Biomedical subjects
B Hohn
Publications and source records attributed to B Hohn.
Isolation and complementation of mutants of Anabaena sp. strain PCC 7120 unable to grow aerobically on dinitrogen.
Mutants of Anabaena sp. strain PCC 7120 unable to grow aerobically on dinitrogen were isolated by mutagenesis with UV irradiation, followed by a period of incubation in yellow light and then by penicillin enrichment. A cosmid vector, pRL25C, containing replicons functional in Escherichia coli and in Anabaena species was constructed. DNA from wild-type Anabaena sp. strain PCC 7120 was partially digested with Sau3AI, and size-fractionated fragments about 40 kilobases (kb) in length were ligated into the phosphatase-treated unique BamHI site of pRL25C. A library of 1,054 cosmid clones was generated in E. coli DH1 bearing helper plasmid pDS4101. A derivative of conjugative plasmid RP-4 was transferred to this library by conjugation, and the library was replicated to lawns of mutant Anabaena strains with defects in the polysaccharide layer of the envelopes of the heterocysts. Mutant EF116 was complemented by five cosmids, three of which were subjected to detailed restriction mapping; a 2.8-kb fragment of DNA derived from one of the cosmids was found to complement EF116. Mutant EF113 was complemented by a single cosmid, which was also restriction mapped, and was shown to be complemented by a 4.8-kb fragment of DNA derived from this cosmid.
Mobilization of T-DNA from Agrobacterium to plant cells involves a protein that binds single-stranded DNA.
Crude protein extracts of induced and uninduced octopine wild-type strain of Agrobacterium tumefaciens, as well as several mutants of the virulence loci virA, -B, -G, -C, -D, and -E, were probed with single- and double-stranded synthetic oligodeoxynucleotides of different sequence and length in an electrophoretic retardation assay. Four complexes involving sequence-nonspecific, single-stranded-DNA-binding proteins were recognized. One inducible complex is determined by the virE locus, two Ti-plasmid-dependent complexes are constitutively expressed, and a fourth one is controlled by chromosomal genes. The protein-DNA complexes were characterized by sucrose density gradient centrifugation and by determination of the length of single-stranded DNA required for their formation. It is hypothesized that the single-stranded-DNA-binding proteins are involved in the production of T-DNA intermediates or have a carrier or protective function during T-DNA transfer.
[Preparatory technical modification and standardization of the Spalteholz microangiographic study method].
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A complete set of overlapping cosmid clones of M-ABA virus derived from nasopharyngeal carcinoma and its similarity to other Epstein-Barr virus isolates.
DNA of the transforming, nondefective Epstein-Barr virus (EBV) strain M-ABA, which is derived from nasopharyngeal carcinoma cells, was cloned as large overlapping pieces into the cosmid pHC79 . The termini were cloned from closed circular virus DNA molecules out of M-ABA cell DNA in phage lambda L47 . The large overlapping clones were used to prepare a library of subclones with inserts of 1-15 kb. A detailed restriction enzyme map of M-ABA virus DNA reveals the close similarity to isolates from other sources. The high number of tandem repeats in EBV DNA stresses the importance of using cloning vectors that can be propagated in recA- Escherichia coli hosts.
Selective isolation of cosmid clones by homologous recombination in Escherichia coli.
A procedure for selection of specific cosmid clones by homologous recombination between cosmid clones from a library and sequences cloned into a plasmid has been developed. Cosmid libraries constructed in a rec- host strain are packaged in vivo into lambda particles. Appropriate aliquots are then introduced into a rec+ host containing the sequence used for selection cloned into a plasmid vector without sequence homology to the cosmid vector. After a short time for recombination, the cosmids are packaged in vivo. Cosmids that have taken up the plasmid by homologous recombination are isolated by plating under conditions selecting for the antibiotic resistance markers carried by both vectors. The recombined cosmids can lose the inserted sequence by another homologous recombination event and, after packaging in vivo, these revertants can be identified on appropriate indicator plates.
Infrequent cleavage of cloned Anabaena variabilis DNA by restriction endonucleases from A. variabilis.
A cosmid vector has been constructed, using a lambda replicon. A library of cosmids from Anabaena variabilis ATCC 29413 based on use of this vector is shown to be highly deficient in sites for the two type II restriction endonucleases found in that organism.
DNA sequences necessary for packaging of bacteriophage lambda DNA.
The extent of DNA flanking the "cohered cohesive end" site of bacteriophage lambda DNA, which is required for packaging, was determined by using defined DNA fragments and a cosmid in vivo packaging assay. From the right end of lambda DNA a 20- to 36-base-pair stretch extending from the center of the cohered cohesive ends is shown to be required, whereas the packaging efficiency of cosmids extending to 70 base pairs into the left lambda arm is reduced to 10% (compared to a fragment extending until about 80 base pairs). A 60-base-pair stretch of the left arm leaves an efficiency of only 1%. The segment thus delineated, by the nature of the assay, is both necessary and sufficient for the binding of packaging proteins to the DNA, the packaging of DNA itself, the DNA cleavage, and successful injection of the DNA into a bacterial host. By contrast, in vitro packaging of restriction fragments of mature lambda DNA directly demonstrated the selectivity of the packaging proteins for the fragment originating from the left end of the DNA. The results of the two complementary experiments are discussed in terms of the various steps before, during, and after packaging for which different sequences flanking and including the cohered cohesive ends might be required.
[Rheumatoid arthritis, obliterating bronchiolitis and D-penicillamine. Apropos of a new case with ultrastructural examination and review of the literature].
Over recent years, several cases have been reported of bronchiolitis obliterans, of very rapid onset and serious clinical course in patients with rheumatoid arthritis being treated with D-Penicillamine. After reporting a new case, the authors stress the unusual features of this bronchiolar condition which is probably of iatrogenic origin and irreversible, with death occurring within a few years. The pathogenesis is poorly understood and many questions still remain concerning the relationships between rheumatoid arthritis, the bronchiolar lesions and D-Penicillamine in this disease of pulmonary collagen. The ultrastructural examination and the morphometric examination of this new case suggest the presence of predominantly type III collagen, which is normally present in the lungs in only small quantities.
DNA mediated transfer and fate of a proviral gene of mouse mammary tumor virus in cultured cells.
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Plasmid vectors for positive selection of DNA inserts controlled by the lambda pL promoter, repressor and antitermination function.
Hybrid plasmids consisting of pBR322 or pOP203-3 and the EcoRI-D fragment of lambda DNA kill their bacterial host upon expression of a lambda gene (probably the kil function) located either between or across the SalI sites. The plasmids from surviving hosts are acquired deletions that remove the lambda kil gene or insertions that block the transcription of the kil gene. Some plasmids probably carry point mutations. Based on these findings, we constructed two vector plasmids, pKL1 and pHA10, which can be used for a direct positive selection of cloned fragments. These plasmids are particularly useful for the cloning and selection of N-unresponsive termination signals using BamHI and its isoschizomers. The DNA fragments cloned into these plasmids are under control of the strong pL promoter, which can be regulated by the lambda repressor, and the antitermination activity of the N gene product.
Infectivities of native and cloned DNA of cauliflower mosaic virus.
Infectivity assays on turnips reveal that (i) cauliflower mosaic virus (CaMV) DNA, whether circular or linear, is as infectious as the complete virus; (ii) linear DNA obtained with restriction enzymes from the native CaMV DNA has the same specific infectivity as when first cloned in plasmid (pBR322) or bacteriophage (lambda gtWES) vectors and then restricted at the cloning site; (iii) in all cases studied mosaic symptoms are accompanied by virus production. DNA isolated from these viruses is again circular and possesses the three "gaps" characteristic of CaMV DNA. The cloned CaMV DNA, when linked to the vector DNA, is noninfectious or exhibits very low infectivity.
A small cosmid for efficient cloning of large DNA fragments.
The production and use of the 6 kb cosmid pHC79, a derivative of pBR322, is described. It can be used for cloning of fragments cleaved by EcoRI, ClaI, BamHI (also BglII, BclI, Sau3A and MboI), SalI (also XhoI and AvaI), EcaI and PstI. Hybrid cosmids containing inserts in the size range of 40 kb are packaged in vitro and transduced with an efficiency of 5 X 10(4) - 5 X 10(5) clones/microgram of insert DNA. Prefractionation of the DNA fragments to be cloned into 40 kb sized fragments ensures the cloning of contiguous stretches of DNA. Proteins produced in vitro by the cosmid pHC79 are identical to the ones produced by its pBR322 parent.
Isolation and characterization of the host protein groE involved in bacteriophage lambda assembly.
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In vitro packaging of lambda and cosmid DNA.
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Identification of a host protein necessary for bacteriophage morphogenesis (the groE gene product).
Mutations in the groE gene of Escherichia coli, which block the correct assembly of the phage lambda head, have been previously described. Many groE mutations exert pleiotropic effects, such as inability to propagate phages T4 and T5 and inability to form colonies at 43 degrees. With the help of the EcoRI and HindIII restrictionenzymes and the appropriate phage vectors, we have constructed two lambda transducing phages, called W3 and H18, that carry the groE+ bacterial gene. Upon lysogenization by phage H18 the groE bacterial mutants recover their gro+ phenotype for both phage growth and the ability to form colonies at 43 degrees. We have identified the groE+ bacterial gene product as a protein of 65,000 molecular weight. Mutants of the W3 transducing phage that were selected on the basis of their ability to propagate on some groE mutant hosts induce the synthesis of a groE protein with altered electrophoretic mobility.
DNA clones containing mouse immunoglobulin kappa chain genes isolated by in vitro packaging into phage lambda coats.
Endonuclease EcoRI-digested DNAs from BALB/c mouse embryos and MOPC 321 (a kappa chain secretor) myeloma were fractionated by agarose gel electrophoresis, and the DNA fragments containing part or all of the MOPC 321 kappa chain structural gene sequences were visualized by the Southern gel blotting technique using as the hybridization probes pCRI plasmids containing all or part of the enzymatically synthesized cDNA transcripts of the MOPC 321 kappa chain mRNA. The clear differences observed in the hybridization patterns of the two DNAs are in agreement with our previously reported results obtained with endonuclease BamHI and confirms that the sequence arrangement of kappa chain genes is different in the embryo and myeloma cells. We have cloned most of the kappa-sequence-positive EcoRI DNA fragments in Charon 4A phage by using the highly efficient in vitro phage lambda DNA packaging method, and we have characterized the cloned mouse DNA sequences by agarose gel blotting and R-loop mapping in electron microscopy. These studies identified, among others, one EcoRI DNA fragment which contains both variable and constant immunoglobulin kappa-gene sequences and is present only in the myeloma DNA. The two sequences are separated by a 2.8-kbase intron. We tentatively conclude that the kappa gene sequences on this DNA fragment underwent somatic rearrangement.
Cosmids: a type of plasmid gene-cloning vector that is packageable in vitro in bacteriophage lambda heads.
Evidence is presented that ColE1 hybrid plasmids carrying the cohesive-end site (cos) of lambda can be used as gene cloning vectors in conjunction with the lambda in vitro packaging system of Hohn and Murray [(1977) Proc. Natl. Acad. Sci. USA 74, 3259--3263]. Due to the requirement for a large DNA molecule for efficient packaging, there is a direct selection for hybrids carrying large sections of foreign DNA. The small vector plasmids do not contribute a large background in the transduced population, which is therefore markedly enriched for large hybrid plasmids (over 90%). The efficiency of the in vitro packaging system is on the order of 10(5) hybrid clones per microgram of foreign DNA for hybrids in the 20--30 million dalton range.