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R DeMars

Publications and source records attributed to R DeMars.

At least 73 records · Page 4Linked to original sources
PubMed1984

The detection of HLA-DR, MB and MT determinants on purified class II molecules by inhibition of microcytotoxicity.

An assay has been developed which makes it possible to determine the HLA allospecificities carried by molecules in purified fractions of detergent lysates from EBV-transformed human lymphocytes. It is based on inhibition of the standard microlymphocytotoxic test used for identifying HLA class I and II antigens with alloantisera. Soluble cell membrane products from EBV-transformed cell lines homozygous for the HLA region gave specific inhibition of standard typing antisera. The test requires preincubation of microliter volumes of soluble antigen preparations maintained in 0.05% NP-40 with selected antisera prior to adding EBV-transformed cells as target cells. It was possible using this assay to follow isolation of the structurally related human class II molecules bearing the MB and DR specificities. Detergent lysates of cells were fractionated on affinity columns prepared from monoclonal antibodies directed against distinct class II antigens. Eluates from these columns contained the expected DR and MB specificities. The assay is easy to perform, highly reproducible and allows multiple determinations.

Antibodies, Monoclonal↗
PubMed1984

Molecular localization of human class II MT2 and MT3 determinants.

The specificities of the monoclonal antibodies I-LR2 and 109d6, which recognize MT2- and MT3-like serologic determinants, respectively, have been confirmed by panel testing. In addition, the relationships of these antibodies to other monoclonal antibodies and alloantisera have been studied by means of cell surface fluorescence, complement-dependent cytotoxicity and immunoprecipitation. Using these monoclonal antibodies, molecules encoded by the HLA-D region have been isolated and characterized by amino acid sequencing and peptide mapping. By these criteria, the major populations of molecules bearing MT2- and MT3-like determinants are indistinguishable from DR molecules.

Amino Acid Sequence↗
PubMed1984

Homozygous deletions that simultaneously eliminate expressions of class I and class II antigens of EBV-transformed B-lymphoblastoid cells. I. Reduced proliferative responses of autologous and allogeneic T cells to mutant cells that have decreased expression of class II antigens.

Mutant human B-lymphoblastoid cell lines, 721.174 and 721.180, that have greatly reduced expressions of known class I and class II HLA antigens were produced by two cycles of gamma-ray mutagenesis followed by selection for HLA antigen loss. Residual binding of monoclonal antibodies directed against class II antigens was negligible except for 10% residual binding of SB-binding antibody ILR1. However, deletion of SB was functionally complete as indicated by failure of the mutants to stimulate proliferation of SB-primed lymphocytes. Residual binding of monoclonal antibodies directed against class I "framework" determinants was reduced by 90-95%. However, the binding of monoclonal antibodies directed against beta 2-microglobulin and against the A2 epitope recognized by monoclonal antibody BB 7.2 was about 20% of normal. The identities of the residual class I-like antigens are unknown. The mutants retained full expression of the EBV-induced EBVCS antigen. Mutants 721.174 and 721.180 have lost most, but definitely not all, of their capacity to stimulate primary allogeneic and autologous lymphoproliferative responses. Therefore, the mutants still express antigens other than those that are presently known to stimulate lymphocyte proliferation. By means of studies of the present sort, and in future studies on expression of transferred HLA genes, mutants 721.174 and 721.180 promise to be useful in molecular and functional analysis of MHC-region-encoded gene products and their genetic regulation.

Antibodies, Monoclonal↗
PubMed1984

Isolation of HLA locus-specific DNA probes from the 3'-untranslated region.

When human class I cDNA clones containing coding sequences are used to probe genomic DNA, 15-20 fragments, each containing a complete class I gene or pseudogene, are seen. Identification of which genomic DNA segments encode the HLA-A and -B antigens has to date required transfection of mouse L cells with cloned class I genes or analysis of HLA loss mutants. In this report we show that under high-stringency conditions, probes constructed from the 3'-untranslated region can be used to specifically identify the segments of DNA that encode the HLA-A and -B antigens in the human lymphoblastoid cell line 721. Examination of DNA from unrelated individuals indicates that these probes are locus specific and will permit identification of HLA-A and -B genes in the population.

Cell Line↗
PubMed1983

Class I-like HLA genes map telomeric to the HLA-A2 locus in human cells.

Among the proteins encoded by the major histocompatibility complex (MHC) are the highly polymorphic class I major transplantation antigens, HLA-A and HLA-B in man and H-2K and H-2D in mouse. Class I loci also include the less polymorphic HLA-C in man and H-2L in mouse. Class I antigens are 45,000-molecular weight (Mr) glycosylated membrane proteins associated on the cell surface with apparently nonpolymorphic beta 2-microglobulin. Linked to the murine MHC are genes encoding the class I-like Qa and Tla antigens, which are closely related structurally to H-2K, D and L, as they are also 45,000-Mr cell surface glycoproteins associated with beta 2-microglobulin. Such class I molecules have also been found on the surface of human T lymphocytes and, in one case, shown to be linked to HLA-A. Recombinant DNA techniques have been used to map DNA fragments to the Tla region in mouse. A similar genetic analysis of the HLA complex is hampered by the lack of congeneic strains and by the small number of intra-MHC recombinants in well studied families. One means of overcoming these problems involves a molecular genetic analysis of human lymphoblastoid cell line (LCL) mutants having gamma-ray-induced physical deletions of HLA DNA to map DNA fragments in the MHC. Here we have applied this method to provide evidence that some human class I-like DNA sequences are telomeric to the A2 locus in LCL 721.

Cell Line↗
PubMed1983

Mapping of class I DNA sequences within the human major histocompatibility complex.

Mutants that had lost expression of alleles of one or more HLA loci were isolated with immunoselection after gamma-irradiation of a human lymphoblastoid cell line LCL 721. DNAs from the mutants were digested with restriction endonucleases and analyzed by Southern blotting using probes for class I HLA genes. Eight polymorphic cut sites for HindIII and PvuII were discovered in class I-associated sequences of LCL 721. Losses of specific fragments generated by restriction enzymes could be associated with losses of specific antigenic expressions and it was possible in this way to assign HLA-A1, HLA-A2, and HLA-B8 to specific DNA fragments. Patterns of gamma-ray-induced segregations of DNA fragments permitted rough linkage alignment of about 30% of the fragments generated by PvuII. The resultant map showed that there are class I HLA genes on the telomeric side of the HLA-A locus. Restriction enzyme site polymorphisms were also examined in a panel of DNAs isolated from peripheral blood lymphocytes (PBLs) of HLA-typed individuals. This panel of PBL DNA complemented the analysis using the HLA deletion mutants.

Alleles↗
PubMed1983

Dissection of the D-region of the human major histocompatibility complex by means of induced mutations in a lymphoblastoid cell line.

This paper describes part of a mutagenic dissection of the human D-region. Twenty-six human lymphoblastoid cell mutants that had lost expressions of HLA-DR were created with a two-step procedure: (i) A mutant from which one entire haplotype had been physically deleted by gamma-rays was isolated by means of immunoselection against cells expressing a specific HLA-B antigen. (ii) This heterozygous deletion mutant was irradiated with gamma-rays or treated with ICR 191, a frameshift mutagen, and mutants that no longer expressed the remaining DR1 antigen were selected with a monoclonal antibody directed against a monomorphic DR determinant. Monoclonal antibody GENOX 3.53 was used to show that four of the gamma-ray induced DR-null mutants did not express the cis-linked MB1/MT1 locus. Since MB1/MT1 was still expressed in the other 16 gamm-ray induced and 6 ICR 191-induced DR-null mutants, the separate loss of expression of MB1/MT1 and DR1 is strong evidence that the DR1 and MB1/MT1 alloantigens are under separate genetic control in the cells we used. Since DR-null mutants bound SB2-specific monoclonal antibody ILR1, whether or not they expressed MB1/MT1, the results mean that gamma-rays resolved the genetic determinants for DR1, MB1/MT1, and SB2. Additional complexity of determinants encoded by D-region genes is indicated by the following results. The amount of MB1/MT1 antigen that was detected with ELISA tests for binding of GENOX 3.53 antibody to cells varied inversely with the number of expressed copies of DR or of a locus near DR. This could result from an increased amount of MB1/MT1 antigen or from increased binding accessibility of GENOZ 3.53-reactive antigen in DR-null mutants. Monoclonal antibodies CC 11.23 and CC 6.4 displayed patterns of binding to parental and diverse mutant cells that differed from that of GENOX 3.53, suggesting the existence of at least one additional D-region antigen that is neither SB, DR, nor MB/MT.

Animals↗
PubMed1983

Genetic mapping of a human class II antigen beta-chain cDNA clone to the SB region of the HLA complex.

A class II antigen beta-chain cDNA clone was isolated from a human B-cell cDNA library by using as a probe the murine I-A beta gene. This cDNA clone, pHA beta, was shown to be distinct from the DC beta- and DR beta-related loci by DNA sequence analysis, thus suggesting that it might correspond to a third polymorphic human class II locus, SB, which encodes secondary B-cell antigens. Genetic mapping of this beta-chain cDNA clone to the SB region was performed by the blot hybridization procedure. We showed that (i) within panels of HLA-DR homozygous human B-cell lines and of unrelated individuals who have been typed for HLA antigens, differential mobility of DNA fragments segregated with distinct SB genotypes; (ii) gamma-ray-induced deletion mutants that have lost the expression of DR or DC/MT antigens but maintain SB expression preserved a pattern consistent with (a) their SB phenotype and (b) the genetic independence of the SB locus with respect to DR and DC/MT; and (iii) within an informative family, two siblings differing only for one allele at the SB locus (because of the occurrence of an internal recombination between DR and GLO) and otherwise HLA identical exhibited a restriction enzyme polymorphism linked to the SB locus. Therefore, all available data are compatible with identity between HA beta and SB beta.

Alleles↗
PubMed1983

Dissociation in expression of MB1/MT1 and DR1 alloantigens in mutants of a lymphoblastoid cell line.

Cytotoxicity tests with alloantisera were used to study the expression of HLA-D region antigens in HLA-DR-null mutants of a human lymphoblastoid cell line. The initial cell line contained just one copy of the MHC as a haplotype that included DR1 and MB1/MT1. Gamma ray mutagenesis of the single haplotype cells followed by selection with complement and an anti-DR monoclonal antibody were then used to isolate DR-null mutants. Two categories of mutants were identified with a panel of alloantisera. Expressions of DR1 and MB1/MT1 were simultaneously lost in four mutants. Nine mutants still expressed MB1/MT1 but had lost the expression of DR1. The dissociated loss of expression of MB1/MT1 and DR1 antigens is evidence for separate genetic control of these alloantigens. The methods used exemplify a versatile approach for conveniently inducing separations of closely linked loci of the MHC.

Cell Line↗
PubMed1982

Serologic identification of the human secondary B cell antigens. Correlations between function, genetics, and structure.

The secondary B cell (SB) antigens are polymorphic HLA-linked antigens on human B cells and macrophages that are identified by primed T cell responses but are genetically distinct from the HLA-DR, MB, and MT antigens. Serologic identification of the SB molecule, using the monoclonal antibody ILR1, now makes it possible to correlate the function of these determinants in human T cell recognition with an Ia-like molecular structure and a genetic locus that marks a new HLA subregion. Three lines of evidence indicate that the ILR1 molecule identifies an epitope on some alleles of the SB gene: (a) the polymorphism of ILR1 -reactivity in the population correlates with SB2 SB3; (b) T cell proliferative response to SB2 and SB3 are specifically inhibited by ILR1; and (c) ILR1 reactivity is exactly concordant with the expression of SB2 in a panel of HLA-deletion mutant lymphoblastoid cell line. Together with previous studies, these results indicate that the SB antigens are on Ia-like molecules. Furthermore, the serologic studies of HLA-deletion mutant cell lines demonstrate that there are two HLA regions centromeric to HLA-B controlling expression of Ia-like molecules: a region toward HLA-B that controls expression of HLA-DR, and a region toward GLO that controls expression of SB.

Antibodies, Monoclonal↗
PubMed1982

Mutations causing deficiency of APRT in fibroblasts cultured from human heterozygous for mutant APRT alleles.

Frequent mutation to adenine analog resistance in diploid human cells reflected heterozygosity for recessive alleles affecting expression of the adenine phosphoribosyltransferase (APRT) locus. Cells from both parents of APRT-deficient sibs were heterozygous and had rates of spontaneous mutation to 2,6-diaminopurine (DAP) resistance of 6.0 x 10(-5) and 16 x 10(-5) per cell generation. Spontaneous DAP-resistant mutants were not observed in cultures of homozygous cells. Almost all mutants of proven heterozygous cultures were APRT deficient and could not use adenine for growth. Frequent DAP-resistant mutations identified heterozygous strain 438, which carried an allele encoding a partially defective form of APRT. All DAP-resistant mutants of strain 438 were partially APRT deficient and could use adenine for growth. The frequency of MNNG-induced DAP-resistant mutants in homozygous strains was approximately the square of the induced frequency in heterozygous strains.

2-Aminopurine↗
PubMed1982

Derepression of genes on the human inactive X chromosome: evidence for differences in locus-specific rates of derepression and rates of transfer of active and inactive genes after DNA-mediated transformation.

Mouse-human hybrid cells that contained an inactive human X chromosome were treated with agents known to alter gene expression and to perturb DNA methylation. 5-Azacytidine greatly increased the rate of derepression of HPRT on the inactive X, while butyrate and dimethyl sulfoxide had smaller effects. Ethionine did not change the rate of derepression. Derepression of two other X-chromosomal loci, PGK and GPD, was also detected. The rate of derepression of PGK was 20-fold higher than the rate for HPRT. Derepression events at the two loci appeared to be independent. Hybrids expressing derepressed X-chromosomal genes had more variable levels of human enzyme activities when compared to control hybrids. HPRT+ clones did not appear after transfer of purified DNA from a cell hybrid containing an inactive human X into HPRT- recipients, but such clones did appear after transfer of DNA from derivative cells in which HPRT had been derepressed.

Animals↗
PubMed1982

Clonal analysis of the stepwise appearance of anchorage independence and tumorigenicity in CAK, a permanent line of mouse cells.

To provide information on the number of steps involved in preneoplastic progression in vitro, the induced and spontaneous appearances of anchorage-independent and tumorigenic variants were studied with clones of a permanent cell line of morphologically transformed, anchorage-dependent, non-tumorigenic pseudodiploid mouse cells (CAK). Tumorigenicity was assayed in the nude mouse by s.c. coinoculation of CAK cell derivatives with 2 x 10(6) human fibroblasts. Under the assay conditions, as few as 10 tumorigenic cells formed tumors. The assay permitted detection of tumorigenic variants soon after their origin and reduced the spontaneous development of new variants that result from extensive proliferation of clonal cell populations prior to testing for tumorigenicity. Anchorage-independent, nontumorigenic variants of CAK cells originated spontaneously at an estimated rate of about 10(-4)/cell/generation. Tumorigenic variants appeared spontaneously during proliferation of an anchorage-independent cell clone at an estimated rate of about 10(-7)/cell/generation but were undetectable among anchorage-dependent CAK cells. In contrast, N-methyl-N'-nitro-N-nitrosoguanidine treatment induced the appearance of tumorigenic variants in both anchorage-dependent and -independent clones with an estimated frequency of about 10(-4)/surviving clone former, which was similar to the induced frequency of ouabain-resistant variants in the same cells. Anchorage independence was expressed without tumorigenicity in new anchorage-independent variants but tumorigenic cells were always anchorage independent. We propose that CAK cells can become tumorigenic by a three-step pathway that includes changes causing morphological transformation, anchorage independence, and tumorigenicity. Our evidence is also consistent with an alternative two-step pathway where anchorage independence and tumorigenicity are acquired in a single step, since anchorage-independent, tumorigenic clones were derived from anchorage-dependent cells soon after a single mutagenic treatment.

Animals↗
PubMed1981

In vitro chemical mutagenesis and viral transformation of a human endothelial cell strain.

We have established and characterized a diploid cell strain of normal human endothelial cells, RuBa 7E. RuBa 7E cells have an average cloning efficiency of 20% during early passages and undergo approximately 50 doublings in vitro before senescing spontaneously. At confluence, RuBa 7E cells form a homogeneous monolayer of flat polygonal cells. RuBa 7E cells react positively with antibody to human endothelial-specific Factor VIII. The toxic and mutagenic effects of N-methyl-N'-nitro-N-nitrosoguanidine on RuBa 7E cells were studied and are similar to those reported for diploid human fibroblasts. Mutant cells lacking hypoxanthine-guanine phosphoribosyltransferase were selected by their resistance to 6-thioguanine. The spontaneous incidence of mutants was less than or equal to 6 X 10(-6), and the induced incidence was 4.4 X 10(-4) at a survival frequency of 0.05. All 17 mutants that were tested lacked detectable hypoxanthine-guanine phosphoribosyltransferase activity, and none grew in medium containing azaserine and hypoxanthine. Autoradiography showed that mutant cells incorporated radioactive adenine but did not incorporate radioactive hypoxanthine. Unlike human fibroblasts, in which the recovery of 6-thioguanine-resistant mutants is reduced by contact feeding when the inoculum size during selection is increased above 10(4) cells per P60 dish, 5 to 10 X 10(4) RuBa 7E cells can be plated per P60 dish without reducing mutant recovery. This apparent lack of plating density suppression of mutant recovery makes RuBa 7E cells a comparatively compact and economical system for quantifying mutagenesis in diploid human cells. In order to determine whether RuBa 7E cells would undergo a distinct morphological transformation toward cancer in vitro, we infected them with SV40. As early as 14 days postinfection, discrete foci of morphologically transformed, mitotically active cells were seen against a monolayer background of normal cells when cultures were maintained in medium with low serum. Seven of the 33 foci which were obtained were studied for SV40-specific viral T-antigen, and all were positive. The facility with which RuBa 7E cells can be mutagenized and the ease with which morphological transformants can be identified make these cells potentially useful for studies comparing the mutagenic and transforming effects of chemicals and other agents on diploid human cells.

Cell Division↗
PubMed1981

A new variant glyoxalase I allele that is readily detectable in stimulated lymphocytes and lymphoblastoid cell lines but not in circulating lymphocytes or erythrocytes.

We describe an allele of the human glyoxalase GLO locus that encodes an enzymatically inactive form of the protein, which would not have been detected if only circulating erythrocytes and lymphocytes had been studied. The new allele is named GLO*3 and its protein product, GLO 3. Circulating blood cells of GLO*2/GLO*3 heterozygotes have just one electrophoretic band that migrates as the normal 2-2 dimer. Lymphoblastoid cell lines and phytohemagglutinin-stimulated lymphocytes from the same individuals have two electrophoretic bands, one with the mobility of the 2-2 dimer and one with the mobility of the 2-1 dimer that is present in GLO*2/GLO*1 heterozygotes, but a band with the mobility of the 1-1 dimer is not present. Therefore, the GLO*3 allele encodes a monomer that has the electrophoretic mobility of GLO 1 but is enzymatically inactive unless it is combined with normal monomers in 2-3 and 1-3 heterodimers. The failure to detect the GLO 3 protein in red cells and unstimulated lymphocytes is attributed to a relatively great instability or small rate of production in those cells. Consistent with this interpretation is the reduction of GLO activity in red cells of GLO*2/GLO*3 and GLO*1/GLO*3 heterozygotes to 65% or less of that in normal homozygotes and heterozygotes, while the activity of GLO*3 heterozygous lymphoblastoid cells is about 80% of normal. In contrast, the GLO activity of lymphoblastoid cells that had one copy of the GLO locus deleted by gamma-irradiation was 50%-60% of normal. Our observations indicate that certain kinds of mutant alleles of the GLO locus, and perhaps other loci, may not be detected in electrophoretic surveys on circulating blood cells only. The segregation of alleles that are not expressed in circulating red and white blood cells could confuse attempts to determine parentage, as they might have in the family described here. The observations also demonstrate the feasibility of mapping human genes by using ionizing radiation to create partial chromosome deletions in cultured cells.

Alleles↗
PubMed1980

Expression of human genes for adenine phosphoribosyltransferase and hypoxanthine-guanine phosphoribosyltransferase after genetic transformation of mouse cells with purified human DNA.

Human DNA purified from HeLa cells and from three strains of skin fibroblasts was precipitated with calcium phosphate and added to mouse cells that were deficient in adenine phosphoribosyltransferase (APRT) and hypoxanthine phosphoribosyltransferase (HPRT). Selection for cells possessing either of the phosphoribosyltransferases was imposed by blocking de novo synthesis of purine nucleotides with azaserine in a medium supplemented with adenine and hypoxanthine. The frequency of colony formation after selection was 1.7 x 10(-7)-3.3 x 10(-6). Excepting some azaserine-resistant colonies that appeared only in the first experiment and infrequent revertants expressing moust APRT, all characterized clones expressed the human forms of APRT or HPRT according to the criteria of specific immunoprecipitation and electrophoretic mobility. The frequency of transfer of the human APRT gene was much greater than that of HPRT. Transfer efficiency was not significantly reduced when HeLa DNA was sheared to 6.5-13.5 kb size or when the donor DNA was isolated from a transferent that expressed human APRT.

Adenine Phosphoribosyltransferase↗