In situ nick translation of metaphase chromosomes with biotin-labeled d-UTP

Summary Conditioned culture medium from Daudi cells was used as a source of soluble H-Y antigen. Concentrated culture medium was labeled with ¹²⁵I and then fractionated by gel filtration. Column fractions were assayed for the presence of H-Y antigen by urease-ELISA. H-Y antigen-containing fractions were then pooled and subjected to an improved immunoprecipitation protocol. Three predominant H-Y antigenic proteins were identified with estimated molecular weights of above 200,000, 50,000, and 20,000.     

Intestinal synthesis of 24-keto-1,25-dihydroxyvitamin D3. A metabolite formed in vivo with high affinity for the vitamin D cytosolic receptor

24-Keto-1,25-dihydroxyvitamin D3 has been identified as an intestinal metabolite of 1,25-dihydroxyvitamin D3 by ultraviolet absorbance, mass spectroscopy, and chemical reactivity. The metabolite was produced from 1,25-dihydroxyvitamin D3 and 1,24R,25-trihydroxyvitamin D3 in rat intestinal mucosa homogenates. 24-Keto-1,25-dihydroxyvitamin D3 is present in vivo in the plasma and small intestinal mucosa of rats fed a stock diet, receiving no exogenous 1,25-dihydroxyvitamin D3, and in the plasma and small intestinal mucosa of rats dosed chronically with 1,25-dihydroxyvitamin D3. 24-Keto-1,25-dihydroxyvitamin D3 has affinity equivalent to 1,24R,25-trihydroxyvitamin D3 for the 3.7 S cytosolic receptor specific for 1,25-dihydroxyvitamin D3 in the intestine and thymus. In cytosolic preparations contaminated with the 5 S vitamin D-binding protein, both metabolites are about 7-fold less potent than 1,25-dihydroxyvitamin D3. In contrast, in cytosolic preparations largely free of the 5 S binding protein, both metabolites are equipotent with the parent compound. No evidence was obtained supporting a substantial presence of 23-keto-1,25-dihydroxyvitamin D3 in vivo; nor was the latter compound generated in detectable amounts from 1,25-dihydroxyvitamin D3 by intestinal homogenates. Thus, C-24 oxidation is a significant pathway of intestinal 1,25-dihydroxyvitamin D3 metabolism that produces metabolites with high affinity for the cytosolic receptor which mediates vitamin D action.     

Oat mesophyll protoplasts: their response to various feeder cultures

Oat (Avena sativa L.) mesophyll protoplasts were recently demonstrated to be capable of dedifferentiation, repeated divisions, and colony formation. Since the development of oat mesophyll protoplasts is decisively influenced by the nature of the used feeder culture (species, variety and concentration), we conducted a systematic study of this parameter. Generally, graminaceous feeders promoted protoplast proliferation, while dicot species repressed protoplast divisions. The beneficial effect of those feeders that promote divisions was counterbalanced by a factor that causes necrosis. The correct balance between promotion of divisions or necrosis depended on the nature of the feeder and its plating density.     

Duplication of the streptokinase gene in the chromosome of Streptococcus equisimilis H46A

The erythromycin resistance plasmid pSM752 carrying the cloned streptokinase gene, skc, was introduced by protoplast transformation into Streptococcus equisimilis H46A from which skc was originally cloned. Cells transiently supporting the replication of pSM752 gave rise to an erythromycin-resistant clone designated H46SM which was plasmid free and produced streptokinase at levels approximately twice as high as the wild type. Southern hybridization of total cell DNA with an skc-containing probe provided evidence for the duplication of the skc gene in the H46SM chromosome. The results, which have some bearing on industrial streptokinase production, can be best explained by a single cross-over event between the chromosome and the plasmid in the region of shared homology leading to the integration of pSM752 in a Campbell-like manner.     

Self-splicing of yeast mitochondrial ribosomal and messenger RNA precursors

We have previously shown linear and circular splicing intermediates resembling intermediates that result from self-splicing of ribosomal precursor RNA of Tetrahymena to be present in mitochondrial RNA. Here we show that splicing of yeast mitochondrial precursor RNA also occurs in vitro in the absence of mitochondrial proteins. The large ribosomal RNA gene, consisting of the intron and part of the flanking exon regions, was inserted behind the SP6 promoter in a recombinant plasmid and was transcribed in vitro. The resulting RNA shows self-catalyzed splicing via incorporation of GTP at the 5'-end of the excised intron, 5'- to 3'-exon ligation, and intron circularization. When purified mitochondrial RNA is incubated under similar conditions with alpha-32P-GTP, the excised ribosomal intron RNA is also labeled, as well as several other RNA species. Some of these RNAs are derived from excised introns from the multiply split gene coding for cytochrome oxidase subunit I.     

In vitro site-directed mutagenesis with synthetic DNA oligonucleotides yields unexpected deletions and insertions at high frequency.

We have used in vitro site-directed mutagenesis with synthetic DNA oligonucleotides to introduce single nucleotide mutations in yeast mtDNA. In addition to the expected DNA alterations we also recovered with high frequency mutants with large deletions and insertions which arose through interaction with the synthetic DNA fragment. Characterization of a number of these by DNA sequence analysis has permitted reconstruction of the mutagenic events. In all cases, the DNA fragment had base paired with non-adjacent DNA sequences sometimes more than 1000 nucleotides apart from each other on the target strand. The products of such interactions cannot be avoided due to the non-stringent annealing conditions during complementary DNA strand synthesis. However, deliberate mispairing can be directed precisely, as shown by our ability to specifically delete the 1143-bp intron from the yeast mitochondrial gene coding for large ribosomal RNA with a synthetic DNA fragment consisting of the sequence of the exon borders flanking the intron.     

Electron spin resonance evidence for the formation of free radicals in plants exposed to ozone

Electron spin resonance (ESR) spectroscopy was used to demonstrate that free radicals are formed in O₃-fumigated plant leaves prior to the formation of visible leaf injury. ESR signals with a g-value of 2.0037 to 2.0043, were observed in pea ( Pisum sativum L. cv. Feltham first) and bean ( Phaseolus vulgaris L. cv. Pinto) plants that had been fumigated for 4 h with 70–300 nl l⁻¹ of ozone after they had been treated with the spin-trap N- t -butyl-α-phenylnitrone (PBN). The size of the ESR signals increased with the concentration of ozone used but the nature of the trapped radicals could not be identified. However, further experiments using an inhibitor of ethylene biosynthesis, arninoethoxyvinyl glycine (AVG), showed that the reaction between ozone and ethylene is the cause for ozone toxicity.