On the identity of dnaP and dnaF genes of Bacillus subtilis

Summary The dnaP strains of Bacillus subtilis are altered in the initiation of DNA replication at high temperature (Riva et al., 1975). Fine mapping of the gene shows that it is located very close to the dnaF gene, described by Karamata and Gross (1970) and mapped by Love et al. (1976) in the polC region. The phenotype of both mutants is indistinguishable: the DNA synthesis stops at non permissive temperature after synthesizing an amount of DNA equivalent to the completion of the rounds of replication already initiated; at permissive temperature they are abnormally sensitive to MMS and are reduced in the ability to be transformed. Both mutants are to be considered as belonging to the dnaF locus.The dnaF gene is very close to the polC gene, which specifies the DNA polymerase III of B. subtilis. The DNA polymerase III of the dnaF mutants is not temperature sensitive in vitro, however, the level of this enzyme is lower by a factor of 4 or 5 in the dnaF mutants, at the permissive temperature. Following shift of dnaF cultures to the non permissive temperature, the level of DNA polymerase III activity specifically decreases further by a factor of at least 10 in the mutant, whereas the DNA polymerase I level is unaffected.The possible roles of the dnaF gene in the control of the cellular level of the DNA polymerase III, and the possibility of a regulatory role of DNA polymerase III in the initiation of DNA replication in bacteria are discussed.Abbreviations and symbols HPUra 6-(p-hydroxyphenylazo)-uracil; mic, minimum inhibitory concentration - MMS methyl-methanesufonate - Pol I Pol II and Pol III: DNA polymerase I, II and III respectively - PCMB parachloro-mercuri-benzoate     

An electron microscope study of the relative positions of the 4S and ribosomal RNA genes in HeLa cell mitochondrial DNA

The 4S RNA genes in HeLa mitochondrial DNA (mtDNA) have been mapped by electron microscopy using the electron-opaque label ferritin. This method is based on the high affinity interaction between the protein, avidin, and biotin. 4S RNA, covalently coupled to biotin, was hybridized to single-stranded mtDNA. The hybrids were then labeled with ferritin-avidin conjugates. The positions of ferritin-labeled 4S RNA genes were determined relative to the rRNA genes on both heavy (H) and light (L) strands of mtDNA. This region was recognized as a duplex segment after hybridization either with rRNA in the case of H strands or with DNA complementary to rRNA in the case of L strands.Our studies suggest that at least nineteen 4S RNA genes are present in the HeLa mitochondrial genome. On the H strand, we have confirmed the nine map positions found in a previous electron microscope mapping study (Wu et al., 1972) and obtained evidence for three additional 4S RNA genes. On the L strand, seven 4S RNA genes have been mapped. The nineteen genes are distributed more or less uniformly around the genome. There is a pair of closely spaced genes, approximately 150 nucleotides apart, on the H strand, and another closely spaced pair on the L strand.     

On the fungitoxicity of some new thiocyanatopyrazole derivatives: Electron microscopical study in trichophyton mentagrophytes

Four thiocyanatopyrazole derivatives were synthesized and their fungistatic activity was demonstrated in vitro against a number of dermatophytic fungi. In Trichophyton mentagrophytes, the most active compound induced an unusual increase of the plasma membrane with production of intra and extracytoplasmic complexes, a deterioration of nuclear and mitochondrial membranes and a formation of autophagic-like vacuoles. Plasmolysis, accompanied by an almost complete disorganization of cytoplasmic structures, seemed to be the final event. A possible mechanism of action of the compounds was discussed.Investigation supported by a grant from Consiglio Nazionale delle Ricerche of Italy (Contract No. 7500536).     

Chromosome constitution of in vitro segregated haploid and diploid cells of the mouse

The composition in segregated haploid sets of paternal and maternal chromosomes has been studied in order to verify whether their composition is uniparental of mixed, fixed or variable. Primary cultures where prepared using kidneys from hybrids of strains of Mus musculus in which the parental chromosomes are distinguishable; the maternal set consists of 20 teleocentric chromosomes, the paternal set of 9 metacentric chromosomes, derived by Robertsonian fusion and 2 telocentrics. Applying Seabright's banding technique, an analysis of segregated haploid and diploid cells, which have originated spontaneously through polyploidisation-segregation processes was carried out. It was concluded that the haploid sets have a variable composition of paternal and maternal chromosomes.     

Fluorogenic detection of primary amines in plant histochemistry with fluorescamine: A comparative study on the effects of coagulant and non-coagulant fixatives

Summary The new highly sensitive method of fluorescamine reaction for the topochemical detection of primary amino groups was studied as a substitude of ninhydrin-Schiff's reaction for the localisation of total proteins in plant tissues. The influence of various coagulant and non-coagulatn fixatives on the induction of fluorescamine fluorescence was examined: ethanol, formaldehyde gas and solution, glutaraldehyde, acrolein, osmium tetroxide, Bouin, Rossman, Clarke and Zenker's fluids and FMA were employed. It was found that the use of the fluorogenic method is conditioned by the fixative ability to keep the amino groups disposable and by its capability to reduce the natural autofluorescence of plant material. A detailed account of the fixation methodology demonstrated that non-coagulant acrolein and coagulant mercuric chloride are the most promising fixatives for the use of the fluorescamine reaction in plant histochemistry.     

Phenyl-substituted aminomethylene-bisphosphonates inhibit human P5C reductase and show antiproliferative activity against proline-hyperproducing tumour cells

In certain cancers, such as breast, prostate and some lung and skin cancers, the gene for the enzyme catalysing the second and last step in proline synthesis, δ¹-pyrroline-5-carboxylate (P5C) reductase, has been found upregulated. This leads to a higher proline content that exacerbates the effects of the so-called proline-P5C cycle, with tumour cells effectively using this method to increase cell survival. If a method of reducing or inhibiting P5C reductase could be discovered, it would provide new means of treating cancer. To address this point, the effect of some phenyl-substituted derivatives of aminomethylene-bisphosphonic acid, previously found to interfere with the catalytic activity of plant and bacterial P5C reductases, was evaluated in vitro on the human isoform 1 (PYCR1), expressed in E. coli and affinity purified. The 3.5-dibromophenyl- and 3.5-dichlorophenyl-derivatives showed a remarkable effectiveness, with IC₅₀ values lower than 1 µM and a mechanism of competitive type against both P5C and NADPH. The actual occurrence in vivo of enzyme inhibition was assessed on myelogenous erythroleukemic K562 and epithelial breast cancer MDA-MB-231 cell lines, whose growth was progressively impaired by concentrations of the dibromo derivative ranging from 10⁻⁶ to 10⁻⁴M. Interestingly, growth inhibition was not relieved by the exogenous supply of proline, suggesting that the effect relies on the interference with the proline-P5C cycle, and not on proline starvation.     

Biogenesis of linear O-alkylfuranocoumarins: A new pathway involving 5-hydroxymarmesin

adioactivity from [³H] 5-hydroxymarmesin was incorporated into 5-methoxypsoralen by administration to leaves of Ficus carica and cut ends of Ruta graveolens. No other furanocoumarins were labelled. Trapping experiments, in which [³H]marmesin together with 5-hydroxymarmesin was administered to fig leaves and to cut ends of rue, provided good evidence that 5-hydroxymarmesin is formed by hydroxylation of marmesin. These results, together with those obtained previously with 8-hydroxymarmesin demonstrate that, in addition to the pathway which involves the hydroxylation of psoralen, the O-alkylfuranocoumarins are also formed by a pathway which involves the hydroxylation of marmesin.