DNA fingerprint analysis for the detection of induced mutations in mammalian cells in culture

A mutation assay in cultured mammalian cells based on the direct analysis of minisatellite DNA was developed. Band pattern variations reflect DNA alterations ranging from single base changes to complex rearrangements. By DNA fingerprinting a large number of autosomal loci throughout the human genome can be simultaneously checked, therefore minimizing the size of the samples of cell colonies to be scored in the absence of phenotypic selection. For the mutation assay chinese hamster cells (V79) were treated with Nitrosoguanidine and 14 independent colonies were isolated and expanded. DNA fingerprints were obtained after digestion of the DNA extracted from each clone with bothHinfI andHae III, and hybridisation with both 33.15 and 33.6 probes. Twelve colonies from untreated cells were also analysed. Several differences in the band pattern of treated colonies were observed when compared with untreated cells; digestion withHae III and hybridisation with 33.15 probe allowed the detection of the highest frequency of induced variants. The results suggest that minisatellite sequences are hypermutable sites that can be used to monitor the mutagenic potential of chemical agents directly at the DNA level, without phenotypic selection. Moreover, with the method herein decribed, it is possible to distinguish between true mutations and epimutations, such as those caused by changes in DNA methylation.     

Resolving genetic relationships with microsatellite markers: a parentage testing system for the swallow Hirundo rustica

Eight polymorphic microsatellite markers from the swallow were isolated and characterized. Extraordinary variability was revealed at the HrU6 locus with 45 different alleles scored among 46 unrelated individuals. The probability that the same genotype combination would occur in two random and unrelated individuals at six selected loci was as low as 1.3 × 10^-8 and the combined exclusion probability was 0.9996. Stable Mendelian inheritance was observed in about 1000 meioses. No significant linkage was revealed and for almost all combinations of marker-pairs, linkage closer than 5 cM could be excluded. At two loci, null (nonamplifying) alleles were encountered. Thirteen (30%) extra-pair offspring were identified in 5 (56%) broods when applying the marker set on a nearly complete swallow colony. We were able to identify a single male from the other families in the colony as the most likely father for nine of the 13 extra-pair offspring.     

Roche Susceptibility Test (RST) medium,a defined formulation for susceptibility testing: I. Nutrient interaction and optimization studies

Roche Susceptibility Test (RST) Medium represents the most completely optimized and convenient fully defined medium described. It requires no post-autoclaving supplementation with vitamins, supports good growth of all common aerobic and anaerobic pathogens and may be used as a broth or agar gel on which the swarming of Proteus spp. is virtually eliminated. The broth as a superior buffering capacity to most complex media and an osmolality and pH close to those of human serum. RST is highly satisfactory for the susceptibility testing of commonly used antibiotics and meets the requirements of the National Committe for Clinical Laboratory Standards of the U.S.A. in almost every respect.     

Vitamin K-dependent carboxylase: Evidence for a semiquinone radical intermediate

Nanosecond laser flash photolysis has been used to produce and identify the vitamin K semiquinone (radical) from vitamin K dihydroquinone and to observe its formation and decay in the presence of vitamin K-dependent carboxylase (epoxidase). The activity of vitamin K-dependent carboxylase is not decreased by exposure to the laser. Absorbance of the semiquinone is proportional to enzyme concentration and is stimulated by a synthetic substrate, PheLeuGluGluIle. Stabilization of the semiquinone is observed in the presence of the enzyme. The semiquinone is rapidly destroyed in the presence of inhibitors of vitamin K-dependent carboxylase and vitamin K epoxidase.     

N-demethylation of p-chloro-N-methylaniline catalyzed by subcellular fractions from the avocado pear (Persea americana)

Subcellular fractions from the avocado pear ( Persea americana) catalyzed formation of p-chloroaniline from p-chloro-N-methylaniline. Fractions prepared by centrifugation of avocado homogenates at 20, 000g for 20 min formed p-chloroaniline (2900 +/- 500 pmol min-1 mg protein-1) with an NADPH-generating system. p-Chloroaniline formation required reduced pyridine nucleotide (NADPH was 6-7 times more effective than NADH) and O2. N-Demethylation was inhibited by CO (55% inhibition at CO:O2 = 1) and was not inhibited by CN. Cytochrome P-450 was detected in the 20, 000g pellet at levels of 300-380 pmol/mg protein. This particulate preparation was also active in catalyzing the NADPH-dependent epoxidation of the chlorinated cyclodiene aldrin. Improvements to a colorimetric procedure for measuring p-chloroaniline increased the sensitivity of the procedure fourfold, and allowed use of samples containing high amounts of lipid. Avocado pear is suitable tissue for further studies on the oxidation of foreign compounds by higher plants.     

The evaluation of pollen quality,and a further appraisal of the fluorochromatic (FCR) test procedure

Summary Methods currently available for evaluating pollen quality in vitro include, (a) tests of germinability; (b) tests of the stainability of the vegetative cell contents; (c) tests for enzyme activity, and (d) the fluorochromatic procedure (FCR), which tests principally the integrity of the plasmalemma of the vegetative cell. Using germinability in vitro as a standard, a comparison has been made between histochemical methods of classes (b), (c) and (d) in application to various pollens, immature, mature, and treated in ways known to affect viability and membrane state. Predictably, the lowest correlation was obtained with tests of stainability. The highest was given by the FCR, which generally provided an excellent guide to potential germinability. The FCR procedure is subject to various limitations, however, (a) A high correlation between FCR and germinability can only be expected when mature, ripe pollen is used; with immature pollen, the FCR will predict excessively high potential germinability. (b) The FCR may also predict a higher potential level of pollen function than in vitro germinability when the germination medium is sub-optimal. In this situation, however, it will generally give a better guide to fertilising capacity, (c) The FCR is not a test of pollen viability. Like germinability in vitro, it can yield a negative score with pollen which is nevertheless capable of functioning. For example, false negatives will be obtained with some species if the pollen is not properly pre-conditioned by rehydration before testing, an important point in monitoring stored pollen. The paper includes a brief discussion of the rationale of pollen testing.     

Binding properties of 23S,25-dihydroxyvitamin D3: an in vivo metabolite of vitamin D3

23$\text{S}$,25-Dihydroxyvitamin D₃ was isolated from the plasma of vitamin D₃-toxic pigs. An ultraviolet absorbance spectrum confirmed its purity. The configuration of the 23-hydroxyl group was determined to be S by comparison of the natural product with synthetic 23$\text{R}$,25- and 23$\text{S}$,25-dihydroxyvitamin D₃ by high-pressure liquid chromatography. The affinity of both 23$\text{S}$,25- and 23$\text{R}$,25-dihydroxyvitamin D₃ for the plasma vitamin D binding protein was similar to vitamin D₃. Thus, with respect to the plasma vitamin D binding protein, 23$\text{S}$,25-dihydroxyvitamin D₃ is the least potent, naturally-occurring, dihydroxylated vitamin D₃ metabolite known.     

1,25-Dihydroxyvitamin D3 receptor in bovine thymus gland

1,25-Dihydroxyvitamin D₃ [1,25-(OH)₂D₃] receptor was characterized after partial purification of thymus cytosol by ammonium sulfate fractionation. The 1,25-(OH)₂D₃ receptor sediments at 3.7S in 5–20% sucrose gradients. The binding of 1,25-(OH)₂D₃ in thymic cytosol was a saturable process with high affinity (Kd = 0.12?0.48 nM) at 4°C. Competition for 1,25-(OH)₂[³H]D₃ receptor by nonradioactive analogs demonstrated the affinities of these analogs to be in order; 1,25-(OH)₂D₃ = 1,24R,25-(OH)₃D₃ = 1,25S,26-(OH)₃D₃ = 1,25R,26-(OH)₃D₃ > 1,25-(OH)₂D₃-26,23 lactone > 25-OHD₃ > 23R,25-(OH)₂D₃ > 24R,25-(OH)₂D₃ > 23S,25-(OH)₂D₃ ? 25-OHD₃-26,23 lactone. The receptor bound to DNA cellulose columns in low salt buffer and eluted as a single peak at 0.21 M KCl. These findings provide evidence that the thymus possesses a 1,25-(OH)₂D₃ receptor with properties indistinguishable from 1,25-(OH)₂D₃ receptors in other tissues.     

cDNA and amino acid sequences of rainbow trout (Oncorhynchus mykiss) lysozymes and their implications for the evolution of lysozyme and lactalbumin

Summary The complete 129-amino-acid sequences of two rainbow trout lysozymes (I and II) isolated from kidney were established using protein chemistry microtechniques. The two sequences differ only at position 86, I having aspartic acid and II having alanine. A cDNA clone coding for rainbow trout lysozyme was isolated from a cDNA library made from liver mRNA. Sequencing of the cloned cDNA insert, which was 1 kb in length, revealed a 432-bp open reading frame encoding an amino-terminal peptide of 15 amino acids and a mature enzyme of 129 amino acids identical in sequence to II. Forms I and II from kidney and liver were also analyzed using enzymatic amplification via PCR and direct sequencing; both organs contain mRNA encoding the two lysozymes. Evolutionary trees relating DNA sequences coding for lysozymesc and α-lactalbumins provide evidence that the gene duplication giving rise to conventional vertebrate lysozymesc and to lactalbumin preceded the divergence of fishes and tetrapods about 400 Myr ago. Evolutionary analysis also suggests that amino acid replacements may have accumulated more slowly on the lineage leading to fish lysozyme than on those leading to mammal and bird lysozymes.