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.
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.
Packaging recombinant DNA molecules into bacteriophage particles in vitro.
Recombinant phage genomes made in reactions with purified enzymes may be recovered directly by packaging into phage heads in vitro. The process is efficient and nonselective and offers containment in initial stages of handling recombinant DNA. Ligase [poly(deoxyribonucleotide):poly-(deoxyribonucleotide) ligase (AMP-forming), EC 6.5.1.1] reaction products can recombine with endogenous phage DNA during packaging, but UV-irradiation eliminates the biological activity of the endogenous DNA.
Capsid transformation during packaging of bacteriophage lambdaDNA.
Assembly pathways of complex viruses might not be simple additions of one protein after another with rigid tertiary structure. It might in fact involve shifts in subunit structure, movement of subunits relative to each other to form new arrangements, transient action of proteins and protein segments, involvement of structure forming 'microenvironments' of the host. Thus morphogenesis of the bacteriophage lambda head starts with the formation of a core-containing DNA-free petit lambda particle. In a first transition, and dependent on a host function, the core is released, minor protein components of the capsid are processed and the particle's structure is altered, as shown by a change of its hydrodynamic properties. The resulting 'prehead' undergoes a second transition triggered by a complex of DNA and recognition protein (A-protein). This transition is more drastic than the first one. The particle doubles its volume without increasing in protein mass, the shell becomes thinner, and the surface structure is changed. Concomitantly with this process, the DNA becomes packaged and the particle becomes able to bind the small 'D-protein' in amounts equimolar to the capsid protein, which it could not do before. The D-protein addition probably causes another shift of the capsid structure. DNA packaging is completed, and the DNA is cut from concatemeric precursors to unit length molecules. Binding sites are created for the tail connector molecules which in turn allow the independently assembled tail to attach. Research on these processes proceeds along several lines: comparison of physical and chemical properties of particles accumulating in mutants; pulse-chase experiments on assembly precursors; morphogenesis in vitro; and model transitions of aberrant lambda polyheads.
Functional empty capsid precursors produced by lambda mutant defective for late lambda DNA replication.
This report described lambda phage morphogenesis in a mutant system in which the normal pathways for late phage DNA (concatemer) synthesis are blocked and early (monomeric circular) DNA replication products accumulate. As shown earlier (Dawson et al., 1975) under these conditions, late proteins are synthesized and assembled into headlike structures. These structures that accumulate in the mutant are empty, suggesting the monomeric circular DNA molecules cannot be encapsulated. The present results show that crude extracts of induced lysogens of the mutant contain the complementation activities of preheads (the empty precursors to DNA-filled heads), tails, and DNA terminigenerating protein(s). Sucrose gradients of these crude extracts yield fractions containing prehead activity in relative amounts expected from the concentration of late proteins and empty structures. Furthermore, the proteins present in these fractions coelectrophorese with the known capsid proteins of preheads, and empty structures that look like preheads are observed in electron microscope examination of samples from the fractions. Based on our biological, biochemical, and electron microscope analyses, we conclude that the empty structures that accumulate in the induced lysogen of the mutant are normal preheads, which could become filled phage heads if DNA of the appropriate structure (i.e., "late DNA") were available.
[Respiratory function in chronic obstructions of the large pulmonary arteries (study of 30 cases].
If diagnosis of chronic thromboses of pulmonary arteries is usually easy at the stage of confirmed chronic pulmonary heart, it is not so when dysponea is the only symptom of the disease. The authors report 30 cases of chronic thrombosis and remark that if respiratory alkalosis is very frequent, hipoxemia is often missing. On the other hand they observe 27 times out of 28 an increase of CO2 alveoloarterial difference; measuring the CO2 difference appears a safe and reliable test for detecting chronic thrombosis. It should take place in every systematic complete examination of unexplained dyspnoea.
DNA as substrate for packaging into bacteriophage lambda, in vitro.
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Petit lambda, a family of particles from coliphage lambda infected cells.
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[Value of the measurement of ventilatory and circulatory components in CO2 ductance].
Authors bring forth the results of the determination of CO2 ductances in 28 patients : 14 cases of pulmonary arterial thrombosis, 7 cases of diffuse interstitial fibrosis recognizing various etiologies, 4 cases of endothoracic sarcoidosis and 3 cases of pure restrictive syndrome due to functional amputation of some territories. The global CO2 ductance is lowered in 24 cases. In each one of these cases the PACO2 determination makes possible to decompose the global ductance into a ventilatory ductance and a circulatory ductance. The circulatory ductance is lowered in the case of pulmonary arterial thrombosis; the ventilatory component is lowered in the case of restrictive syndromes and in the case of diffuse interstitial fibrosis. For what concerns the pulmonary arterial thrombosis, authors discuss then the value of the information contained in the expression of the CO2 arterial-alveolar gradient on the one hand, and in the circulatory component of the ductance on the other hand. At last, the determination of the ductances appears interesting in the supervision of the course of various diseases.
[Value of ventilatory assistance through the mouth in readaptation of patients with chronic respiratory insufficiency. Report of 20 cases (author's transl)].
Twenty patients with severe and chronic respiratory insufficiency (SaO2 less than or equal to 80% - PaCO2 greater than or equal to 50 mmHg) were treated with intermittent ventilatory assistance. They used volumetric apparatus 2 to 8 hours a day, during at least 6 months (22 months +/- 13). Respiratory status never improved strikingly, on the contrary most of the time it worsened and patients needed other ventilatory technics and/or died (11 of 20). This technic of ventilatory assistance does not seem to really change the prognosis of these patients in a severe and chronic state.
[Chronic thrombosis of the major pulmonary arterial trunks. Apropos of 30 cases].
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