Donald Pious

The firstH- 2 mutant workshop.

ConclusionThe major accomplishment of the Workshop was probably the realization of many of its participants that most of the 21 availableH- 2 variants aretrue mutations very likely derived from single nucleotide substitutions. Any theory of the pleiotropic effect of theH- 2 genes must now take this fact into account; such theories must also consider the observation that a wide variety of immunological phenomena are affected byH- 2 mutations and thus, apparently, are controlled by a single gene.

Cyclic AMP dependent regulation of mitosis in human lymphoid cells

Abstract Intracellular levels of cyclic AMP (cAMP), cAMP-dependent phosphodiesterase activity, and adenylate cyclase activity are examined in an established line of human lymphoid cells synchronized by either excess thymidine or by colcemid treatment. cAMP levels and adenylate cyclase activities during the two G periods are high when compared with the values in M. cAMP-dependent phosphodiesterase activity, which is low during early G 2, is shown to increase during G 2 and reach a maximum activity during M. Agents such as dibutyryl cAMP, 1-methyl-3-isobutyl xanthine, noradrenaline, and isopropyl noradrenaline, which increase the levels of intracellular cAMP were examined to determine their effects on mitosis and on DNA synthesis. In thymidine-synchronized cells the onset of mitosis is prevented by increasing or maintaining high levels of cAMP during G 2. The specificity of inhibition of DNA synthesis or mitosis by dibutyryl cAMP is a function of the time, during the cell cycle, when the analogue is added. The elevation of cAMP by methyl xanthine results in a more general inhibition of nucleic acid synthesis and mitosis. Although both catecholamine hormones inhibit mitosis, isopropylnoradrenaline also inhibits DNA synthesis while noradrenaline treatment does not result in such inhibition.

Identification of a Trans-Acting Function Regulating HLA-DR Expression in a DR-Negative B Cell Variant

Somatic cell hybridizations were performed between an HLA-DR negative variant of a human B lymphoid cell line (B-LCL) and normal unrelated B-LCLs. The HLA-DR codes for polymorphic determinants on a heterodimeric cell surface lymphocyte differentiation glycoprotein. A variant subline which was selected in a single step from a diploid heterozygous DR-1 DR-3 B-LCL had lost expression of both DR-1 and DR-3 and the heterodimer; it has been described earlier. In a fusion with a DR-2 B-LCL, the hybrids expressed DR-2 and reexpressed the DR-1 and DR-3 alleles. Similar results were seen in a fusion with a different normal B-LCL. Hybrid clones from both fusions were tested with a large number of alloantisera and essentially all informative sera showed reexpression. The results show that (1) the variant did not arise by mutations in the structural genes for DR-1 and DR-3; (2) the normal cells are supplying a missing gene product needed for expression of DR; (3) this gene product is capable of acting in trans. Chromosome counts showed that the apparent recessiveness of the variant in the hybrids was not due to chromosomal segregation.

Cyclic AMP in cultured human lymphoid cells: Relationship to mitosis

Abstract Cultures of human lymphoid cells, synchronized in S by a single thymidine block, demonstrated high levels of intracellular cyclic AMP in G 1 and G 2 when compared with levels in S and M. Further, the addition of dibutyryl cyclic AMP to such cultures resulted in a delay of the mitotic peak without affecting the amount of DNA synthesis. When dibutyryl cyclic AMP was washed from the cultures at the end of the S period, the mitotic peak was delayed, but to a lesser degree. It appears that cyclic AMP plays a role in the regulation of the cell cycle through its effect on mitosis.

Importance of HLA-DQ and-DP restriction elements in T-cell responses to soluble antigens: mutational analysis

We describe a new approach to delineating the restriction elements used by antigen-specific human T-cell lines. EBV-transformed B cell lines and congenic HLA class II antigen-loss mutants are used to present soluble antigen to immune T cells. In this way it is possible to assess the independent contribution of individual class II loci to the restriction repertoire of the T cells. In contrast to results obtained with other methods of restriction element analysis, we find that approximately 40% of the T-cell response to several antigens is restricted by non-DR class II molecules. Both mutational analysis and blocking by class II specific monoclonal antibodies demonstrate that the non-DR restricted responses derive from DQ and DP-encoded determinants. We also find specific DR/DQ haplotype preferences for the presentation of some but not all antigens. Using a mutant that expresses only the DQ1 molecule, and is derived from a DR1, DQ1 parent line, we demonstrate a functional split of serologically defined DQ1 molecules consistent with the electrophoretic variation reported between DQ1 molecules linked to DR1 and those linked to DR2. Pairs of mutants that differ by expression of a single class II protein reveal a much broader use of available class II restriction elements than previously recognized.

Induction of HLA mutations by chemical mutagens in human lymphoid cells

We studied the effects of mutagens on the frequency of HLA “loss” variants in the diploid human lymphoid cell line T5-1, as well as the stability of mutagen-induced variants and the extent of the genetic lesions induced. Mutagens used were of different types, and included ethyl methanesulfonate (EMS), ICR-191 (ICR), and mitomycin C. Variant frequency was determined seven days after a 24-hour exposure to mutagen, by which time the cloning efficiency of exposed cultures, which was reduced following mutagenesis, had returned to normal. ICR and EMS induced dose-related increases in variant frequency of greater than two orders of magnitude at the highest concentrations tested. Mitomycin C increased variant frequency by tenfold at the one concentration tested. Seventeen of 18 induced variants showed persistence of the variant phenotype during prolonged culture following isolation. Genetic lesions induced by ICR and EMS did not extend as far as the distance between theHLA-A and -B loci (0.8 map units) in any of 21 clones tested. These data suggest a mutational origin for most mutagen-induced HLA variants of diploid cells (except for some induced by mitomycin C). Mutagenesis and mutational analysis are promising probes for studies of theHLA region.

Genetics of mammalian cell cultures

The long generation time for man and the difficulty of experimentation have restricted the progress of human genetics. The same difficulties to varying degrees apply to other mammalian forms. I t is not surprising then, that with the rapid spread of cell culture techniques, a number of investigators considered substituting rapidly growing in vitro mammalian cell cultures for intact organisms in their genetic studies. In some respects, animal cell cultures may be compared with microbial cell populations, and it was hoped that genetic progress with in vitro mammalian cell systems might be comparable with the advances in microbial genetics these past 20 years. In an absolute sense this young field has not made such progress. However, considering that rapidly growing in vitro mammalian cells have a generation time some 50 to 100 times longer than most microbial systems of genetic interest, the progress made in the genetics of mammalian cell cultures these past several years has been satisfying and at times exciting. The cell culture literature of genetic interest has already reached the point where a comprehensive review seems neither possible nor desirable; and in this review, we shall be concerned primarily with studies relevant to the question of genetic structure or linkage in mammalian forms. For more comprehensive reviews than this one, the reader is referred to the general reference section in the bibliography.

Oxidative phosphorylation in mitochondria isolated from human fibroblasts

Abstract Mitochondria were isolated from several strains of human fibroblasts in order to evaluate their potential for use in screening for inherited defects in mitochondrial function. The mitochondria were purified 10-fold from whole fibroblasts as determined by the increase in the specific activity of cytochrome oxidase. Polarographic studies of respiration at 26 °C, in mitochondria isolated by enzymatic digestion, revealed that these mitochondria contained an intact electron-transport system and three sites for the oxidative generation of ATP. Further, they showed tight coupling of respiration with phosphorylation. It appears that fibroblasts will be suitable for use in studying individuals suspected of having inherited abnormalities of mitochondrial function.

Different roles for cytosine methylation in HLA class ii gene expression

We studied the role of cytosine methylation in the control of HLA class II gene expression in isogenic sets of cells whose members differ in their expression of HLA class II genes. These included: (1) T5-1, 6.1.6, and P30, which are a class II expressing B-cell line, a class II nonexpressing mutant derived from T5-1, and an HLA-DR expressing partial revertant derived from 6.1.6, respectively; (2) the class 11 expressing B-cell line, SB, and the class II non-expressing T-cell line, HSB, from the same individual. The use of sets of cells that differ in the way their class II genes are regulated allows us to study how that difference is reflected in the methylation state of their class II genes. At least five out of six class II genes in nonexpressing cells have a CpG site that is demethylated, when compared with the same class II gene in the respective expressing cells. The results presented in this paper indicate that most methylation changes in and around class II genes have a correlation with their state of expression. Some of these changes reflect rather than determine the state of expression. Other methylation changes appear to directly affect expression, whereas some methylation differences neither correlate with nor influence gene expression. Although 5-azacytidine does not affect class II expression in T5-1 or 6.1.6, it does induce expression in HSB. This indicates that the basis for nonexpression of class II genes is different in 6.1.6 and HSB.

Control of HLA-DR antigen gene expression at the pretranslational level: Comparison of an HLA-DR-positive B lymphoblastoid cell line and its HLA-DR-Negative variant

An HLA-DR-positive human B lymphoblastoid cell line, T5-1, and its HLA-DR-negative variant, 6.1.6, were studied to elucidate mechanisms resulting in the nonexpression ofHLA-DR genes in 6.1.6. The cell lines were labeled with35S-methionine in vivo, their proteins immunoprecipitated with a monoclonal HLA-DR-specific antibody, and their two-dimensional gel electrophoresis patterns compared. The T5-1 map showed DR-antigen heavy and light chains, while the 6.1.6 map showed neither chain. When the cells were labeled in the presence of tunicamycin, the two-dimensional map of T5-1 showed nonglycosylated heavy and light chains of DR antigen while that of 6.1.6 did not. RNA was extracted from T5-1 and 6.1.6 cells and translated in rabbit reticulocyte lysates. Two-dimensional gel analysis of the immunoprecipitated proteins from T5-1 revealed spots which were identified as HLA-DR light chain and I invariant on the basis of their precipitation by monoclonal and specific allo- and heteroantibodies, and their molecular weight and pl values. These spots were absent in the 6.1.6 maps, indicating that 6.1.6 has no detectable translatable messenger RNA for HLA-DR light chains. The addition of dog pancreas microsomes to the T5-1 cell-free translation mixture resulted in an increase in the molecular weight of the precursor HLA-DR proteins consistent with glycosylation. Together with earlier cell fusion studies showing thatDR structural genes were intact in 6.1.6, these data suggested that the lesion in 6.1.6 is an alteration in a regulatory element required for transcription ofDR genes or mRNA processing.