphoblastoid interferon.

Automated chemical synthesis of a protein growth factor for hemopoietic cells, interleukin-3.

Interleukin-3 (IL-3), a protein of 140 amino acids, was chemically synthesized by means of an automated peptide synthesizer and was shown to have the biological activities attributed to native IL-3. Assays of synthetic analogues established that an amino terminal fragment has detectable IL-3 activity, but that the stable tertiary structure of the complete molecule was required for full activity. The results demonstrate that automated peptide synthesis can be applied to the study of the structure and function of proteins.

Location and chemical synthesis of a binding site for HIV-1 on the CD4 protein.

The human immunodeficiency virus type 1 (HIV-1) uses the CD4 protein as a receptor for infection of susceptible cells. A candidate structure for the HIV-1 binding site on the CD4 protein was identified by epitope mapping with a family of eight functionally distinct CD4-specific monoclonal antibodies in conjunction with a panel of large CD4-derived synthetic peptides. All of the seven epitopes that were located reside within two immunoglobulin-like disulfide loops situated between residues 1 and 168 of the CD4 protein. The CD4-specific monoclonal antibody OKT4A, a potent inhibitor of HIV-1 binding, recognized a site between residues 32 and 47 on the CD4 protein. By analogy to other members of the immunoglobulin superfamily of proteins, this particular region has been predicted to exist as a protruding loop. A synthetic analog of this loop (residues 25 to 58) showed a concentration-dependent inhibition of HIV-1-induced cell fusion. It is proposed that a loop extending from residues 37 to 53 of the CD4 protein is a binding site for the AIDS virus.

Simian sarcoma virus onc gene, v-sis, is derived from the gene (or genes) encoding a platelet-derived growth factor.

The transforming protein of a primate sarcoma virus and a platelet-derived growth factor are derived from the same or closely related cellular genes. This conclusion is based on the demonstration of extensive sequence similarity between the transforming protein derived from the simian sarcoma virus onc gene, v-sis, and a human platelet-derived growth factor. The mechanism by which v-sis transforms cells could involve the constitutive expression of a protein with functions similar or identical to those of a factor active transiently during normal cell growth.