Transformation of distinct mycobacterial species by shuttle vectors derived from theMycobacterium fortuitum pAL5000 plasmid

Mycobacterium tuberculosis H37Ra,M. smegmatisATCC 607,M. smegmatis MC²155,M. aurum A +,M. aurum A11, and one representative strain ofM. flavescens were transformed by electroporation with plasmid pMY10 and cosmid pDC100. Plasmid pMY 10 contained the origin of replication of pAL5000, the origin of replication of pBR322, a kanamycin resistance gene, and the origin of transfer of the Inc plasmid RK2; the cosmid pDC100 contained the pHC79 SS cosmid, the origin of replication of pAL5000, and a kanamycin resistance gene. The efficiency of transformation varied with the recipient cells used and was in decreasing order: 7×10⁵ forM. smegmatis MC²155, 6×10³ forM. tuberculosis H37Ra, 10³ forM. aurum, 50 forM. smegmatis ATCC 607, and 5 forM. flavescens. A rapid protocol for plasmid extraction from mycobacteria was developed.The satisfactory transformation of the nonvirulentM. tuberculosis strain H37Ra was of interest for future studies on cloning of virulence genes, while the satisfactory transformation ofM. aurum was of interest for future studies on the genetics of drug resistance because these bacteria are sensitive to drugs specifically used in the treatment of tuberculosis and leprosy. However, neither vector was stably maintained inM. smegmatis, indicating that further investigations are still necessary to resolve this difficulty.     

A chloramphenicol-streptomycin-resistance plasmid from a clinical strain of Staphylococcus sciuri and its structural relationships to other staphylococcal resistance plasmids

A 5.1-kb plasmid, designated pSCS12, isolated from a naturally occurring Staphylococcus sciuri conferred resistance to chloramphenicol (CmR) and streptomycin (SmR). Restriction endonuclease analyses of pSCS12 revealed partial structural homologies to the CmR-plasmids pC221 from S. aureus and pSCS1 from S. intermedius, to the SmR-plasmids pSAI-1 from S. hyicus and pS194 from S. aureus, as well as to the CmR/SmR plasmid pSK68 from S. aureus. Southern-blot hybridization with specific CmR- and SmR-gene probes confirmed these similarities and allowed the mapping of the CmR- and SmR-determinants in the S. sciuri plasmid pSCS12. These observations lead to the suggestion that CmR/SmR-plasmids, such as pSCS12, may have evolved from CmR- and SmR-plasmids by interplasmidic recombination.     

Polyester Wadding for Specimen Orientation During Embedding in Methacrylates

Polyester fibers are not dissolved by either glycol methacrylate or methyl methacrylate. Commercial polyester wadding is consequently an advantageous material to use in getting precise orientation of tissue specimens during embedding in methacrylate.     

Permanent Preservation of Whole Alizarin Red S Skeletons by Clearing and Embedding in Polyester Resins

A one-step clearing and embedding procedure for alizarin red S stained skeletons is described. Embryos are fixed in formalin, skinned and eviscerated. After staining in a 10 mg/liter solution of alizarin red S in 5% aqueous KOH, specimens are dehydrated in a graded series of acetone-polyester monomer solutions. Finally, the specimens are embedded at room temperature in the polyester resin. A special reusable metallic mold is described for embedment of large fetuses. Specimens previously cleared in glycerol can be processed with this method.     

Characterization of mitoses with sister chromatid differentiation (SCD) and consequences for the analysis of proliferation kinetics and sister chromatid exchanges in asynchronously growing cells

Summary Labeling cells with bromodeoxyuridine (BrdU) permits the differentiation of mitoses of the first, second, and third generation after the addition of BrdU. The term second mitoses is used for those cells which have incorporated BrdU for two-S-phases and which exhibit sister chromatid differentiation (SCD). However, SCD can also be obtained if the cell was in S-phase at the time of BrdU-addition and had already replicated part of its DNA. Such cells with incomplete BrdU-substitution in the first S-phase can only be differentiated from completely substituted ones by the quality of the SCD and are usually also grouped as second mitoses in the evaluation of experiments. Due to the heterogeneity of the evaluated second mitoses, the determination of proliferation delay and the incidence of sister chromatid exchange-induction can depend on the time of chromosome preparation.     

Thylakoid differentiation in endocyanelles

Cyanophora paradoxa Korshikov synchronized autotrophically in a light-dark regime of 14 h light and 10 h dark divides in the last two hours of the dark period. The division rate of the free-living blue-green alga, Synechococcus leopoliensis Raciborski, at identical culture conditions (24°C; 32 W m⁻²) is only slightly lowered in the light period. The comparison of thylakoid differentiation in the endocyanelles of Cyanophora paradoxa and in Synechococcus leopoliensis during the light-dark regime yields (1) the same ensemble of pigment-protein complexes in both organisms, (2) comparable syntheses of chlorophyll and phycobilins of Cyanophora paradoxa grown under 32 W m⁻² and of Synechococcus leopoliensis grown under light intensities below 9.2 W m⁻², and (3) identical photosynthetic oxygen evolution during the light period of the light-dark regime with minima at the beginning, in the middle (6th–7th h), and at the end of the light period. In both organisms this stage-specific oxygen evolution is inhibited by treatment with chloroamphenicol. Cycloheximide, however, causes no significant alterations. Results are discussed in view of the endosymbiotic theory.