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Biomedical subjects

B Mach

Publications and source records attributed to B Mach.

At least 163 records · Page 9Linked to original sources
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↗
PubMed1976

Stepwise biosynthesis in vitro of globin genes from globin mRNA by DNA polymerase of avian myeloblastosis virus.

Two approaches have been explored for the synthesis of double-stranded DNA from single-stranded DNA template complementary to rabbit 9S globin mRNA (cDNA). (i) cDNA was elongated with dCMP or dTMP homopolymeric tracts using terminal deoxynucleotidyltransferase (EC 2.7.7.31; nucleosidetriphosphate:DNA deoxynucleotidylexotransferase). cDNA-dC, in the presence of an oligo(dG)10 primer, was an efficient template with either DNA polymerase of Escherichia coli (EC 2.7.7.7; deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase) or RNA-directed DNA polymerase of avian myeloblastosis virus. cDNA-dT [ with an oligo(dA)10 primer] functioned as template only with E. coli polymerase. (ii) cDNA, without homopolymeric tails, was also efficiently copied in the absence of oligonucleotide primer, by DNA polymerase of avian myeloblastosis virus or of E. coli. The product of the reaction consisted of long hairpin molecules which could be converted into DNA duplex (melting temperature, 93 degrees) by digestion with single-strand nuclease S1. The data indicate that a loop structure on the 3' end of cDNA allowed DNA synthesis to take place by a "self-priming" mechanism. Some of the double-stranded DNA synthesized corresponded to the entire sequence of the 9S mRNA template. The synthesis of full-length double-stranded DNA from mouse globin mRNA and immunoglobulin light chain mRNA is also discussed.

Avian Myeloblastosis Virus↗
PubMed1976

No detectable reiteration of genes coding for mouse MOPC 41 immunoglobulin light-chain mRNA.

RNA fractions rich in immunoglobulin light (L)-chain mRNA were isolated from mouse myeloma MOPC 41 by procedures previously described, and chemically labeled with 125I. These RNA fractions were hybridized with MOPC 41 DNA under conditions of DNA excess. Hybridization conditions were chosen under which the entire sequence of the L-chain mRNA probe, thus including the variable region, remains available for hybridization throughout the reaction. The hybridization (C0t) curve showed double transition kinetics, with one component corresponding to about 250 gene copies and the other to about two to four copies. In contrast, when MOPC 41 L-chain mRNA was further purified as a single band by gel elecptrophoresis in 99% formamide, the hybridization curve showed only a single transition, corresponding to about two to four genes, with the disappearance of the "reiterated" component. That component resulted therefore from contaminating RNA species. The data indicate that no reiteration can be detected by RNase or by hydroxylapatite for the genes corresponding to the entire sequence of MOPC 41 L-chain mRNA, including the untranslated segments, within the limits of detectability of short reiterated segments. It thus appears that there is only one or very few genes corresponding to the 41 L-chain variable region "subgroup" in MOPC 41 DNA. The possibility that the variable genes of plasmocytes might result frm a combination of several nonreiterated germline genes is discussed.

Alleles↗
PubMed1975

Insertion of a rabbit beta-globin gene sequence into an E. coli plasmid.

Double stranded DNA has been synthesized in vitro from rabbit globin messenger RNA and elongated with homopolymeric dG tails. An E. coli plasmid was cleaved by EcoRI. The cohesive ends were repaired and dC tails added, to permit reconstitution of the EcoRI sites upon annealing with the dG elongated globin DNA. Transformation of E. coli with the globin-plasmid DNA hybrid has yielded a clone which harbours a recombinant plasmid (pCR1-betaG1), as demonstrated by hybridization experiments with radioactive globin cDNA. The sequence carried by the recombinant plasmid corresponds to part of the gene sequence coding for the beta chain of rabbit globin. Circular DNA of the purified recombinant plasmid exhibits sensitivity to EcoRI.

Animals↗
PubMed1974

Estimation of the number of genes coding for the constant part of the mouse immunoglobulin kappa light chain.

As previously shown, purified 14S RNA of mouse myeloma MOPC-41 forms a single peak on sucrose gradients and gel electrophoresis and codes for a single polypeptide chain, the immunoglobulin light chain produced by the same myeloma in vivo. This 14S mRNA was used for the enzymatic synthesis of DNA (cDNA) which is complementary to the RNA template. A DNA fraction was isolated which has an average size of 300 nucleotides. Kinetic studies of the hybridization of the 14S RNA with the cDNA indicate that about 40% of the RNA consists of a single RNA sequence. From the size of the cDNA fraction and from the direction of DNA synthesis, it can be concluded that the cDNA includes the sequence complementary to the constant region of light chain mRNA. This radioactive cDNA was used for DNA.DNA reannealing experiments with unlabeled DNA from mouse liver or myeloma tumor in 3 x 10(6)-fold excess. This allowed the determination of the number of copies in the mouse genome of those sequences represented in the cDNA. The data show no significant reiteration in either liver or myeloma DNA and suggest that the gene coding for the constant part of immunoglobins is present in the haploid genome in one to five copies. Furthermore, this gene is not "amplified" in nuclear DNA of myeloma plasmocytes.

Animals↗