Recent reports suggested a correlation between decreased expression of tumor cell MHC class I Ag and increased susceptibility to NK cells. These studies led to the hypothesis that tumor cells displaying reduced levels of MHC class I Ag have reduced tumorigenicity in vivo because they are eliminated from the host by endogenous NK cells. The present studies use the murine hepatoma BW7756 and a spontaneous H-2Kb loss variant, Hepa-1, to test this hypothesis. The parental BW7756 tumor is highly malignant in syngeneic C57L/J hosts while Hepa-1 cells do not give rise to tumors, suggesting that the loss of H-2Kb Ag expression correlates with decreased tumorigenicity and NK susceptibility. Hepa-1 cells were therefore transfected with an H-2Kb gene to generate H-2Kb Ag expressing clones. The resulting clones were tested for tumorigenicity. Syngeneic or NK-deficient C57BL/6-beige/beige mice challenged with Hepa-1 or the H-2Kb transfectants rejected the cells, suggesting that reexpression of H-2Kb Ag does not restore tumorigenicity and that NK cells are not involved in Hepa-1 rejection. In vitro H-2Kb Ag-negative and -positive Hepa-1 cells are equally susceptible to tilorone-boosted NK cells, indicating that MHC class I Ag expression also does not affect in vitro NK susceptibility. Tumor challenged athymic nude and sublethally irradiated syngeneic mice develop tumors demonstrating that T cells are probably responsible for rejection of the Hepa-1 tumor, and that H-2Kb Ag expression has no effect on rejection. Inasmuch as the expression of H-2Kb Ag on Hepa-1 cells does not effect tumorigenicity or in vitro NK susceptibility, the previously reported association between reduced MHC class I Ag levels and increased NK susceptibility is not universally applicable.

Animals↗

PubMed1988

Relaxation intervals alter the mobility of large DNA molecules in pulsed field gel electrophoresis.

Explore the source record for details and available documents.

Batroxobin↗

PubMed1988

DNA diagnostics--molecular techniques and automation.

Molecular biology has revolutionized the understanding of many aspects of human disease. Ongoing developments in DNA diagnostics--the analysis of disease at the nucleic acid level--will soon provide automated, rapid, and inexpensive analyses for DNA or RNA sequences associated with genetic, malignant, and infectious diseases. DNA diagnostics will also facilitate the identification of disease-associated genes at birth, thus creating new opportunities for preventive medicine.

Automation↗

PubMed1988

A T cell clone expresses two T cell receptor alpha genes but uses one alpha beta heterodimer for allorecognition and self MHC-restricted antigen recognition.

All of the T cell receptor alpha- and beta-chain rearrangements present in a dual reactive T cell clone were characterized. This clone exhibits allelic exclusion of its beta-chain genes in that only one of the two alleles is productively rearranged. Unexpectedly, it displays two productive V alpha-gene rearrangements, which are both transcribed into 1.5 kb mRNA. The contribution of each of the two productive alpha genes to the dual recognition was analyzed by gene transfer. To this end, each of the two alpha genes was separately transfected with the single productively rearranged beta gene. Transfer of only one of the two alpha beta combinations restored both allogeneic MHC recognition and self MHC-restricted antigen recognition. Thus, T cell dual recognition results from the cross-reactive recognition of an allo-MHC product by a single antigen-specific and MHC-restricted alpha beta T cell receptor. Furthermore, the presence of two productively rearranged alpha-chain genes in a T cell clone raises questions concerning the level at which allelic exclusion operates in T cells.

Animals↗

PubMed1988

Zinc-dependent structure of a single-finger domain of yeast ADR1.

In the proposed "zinc finger" DNA-binding motif, each repeat unit binds a zinc metal ion through invariant Cys and His residues and this drives the folding of each 30-residue unit into an independent nucleic acid-binding domain. To obtain structural information, we synthesized single and double zinc finger peptides from the yeast transcription activator ADR1, and assessed the metal-binding and DNA-binding properties of these peptides, as well as the solution structure of the metal-stabilized domains, with the use of a variety of spectroscopic techniques. A single zinc finger can exist as an independent structure sufficient for zinc-dependent DNA binding. An experimentally determined model of the single finger is proposed that is consistent with circular dichroism, one- and two-dimensional nuclear magnetic resonance, and visual spectroscopy of the single-finger peptide reconstituted in the presence of zinc.

Circular Dichroism↗

PubMed1988

A novel instrument for separating large DNA molecules with pulsed homogeneous electric fields.

A new instrument has been developed for the electrophoretic separation of large DNA molecules that can independently regulate the voltage of each of 24 electrodes and allow the magnitude, orientation, homogeneity, and duration of the electric field to be precisely controlled. Each parameter can be varied at any time during the electrophoretic process. Thus distinct sets of conditions can be combined to optimize the separation of various fragment sizes in a single run. Independent control of electrode voltage allows all of the fields to be generated with electrodes arranged in a closed contour, independent of a particular geometry. This device increases both the resolution in any size range and the speed of separation, especially for DNA molecules larger than 3 megabases.

DNA↗

PubMed1988

A ligase-mediated gene detection technique.

An assay for the presence of given DNA sequences has been developed, based on the ability of two oligonucleotides to anneal immediately adjacent to each other on a complementary target DNA molecule. The two oligonucleotides are then joined covalently by the action of a DNA ligase, provided that the nucleotides at the junction are correctly base-paired. Thus single nucleotide substitutions can be distinguished. This strategy permits the rapid and standardized identification of single-copy gene sequences in genomic DNA.

Amino Acid Sequence↗

PubMed1988

Restricted use of T cell receptor V genes in murine autoimmune encephalomyelitis raises possibilities for antibody therapy.

Experimental allergic encephalomyelitis (EAE) is a paralytic autoimmune disease induced in susceptible animals by active immunization with myelin basic protein (MBP) or by passive transfer of MBP-specific T helper (TH) lymphocytes. We have analyzed the T cell receptor genes of 33 clonally distinct TH cells specific for a nonapeptide of MBP inducing EAE in B10.PL (H-2u) mice. All 33 TH cells used two alpha variable gene segments (V alpha 2.3, 61%; V alpha 4.2, 39%), the same alpha joining gene segment (J alpha 39), and two V beta and J beta gene segments (V beta 8.2-J beta 2.6, 79%; V beta 13-J beta 2.2, 21%). The anti-V beta 8 monoclonal antibody F23.1 was found to block completely recognition of the nonapeptide by V beta 8 TH cells in vitro and to reduce significantly the susceptibility of B10.PL mice to peptide-induced EAE.

Animals↗

PubMed1988

Optimized conditions for pulsed field gel electrophoretic separations of DNA.

Quantitative measurement of DNA migration in gel electrophoresis requires precisely controlled homogeneous electric fields. A new electrophoresis system has allowed us to explore several parameters governing DNA migration during homogeneous field pulsed field gel (PFG) electrophoresis. Migration was measured at different switch times, temperatures, agarose concentrations, and voltage gradients. Conditions which increase DNA velocities permit separation over a wider size range, but reduce resolution. We have also varied the angle between the alternating electric fields. Reorientation angles between 105 degrees and 165 degrees give equivalent resolution, despite significant differences in DNA velocity. Separation of DNA fragments from 50 to greater than 7000 kilobases (Kb) can easily be optimized for speed and resolution based on conditions we describe.

Chromosomes↗

PubMed1988

Two minor determinants of myelin basic protein induce experimental allergic encephalomyelitis in SJL/J mice.

Experimental allergic encephalomyelitis (EAE) is an autoimmune demyelinating disease of the central nervous system (CNS) that occurs after immunization of animals with myelin basic protein (MBP). The disease is a prototype model for the study of antigen-specific T helper cell-mediated autoimmune disease. In SJL/J mice, EAE is mediated by T helper cells directed against a 40-amino acid COOH-terminal peptic fragment of mouse small MBP. To identify the minimal T cell epitopes of MBP responsible for EAE, overlapping peptides completely encompassing the epitopes within this region were synthesized. A 28-residue peptide of mouse MBP spanning residues 87-114 (pM87-114) was able to elicit both a strong T cell response and chronic relapsing disease. To better localize the T cell epitopes, shorter peptides within this region were synthesized and two overlapping peptides, pM87-98 and pM91-104, were able to induce EAE. T cell clones and bulk lymph node cell populations reactive with pM87-98 did not respond to pM91-104. However, lymph node cells reactive with pM91-104 also reacted with pM87-98, thus showing that these two peptides represent contiguous, but distinct encephalitogenic epitopes and that both these epitopes may be contained within pM87-98. In addition, pM87-114 and pM87-98 were found to be minor determinants of the total T cell response to rat and rabbit MBP. The restricted response to MBP in SJL/J mice is similar to that of the PL/J mice in that each appears to have only a single peptide region in MBP that elicits encephalitogenic T cells. However, within the region studied, there were two if not more T cell epitopes. This differs from the single encephalitogenic PL/J epitope. These findings of a single encephalitogenic peptide region with multiple T cell epitopes and the fact that encephalitogenic T cell epitopes may be subdominant have implications for the design of treatments directed at the T cell receptor-MHC-peptide epitope complex in autoimmune disease.

Animals↗

PubMed1988

Mapping genomic organization by field inversion and two-dimensional gel electrophoresis: application to the murine T-cell receptor gamma gene family.

A new two-dimensional gel electrophoresis technique has been developed for the mapping of multigene families. Resolution in the first dimension is based on the generation of large size DNA fragments by infrequently-cutting restriction enzymes, and separation of these fragments by field inversion gel (FIG) electrophoresis. A second restriction enzyme digestion is then carried out with the separated DNA fragments in the agarose gel. Standard gel electrophoresis in the second dimension allows one to estimate the number of hybridizing genes contained in each large DNA fragment. We have also developed a novel method to increase the separation, resolution and hybridization signal in the second dimension by condensing the bands from the first dimension into spots. As an example, we have applied these techniques to determine the organization of the murine T-cell receptor gamma locus. The murine gamma gene family was found to be contained on two DNA fragments encompassing 195 kilobases of DNA. The two-dimensional gel electrophoresis method is particularly useful in the analysis of the organization of multigenic families where single copy probes are not readily available, and should extend the potential usefulness of field inversion gel electrophoresis in gene mapping.

Animals↗

PubMed1988

Biotechnology and medicine of the future.

The practice of biology and medicine has been revolutionized during the past ten years by the advent of three biotechnologies--recombinant DNA techniques, the monoclonal antibody technology, and the development of microchemical instrumentation, machines that permit the rapid and effective synthesis and sequence analysis of proteins and genes. In this article, these powerful biotechnologies will be discussed, with particular emphasis on microchemical instrumentation, a major focus of my efforts for the past 15 years. I will also discuss two fundamental problems in modern medicine that are being explored in my laboratory using these techniques--genetic engineering of the nervous system and the mapping and sequencing of the human genome.

Animals↗

PubMed1988

Regulation of gene expression by interferons: control of H-2 promoter responses.

The magnitude of the response to interferons and the requirement for individual elements in the promoter of the H-2Dd gene were shown to be cell-specific and dependent on the type of interferon used. Three DNA sequences in the promoter were found to bind murine nuclear factors. Two of these sequences are in functionally defined enhancer regions and also bind to the transcription factor AP-1. The third sequence is part of the region involved in interferon regulation and is homologous to the enhancer element of the interferon beta gene. A model for interferon regulation of H-2 promoters is discussed.

Animals↗

PubMed1988

Conserved organization of the human and murine T-cell receptor beta-gene families.

Generation of an immune response depends on the interaction of haematopoietic cell types, among which T cells and their receptors are of central importance. The T-cell receptor is a heterodimer consisting of disulphide-linked alpha and beta-chains, each chain divided into variable (V) and constant (C) regions. The beta-chain is encoded by the rearrangement of separate variable (V beta), diversity (D beta) and joining (J beta) gene segments during T-cell differentiation. To examine the mechanisms of somatic DNA rearrangement and evolution of the beta-gene segments, we have constructed a physical map of the human T-cell receptor beta-chain family containing 40 V beta gene segments as well as both C beta gene clusters. A comparison of the published nucleotide sequences of human and murine V beta gene segments reveals 12 examples of gene segments sharing 65% or more interspecies homology. The relative order of these human and murine V beta gene segment homologues is also conserved along the chromosome, apart from more extensive human gene duplication, presumably as a consequence of constraints imposed on evolutionary mechanisms operating to diversify these gene families or of selective pressures operating to maintain order.

Animals↗