Only 10 different V beta gene segments were found when the sequences of 15 variable (V beta) genes of the mouse T-cell receptor were examined. From this analysis we calculate that the total number of ...expressed V beta gene segments may be 21 or fewer, which makes the expressed germline V beta repertoire much smaller than that of immunoglobulin heavy-chain or light-chain genes. We suggest that beta-chain somatic diversification is concentrated at the V beta-D beta-J beta junctions.
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.
Egg-laying hormone (ELH), a neuropeptide synthesized by the bag cell neurons, induces egg laying and its correlated behavior in Aplysia californica. In the present study, ELH has been purified to ...homogeneity and its primary structure has been determined. We find this molecule to have 36 amino acid residues with a M$_{\text{r}}$ of 4385 and a calculated isoelectric point of 9.7. Direct microsequence analysis revealed a single amino acid sequence that is in agreement with the amino acid composition determined after acid hydrolysis of ELH: H-Ile-Ser-Ile-Asn-Gln-Asp-Leu-Lys-Ala-Ile-Thr-Asp-Met-Leu-Leu-Thr-Glu-Gln-I le -Arg-Glu-Arg-Gln-Arg-Tyr-Leu-Ala-Asp-Leu-Arg-Gln-Arg-Leu-Leu-Glu-Lys-OH. Enzyme data indicate that the COOH-terminal lysine may be modified but its exact nature remains to be determined. There is no similarity between the amino acid sequence of ELH and that of presently known vertebrate neuropeptides. The two-step purification procedure, starting with a homogenate of bag cell clusters, consisted of cation exchange chromatography on SP C25 (Sephadex) followed by gel filtration on Bio-Gel P-6. Our purification results in a 100-fold enrichment of ELH from bag cell homogenates and a 36% recovery of purified radiolabeled marker ELH. Analysis of purified ELH radiolabeled with $^{35}$Smethionine or $^{3}$Hleucine on isoelectric focusing gels and on 8 M urea/sodium dodecyl sulfate gels showed only a single peak containing 90% of the radiolabel. Radiolabeled ELH migrated with a pI of 9.0-9.2 and an apparent M$_{\text{r}}$ of 3500-5700. ELH retained egg-laying bioactivity when eluted from this segment of the gel. We find that 2.5 nmol of pure ELH consistently induces egg laying at 20 degrees C.
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.
The previously assigned structure of human big gastrin is revised as a result of sequencing and immunological studies on synthetic peptides. A nonadecapeptide has been synthesized and found to have ...full immunochemical potency compared with natural human G34 in a radioimmunoassay which is specific for the N-terminal sequence. Syntheses of the peptides were achieved using the stepwise procedure with benzyloxycarbonyl-amino acids and fragment couplings mediated mainly by the dicyclohexylcarbodiimide procedure in the presence of either N-hydroxysuccinimide or 1-hydroxybenzotriazole. Purification of the peptide fragments was by Sephadex LH-20 chromatography and removal of protecting groups was effected using 90% trifluoroacetic acid in the presence of scavengers. Purification of the nonadecapeptide was achieved by high performance liquid chromatography.
From a library of mouse sperm DNA, we have isolated two overlapping clones which contain the Cδgene. One of these clones also contains the Cμgene. The Cδgene is separated from the Cμmembrane exons by ...approximately 2 kilobases (kb) of DNA. The Cδgene was identified by (a) hybridization to poly(A)+RNA prepared from the IgD-producing rat plasma cell tumor IR731, and (b) homology of a translated nucleotide sequence to the amino acid sequence of the human δ chain. The Cδgene spans 8 kb of DNA in the germ line. Plasmid subclones of the Cδgene were used as probes in Southern and RNA blot experiments. RNA blot analysis of cytoplasmic poly(A)+RNA from IR731 and a μ+δ+B-cell hybridoma revealed 1.6- and 2.7-kb δ mRNA species with different 3′ends, which presumably encode the secreted and membrane-bound forms, respectively, of the δ chain. Southern blot analysis of DNA from two μ+δ+lymphomas revealed that the Cδgene is in the germ-line configuration in each case. Restriction map analysis of Cμand Cδgenomic clones isolated from a library of normal μ+δ+B-cell DNA also gave no evidence for DNA rearrangement in the region between the Cμand Cδgenes. Taken together, these data suggest that IgD expression in μ+δ+B cells does not involve a VH-to-CδDNA switch rearrangement. We propose that simultaneous expression of Cμand Cδwith a single VHgene is mediated by two alternative routes of RNA processing of a primary nuclear transcript which contains the VH, Cμ, and Cδgenes. In contrast, analogous experiments with myeloma IR731 DNA revealed that the Cμgene has been deleted from the myeloma DNA and that the Cδgene has undergone DNA rearrangement, presumably including a switch recombination of the VHgene from the Cμto the Cδgene. These results indicate that two alternative mechanisms may be used in the expression of IgD molecules--RNA splicing in B cells and DNA rearrangement in plasma cells.
The helper T cell clone 3H.25 is specific for hen egg white lysozyme and the class II MHC molecule I-Ab. This TH cell has three rearrangements in the beta-chain gene family-a V beta-D beta-J beta 1 ...and a D beta 2-J beta 2 rearrangement on one homolog and a D beta 1-J beta 2 rearrangement on the other. These observations demonstrate that this functional T lymphocyte expresses only a single V beta gene segment and, accordingly, exhibits allelic exclusion of beta-chain gene expression. The rearranged 3H.25 V beta gene segment is the same as that expressed in a T helper cell specific for cytochrome c and an I-Ek MHC molecule. Thus, there is no simple correlation between the V beta gene segment and antigen specificity or MHC restriction.
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.
DNA sequences of the fifth exon, which encodes the transmembrane domain, were determined for the BALB/c mouse class I MHC genes and used to study the relationships between them. Based on nucleotide ...sequence similarity, the exon 5 sequences can be divided into seven groups. Although most members within each group are at least 80% similar to each other, comparison between groups reveals that the groups share little similarity. However, in spite of the extensive variation of the fifth exon sequences, analysis of their predicted amino acid translations reveals that only four class I gene fifth exons have frameshifts or stop codons that terminate their translation and prevent them from encoding a domain that is both hydrophobic and long enough to span a lipid bilayer. Exactly 27 of the remaining fifth exons could encode a domain that is similar to those of the transplantation antigens in that it consists of a proline-rich connecting peptide, a transmembrane segment, and a cytoplasmic portion with membrane-anchoring basic residues. The conservation of this motif in the majority of the fifth exon translations in spite of extensive variation suggests that selective pressure exists for these exons to maintain their ability to encode a functional transmembrane domain, raising the possibility that many of the nonclassical class I genes encode functionally important products.
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