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H Erlich

Publications and source records attributed to H Erlich.

49 records · Page 3Linked to original sources
PubMed1989

Prenatal diagnosis of beta-thalassaemia in Mediterranean populations by dot blot analysis with DNA amplification and allele specific oligonucleotide probes.

In this study, we describe a simple strategy to detect beta-thalassaemia mutations in prospective parents and to make prenatal diagnosis in pregnancies at risk in the Mediterranean population. Screening of prospective parents is carried out by dot blot analysis on enzymatically amplified DNA with a set of oligonucleotide probes complementary to the most common mutations in this population. Prenatal diagnosis is accomplished by the same procedure on enzymatically amplified amniocyte or trophoblast DNA. The main advantages of this procedure are the simplicity, sensitivity (0.05 micrograms of DNA), and rapidity (12-24 h). Further simplification is obtained by amplification of the DNA from crude amniotic cell lysate. The very low amount of fetal material necessary for this analysis eliminates the need to culture amniotic fluid cells and may decrease the fetal loss rate associated with trophoblast sampling. The number of specific DNA sequences obtained by the amplification procedure allowed us to use non-radioactive labelled oligonucleotide probes, which have several advantages compared to radioactive probes.

Alleles↗
PubMed1989

In utero gene transfer and expression: a sheep transplantation model.

Retroviral-mediated gene transfer was used to insert a Neo R gene into fetal sheep hematopoietic cells obtained by exchange transfusion from lambs in utero. After gene transfer the cells were returned to the donor fetus. The lambs were examined after birth for the presence of a functioning Neo R gene. Of ten analyzable animals, six were positive for G418 resistant progenitor cells (CFU-Mix, CFU-C, BFU-E, CFU-E). Two animals were studied for extended periods of time: 8 and 24 months. Each has demonstrated a pattern wherein positive periods are interspersed with times when there were no detectable G418-resistant cells. We conclude that retroviral-mediated gene transfer can be used to insert genes into early progenitor cells of fetal sheep in utero and that the animals can continue to demonstrate blood cells expressing the gene for more than 2 years after birth. This is a US government work. There are no restrictions on its use.

Adenosine Deaminase↗
PubMed1988

A single amino acid substitution in the human histocompatibility leukocyte antigen DR3 beta chain selectively alters antigen presentation.

Activation of T lymphocytes by immunogenic peptides bound to HLA molecules is a central event in the generation of an immune response. To determine the sites on HLA molecules involved in this process, we isolated mutant EBV-transformed B cell clones that express altered HLA-DR3 molecules. One mutant has lost the ability to stimulate a T cell clone specific for a mycobacterial protein, but retains the ability to stimulate other antigen-specific T cells. The DNA sequence of the complete DR alpha and beta coding regions revealed a single nucleotide change resulting in a glutamic acid to lysine substitution at amino acid 9 in the first hypervariable region of the DR beta chain. These results are discussed in relation to a recently proposed model of class II molecule structure.

Amino Acid Sequence↗
PubMed1987

HLA-DP and HLA-DO genes in presumptive HLA-identical siblings: structural and functional identification of allelic variation.

We analyzed HLA class II genomic polymorphisms in three families in which bone marrow transplantation was performed between individuals presumed to be HLA identical, but in which unexplained mixed lymphocyte culture reactivity was observed. These families were characterized by classical HLA serology, MLC, and DP typing. In each family, a pair of "HLA-identical" siblings demonstrated a small proliferative response in bidirectional MLC. Southern blotting analysis performed with cDNA probes for DQ alpha, DP alpha, and DP beta identified DP genomic differences in each case. Hybridization of Bgl II-digested genomic DNA with a DP alpha cDNA probe revealed three prominent polymorphic fragments (7.7, 5.8, and 3.7 kb), which discriminated between presumptive identical siblings and indicated crossover events within HLA. Similarly, hybridization of SstI-digested genomic DNA with a DP beta cDNA probe, although resulting in a more complex pattern, identified DP genomic disparity between the presumed HLA identical siblings. Hybridization of SstI-digested DNA from two families with evidence of DP recombination was performed by using an oligonucleotide probe specific for the newly described HLA class II gene DO beta. Two major polymorphic fragments, at 6.2 and 3.3 kb, segregated in these families and localized the crossovers flanking the DO beta gene between the DQ and DP loci. The contribution of the antigenic differences marked by these HLA DP and DO DNA polymorphisms to allorecognition in MLR and in graft-vs-host disease are discussed.

Alleles↗
PubMed1986

Molecular analysis of HLA class I and class II antigen loss mutants reveals a homozygous deletion of the DR, DQ, and part of the DP region: implications for class II gene order.

The mutant human B-lymphoblastoid cell lines, 721.174 and 721.180, previously reported to exhibit greatly reduced expression of human HLA class I and II antigens (DeMars et al., Hum Immunol 11:77, 1984), were analyzed by Southern blotting using class II cDNA and genomic clones as hybridization probes. All genomic sequences complementary to DR alpha, DR beta, DQ alpha, and DQ beta probes were absent from these mutants. DZ alpha genomic sequences were deleted as were the DP alpha 1 and DP beta 1 loci but the DP beta 2 and most, if not all, of the DP alpha 2 locus were retained. However, no RNA transcripts for either DP alpha 2 or DP beta 2 could be detected. The mapping of the deletion breakpoint within the DP cluster allows the orientation of the loci in the DP region with respect to the centromere as follows: centromere, DP beta 2, DP beta 1, DP alpha 1, (DQ, DR). In addition, the analysis of a set of DR-, DQ-, DP+ homozygous deletion mutants (721.82, 721.84, and 721.101) reveals a deletion breakpoint between the DQ alpha 1/DQ beta 1 loci and the DQ alpha 2/DQ beta 2 loci. These mutants retain DZ alpha genomic sequences, tentatively mapping the DZ alpha locus between the DQ and the DP region. The residual ability of the DR-, DQ-, DP- mutants (174 and 180)* to stimulate allogeneic and autologous lymphoproliferative responses must be attributed to expression of as yet unidentified class II antigens, or to non-class II antigens.

Cell Line↗
PubMed1985

Deletion mapping of HLA and chromosome 6p genes.

We have analyzed a set of heterozygous mutants with deletions that encompass parts of HLA and surrounding regions of chromosome 6p. By a combination of Southern blotting, serologic, enzymatic, and cytogenetic analyses, we have ordered eight independent deletion break points into a sequence that divides chromosome 6p into six regions. The deletion mutants have been used in conjunction with the Southern blot technique to map HLA and other 6p gene probes into those regions. On the basis of these and other data we propose a genetic and physical map of HLA and surrounding regions of chromosome 6p. We find that for HLA probes, most of which hybridize with more than one gene, the multiple copies recognized by single probes map to single regions. Any chromosome 6p gene can now be regionally mapped by using these mutants.

Chromosome Deletion↗
PubMed1985

Use of pooled DNA samples to detect linkage disequilibrium of polymorphic restriction fragments and human disease: studies of the HLA class II loci.

A rapid method has been developed and used to search for restriction fragment length polymorphisms (RFLPs) that are in linkage disequilibrium with disease-associated loci. By using genomic blot-hybridization analysis with DQ beta-chain and DR beta-chain cDNA probes, we examined DNA polymorphisms within the HLA class II loci associated with susceptibility to insulin-dependent mellitus (IDDM). To facilitate the search for informative RFLPs, we compared pooled DNA samples from IDDM patients with pooled DNA samples from randomly selected control individuals, instead of using the conventional approach of examining DNA samples from individuals in two groups. (The conditions under which this approach is useful are treated theoretically in the Appendix.) Several specific polymorphic restriction fragments associated with IDDM were revealed by using this economical and rapid approach. The restriction enzymes and probes identified as informative in this screening were then used to analyze HLA-DR-typed IDDM families, homozygous typing cells, and unrelated individuals to determine the association of the specific restriction fragments with HLA-DR serological type and the frequency in control and IDDM populations. Some individual polymorphic fragments for which the IDDM population was enriched correlated strongly with HLA-DR3, whereas others correlated strongly with HLA-DR4. Some fragments (e.g., a 10-kilobase Taq I fragment detected with the DR beta probe) that were more prevalent in the IDDM population subdivided the serologically defined HLA-DR type and may be informative markers for IDDM susceptibility.

Cloning, Molecular↗
PubMed1984

Analysis by molecular cloning of the human class II genes.

The HLA class II genes control immune responsiveness to defined antigens; they encode cell surface heterodimers composed of alpha and beta glycopeptides. Recently, cDNA and genomic clones encoding these chains have been isolated, which allows molecular analysis of the class II genes. cDNA clones encoding the alpha chain of the HLA-DR antigen as well as that of another HLA class II antigen have been identified and characterized by nucleotide sequence analysis. These clones have been used as probes to isolate additional class II alpha cDNA clones in cDNA libraries and to identify polymorphisms in genomic DNA. Polymorphic restriction sites have been localized within the HLA-DR alpha gene and used as genetic markers in the analysis of families and of disease (insulin-dependent diabetes mellitus) and control populations. In addition, cDNA clones encoding the DR beta and DC beta chains were used as hybridization probes to identify DNA polymorphism. cDNA clones encoding the DR gamma (Ii) chain have also been identified; unlike the DR alpha and DR beta loci, the DR gamma gene is located on some chromosome other than chromosome 6. The genetic complexity of the human class II alpha and beta loci, as revealed by analysis with cDNA and genomic clones, is greater than that of the murine class II genes. The extent of that complexity will be defined by future work in this area.

Amino Acid Sequence↗
PubMed1982

Nonsense suppression in aminoacyl-t-RNA limited cells.

A number of nonsense alleles of lacZ exhibit phenotypic suppression (as much as a sixteen-fold increase in leakiness) during partial limitation for certain aminoacyl-tRNA species in relA mutant cells. Each responsive allele has its individual pattern of response to limitation for one or more amino acids or aminoacyl-tRNA's. The phenotypic suppression occurs only during limitation, and ceases once limitation is reversed. Suppression is much reduced by the presence of the relA+ allele or an allele of rpsL which restricts ribosomal ambiguity. In one case, the suppressed product has been identified by radioimmune assay and gel electrophoresis, and is a full-length lacZ protomer. Mechanisms are discussed whereby aberrations of translation at codons calling for an aminoacyl-tRNA species in short supply might lead to readthrough of a nearby nonsense codon.

Escherichia coli↗
PubMed1975

Synthesis and turnover of ribosomal ribonucleic acid in guanine-starved cells of Escherichia coli.

The relationship between the rate of RNA accumulation and the level of guanosine triphosphate was examined. Cells auxotrophic for guanine show a 6-fold drop in the intracellular level of GTP in response to the exhaustion of the exogenous guanosine supply. Contraction of the GTP pool results in a 2.5-fold reduction in the rate of RNA synthesis and the cessation of RNA accumulation. The decrease in the rate of RNA synthesis is seen to occur at the level of chain elongation. Analysis of RNA made in guanine-starved cells by competition-hybridization and sucrose gradient sedimentation suggests that the turnover of newly synthesized ribosomal RNA accounts for the observed failure of RNA to accumulate.

Escherichia coli↗
PubMedDate not supplied

HLA-DR, DQ and DP typing using PCR amplification and immobilized probes.

A simple, rapid, and precise method of typing HLA class II polymorphism would be valuable in the areas of disease susceptibility, tissue transplantation, individual identification and anthropological genetics. Here we describe a method of analysing class II sequence polymorphism based on polymerase chain reaction (PCR) amplification and hybridization with oligonucleotide probes. One valuable property of sequence-based HLA typing strategies, like oligonucleotide probe hybridization, is that they reveal how and where two alleles differ, not simply that they can be operationally distinguished. The nature and location of HLA polymorphisms appears to be critical in disease association studies and are likely to be important in tissue typing for transplantation. New alleles at the DRB1, DPB1 and DQB1 loci are likely to be identified as this technology is applied to more and more samples, particularly in non-Caucasian ethnic groups. A new allele is uncovered as an unusual pattern of probe binding and then confirmed by sequencing. This pattern is observed because class II polymorphism is localized to specific regions and virtually all 'new' alleles have polymorphisms in the region of probe binding. Obviously, any new allele with a new polymorphic sequence in a region for which typing probes are not available would not be revealed by oligonucleotide typing. With the PCR primers and probes described here, 7 DQA1 alleles, 15 DQB1 alleles, 18 DPB1 alleles, and 32 DRB1 alleles are distinguished. Additional primers and/or probes can, of course, increase the allelic discrimination of oligonucleotide dot blot typing. These horseradish peroxidase (HRP)-labelled oligonucleotide probes are stable (greater than 2 years when stored at 4 degrees C) and the typing system is simple and robust. Over 500 samples from the CEPH pedigrees (unpublished data; A. B. Begovich, et al., manuscript in preparation) and greater than 1000 unrelated samples have been typed by this procedure. Although this dot blot/oligonucleotide hybridization procedure is a powerful and precise method of HLA class II typing, the complexity of the procedure increases as the number of probes required for analysis increases. The reverse dot blot method, based on an array of immobilized probes, allows the typing of individual samples in one single hybridization reaction. In this approach, a panel of unlabelled oligonucleotides are immobilized to a nylon membrane. The PCR product is labelled during the amplification reaction by using biotinylated primers and hybridized to the membrane. The presence of bound PCR product specifically hybridized to a given probe is detected using streptavidin-HRP conjugates and either chromogenic or chemiluminescent substrates.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗