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Biomedical subjects

B Mach

Publications and source records attributed to B Mach.

At least 109 records · Page 6Linked to original sources
PubMed1983

The absence of beta 2-microglobulin in Daudi cells: active gene but inactive messenger RNA.

The Daudi cell line is characterized by an absence of HLA antigen on its surface. This has been attributed to a lack of beta 2-microglobulin (beta 2m) while the heavy chain of HLA is present intracellularly. Karyotype analysis of Daudi cells has shown a deletion involving one of the beta 2-microglobulin alleles. It was generally believed that the absence of beta 2-microglobulin in that cell line resulted from an absence of expression of the remaining gene. We report here the unexpected finding of a normal amount of beta 2-microglobulin messenger RNA in Daudi cells. This was demonstrated by "Northern blot" hybridization with cDNA plasmid clones as a probe. This mRNA, however, when purified by hybridization-selection with beta 2-microglobulin plasmid DNA, is unable to function as messenger in protein synthesis and is therefore an inactive mRNA. The finding of a translationally inactive beta 2-microglobulin mRNA provides a new explanation for the absence of beta 2-microglobulin and therefore of HLA antigens in Daudi cells.

Animals↗
PubMed1983

Molecular analysis of the genes for human class II antigens of the major histocompatibility complex.

Different cDNA clones have been isolated that encode each of the three chains of HLA-DR antigens: alpha, intermediate and beta, as well as another beta chain, most likely DC. Whereas the DR alpha and intermediate chains seem encoded by single genes, the DR and DC beta chains are most likely encoded by multiple genes; furthermore, their polymorphism can be readily detected by restriction analysis of cellular DNA. Several genomic DNA clones were isolated for the DR and DC beta chain genes and for the intermediate chain gene. The sum of all distinct cDNA clones and genomic DNA clones for HLA-DR beta chains, isolated from a heterozygous cell line, represent five genes. This implies the existence of at least three nonallelic DR beta chain genes in addition to the DC beta chain genes. The complete sequence of one of the DR beta chains is presented. A genomic DNA clone for a DR beta chain was transferred into mouse L cells and found to be expressed into RNA of the same size as DR beta mRNA. The finding, among the genes for class II antigens, of multiple genes for the beta chain of HLA-DR, distinct from those of other known subregions such as DC, emphasizes the importance of gene transfer experiments, where individual genes can be expressed and tested for their functional role in the immune response.

Alleles↗
PubMed1983

Isolation of cDNA clones for the p33 invariant chain associated with HLA-DR antigens.

HLA-DR antigens are polymorphic cell surface glycoproteins involved in the control of the immune response in man. They consist of two subunits, the alpha and the beta chains. In addition, an invariant glycoprotein of Mr 33,000 (DRp33) is associated intracellularly with HLA-DR antigens. A cDNA clone for DRp33, called 33-10, was isolated. Because no amino acid sequence has yet been determined for DRp33 the identification of cDNA clone 33-10 was based on selection of mRNA by hybridization, subsequent translation in a rabbit reticulocyte lysate supplemented with microsomes, and translation in microinjected Xenopus oocytes followed by immunoprecipitation with an anti-DR antiserum. The translation products assembled with DR alpha and beta chains in oocytes coinjected with all three mRNAs. Assembly of DR alpha and beta chains was also observed in the absence of DRp33 mRNA. Furthermore, when compared with DRp33 immunoprecipitated from a human B-cell line, translation products of the hybrid-selected mRNA showed (i) identical migration in two-dimensional gel electrophoresis, (ii) identical apparent molecular weight in the absence of N-linked glycosylation, and (iii) a very similar two-dimensional peptide map. Transcription of the DRp33 gene into a mRNA 1,400 nucleotides long was observed in B cells but was undetectable in T-cell lines and was very low in liver. Thus, DRp33 appears to be coordinately expressed with DR alpha and beta chains. Hybridization to DNA of mouse-human somatic cell hybrids showed that DRp33 is encoded by a gene that is located outside the major histocompatibility complex.

Animals↗
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

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↗
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↗
PubMed1977

Cloning and amplification of alpha and beta mouse globin gene sequences synthesised in vitro.

New chimeric Escherichia coli plasmids containing alpha or beta globin gene sequences of the mouse were constructed. Double-stranded DNA, synthesised in vitro in a 2-step reaction from mouse globin mRNA was inserted into E. coli plasmid pCR1, after tailing of the 2 DNAs with dG and dC respectively. Some of the mouse globin plasmids described contain at least 90% of the globin mRNA sequence and therefore contain the entire translated sequence of the globin genes. Some possible uses of these recombinant plasmids are described.

Animals↗