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Biomedical subjects

H A Erlich

Publications and source records attributed to H A Erlich.

At least 217 records · Page 12Linked to original sources
PubMed1983

Segregation and mapping analysis of polymorphic HLA class I restriction fragments: detection of a novel fragment.

An HLA-B7 complementary DNA clone was used as a hybridization probe to analyze the segregation pattern of polymorphic class I restriction fragments in several families whose HLA types had been determined by serological techniques. In one family in which a crossover in the HLA region had occurred, a specific genomic fragment was mapped with respect to the crossover. In another family, a novel genomic fragment present in one child and absent in all other family members was observed. With the exception of this novel fragment, all polymorphic class I fragments observed in this study segregated with a serologically defined parental haplotype, a result consistent with HLA linkage.

Chromosome Mapping↗
PubMed1983

Biochemical characterization of an invariant polypeptide associated with Ia antigens in human and mouse.

Ii, a 31,000 mol. wt polypeptide chain associated with murine and human Ia antigens was investigated for its labeling pattern, carbohydrate content and structural polymorphism. Two-dimensional gel electrophoretic analysis of tunicamycin treated cells from mouse and human lymphocytes shows that Ii contains two N-linked carbohydrate chains. Ii is a methionine rich polypeptide. Tryptic and chymotryptic two dimensional peptide maps of Ii chain associated with I-A and I-E subregion products are identical. This absence of polymorphism holds true when Ii chain is isolated from different mouse haplotypes. Human Ii chains from different HLA-DR types appear also invariant by peptide map analysis. By molecular weight, carbohydrate content, charge and tryptic and chymotryptic maps criteria, Ii of mouse and human are strikingly homologous.

Animals↗
PubMed1983

Use of synthetic oligonucleotide probes complementary to genes for human HLA-DR alpha and beta as extension primers for the isolation of 5'-specific genomic clones.

We have synthesized 175-nucleotide-long probes for the DNA of human histocompatibility antigens HLA-DR alpha and beta by extending on poly(A)+ mRNA from B-cell lines with short synthetic deoxyribonucleotide primers complementary to the predicted nucleotide sequence of the NH2 terminus of both polypeptides. The synthesis of the probe for the alpha-chain DNA was a two-step process starting with 11-mers which were extended by dideoxynucleotide chain termination experiments to a 20-mer of predicted sequence. The synthesized 20-mer was then used to generate a 175-nucleotide cDNA probe which was shown to encode the appropriate amino acids for the alpha chain and was used to select a human genomic DNA clone containing the coding sequences for HLA-DR alpha. For the beta polypeptide an 18-mer homologous to the NH2-terminal sequence of a cDNA clone from another B-cell line was used to extend on poly(A)+ mRNA isolated from a B-cell line. Preliminary sequence analysis of a 175-base-long extension product indicates a match of the cDNA sequence to the published sequence of a clone for HLA-DR beta. Information from these extension experiments helps to establish the sensitivity and specificity of the primer extension method.

Amino Acid Sequence↗
PubMed1983

Mapping of the genes encoding the HLA-DR alpha chain and the HLA-related antigens to a chromosome 6 deletion by using genomic blotting.

We have used genomic blotting with DNA from a human cell line that has a small deletion on chromosome 6 (6.3.6) and from its parent cell line (T5-1) to map DNA fragments complementary to cloned DNA sequences encoding the HLA-B7 antigen (class I) and the alpha chain of the HLA-DR antigen (class II). The 6.3.6 variant fails to express the HLA-A, -B, -C, and -DR and MB specificities associated with one of the parental T5-1 haplotypes and has a visible deletion in the short arm of one chromosome 6 (1). The gene locus assignment was based on the expectation that, if the chromosomal location of the DNA sequences used as a hybridization probe were within the deletion, then the relative amount or size (or both) of genomic restriction fragments that hybridize to the probe in T5-1 and in 6.3.6 DNAs should differ predictably. By comparing the genomic blot patterns from T5-1 and 6.3.6 DNAs, we have shown directly that the loss of haplotype expression was due to deletion of the structural genes and have mapped the structural gene for the HLA-DR alpha chain to the chromosomal location (6p2105-6p23) defined by the 6.3.6 deletion. A cDNA clone encoding the alpha chain of the HLA-DR antigen hybridized to two genomic fragments, 4.2 and 3.8 kilobases long, generated by Bgl II digestion of T5-1 DNA. The 4.2-kilobase fragment was absent from DNA derived from the 6.3.6 deletion variant. Thus, this fragment could be assigned to the parental chromosome 6 with the A1, B8, DR3 haplotype, and the 3.8-kilobase fragment, to the chromosome 6 with the A2, B27, DR1 haplotype. In addition, comparison of the T5-1 and 6.3.6 genomic blot patterns obtained with the HLA-B7 probe revealed dosage differences for all of the class I genomic fragments generated by BamHI digestion, suggesting that all of the class I loci map to the region 6p2105-6p23.

Chromosome Deletion↗
PubMed1983

Monoclonal antibodies directed against human fibroblast interferon: characterization and functional studies.

Spleen cells from BALB/c mice, immunized with SDS-acrylamide gel purified human fibroblast interferon (HuIFN-beta) were fused with mouse myeloma cells. Culture fluids from the resulting hybrid cells were screened for HuIFN-beta specificity by the following tests: a solid-phase RIA using partially purified HuIFN-beta, a protein-transfer RIA using electrophoretically resolved and immobilized HuIFN-beta, and a bioassay which tests residual HuIFN-beta activity in the supernatant following immunoprecipitation. Six HuIFN-beta-specific hybridomas were identified; two were capable of partially neutralizing HuIFN-beta activity. All six antibodies bind to the beta 1-IFN polypeptide synthesized in E. coli cells containing a cloned beta 1-IFN DNA sequence. All six monoclonal antibodies were found to be IgG3/kappa.

Animals↗
PubMed1983

Identification of an antigen-specific immunoglobulin M antibody associated with acute Toxoplasma infection.

We used the protein gel transfer technique (Western blotting) to analyze the immune response to individual electrophoretically resolved Toxoplasma antigens. Toxoplasma antigens were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The array of resolved proteins was electrophoretically transferred to CNBr-activated filter paper and incubated with serum samples from patients with either acute or chronic Toxoplasma infections or with serum samples from seronegative controls. Serum immunoglobulin G and immunoglobulin M bound to individual Toxoplasma antigens were detected by radioiodinated staphylococcal protein A and a goat anti-human immunoglobulin M antibody, respectively. A complex array of Toxoplasma antigens was revealed by this procedure. Immunoglobulin M directed against a low-molecular-weight antigen was detected in sera from all acutely infected adults (19 sera tested) and was absent from all sera from individuals with chronic infections (9 sera tested) and from sera from seronegative controls (9 sera tested).

Acute Disease↗
PubMed1983

Western Blot analysis of the antigens of Toxoplasma gondii recognized by human IgM and IgG antibodies.

Western Blot analysis revealed that both IgM and IgG antibodies present in the sera of humans infected with Toxoplasma gondii recognize three major antigens with apparent m.w. of 32,000, 22,000, and 6000, respectively. In addition, IgG antibodies recognized at least 17 other antigenic components. After subcellular fractionation, enrichment of the three major antigens recognized by IgM and IgG antibodies by the membrane fraction was observed. Solubilization of membrane-enriched preparations with a mixture of sodium dodecyl sulfate and sodium deoxycholate did not reveal any new antigenic structures that reacted with IgM or IgG antibodies. Treatment of Toxoplasma lysate preparations and various fractions obtained after differential centrifugation with NaIO4 diminished the reactivity of the antigens with both IgM and IgG antibodies. Lipase treatment had no effect on the number or nature of antigens recognized by IgM antibody. Treatment with pronase and trypsin eliminated the 32,000 and 22,000 m.w. antigenic components detected by IgM antibodies, whereas such treatment had no effect on the 6000 m.w. component. Periodic acid-Schiff staining of polyacrylamide gels of Toxoplasma sonicates revealed the presence of three components corresponding to m.w. of 62,000, 45,000, and 6000, respectively. At least 15 components, including the 6000 m.w. component, directly bound concanavalin A.

Animals↗
PubMed1982

Isolation of a cDNA clone for the human HLA-DR antigen alpha chain by using a synthetic oligonucleotide as a hybridization probe.

We have used a synthetic 20-nucleotide hybridization probe to isolate a cDNA clone encoding the alpha chain of the HLA-DR antigen from a cDNA library constructed from membrane-bound poly(A)+ mRNA. A set of synthetic 11-nucleotide fragments, potentially complementary to the codons for amino acids 11-14 of the HLA-DR alpha chain, were used to prime a cDNA synthesis reaction on various poly(A)+ mRNA templates. Extension of the primers in the presence of a single dideoxynucleotide triphosphate resulted in an 18-nucleotide cDNA product whose sequence corresponded to the NH2-terminal amino acids of the HLA-DR alpha chain. An oligonucleotide was synthesized based on this sequence information and its specificity for HLA-DR alpha mRNA was confirmed by primer extension and blot analysis. The cDNA library made from mRNA from the lymphoblastoid cell line CA-SC was probed with 32P-labeled cDNA synthesized on poly(A)+ mRNA from a B-cell line (CA-SC) or from a T-cell line (Molt-4) to enrich for B-cell-specific clones. A set of cDNA clones that hybridized preferentially with the B-cell probe was screened with the 32P-labeled 20-nucleotide probe. The cDNA clone isolated by this procedure is 1,100 nucleotides long; the nucleotide sequence of the 5' end of the cDNA insert corresponds to the amino acid sequence of the HLA-DR alpha chain. Hybridization of this cDNA clone to genomic blots suggests that the HLA-DR alpha chain is encoded by a single-copy gene. One of the restriction endonucleases used in genomic DNA digests reveals a restriction fragment polymorphism.

Amino Acid Sequence↗
PubMed1980

Interaction of an Escherichia coli mutator gene with a deoxyribonucleotide effector.

Strains of Escherichia coli carrying mutD5 display very high mutation rates (about 10(4)-fold above wild-type) when grown in rich medium, but relatively low mutation rates (about 10- to 50-fold above wild-type) in minimal medium. Thymidine, deoxycytidine, and deoxyuridine are all capable of stimulating mutation when added to minimal medium. Studies with mutants blocked in various steps of thymidine metabolism implicate a phosphorylated thymidine effector which mediates the mutagenic action of the added deoxyribonucleotides. In addition, an unidentified compound or compounds other than thymidine present in rich medium (L-broth) can also increase the mutation rate.

Culture Media↗
PubMed1980

Initiation of protein synthesis in bacteria at a translational start codon of mamalian cDNA: effects of the preceding nucleotide sequence.

Plasmids containing a mouse cDNA sequence encoding the enzyme dihydrofolate reductase (DHFR; tetrahydrofolate dehydrogenase; 5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase, EC 1.5.1.3) have been used to study the efficiency of initiation of protein synthesis at an ATG (AUG) translational start codon indigenous to the eukaryotic CDNA. differences in DHFR production assayed phenotypically, enzymatically, and immunologically were correlated with the primary structure of the DNA segment that precedes the translational start codon. Our results indicate that initiation of a structurally discrete and biologically functional eukaryotic protein can occur in bacteria on a fused mRNA molecule, and that the efficiency of expression is strongly affected by: (i) the extent of homology of the translational control region with the 3'-OH end of 16S ribosomal RNA, and (ii) the distance between the protein start codon and the ribosome-binding sequence on the mRNA.

Animals↗
PubMed1978

Phenotypic expression in E. coli of a DNA sequence coding for mouse dihydrofolate reductase.

The construction and analysis of bacterial plasmids that contain and phenotypically express a mammalian genetic sequence are described. Such plasmids specify a protein that has enzymatic properties, immunological reactivity and molecular size characteristic of the mouse dihydrofolate reductase, and render host cells resistant to the antimetabolic drug trimethoprim.

Animals↗
PubMedDate not supplied

Genetic prediction of type 1 diabetes in a population with low frequency of HLA risk genotypes and low incidence of the disease (the DIABFIN study).

BACKGROUND: To develop a sensitive, specific screening strategy for predicting genetic risk for type 1 diabetes mellitus (T1DM) in the low-incidence continental Italian population, and to define with this tool, a cohort of high-to-moderate risk infants for an immunological follow-up study aimed at identifying environmental risk factors for T1DM. METHODS: 4855 newborns in three regions of continental Italy were screened for T1DM HLA-DRB1-DQB1 risk genotypes using a reverse line blot typing method. Risk classification was based on odds ratios (OR) found in a preliminary case-control study (356 T1DM patients, 412 controls). Screening efficiency was optimized by allele subtyping. RESULTS: Screening for well-known T1DM susceptibility genotypes [DRB1*03/*04-DQB1*0302; DRB1*03/*03; DRB1*04/*04-DQB1*0302; DRB1*04-DQB1*0302/X where X is not equal to DRB1*03, DRB1*04-DQB1*0302, DQB1*0602 or DQB1*0603] was associated with <60% sensitivity due to their low frequencies in the general Italian population. Inclusion of an additional genotype from which protective DRB1 and DQB1 alleles had been excluded [DRB1*03/X degrees where DQB1 is not equal to *0301, *0503, *0602, or *0603 and X degrees not equal DRB1*03, DRB1*04-DQB1*0302 or DRB1*07] increased screening sensitivity to 75% (specificity: 85%). Among 4855 newborns, we have found the high-risk genotype [DRB1*03/*04-DQB1*0302; estimated absolute risk (AR) 1/23] to be present in only 0.9%. The moderate-risk genotypes were found in 13.8% of newborns (estimated AR 1/177). CONCLUSIONS: Risk classification must be tailored to the characteristics of the individual population, in particular, the allelic frequencies in the background population and T1DM prevalence. We have developed a screening strategy with good levels of sensitivity that should prove effective for use throughout the Italian peninsula.

Adult↗
PubMedDate not supplied

Analysis of enzymatically amplified beta-globin and HLA-DQ alpha DNA with allele-specific oligonucleotide probes.

Allelic sequence variation has been analysed by synthetic oligonucleotide hybridization probes which can detect single base substitutions in human genomic DNA. An allele-specific oligonucleotide (ASO) will only anneal to sequences that match it perfectly, a single mismatch being sufficient to prevent hybridization under appropriate conditions. To improve the sensitivity, specificity and simplicity of this approach, we used the polymerase chain reaction (PCR) procedure to enzymatically amplify a specific segment of the beta-globin or HLA-DQ alpha gene in human genomic DNA before hybridization with ASOs. This in vitro amplification method, which produces a greater than 10(5)-fold increase in the amount of target sequence, permits the analysis of allelic variation with as little as 1 ng of genomic DNA and the use of a simple 'dot blot' for probe hybridization. As a further simplification, PCR amplification has been performed directly on crude cell lysates, eliminating the need for DNA purification.

Alleles↗
PubMedDate not supplied

Characterization of beta-thalassaemia mutations using direct genomic sequencing of amplified single copy DNA.

Direct sequencing of specific regions of genomic DNA became feasible with the invention of the polymerase chain reaction (PCR) which permits amplification of specific regions of DNA. Recently, human mitochondrial DNA was amplified and directly sequenced. Using a thermostable DNA polymerase of T. aquaticus (Saiki, R.K. et al., manuscript in preparation) in the PCR, we have applied a combination of PCR and direct sequence analysis of the amplified product to a human single-copy gene. We studied the genomic DNA of five patients with beta-thalassaemia whose mutant alleles were uncharacterized, and found two previously undescribed mutations, along with three known alleles. One new allele is a frameshift at codons 106-107 and the other is an A-C transversion at the cap site (+1) of the beta-globin gene. This latter is the first natural mutation observed at the cap site and it occurs in a gene which is poorly expressed.

Base Sequence↗