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Products of interaction between DNA and an antitumour drug N, N', N''-triethylenethiophosphoramide (thiotepa) have been observed for the first time by the fast atom bombardment mass spectrometry. The sites of alkylation are detected as N7 (Gua) and N3 (Ade), and yields of the products are evaluated.
The establishment of an artificial endoassociation between Escherichia coli (JC 5466, trp, his, recA 56, lac delta X 74, SpcR, harbouring the plasmid pRD1 which confers on it the capacity to produce penicillinase), and Saccharomyces cerevisiae (3.2, a, ade, ura, lys) was carried out in order to study its behaviour and stability. The pattern of protoplast reversion to whole cells, the penicillinase production capacity, the stability without selective pressure and the bacterial localization in the yeast cells, is described and discussed.
The influx of 5'-deoxy-5'-methylthioadenosine (MeSAdo) into human HL-60 leukemia cells and erythrocytes was characterized in order to determine whether it is facilitated by the nonspecific nucleoside carrier system or by a separate transporter, as suggested by other reports. Initial velocities were measured at room temperature by means of inhibitor-stop and oil-stop assays. MeSAdo influx was strongly inhibited by Ado, dAdo, and nucleoside transport inhibitors including nitrobenzylthioinosine and dipyridamole. Ade was inhibitory only at concentrations in excess of 1 mM. Loss of nucleoside transport capacity during differentiation of HL-60 cells was accompanied by a corresponding decrease in MeSAdo influx rates. These results indicate that MeSAdo influx was mediated by the nonspecific nucleoside transport system. The kinetic data were consistent with a single saturable carrier and yielded Km values of 74 and 184 microM and Vmax values of 424 and 48 pmols/10(6) cells/min with HL-60 cells and erythrocytes, respectively, after correction for a substantial passive diffusion component, which accounted for over 50% of the influx of 1 mM MeSAdo. The passive diffusion of MeSAdo in the presence of a transport inhibitor was not rate-limiting for the salvage of 50 microM MeSAdo to methionine when HL-60 cells were cultured in methionine-deficient medium. The large contribution of passive diffusion to the influx of MeSAdo is consistent with its unusually high octanol/water partition ratio (5.7-fold greater than that of Ado).
Red adenine-dependent mutants of Hansenula polymorpha, Pichia guilliermondii, Williopsis saturnus yeasts have been transformed by the plasmid pYE (ADE2) 2 DNA containing ADE2 gene from Saccharomyces cerevisiae. The analysis of two P. guilliermondii Ade+-transformants has revealed the integration of pYE (ADE2)2 sequence into the recipient strain genome and partial restoration of the deficient function.
Enthalpies of solution in water, delta H0sol, and vant'Hoff enthalpies of sublimation, delta H0subl, were determined experimentally for a number of crystalline 2-alkyl derivatives of 9-methyladenine: m2(2,9)Ade, e2m9Ade, pr2m9Ade and but2m9Ade. Standard enthalpies of hydration, delta H0hydr derived from these data were corrected for the calculated cavity terms, delta H0cav, to yield enthalpies of interaction, delta H0int, of the solutes with their hydration shells. The apparent residual contribution of alkyl groups, R, to the enthalpy of interaction delta delta H0int (R) was found to increase linearly with the number of CH2 groups added upon alkyl substitution, whereas this contribution calculated per unit area of the water-accessible molecular surface, SB, of alkyl residues delta delta H0int (R): delta SB(R) appeared constant over the whole series of the compounds investigated. This indicates that alkyl groups substituted at the C(2) carbon atom of the adenine contribute additively to the van der Waals' part of the enthalpy of interaction and do not affect the electrostatic part of the energy of interaction of the solutes with their hydration shells.
Recombinants between B. subtilis and B. licheniformis were prepared by fusion of the bacterial protoplasts. Genetically marked strains SB25 trp C hisH and 168 ade-met-leu- of B. subtilis and 1001 ura-thr- and 1001 met- of B. licheniformis were used as the parent strains. The recombinants were selected with the indirect method followed by analysis of their nutrient requirements and cultural and morphological features. All the hybrids acquired the specific properties of B. subtilis. Apparently, their formation was based on the whole chromosome of B. subtilis and recombination of separate fragments of B. licheniformis with it. Hybrids with prototrophic properties with respect to one, two or three markers of the initial strains were detected independent of the genotype of the B. subtilis parent strains. Moreover, the protoplast fusion resulted in formation of hybrids which were prototrophic with respect to the amino acid markers of B. subtilis and deficient with respect to homoserine and thiamine or only thiamine, whereas the initial strains were not auxotrophic with respect to homoserine and thiamine. Thi-Hom- and a number of the prototrophic recombinants were characterized by the capacity for increased synthesis of riboflavin lacking in the initial cultures. Homologous and heterologous transformation appeared to be possible in the recombinants of the Thi-Hom- phenotype, while transformation of the initial strain SB25 by the intergenocytic markers was possible in reciprocal crossings. It is concluded that contrary to transformation of isolated DNA, protoplast fusion may result in formation of interspecies recombinants of B. subtilis and B. licheniformis with respect to different operones of amino acid synthesis.
A survey of the efficiency of production of Ontario beef cow-calf herds was conducted using a stratified systematic random sample of Ontario producers. In general, about 87% of females exposed to breeding produced a live calf and 6% of these died before reaching four weeks of age. The herd to herd variation in these rates was quite large, the coefficient of variation being about 17%. The stillbirth rate was 1.7% and the abortion rate 1.2%. In general, herds in northern Ontario and herds whose owners kept breeding and calving records, had reduced livebirth rates, the latter probably reflecting accuracy of data. Herds with a restricted (less than three months) breeding season had increased livebirth rates. Herds using injectable vitamins ADE, and prophylactic antibiotics, had increased neonatal losses. Herds with a restricted calving season (less than or equal to 3 months) and/or feeding free choice salt to cows had decreased neonatal losses. Herdsize and calf mortality rate were directly related, but this did not appear to be due to increased density of cows at calving time. In herds, where calving occurred during the spring, using scour vaccines in calves was associated with increased calf mortality.
The frequency of prototrophic recombinants produced by mitotic gene conversion at the trp5 and ade2 loci were observed using the plate test. The yeast cells were exposed in the concentration gradient of ethyl methanesulphonate--EMS (10 microliter/plate). Selective media with addition of a small amount of tryptophan (for the selection of the convertants trp+) or adenine (for the selection of the convertants ade+) were used. The use of medium with addition of tryptophan or adenine considerably increased the sensitivity of the plate method as compared with the use of the medium deficient in either tryptophan or adenine. The above results suggest possible usefulness of testing for mutagenic activity various chemical using growing cells of yeast.
The ultrastructural changes taking place in the diaphragm and plantaris of rat are followed up from the 3rd hour to 24th week after section of the peripheral nerve. The latter is either sectioned, or crushed. It is found out that the initial changes, consisting in an increase of the number of ribosomes and certain degree of mitochondrial destruction, are observed as early as the first 24 hours after denervation. The postdenervational changes are traced up to the occurrence of reinnervation with restoration of the myoneural junctions. A description is ade of the changes in the various organelles of the muscle cell, and of the appearance of spheromembranous bodies, hellical complex and destruction of the contractile apparatus. Differences in the course of the post-denervational process in the diaphragm and plantaris are outlined. The results obtained are discussed and comparatively studied with literature data.
Dialyzed extracts of the mutants of Pichia guilliermondii RG80 (rib3, his-) and RA39 (rib3, ade-) in which the third step of flavinogenesis was genetically blocked converted GTP into 2,5-diamino-6-hydroxy-4-ribitylaminopyrimidine phosphate and 2,5-diamino-6-hydroxy-4-ribitylaminopyrimidine in the presence of NADPH2. Apparently, 2,5-diamino-6-hydroxy-4-ribitylaminopyrimidine phosphate is a true precursor of riboflavin whereas 2,5-diamino-6-hydroxy-4-ribitylaminopyrimidine is formed upon its hydrolysis.
This is a studym ade by pulmonary perfusional scintigraphy and angiopneumography on 60 patients suffering from tuberculosis of the lung; they were cured by chemoantibiotic treatment. They were recent tubercular diseases with limited extension (nodular infiltrations, former phthisiogenic lobitises, nodular and miliarich disseminations), so their outcomes were extremely modest or not worthy on radiologic plane. The results acquired with these methods firmly showed a larger ematoperfusive injury as to the affected area.
The enzyme which confers resistance to erythromycin in the producing organism Streptomyces erythraeus dimethylates a single adenine residue in Bacillus stearothermophilus 23 S rRNA. This corresponds to residue Ade 2058 in Escherichia coli 23 S RNA. The methylase responsible for resistance to macrolides, lincomycin, and streptogramin B-related antibiotics in Staphylococcus aureus also acts at this site.
Transformation procedures were used to study the residual activity of various genetic markers belonging to both Bac. subtilis and Bac. natto after treating their DNAs with EcoRI endonuclease. It is shown that in case of Bac. natto DNA the residual transforming activity of ade, thy and rib markers is 5,10 times higher in comparison with Bac. subtilis DNA. Bac. natto is suggested to be used as a source of genes in gene engineering.
We describe methods for the complete analysis of cellular nucleotides from as few as 10(6) 32Pi-labeled cells in a simple 2-day experiment. Nucleotides are extracted with acid, neutralized, and resolved by two-dimensional thin layer chromatography on polyethyleneimine cellulose. In the first dimension the nucleotides are separated based on the negative charge of their phosphate groups (i.e. cyclic, mono-, di, and triphosphates) and in the second dimension on their content of nucleobases (i.e. Ura, Cyt, Thy, Gua, and Ade). Because the separation is logical, one can predict the chromatographic migration of most nucleotides. By running standards we have determined the chromatographic location of over 90 biologically important nucleotides, nucleotide derivatives, and modified nucleotides from tRNA. We also developed a set of enzymatic and chemical methods to be used in conjunction with the chromatographic separations for verifying the identity of nucleotides and characterizing novel nucleotides. In this paper we use these methods to analyze and inventory the nucleotide content of Salmonella typhimurium in balanced log phase growth. Other potential uses of the method are also described.
A clinical-immunologic study of 133 patients was carried out to elucidate the status of the T- and B-immune systems in atherosclerotic dyscirculatory encephalopathy (ADE) in the acute period of cerebral infarction and hemorrhagic stroke. A similar pattern of elevation in humoral and tissue immunity, with quantitative fluctuations in the intensity of individual parameters in various dyscirculatory disorders, was found. The changes indicate that cerebral stroke develops in the body already sensitized in the prestroke period and its development involves the central and peripheric links of the immune system.
It was shown that the rate of segregation of variants sensitive to tetracycline by Str. coelicolor A3(2) was high, i. e. 0.1 per cent. The tetracycline sensitive variants in their turn had an ability to reverse tetracycline resistance at a rate of 10(-2)--10(-4). Such a genetically unstable determinant of tetracycline resistance is localized on the chromosome between markers ade C (V10) and arg A1. The data are indicative of the fact that formation of tetracyctine sensitive variants could not be associated with elimation of the plasmid carrying the tetracycline resistant determinant or deletion. Formation of such variants as a result of point mutations is also hardly probable. It is suggested that the genetic instability of tetracycline resistance is due to recombination events (transposition of the controlling element) involving and expelling expression of the tetracycline resistance gene(s).
The potential of actinomycin D for induction of forward mutation (ADE-), reversion (TRP+), gene conversion, and mitotic recombination, was examined using haploid and diploid strains of yeast Saccharomyces cerevisiae. No increase in forward or reverse mutations or gene conversion was detected, but actinomycin D induced up to 13-fold increases in mitotic recombinants and a 2-fold increase in numbers of aberrant colonies, in a non-selective assay for genetic activity. Actinomycin D was non-mutagenic in a fluctuation test using Escherichia coli strain WP2 UvrA-. This furnishes an example of a mutagen which is negative in bacteria but has genetic activity in yeast, emphasizing the need for using a battery of microbial tests for determining the genetic activity of any given chemical.
The absorption bands of the charge transfer complexes between nucleic acid, purine components and some of their derivatives (9CH3--Ade, Ado, dAdo, pA, App, Guo, Gpp, Gppp, Cof, Hyp) as electron donors and p-benzoquinone as the acceptor were obtained by the differential method in water solution at 293 K. These bands lie in the range 330--430 nm and have all characteristic properties of the charge transfer bands. All points of the relationships between the energy corresponding to the charge transfer band maximum (hvct) and the donor ionization potential (Id) for the nucleic acid components and their derivatives fall on the same straight line for the pi-donors (the aromatic molecules), known from literature data, which differ markedly from the same straight line for n-donors (the alifatic amines), obtained in this work. It shows that the investigated nucleic acid components and their derivatives are the pi-donors in the charge transfer complexes with p-benzoquinone.