Relaxed control of RNA synthesis during nutritional shiftdowns of hisU mutant of Salmonella typhimurium.

Among the classes of histidine regulatory mutants isolated in Salmonella typhimurium, three of these mutants (hisT, hisW and hisU) exhibit pleiotrophic effects on the regulation of expression of other amino acid biosynthetic operons (1, 2). While the regulatory patterns of the hisT mutants are explained by the defective tRNA pseudouridylate synthetase (3), the exact function of the hisW and hisU loci are not as clearly defined, although both mutants exhibit reductions in the relative amino acid acceptance activity of several tRNA's (4). In studies of tRNA synthesis and processing in one such hisU mutant (hisU1820), we unexpectedly observed continued RNA synthesis during nutritional (carbon and energy source) transitions. It was also shown that this relaxed control of stable RNA formation is independent of the relA gene product.     

Alanine metabolism in skeletal muscle in tissue culture.

Alanine production by skeletal muscle in tissue culture was studied using an established myogenic line (L6) of rat skeletal muscle cells. Correlation analyses were performed on rates of metabolism of alanine, glucose, lactate and pyruvate over incubation periods up to 96 h. Alanine production did not correlate significantly with glucose utilization (r = 0.24, P less than 0.20). Alanine production, however, did correlate with lactate production (r = 0.72, P less than 0.0005) as well as medium (r = 0.50, P less than 0.025) and intracellular (r = 0.85, P less than 0.0005) pyruvate concentrations. The intercepts of the latter two correlation analyses indicated that when medium or cell pyruvate fell below 0.28 mM or 1 nmol/mg protein, respectively, net alanine consumption occurred. Alanine synthesis also correlated (r = 0.71, P less than 0.0005) with the percent change in the cell mass action ratio for the sum of the alanine and aspartate aminotransferase reactions, i.e., [alanine] [malate]/[aspartate] [lactate]. These results suggest that alanine production is not necessarily linked to the rate of glucose utilization but rater to pyruvate overflow above a critical intracellular level; under conditions of pyruvate overflow, alanine synthesis is driven by the tendency to establish equilibrium between metabolites of the linked amino acid transaminases in skeletal muscle.     

Improved methods for the formation and stabilization of R-loops

Improved methods for the formation and stabilization of R-loops for visualization in the electron microscope are presented. The two complementary strands of a duplex DNA are photochemically crosslinked once every 1 to 3 kb using 4, 5', 8 trimethylpsoralen. R-loops are then formed by incubation with RNA in 70% formamide at a temperature above the DNA melting temperature. Finally, the R-loops are stabilized by modifying the free single strand of DNA with glyoxal, thus minimizing the displacement of the hybridized RNA by branch migration. In this manner R-loops can be formed and visualized at a high frequency irrespective of the base composition of the nucleic acid of interest.     

Development of a cushion to prevent ischial pressure sores.

A study was carried out jointly by nursing staff and technologists in an attempt to develop a cushion based on scientific principles and measurement that might prevent pressure sores. At each stage in the development clinical trials were carried out, and using the results of these together with the opinions of medical staff and patients who used the cushion the design was suitably modified. Over four years a seat was evolved that was simple to construct and fulfilled the clinical requirements for a wide range of patients while providing maximum relief of high-pressure points. The design was subsequently taken up commercially.     

Structural gene sets active in embryos and adult tissues of the sea urchin

Structural gene sequences active in a variety of sea urchin adult and embryo tissues are compared. A single-copy ³H-DNA fraction, termed mDNA, was isolated, which contains sequences complementary to the messenger RNA present on gastrula stage polysomes. Gastrula message sequences are 50 fold concentrated in the mDNA compared to total single-copy DNA. mDNA reactions were carried out with excess mRNA from blastula, pluteus, exogastrula, adult ovary, tubefoot, intestine, and coelomocytes, and with excess total mature oocyte RNA. A single-copy ³H-DNA fraction totally devoid of gastrula message sequences, termed null mDNA, was also reacted with these RNAs. Large differences in the extent of both mDNA and null mDNA reaction with the various RNAs were observed, indicating that in each state of differention a distinct set of structural genes is active, generally characterized by several thousand specific sequences. The complexity of gastrula mRNA was shown in previous work to be about 17 × 10⁶ nucleotides. In units of 10⁶ nucleotides, the complexities of the RNA sequence reacting with mDNA and with null mDNA in each tissue are, respectively, as follows: intestine mRNA; 2.1 and 3.7; coelomocyte mRNA: 3.5 and ≤1.4; tubefoot mRNA: 2.7 and ≤0.4; ovary mRNA: 13 and 6.7; oocyte total RNA: 17 and 20; blastula mRNA: 12 and 15; pluteus mRNA: 14 and ≤0.6; exogastrula mRNA: 14 and ≤0.6. The total complexity of each mRNA population is the sum of these values, as verified for several cases by reactions with total single-copy DNA. A relatively small set of mRNAs, the complexity of which is about 2.1 × 10⁶ nucleotides, appears to be shared by several of the tissues studied.     

Immune competence in breast cancer — Relationship of pretreatment immunologic tests to diagnosis and tumor stage

Summary The immune competence of a group of 276 patients with suspected breast cancer has been assessed using a spectrum of tests: the peripheral lymphocyte count, serum immunoglobulin levels, lymphocyte response to phytohemagglutinin (PHA), Mantoux test, and dinitrochlorobenzene (DNCB) skin test. All tests were completed prior to any form of treatment as the initial part of an ongoing, long-term assessment which will ultimately relate immune competence to prognosis. 225 patients with breast cancer were allocated into stages based on their TNM status. The remaining 51 patients proved to have benign breast disease and made up the control group. In analysis, control patients were compared with early breast cancer patients, while the effect of advancing disease was assessed by betweenstage comparisons in the cancer group.There were no significant differences between early breast cancer and control patients or between cancer stages in peripheral lymphocyte count, serum immunoglobulin levels, lymphocyte response to PHA, or Mantoux responses. Age was found to have a crucial effect on some of these parameters and some apparent differences between the various groups lost significance after appropriate allowances were made for age.Important differences seen with the DNCB test persisted after allowing for age effects. Responses to DNCB were significantly depressed in patients with early breast cancer compared to controls. Patients with disseminated cancer showed greater depression than early breast cancer patients, but surprisingly, patients with locally advanced tumors had good responses to DNCB. Possible reasons for the paradoxical preservation of DNCB reactivity in patients with locally advanced cancer are discussed.The DNCB test is the most discriminating of the five tests of immune function studied.     

DNA sequence organization in the genomes of five marine invertebrates

The arrangement of repetitive and non-repetitive sequence was studied in the genomic DNA of the oyster (Crassostrea virginica), the surf clam (Spisula solidissima), the horseshoe crab (Limulus polyphemus), a nemertean worm (Cerebratulus lacteus) and a jelly-fish (Aurelia aurita). Except for the jellyfish these animals belong to the protostomial branch of animal evolution, for which little information regarding DNA sequence organization has previously been available. The reassociation kinetics of short (250-300 nucleotide) and long (2,000-3,000 nucleotide) DNA fragments was studied by the hydroxyapatite method. It was shown that in each case a major fraction of the DNA consists of single copy sequences less than about 3,000 nucleotides in length, interspersed with short repetitive sequences. The lengths of the repetitive sequences were estimated by optical hyperchromicity and S1 nuclease measurements made on renaturation products. All the genomes studied include a prominent fraction of interspersed repetitive sequences about 300 nucleotides in length, as well as longer repetitive sequence regions.     

A new method of in situ hybridization

A new method for gene mapping at the chromosome level using in situ hybridization and scanning electron microscopy is described and has been applied to mapping the rRNA genes of Drosophila melanogaster. Biotin is covalently attached to Drosophila rRNA via a cytochrome c bridge at a ratio of one cytochrome-biotin per 130 nucleotides by a chemical procedure. Polymethacrylate spheres with a diameter of ca. 60 nm are prepared by emulsion polymerization and are covalently attached to the protein avidin at a ratio of 5–20 avidins per sphere. The biotin-labeled rRNA is hybridized to denatured DNA in a chromosome squash. Upon incubation with a sphere solution, some of the biotin sites become labeled with spheres because of the strong non-covalent interaction between biotin and avidin. The chromosome squash is examined in the scanning electron microscope (SEM). Polymer spheres, which are visible in the SEM, are observed to label the nucleolus, where the rRNA genes are located.Contribution number 5121 from the Department of Chemistry.     

Xanthine dehydrogenase accumulation in developing Drosophila eyes

Xanthine dehydrogenase activity is assayed by following the oxidation of pterin to isoxanthopterin by spectrofluorometry at the reaction's wavelength peaks: excitation, 344 nm; emission, 412 nm. The method is sensitive to less than 0·1 μU of activity (0·1 pmol/min) and allows the assay of Drosophila imaginal disk homogenates.While the larval eye disk contains less than 0·1 per cent of the individual's XDH, the developing eye becomes a major store, with 30 per cent of the individual's activity by the time of eye pigmentation. The data suggest a basis for the well-known non-autonomous action of the gene rosy, the structural gene for XDH: the enzyme is synthesized in an organ of primary gene expression, and transported through the haemolymph to the eye of the pupa and pharate adult.     

Severe parasitism in an opossum.

Chronic debilitation and anemia were observed in a free-living opossum (Didelphis marsupialis) heavily parasitized by Physaloptera turgida, Brachylaima virginianum, and Cruzia americana. Chronic interstitial pneumonia associated with Capillaria aerophila and a metastrongyloid nematode also was present.