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Related post: Project No. ZOl AI 00006-09 LSD established plasmids are amplifiable with chloramphenicol. Because of the interest in streptococci as cloning vehicles, I plan to continue this work and hope to elucidate the plasmid transformation process. Part II Spiroplasma plasmids . Prior work with W. O. Mitchell, T. J. Popkin, and R. M. Cole (See Flutamide Tablets Projects ZOl AI 00007-06 LSD and ZOl AI 00004-20 LSD) was extended, confirmed, and published. Present data show that 12 Flutamide Hair Loss of 14 spiroplasmas carry from 1 to 8 cryptic plasmids per strain. Sizing by both electron microscopy and agarose gel electrophoresis (the latter using new standards) g showed a range of molecular weights between 1 x 10 and 26 x 10 daltons. Several size classes occurred in common in different spiroplasma strains. The amount of plasmid DNA per strain was as much as 12% of total cellular DNA, as determined by radioactive labelling. No such multiplicity of plasmids, with some in common among several strains, were previously known in any mycoplasmas, and their newly-recognized presence in spiroplasmas raises questions of their relation to phenotypic traits, that have yet to be answered. Preliminary studies showed that a non-helical "mutant" with a distinctive colonial morphology lacks several plasmids present in the parent strain, but trials of plasmid curing of the letter by use of intercalating agents to produce "mutant" colonies have not yet been successful. Future work will be directed to this and other approaches to determining the functions of spiroplasma plasmids, and to their detection and characterization in strains not yet examined. Streptococcal bacteriophages . Because of the paucity of current information. Dr. Moynet (in collaboration with A. colon-Whitt: ZOl AI 00005-15; See also ZOl AI 00004-20) initiated studies to characterize several phages. Nine phages, of which 3 are virulent and 6 temperate, from streptococci of serologic groups A, C, G, E, and H, are being compared. Seven are long-tailed polyhedral phages (Type B) as are most streptococcal phages, but 2 are short- tailed with accessory fibers. DNAs from all have been prepared, and are being analyzed directly for size, Flutamide 250 Mg after denaturation and renaturation for the presence of circular permutations, and after exonuclease III treatment for the presence of terminal repetitions of nucleotide sequences. Early results suggest smaller molecular size and absence of permutation in DNA of short- tailed phages. Other studies in progress include comparison of restriction enzyme cleavage patterns to determine regions of homology in the DNAs, and gel electrophoretic comparison of capsid proteins. Dr. Moynet has also continued physical mapping of the genome (a 24.1 Mdal linear ds-DNA) of Group H streptococcal phage 042. 26-29 Project No. ZOl AI 00006-0 9 LSD Because of the previously shown presence of circular permutation, the map — when completed — will be circular. By the use of single and double digests with restriction endonucleases (chiefly Sal I and Pvu II) , followed by ordering and sizing of resultant fragments by agarose gel electrophoresis, and of ^treatments with large fragment Polymerase I in the presence of [ P] -thymidine triphosphate, it was shown that ^42 DNA possesses 2 Sal I sites that encompass a Pvu II site. One of these now- identified Sal I sites has been chosen arbitrarily as the origin, and construction of the map will proceed. Hemophilus bacteriophage Physical mapping of the genome of 0N3 of H. influenzae was partially completed in 1977-78 (E. Jablonska, Visiting Fellow: See Project No. ZOl AI 0006-7 LSD) , but ambiguities existed among small fragments, in the middle of the genome, generated by restriction endonucleases EcoRl, Xhol, and Xbal. Resolution of this problem is being approached by Dr. Moynet and Dr. Jablonska (as a short-term Guest Worker) by filter hybridization of EcoRl and Xhol fragments with Xbal fragments, and is expected to lead shortly to completion of the map. Publications Ranhand, J. M. : DNA binding and uptake by competent Streptococcus sanguis Wicky cells. In Glover, S., and Butler, L. O. (eds.). Proc. of the 4th European Meeting on Transformation and Trans- fection. Cotswold Press, Oxford, 1979, pp. 60-72. Ranhand, J. M. : Interactions of competent Streptococcus sanguis (Wicky) cells with native or denatured, homologous or heterologous deoxyribonucleic acids. J. Bacteriol. 142: 568-580, 1980. Ranhand, J. M. , Mitchell, W. O. , Popkin, T. J., and Cole, R, M. : Covalently-closed circular Deoxyribonucleic acids in spiroplasmas . J. Bacteriol. (In press: Vol. 143, Sept. 1980) 26-30 SMITHSONIAN SCIENCE INFORMATION EXCHANGE PROJECT NUMBER (Oo NOT use this space} PERIOD COVERED October 1, 197 9 to September 3 0,198 TITLE OF PROJECT (80 characters or less) Viruses of Spiroplasmas and Mycoplasmas PI: Other; U.S. DEPARTMENT OF HEALTH, EDUCATION, AND WELFARE PUBLIC HEALTH SERVICE NOTICE Buy Flutamide OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl AI 00007-05 LSD
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