Complexity of T cell receptor recognition sites for defined alloantigens

Three monoclonal antibodies react with the T cell receptor on the tumor line HPB-ALL and in addition with 3 to 13% of human peripheral blood T cells of normal donors. These antibodies are shown to react with an epitope encoded by the V beta 5 family of T cell receptor beta-chain variable region gene segments. Cells expressing V beta 5 gene segments can have cytotoxic or helper function, be of the T4+ or T8+ phenotype, and have specificity for either class I or class II major histocompatibility complex alloantigens. Seven T cell clones were generated, which express V beta 5 and are specific for the HLA-A2 molecule. With the use of these clones, we illustrate how isoelectric focusing can be used to analyze T cell receptor alpha- and beta-chain structure. The seven clones recognize five distinct conformational determinants on HLA-A2. They procure different binding sites by the use of different alpha-chains, J beta sequences, or both.     

The topogenic signal of the glycosomal (microbody) phosphoglycerate kinase of Crithidia fasciculata resides in a carboxy-terminal extension.

To determine how microbody proteins enter microbodies, we have previously compared the genes for the cytosolic and glycosomal (microbody) phosphoglycerate kinases (PGKs) of Trypanosoma brucei and found the microbody enzyme to differ from other PGKs and the cytosolic form in two respects: a high net positive charge and a C-terminal extension of 20 amino acids (Osinga et al., 1985). Here we present the comparison of the genes for the cytosolic and glycosomal PGKs of Crithidia fasciculata, another kinetoplastid organism. The amino acid sequences of the two Crithidia isoenzymes are virtually identical, except for a C-terminal extension of 38 amino acids. We conclude that this extension must direct the glycosomal PGK to the glycosome. The extensions of the Crithidia and Trypanosoma enzymes are both rich in small hydrophobic and hydroxyl amino acids.     

The H circles of Leishmania tarentolae are a unique amplifiable system of oligomeric DNAs associated with drug resistance

We have induced drug resistance against methotrexate, an inhibitor of dihydrofolate reductase, and CB3717, an inhibitor of thymidylate synthetase in a strain of Leishmania tarentolae. The drug-resistant strains contain extrachromosomal DNA circles of 68 kilobases with a 30-kilobase inverted duplication flanked by 4- and 5 kilobase unique segments. We show that these circles are highly homologous to the drug-induced H circles of L. tropica (1). All three L. tarentolae strains analyzed contain a chromosomal copy of the H region without duplication, but two of the three strains contain extrachromosomal H circles as well, predominantly present as H circle dimers in one strain and as tetramers in the other. After induction of methotrexate resistance, monomeric circles, presumably derived from the oligomers, become the major type of circle. Our results indicate that the H region represents a genomic region that can be copied at very low frequency to yield circles by a precise, but unusual mechanism under natural conditions in wild-type cells. Although superficially analogous to the episomes of bacteria, the system is without precedent in nature.     

A novel DNA nucleotide in Trypanosoma brucei only present in the mammalian phase of the life-cycle.

The existence of an unusual form of DNA modification in the bloodstream form of the African trypanosome Trypanosoma brucei has been inferred from partial resistance to cleavage of nuclear DNA with PstI and PvuII (Bernards et al, 1984; Pays et al, 1984). This putative modification is correlated with the shut-off of telomeric Variant-specific Surface Glycoprotein (VSG) gene expression sites (ESs). The modification only affects inactive VSG genes with a telomeric location, and it is absent in procyclic (insect form) trypanosomes in which no VSG is made at all. Previous attempts to detect unusual nucleosides in T.brucei DNA were unsuccessful, but we now report the detection of two unusual nucleotides, called pdJ and pdV, in T.brucei DNA, using the 32P-postlabeling technique. Nucleotide pdV was present in both bloodstream form and procyclic T.brucei DNA and co-migrated in two different two-dimensional thin layer chromatography (2D-TLC) systems with hydroxymethyldeoxyuridine 5'-monophosphate (pHOMedU). In contrast, nucleotide pdJ was exclusively present in bloodstream form trypanosomal DNA. Levels of pdJ were higher in DNA enriched for telomeric sequences than in total genomic DNA and pdJ was also detected in other Kinetoplastida species exhibiting antigenic variation. Postlabeling and 2D-TLC analyses showed base J to be different from the known eukaryotic unusual DNA bases 5-methylcytosine, N6-methyladenine and hydroxymethyluracil, and also from (glucosylated) hydroxymethylcytosine, uracil, alpha-putrescinylthymine, 5-dihydroxypentyluracil and N6-carbamoylmethyladenine. We conclude that pdJ is a novel eukaryotic DNA nucleotide and that it is probably responsible for the partial resistance to cleavage by PvuII and PstI of inactive telomeric VSG genes. It may therefore be involved in the regulation of ES activity in bloodstream form trypanosomes.     

Properties of a panel of monoclonal antibodies which react with the human T cell antigen receptor on the leukemic line HPB-ALL and a subset of normal peripheral blood T lymphocytes

Five Mab raised against the T cell antigen receptor of the human T cell line HPB-ALL which react with a subpopulation of normal peripheral blood T cells are described. Three Mab, 3D6, 1C1, and 1C2, react with 3 to 5% of normal PBL and stimulate proliferation of the cells with which they react. An increase in the number of cells which react with all five Mab occurs. Two Mab, 2D4 and 65, react with subsets of the cells which bind 1C1, 1C2, and 3D6 and divide the family into four subgroups, 2D4+ 65+, 2D4+ 65-, 2D4- 65+, and 2D4- 65-. Functional T cell clones in all four subfamilies have been observed. Cytolytic function can be correlated with the TcR phenotype expressed because all of the Mab which react with a particular clone inhibit its ability to lyse a specific target. The epitopes recognized by the panel are closely related because all five block each other's binding to HPB-ALL. In addition, the determinants recognized by 3D6, 1C1, and 1C2 on normal lymphocytes are probably very closely related because all clones examined react with all three Mab.