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Biomedical subjects

B Mach

Publications and source records attributed to B Mach.

At least 199 records · Page 11Linked to original sources
PubMed1982

A molecular map of the immune response region from the major histocompatibility complex of the mouse.

A stretch of 200 kilobases (kb) of DNA from the I region of the mouse major histocompatibility complex has been cloned and characterized. It contains the genes for the biochemically defined class II proteins E alpha, E beta and A beta. DNA blot analyses suggest that the I region may contain only 6-8 class II genes. Correlation of our molecular map with the genetic map of the I region confines two of the five I subregions, I-J and I-B, to less than 3.4 kb of DNA at the 3' end of the E beta gene where a hotspot for recombination has been observed. Indeed, the I-A and I-E subregions may be contiguous. If so, the I-B and I-J subregions are not encoded in the I region between the I-A and I-E subregions.

Animals↗
PubMed1982

Translation and assembly of HLA-DR antigens in Xenopus oocytes injected with mRNA from a human B-cell line.

HLA-DR antigens are polymorphic cell surface glycoproteins, expressed primarily in B lymphocytes and macrophages, which are thought to play an important role in the immune response. Two polypeptide chains, alpha and beta, are associated at the cell surface, and a third chain associates with alpha and beta intracellularly. RNA isolated from the human B-cell line Raji was injected in Xenopus laevis oocytes. Immunoprecipitates of translation products with several monoclonal antibodies revealed the presence of HLA-DR antigens similar to those synthesized in Raji cells. One monoclonal antibody was able to bind the beta chain after dissociation of the three polypeptide chains with detergent. The presence of all three chains was confirmed by two-dimensional gel electrophoresis. The glycosylation pattern of the three chains was identical to that observed in vivo, as evidenced in studies using tunicamycin, an inhibitor of N-linked glycosylation. The presence of alpha chains assembled with beta chains in equimolar ratio was further demonstrated by amino-terminal sequencing. An RNA fraction enriched for the three mRNAs, encoding alpha, beta, and intracellular chains, was isolated. This translation-assembly system and the availability of monoclonal antibodies make it possible to assay for mRNA encoding specific molecules among the multiple human Ia-like antigens.

Animals↗
PubMed1982

Origin of transcription of a mouse immunoglobulin light chain gene.

Although the organization, the structure, and the somatic rearrangement of immunoglobulin genes have been studied in detail, there is no information regarding the precise origin of transcription of immunoglobulin genes. We have analyzed the 5'-flanking region of an expressed mouse V kappa light chain gene and the pre-mRNA transcript of that light chain gene, as it is expressed in vivo. Study of the pre-mRNA and of the DNA sequence by the procedure of "S1 mapping" establishes that the 5' end of the pre-mRNA transcript is 25-26 nucleotides upstream from the initiation codon. A CATATA sequence is found 22-23 nucleotides upstream from the origin of transcription. Although several other TATA-like sequences are found further upstream, only a single origin of transcription can be documented. From the S1 protection experiments, the 5' end of the mature mRNA was found to be the same as that of the pre-mRNA, indicating conservation of that sequence during RNA processing. Finally, a conspicuous pentanucleotide repeat CATTG - CATTG has been identified at the position of transcription initiation.

Amino Acid Sequence↗
PubMed1982

Isolation of cDNA clones encoding HLA-DR alpha chains.

HLA-DR antigens, the human equivalent of mouse Ia antigens, are multimeric surface glycoproteins characterized by a high degree of allelic polymorphism. They are expressed specifically on macrophages and lymphocytes and they play a key role in the regulation of the immune response. We have investigated this complex genetic system by a direct study of the genes involved through molecular cloning. This paper deals with the cloning, in plasmids, of full-length cDNA sequences for the HLA-DR alpha chain from the human B-cell line Raji. The approach relies on a translation assay of mRNA injected into frog oocytes and recognition of translation products by polyclonal and monoclonal antibodies. After enrichment of specific mRNA and cloning of cDNA, plasmid clones were analyzed by hybridization-selection of mRNA and translation in oocytes. A clone was identified and used to screen a cDNA library from which several full-length HLA-DR alpha chain plasmids were isolated. DNA sequence determination of one such clone confirmed its identity and also established the amino acid sequence of the NH2-terminal signal sequence of HLA-DR alpha chains. The translation product of HLA-DR alpha chain mRNA purified by hybridization-selection gives a single alpha chain spot on two-dimensional gels, whereas the alpha chain released from the alpha/beta HLA-DR complex gives about seven distinct spots. Finally, the results of analysis of genomic DNA by Southern blotting are compatible with the existence of a single nonpolymorphic alpha chain gene and indicate extensive cross-hybridization with a homologous gene in mouse DNA.

Animals↗
PubMed1982

Isolation of distinct cDNA clones encoding HLA-DR beta chains by use of an expression assay.

cDNA clones encoding different human Ia antigen beta chains were isolated by use of a complementation-expression assay in Xenopus oocytes. The assay was based on two previous findings. First, oocytes injected with mRNA from a human B-cell line express HLA-DR antigen. The three intracellular DR chains are assembled in oocytes and can be immunoprecipitated with anti-DR monoclonal antibodies. Second, we have isolated cDNA clones encoding DR alpha and intermediate chains. In order to identify beta-chain cDNA clones, mRNA was hybrid-selected with pools of cDNA clones, mixed with mRNA for the alpha and intermediate chains, and injected into oocytes. We isolated two distinct clones that could select DR beta-chain mRNA as demonstrated by assembly of the translation product with DR alpha chains and immunoprecipitation with DR-specific monoclonal antibodies. One clone is specific for a beta chain of the DR locus. The other clone, much weaker in its ability to select DR mRNA, encodes another Ia-like beta chain. Full-length cDNA clones corresponding to the DR and Ia-like beta chains were isolated and compared. Cross-hybridization was detectable in the coding regions but not in the 3' untranslated regions. Distinct RNAs homologous to the DR and the Ia-like beta-chain clones were present in B cells but were undetectable in three T-cell lines.

Base Sequence↗
PubMed1981

Cloning of multiple copies of immunoglobulin variable kappa genes in cosmid vectors.

The possibility of cloning large segments of DNA in cosmid vectors offers distinct advantages, in particular for the study of multigene families. Large size fragments of mouse embryo DNA were successfully cloned in the cosmid pHC 79. Twelve recombinants hybridizing specifically to an immunoglobulin kappa chain variable region probe were identified. In 9 of these recombinants, the size of the insert ranges from 30 to 43 kilobases. Factors affecting the cloning efficiency of a complex mammalian genome in cosmids were studied. The stability of these recombinant cosmids and the preparation of recombinant cosmid DNA are also discussed.

Animals↗
PubMed1979

Cloning of immunoglobulin kappa light chain genes from mouse liver and myeloma MOPC 173.

The organization of the kappa chain constant region gene was compared in DNA from an immunoglobulin-producing mouse myeloma (MOPC 173) and from liver. In situ hybridization using the Southern blotting technique revealed constant region gene-containing EcoRI-DNA fragments of 14 and 20 kb in the myeloma tissue whereas one EcoRI-DNA fragment with a length of 15 kb was found in liver DNA. After enrichment by RPC-5 chromatography and preparative electrophoresis the 14 kb fragment from MOPC 173 DNA and the 15 kb fragment from liver DNA were cloned in the bacteriophage lambda vector Charon 4A using in vitro packaging. Extensive characterization of the two fragments by restriction endonuclease mapping, in situ hybridization, and electron microscopy (R-loop and heteroduplex) showed that both fragments contain the constant region but no MOPC 173 variable region gene. Both fragments are homologous over a length of 12.5 kb including the constant region but differ from one another starting about 2.7 kb from the 5' end of the constant region gene. This indicates that the 14 kb EcoRI-DNA fragment from the myeloma tissue clearly resulted from somatic DNA rearrangement although it does not seem to carry the MOPC 173 variable region gene. These observations suggest that somatic DNA rearrangement of immunoglobulin light chain genes can involve both homologous chromosomes.Images

Animals↗
PubMed1979

A precise quantitation of gene number by saturation hybridization using cloned DNA.

This paper describes a precise method of gene titration as applied to the alpha- and beta-globin genes in the mouse. The three salient features of the method are: (i) the use of saturation hybridization in probe cDNA excess, (ii) the use of highly purified cDNA probes prepared by preparative hybridization with cloned globin sequences (Longacre and Mach (1978) J. Biol. Chem. 253, 7500) and (iii) the use of cloned globin sequences to calibrate the system internally. The results indicate that there are two genes for alpha-globin and two genes for beta-globin in the BALB/c mouse. The significance of these results are discussed in relation to other data regarding adult and embryonic globin genes.

Animals↗
PubMed1978

Purification of specific DNA sequences by sulfhydryl-Sepharose chromatography of mercurated polynucleotides.

Recombinant plasmid DNA has been used to purify complementary cDNA by hybridization using a modification of sulfhydryl-Sepharose chromatography described by Dale and Ward ((1975) Biochemistry 14, 2458). Plasmid DNA containing cloned mouse globin or immunoglobulin sequences was mercurated and hybridized in solution to unpurified cDNA. The resulting hybrids were passed over a sulfhydryl-Sepharose column where mercurated polynucleotides are retained. After washing, cDNA hybridized to the mercurated plasmid DNA was melted in situ and eluted while the mercurated plasmid DNA remained bound to the column. The conditions for purification of DNA and RNA sequences are described. The purity of the cDNAs obtained by this method is analyzed by polyacrylamide gel electrophoresis and by hybridization. In addition, this nucleic acid purification procedure has been applied to two problems of general interest: (i) the sensitive titration of specific genes by saturation hybridization; (ii) the purification of DNA fragments bearing specific sequences from restriction endonuclease digests of total cellular DNA. The procedure is generally applicable to the purification by hybridization of any DNA or RNA sequence complementary to an available probe.

Base Sequence↗
PubMed1978

Electron microscope analysis of mouse and rabbit globin and immunoglobulin gene sequences.

Mouse and rabbit globin and immunoglobulin gene sequences, which had been synthesized in vitro from eukaryotic mRNAs and inserted into plasmids, have been examined in the electron microscope. The size of the inserted beta rabbit and alpha and beta mouse globin DNA sequences has been estimated as 620 base pairs while the size of the inserted alpha rabbit globin DNA sequences was found to be about 490 base pairs. Heteroduplex analysis has revealed no structural abnormalities at the insertion sites of the chimeric plasmids except in the case of a plasmid containing an immunoglobulin light chain gene sequence of about 830 bases, in which 3 kb deletion adjacent to the insertion site was observed.

Animals↗
PubMed1978

Genetic engineering.

The field of genetic engineering is reviewed with a special emphasis on in vitro DNA recombinant technology. The basic principles of the biochemistry of DNA splicing and of gene transfer are described. An important distinction is made between the insertion and cloning of genes derived from genomic DNA ("natural" genes) and of DNA synthetized in vitro ("synthetic" genes). Cloning of genes synthetized from mRNA has provided the probes necessary for the identification of genomic clones, and recently it has made possible the synthesis of specific mammalian proteins in bacteria.

DNA, Recombinant↗
PubMed1977

Cloning and amplification of rabbit alpha- and beta-globin gene sequences into Escherichia coli plasmids.

cDNA, synthesized from rabbit globin mRNA, was used in a self-priming reaction, with avian myeloblastosis virus DNA polymerase, for the synthesis of double-stranded DNA. Globin DNA ranging from about 400 to 650 base pairs was elongated with dG tails using deoxypolynucleotide transferase and was annealed to linear Escherichia coli plasmic pCR1, elongated with dC tails. Preparation of the plasmid DNA involved an enzymatic reconstruction of one EcoRI-specific site on each side of the molecule. After transformation of E. coli cells to kanamycin resistance with the hybrid molecules, bacterial clones harboring recombinant plasmids were studied for the presence of globin-specific DNA. Plasmids containing either alpha or beta rabbit globin gene sequences were obtained. There was a 4-fold excess of recombinant plasmids containing beta-globin sequences over those with alpha-globin DNA. The longest beta-globin sequences found in plasmids were about 550 to 600 pairs long, and correspond therefore to the entire beta-globin structural gene and to some of the untranslated regions. The alpha-globin sequences were 400 to 450 base pairs long. Treatment of clone pCR1betarG 19 with EcoRI endonuclease released two DNA fragments (410 and 210 base pairs) resulting from cleavage at two reconstructed external EcoRI sites and at one internal EcoRI site within the rabbit globin gene. The same treatment of pCR1alpharG 11 released one fragment. In most other recombinant plasmids studied however, no fragment was released by EcoRI digestion.

Amino Acid Sequence↗