Joan Goverman

Transgenic mice that express a myelin basic protein-specific T cell receptor develop spontaneous autoimmunity

We constructed a transgenic mouse model that mimics the human autoimmune disease multiple sclerosis in its spontaneous induction and pathology. Transgenic mice were constructed expressing genes encoding a rearranged T cell receptor specific for myelin basic protein (MBP). T cell tolerance was not induced in the periphery, and functional, autoreactive T cells were found in the spleen and lymph nodes of these mice. Transgenic mice developed experimental allergic encephalomyelitis (EAE) following immunization with MBP and adjuvant plus pertussis toxin as well as with administration of pertussis toxin alone. Spontaneous EAE can develop in transgenic mice housed in a non-sterile facility but not in those maintained in a sterile, specific pathogen-free facility. This model system affords a unique opportunity to dissect the genetic and environmental variables that may contribute to the development of spontaneous autoimmune disease.

Mouse T cell antigen receptor: Structure and organization of constant and joining gene segments encoding the β polypeptide

The germ-line joining (J) gene segments and constant (C) genes encoding the beta chain of the mouse T cell antigen receptor have been isolated on a single cosmid clone. There are two constant genes, C beta 1 and C beta 2, each associated with a cluster of J beta gene segments. The nucleotide sequences of the C beta 2 gene and of the J beta 2 cluster gene segments have been determined. The coding sequence of the C beta 2 gene is very similar to the sequence of a cDNA clone encoded by the C beta 1 gene. The C beta 2 gene has four exons; exon-intron structure does not obviously correspond to the functional domains of the protein. The J beta 2 gene segment cluster contains six functional J gene segments. We have isolated specific probes for the C beta 1, C beta 2, J beta 1, and J beta 2 regions to examine DNA rearrangements in T lymphocytes. DNA rearrangements can occur in both J beta gene segment clusters, and both C beta genes appear functional.

The T cell receptor β chain genes are located on chromosome 6 in mice and chromosome 7 in humans

Homologous clones that encode the beta chain of the T cell antigen receptor have been isolated recently from both murine and human cDNA libraries. These cDNA clones have been used in connection with interspecies hybrid cell lines to determine that the murine T cell receptor gene is located on chromosome 6 and the human gene on chromosome 7. In situ hybridization confirms these data and further localizes these genes to band B of chromosome 6 in the mouse and bands 7p13-21 in the human genome. The organization of the T cell antigen receptor J beta gene segments and C beta genes appears to be conserved, since very few intraspecies polymorphisms of restriction fragment length have been detected in either mouse or human DNA.

Gene transfer of h-2 class ii genes: antigen presentation by mouse fibroblast and hamster b-cell lines.

We have transferred the mouse Ak alpha and Ak beta genes, which encode the class II I-Ak molecule, into mouse L-cell fibroblasts and hamster B cells. I-Ak molecules are expressed on the surface of both cell types. The L-cell and hamster B-cell I-Ak molecules appear normal by serological analyses and two-dimensional gel electrophoresis. Furthermore, the I-Ak molecules on L cells can act as targets for the allogenic T-cell killing of the transformed L cells. The I-Ak molecules in both mouse fibroblasts and hamster B cells can present certain antigens to T-cell helper hybridomas. Thus only class II molecules are required to convert the nonantigen-presenting cell. Accordingly, it will be possible to dissect the structure-function relationships existing between Ia molecules, foreign antigen, and T-cell receptor molecules by in vitro site-directed mutagenesis and gene transfer.

Chimeric immunoglobulin-T cell receptor proteins form functional receptors: Implications for T cell receptor complex formation and activation

We constructed chimeric receptor chains in which an immunoglobulin heavy chain variable region (VH) from a phosphorylcholine-specific antibody is substituted for T cell receptor (Tcr) alpha and beta V regions. We demonstrate that the VH region joined to either the C alpha or the C beta region can form stable chimeric proteins in EL4 T cells. Both chimeric receptor chains associate with CD3 polypeptides in functional receptor complexes and respond to phosphorylcholine coupled to Sepharose beads. The VH-C alpha chimeric chain associates with the EL4 beta chain, while the VH-C beta chimeric protein appears to form either a homodimer or a heterodimer with the native EL4 beta chain. Thus, functional receptor complexes can be formed using two C beta regions, and the C alpha region may not be required for CD3 association and surface expression of Tcr complexes.

Rearranged β t cell receptor genes in a helper t cell clone specific for lysozyme: No correlation between Vβ and MHC restriction

Abstract The helper T cell clone 3H.25 is specific for hen egg white lysozyme and the class II MHC molecule I-A b . This T H cell has three rearrangements in the β-chain gene family-a V β -D β -Jp β1 and a D β2 -J β2 rearrangement on one homolog and a D β1 -J β2 rearrangement on the other. These observations demonstrate that this functional T lymphocyte expresses only a single V β gene segment and, accordingly, exhibits allelic exclusion of β-chain gene expression. The rearranged 3H.25 V β gene segment is the same as that expressed in a T helper cell specific for cytochrome c and an I-E k MHC molecule. Thus, there is no simple correlation between the V β gene segment and antigen specificity or MHC restriction.

Model genomes: The benefits of analysing homologous human and mouse sequences

The human genome initiative has provided the motivating force for launching sequencing projects suitable for testing various DNA-sequencing strategies, as well as motivating the development of mapping and sequencing technologies. In addition to projects targeting selected regions of the human genome, other projects are based on model organisms such as yeast, nematode and mouse. The sequencing of homologous regions of human and mouse genomes is a new approach to genome analysis, and is providing insights into gene evolution, function and regulation which could not be determined so easily from the analysis of just one species.

Separation of disulfide-bonded polypeptides using two-dimensional diagonal gel electrophoresis

A simple method for the separation of disulfide-bonded polypeptides using two-dimensional diagonal gel electrophoresis is described. The first dimension involves standard SDS-PAGE of nonreduced proteins. The separated proteins are then subjected to reduction before electrophoresis in the second dimension. This technique has several applications. It can be utilized to detect the presence of disulfide-bonded polypeptides within a protein, as an assay to detect the presence of a protein known to contain disulfide-bonded polypeptides in a mixture of proteins, and as a means of separating disulfide-bonded polypeptides from proteins of similar molecular weight.