Genetic recombination in mutants of the anthracycline-producer Streptomyces griseus

It was found that genetic recombination occurs if two marked strains of Streptomyces griseus (leukaemomycin-producing strains IMET JA 3933 and IMET JA 5142) are grown together in mixed cultures on semisolid media. The crossing techniques used and the method for carrying out selective analysis were essentially the same as those described by HOPWOOD (1967, 1972). The parent strains used for crosses were marked with single or double nutritional requirements and with mutations for drug resistance. The crosses are quite self-sterile, yielding only in one combination stable prototrophic recombinants at a low frequency (10(-5) to 10(-6)). The majority of recombinants behaved as stable haploid genotypes. A series of four-point crosses of different types of auxotrophs was carried out. The results of these experiments do not provide sufficient data for constructing a chromosome map, but provide basic information on the possibilities of genetic analysis of the production of anthracycline antibiotics. The majority of crosses performed were not fertile at 28 degrees C but, surprisingly, in some crosses carried out at 34 degrees C viable colonies were detected on minimal media at frequencies from 10(-3) to 10(-2).     

Flow cytogenetics of uncloned and cloned Chinese hamster cells

Flow cytometry has greatly facilitated the routine use of DNA content as a cellular indicator of the stages of the cell cycle and ploidy. DNA content can also be used to distinguish individual chromosomes. Fluorescent staining of chromosome DNA was done with a combination of ethidium bromide and mithramycin in hypotonic solution. Subsequent detergent treatment of the cells with Triton X-100 facilitated chromosome isolation. DNA flow cytometry of chromosomes of four established uncloned Chinese master cell lines showed 10 to 12 major subpopulations of chromosomes with varying degrees of overlap in the range of low and intermediate DNA content. Cloning of B14F28 cells, the line with the largest heterogeneity in chromosome number and DNA content, considerably reduced the dispersion in chromosome number and improved the resolution of DNA content distributions. Thus, cloned cells with a relatively homogeneous karyotype permit better discrimination of chromosome subpopulations by DNA content than uncloned cells and provide a more sensitive system to study mutagenic effects.     

Studies on Possible Mechanisms of Early Functional Compensatory Adaptation in the Remaining Kidney

Two to 4 hours after unilateral renal exclusion in rats, urine flow rate from the remaining kidney had increased to twice the control level, whereas the filtration rate remained unchanged. After contralateral nephrectomy, NGFR was similar to that of controls, but fractional water reabsorption along proximal tubules decreased. Protein concentration in efferent arteriolar plasma, and hydrostatic pressure gradient between proximal tubules and peritubular capillaries were similar in experimental and control kidneys. Unilateral renal exclusion was followed by a rapid increase of blood pressure. Prevention of this rise depressed but did not abolish functional compensatory adaptation. The occurrence of compensatory adaptation was not affected by decreased renal perfusion pressure.     

Characterization of exoglucanase and synergistic hydrolysis of cellulose in Clostridium stercorarium

Abstract A cellobiohydrolase component was isolated from an anaerobic thermophilic cellulolytic bacterium, Clostridium stercorarium . When acting alone, the enzyme showed minimal activity towards ordered substrates such as cellulose and filter paper but it has been shown to attack phosphoric-acid swollen cellulose giving cellobiose as principal product. When recombined with endoglucanase it did allow an extensive hydrolysis demonstrating a marked synergism in the action of those two components; the addition of β-glucosidase resulted in a further increase in activity.     

Influence of dicarboxylic phosphatidylcholines on phosphatidylcholine liposomes as revealed by gel chromatography and electron microscopy

The effect of dicarboxylic phosphatidylcholines (glutarylphosphatidylcholine) on the structural changes of phosphatidylcholine liposomes is examined by using multilamellar liposomes prepared with egg phosphatidylcholine or dipalmitoylphosphatidylcholine and by varying the surface charge by addition of dicetyl phosphate. Investigations are performed by gel chromatography and electron microscopy. Glutarylphosphatidylcholine is in micellar form (rod-like micelles or globular micelles). The structures obtained depend on the fatty acid saturation of liposomes and on the charge of liposome (addition or not of dicetyl phosphate). With egg phosphatidylcholine/glutarylphosphatidylcholine dispersions, an aspect more similar to myelinic figures than liposomes is observed, while in the presence of dicetyl phosphate, liposomes similar to control egg phosphatidylcholine liposomes are obtained. Gel chromatography on Sepharose 4B and turbidity measurements prove that dicetyl phosphate increases the stability of egg phosphatidylcholine/glutarylphosphatidylcholine mixtures. On the other hand, in dipalmitoylphosphatidylcholine/glutarylphosphatidylcholine dispersions, incorporation of dicetyl phosphate destabilizes bilayer structure and the formation of mixed micelles occurs. Viscosity measurement shows, in the presence of dicetyl phosphate, an increased fluidity for dipalmitoylphosphatidylcholine/glutarylphosphatidylcholine dispersions, in agreement with the micellar organization. These data confirm that the disorganization of liposomal membranes by dicarboxylic phosphatidylcholine depends on the fatty acid composition of phosphatidylcholine and on the presence of dicetyl phosphate.     

Transfer RNA genes from Dictyostelium discoideum are frequently associated with repetitive elements and contain consensus boxes in their 5' and 3'-flanking regions.

A total of 68 different tRNA genes from the cellular slime mold Dictyostelium discoideum have been isolated and characterized. Although these tRNA genes show features common to typical nuclear tRNA genes from other organisms, several unique characteristics are apparent: (1) the 5'-proximal flanking region is very similar for most of the tRNA genes; (2) more than 80% of the tRNA genes contain an "ex-B motif" within their 3'-flanking region, which strongly resembles characteristics of the consensus sequence of a T-stem/T-loop region (B-box) of a tRNA gene; (3) probably more than 50% of the tRNA genes in certain D. discoideum strains are associated with a retrotransposon, termed DRE (Dictyostelium repetitive element), or with a transposon, termed Tdd-3 (Transposon Dictyostelium discoideum). DRE always occurs 50 (+/- 3) nucleotides upstream and Tdd-3 always occurs 100 (+/- 20) nucleotides downstream from the tRNA gene. D. discoideum tRNA genes are organized in multicopy gene families consisting of 5 to 20 individual genes. Members of a particular gene family are identical within the mature tRNA coding region while flanking sequences are idiosyncratic.     

Mitochondrial DNA evolution in lagomorphs: Origin of systematic heteroplasmy and organization of diversity in European rabbits

Summary A characterization was conducted on mitochondrial DNA (mtDNA) molecules extracted separately from 107 European rabbits (Oryctolagus cuniculus) both wild and domestic, 13 European hares (Lepus capensis), and 1 eastern cottontail (Sylvilagus floridanus). Experimentally this study took into account restriction site polymorphism, overall length variation of the noncoding region, and numbers of repeated sequences. Nucleotide divergences indicate that the mtDNAs from the three species derived from a common ancestor some 6–8 million years (Myr) ago. Every animal appeared heteroplasmic for a set of molecules with various lengths of the noncoding region and variable numbers of repeated sequences that contribute to them. This systematic heteroplasmy, most probably generated by a rate of localized mtDNA rearrangements high enough to counterbalance the cellular segregation of rearranged molecules, is a shared derived character of leporids.The geographic distribution of mtDNA polymorphism among wild rabbit populations over the western European basin shows that two molecular lineages are represented, one in southern Spain, the second over northern Spain, France, and Tunisia. These two lineages derived from a common ancestor some 2 Myr ago. Their present geographical distribution may be correlated to the separation of rabbits into two stocks at the time of Mindel glaciation.Finally the distribution of mtDNA diversity exhibits a mosaic pattern both at inter- and intrapopulation levels.     

Fibronectin gene expression in proliferating, quiescent, and SV40-infected mouse kidney cells

To study alterations in cellular gene expression in mouse kidney cell cultures infected with simian virus 40 (SV40) or polyomavirus, we performed a differential screening of a mouse kidney cDNA library with probes prepared from mRNAs of virus-infected and mock-infected cells. We isolated and characterized cDNA recombinant pKT13 which detected increased mRNA levels in infected cells. Sequence analysis of pKT13 revealed close to 100% homology with the 3'-end of mouse fibronectin (FN) mRNA. Since primary cultures of baby mouse kidney cells have been extensively characterized in our laboratories, we studied FN gene expression at different stages of uninfected and virus-infected cultures. High levels of FN and of its mRNA were found in the kidneys of suckling mice, while in primary cultures of proliferating epithelial kidney cells the expression of FN was very low until the cultures became confluent. Thereafter FN increased and reached high levels in cells which were irreversibly arrested in phase Go and which had apparently exhausted their finite division potential. Infection of confluent cultures with polyomavirus or SV40 resulted in a further stimulation of FN gene expression. However, during abortive infection with SV40, FN mRNA and FN levels decreased with emergence of transformed cells and were low in an established SV40-transformed mouse kidney cell line. These changes in FN gene expression suggest that high levels of FN might be indicative in vivo for terminal differentiation and in vitro for cellular senescence.