Clustering of cytokine genes on mouse chromosome 11.

The presence of positionally conserved amino acid residues suggests that the mouse proteins TCA3, P500, MIP1-alpha, MIP1-beta, and JE are members of a single gene family. These proteins are activation specific and can be expressed by both myeloid and lymphoid cells. MIP1-alpha/MIP1-beta and MCAF (the putative human homologue of JE) act as chemotactic and activating agents for neutrophils and macrophages, respectively. The functions of TCA3 and P500 are unknown. We have used interspecies somatic cell hybrids and recombinant inbred mouse strains to show that the genes encoding TCA3, MIP1-alpha, MIP1-beta, and JE (provisionally termed Tca3, Mip-1a, Mip-1b, and Sigje, respectively) map as a cluster on the distal portion of mouse chromosome 11 near the Hox-2 gene complex. DNA sequence analysis indicates that the P500 and TCA3 proteins are encoded by alternative splicing products of one genomic gene. Additionally, the genes encoding TCA3 and JE are found to be strikingly similar with respect to the positions of intron-exon boundaries. Together, these data support the model that the cytokines TCA3, P500, MIP1-alpha, MIP1-beta, and JE are encoded by a single cluster of related genes. The gene encoding IL-5 (Il-5), which acts as a T cell-replacing factor, a B cell growth factor, and an eosinophil differentiation factor, is also mapped to mouse chromosome 11.Il-5 maps approximately 25 cM proximal to the Tca-3 gene and appears tightly linked to a previously described gene cluster that includes Il-3, Il-4, and Csfgm. We discuss the potential relevance of the two cytokine gene clusters described here with particular attention to specific human hematologic malignancies associated with chromosomal aberrations at corresponding locations on human chromosomes 5 and 17.

The genetic analysis of human behavior: a new era?

Recent developments in DNA-based techniques may revolutionize the study of human behavioral genetics. However, unless these methods are used with great care, many of the same mistakes which have plagued non-molecular genetic analyses of behavior will reoccur. Errors in the application of genetic approaches and in the interpretation of results have been a common feature of published studies in this field. We review studies in human behavioral genetics, focusing on those using identical twins and DNA-based linkage techniques in order to draw attention to recurrent problems in molecular and non-molecular studies. We suggest possible guidelines for future research in the area of the biological basis of human behavior.

Genetic discrimination and screening for hemochromatosis.

Recent advances in tests for the genotype for hemochromatosis and suggestions that the tests be used in mass screening programs for the disease raise the possibility of a large increase in the incidence of discrimination against people who are found to be homozygous for hemochromatosis. This paper presents cases of genetic discrimination drawn from a study of discrimination against people with a variety of genetic conditions. The cases discussed here involve employment and several types of insurance discrimination against people diagnosed with hemochromatosis who either are currently asymptomatic or whose condition is controlled by means of phlebotomies. There is no justification for these types of discrimination since people with controlled hemochromatosis suffer no excess mortality or morbidity. Our study suggests that genetic discrimination is already a serious problem and that any proposed screening program for hemochromatosis or other genetic condition must consider and attempt to mitigate its effects.

New techniques for physical mapping of the human genome.

We describe improvements in techniques and strategies used for making maps of the human genome. The methods currently used are changing and evolving rapidly. Today's techniques can produce ordered arrays of DNA fragments and overlapping sets of DNA clones covering extensive genomic regions, but they are relatively slow and tedious. Methods under development will speed the process considerably. New developments include a range of applications of the polymerase chain reaction, enhanced procedures for high resolution in situ hybridization, and improved methods for generating, manipulating, and cloning large DNA fragments. More detailed genetic and physical maps will be useful for finding genes, including those associated with human diseases, long before the complete DNA sequence of the human genome is available.

Hypertension and the genetics of red cell membrane abnormalities.

Hypertension represents the upper 15-25% of the blood pressure distribution in industrialized countries. The trait is practically absent in primitive societies and is made manifest by diet and lifestyles in industrialized countries. High blood pressure is an important risk factor for strokes, heart disease and renal disease. The frequency of hypertension is higher among blacks than among whites in the USA. Various twin, family and adoption studies indicate a strong genetic effect on blood pressure. The genetic mechanisms are unknown. Membrane transport variability has been studied in red cells as a surrogate for analogous alterations in smooth muscle or renal cells. Among the various transport systems, erythrocyte sodium-lithium countertransport (CT) has been consistently elevated in variable proportions of Caucasian hypertensives. Genetic studies of countertransport levels have shown familial aggregation and higher concordance for monozygotic than dizygotic twins. Complex segregation analysis suggests the action of a major gene superimposed on a polygenic background. The postulated gene (B) raises CT activity and has a population frequency of 0.25. CT levels of the common AA homozygotes and AB heterozygotes cannot be distinguished from each other, whereas CT activity of BB homozygotes (6% of the population) is significantly elevated. Although the CT gene contributes only 2.7% to 3.5% of the variability of blood pressure over its entire range, 14% to 20% of persons with systolic hypertension (greater than 140 mmHg) are BB homozygotes rather than the expected 6% to 7%. A much lower frequency of elevated countertransport activity among black hypertensives suggests genetic heterogeneity in the pathogenesis of high blood pressure. Further investigations on the mechanism and genetic linkage relationships of the putative CT gene may aid in elucidating an important mechanism of blood pressure elevation and will allow molecular approaches in the future.

Expression of CD3-associated antigen-binding receptors on suppressor T cells.

Three suppressor T (Ts)-cell hybridomas specific for 4-hydroxy-3-nitrophenyl acetyl (NP) hapten were selected for surface expression of cluster determinant 3 (CD3) by using antibody (anti-CD3) or antigen (NP-bovine serum albumin) panning procedures followed by cloning at limiting dilution. The CD3-selected Ts hybridomas showed a 1-2 logarithmic enrichment in suppressor activity when compared to the parent lines; they also specifically bound NP-coupled sheep red blood cells in rosette assays. This antigen-binding ability could be down-modulated by anti-CD3 antibody. Similarly, surface expression of CD3 was specifically down-modulated by preincubation of these hybridomas with antigen. Anti-CD3 monoclonal antibody under reducing conditions coprecipitated a broad band of 38-50 kDa associated with two CD3 (25 and 16 kDa) bands. T-cell receptor, anti-alpha-specific monoclonal antibody also immunoprecipitated a broad band in the 41 to 49-kDa region. The combined results suggest that, like helper and cytotoxic T lymphocytes, Ts cells also bear antigen-specific receptors associated with CD3 molecules.

The expression and regulation of a potential lymphokine gene (TCA3) in CD4 and CD8 T cell clones.

The TCA3 gene was originally isolated from a cDNA library derived from a TH1 (inflammatory) T cell clone. Expression of TCA3 RNA was limited to cells in the activated state. Based on its expression profile and the existence of a hydrophobic leader sequence with a predicted cleavage site, we proposed that TCA3 encodes a new lymphokine. In the present study, we examine the subset distribution, kinetics, and regulation of TCA3 RNA expression. We show that TCA3 is expressed in response to selected T cell-activating stimuli. TCA3 is transcribed to peak steady state levels by 4 h after stimulation in TH1, TH2 (helper), and CTL clones. Two intracellular signals, supplied in vitro by phorbol ester and one of several agents capable of increasing intracellular free calcium concentrations, are required for the initiation of TCA3 transcription. In addition, TCA3 transcription is blocked by anti-L3T4 mAb, suggesting that prior signaling through L3T4 can inhibit expression of TCA3 and other lymphokines. In contrast to IL-2, IL-4, and IFN-gamma TCA3 is uniformly expressed at high levels among all individual T cell clones examined, including four TH1, three TH2, and three CTL clones. Furthermore, activation-specific TCA3 expression can be dissociated from T cell proliferation.

Antigen-specific and antibody-mediated growth inhibition of suppressor T cell hybridomas. Roles of H-2 and the CD3-T cell receptor-alpha/beta complex.

Eight different Ts cell hybridomas (including inducer (Ts1) and effector (Ts3) suppressor cells) specific for the 4-hydroxy-3-nitrophenyl acetyl (NP) hapten were tested for their ability to respond to Ag or anti-CD3 antibody in a growth-inhibition assay. Results suggest that the expression of the TCR-CD3 complex on Ts hybridomas is required for the Ag or anti-CD3-mediated growth inhibition. One of the CD3+, Ts hybridomas (CKB-Ts3-9.H3) was tested in detail; this CD4- effector suppressor cell hybridoma showed specific inhibition of growth in the presence of NP or NIP-coupled protein conjugates but not in the presence of other irrelevant hapten-protein conjugates. In addition, growth of this hybridoma was specifically inhibited by anti-CD3 and anti-TCR-alpha/beta antibodies but not by control hamster antibodies. In order to study the role of MHC molecules in Ag-mediated growth inhibition, Ts cell hybridomas were incubated with Ag (NP-keyhole limpet hemocyanin) in the presence of spleen cells from various H-2 congenic strains. The results suggest that the Ts hybridomas that express donor Ts-derived TCR beta-chain recognize Ag in an MHC-restricted manner, whereas the two Ts3 hybridomas that utilize BW5147-derived TCR-beta recognize Ag in H-2 unrestricted way. Co-incubation of anti-CD3 and anti-TCR-alpha/beta antibodies with specific Ag enhanced the Ag-mediated growth inhibition, whereas anti-LFA-1 antibody completely blocked the Ag-mediated effect. The combined data suggest that, like Th hybridomas, expression of CD3-associated-TCR complex is essential for the Ag responsiveness of Ts cell hybridomas.

Discrimination as a consequence of genetic testing.

Genetic discrimination refers to discrimination directed against an individual or family based solely on an apparent or perceived genetic variation from the "normal" human genotype. We describe here the results of a case history study designed to assess whether or not genetic discrimination exists. Using the above definition of genetic discrimination and applying stringent criteria for case selection, we find that genetic discrimination exists and is manifested in many social institutions, especially in the health and life insurance industries. Stigmatization, and denial of services or entitlements to individuals who have a genetic diagnosis but who are asymptomatic or who will never become significantly impaired, is noted. Follow-up comprehensive studies on the significance and varieties of genetic discrimination are needed. In order to avoid creating a new social underclass based on genetic discrimination (the "asymptomatic ill"), existing and future genetic testing or screening programs need review by medical, scientific, legal, and social policy experts, as well as the public, and may require modification.

Cloning and characterization of a novel T cell activation gene.

We have used the technique of subtractive hybridization to identify a T cell gene selectively expressed during activation via the antigen-receptor pathway. This gene, termed TCA3 (for T cell activation) encodes a mRNA which is expressed following concanavalin A (Con A) activation of T cell clones at levels of approximately 1% total poly(A)-containing mRNA. The cDNA isolate, termed TCA3.0, is 512 bases in length excluding poly(A) and encodes a predicted 92-amino acid protein having the characteristics of a secreted polypeptide of approximately 69 amino acids. The genomic organizations of TCA3 was determined for two lambda phage clones and was found to be a single copy gene containing at least three exons dispersed over less than 4.7 kb. The temporal appearance of TCA3 mRNA in response to several activating agents was examined. It is not transcribed in response to interleukin 2 stimulation, but is transcribed in response to either antigen or Con A stimulation and can be detected as early as 1 hr poststimulation. Expression TCA3 in response to Con A is blocked by cyclosporin A treatment. The combined data suggest that TCA3 may represent a new lymphokine.