Genetic deletions between directly repeated sequences in bacteriophage T7

Summary DNA sequence analysis of genetic deletions in bacteriophage T7 has shown that these chromosomal rearrangements frequently occur between directly repeated DNA sequences. To study this type of spontaneous deletion in more quantitative detail synthetic fragments of DNA, made by hybridizing two complementary oligonucleotides, were introduced into the non-essential T7 gene 1.3 which codes for T7 DNA ligase. This insert blocked synthesis of functional ligase and made the phage that carried an insert unable to form plaques on a host strain deficient in bacterial ligase. The sequence of the insert was designed so that after it is put into the T7 genome the insert is bracketed by direct repeats. Perfect deletion of the insert between the directly repeated sequences results in a wild-type phage. It was found that these deletion events are highly sensitive to the length of the direct repeats at their ends. In the case of 5 bp direct repeats excision from the genome occurred at a frequency of less than 10⁻¹⁰, while this value for an almost identical insert bracketed by 10 bp direct repeats was approximately 10⁻⁶. The deletion events were independent of a hostrecA mutation.     

Analysis of phylogenetic relations of durum,carthlicum and common wheats by means of comparison of alleles of gliadin-coding loci

Summary Polymorphism and inheritance of wheat storage protein, gliadin, of durum (macaroni) and carthlicum wheats have been studied. Analysis of gliadin in 78 cultivars and in F₂ seeds of intercultivar crosses of durum wheat revealed three different chromosome 1A-encoded blocks of components similar to those found in common wheat (GLD1A2, GLD1A18, GLD1A19). Most of the durum cultivars studied had these three blocks; GLD1A2 was also frequent in common wheat. In contrast, all chromosome 1B-encoded blocks of durum clearly differed in component composition from those found in common wheat. Therefore, durum could not be an ancestor or a derivate of recent bread wheat. Analysis of gliadin in the collection of carthlicum wheat (14 accessions) revealed several suspected chromosome 1A, 1B, and 6A-controlled blocks, some of which were similar to those in common wheat, while others were different. Therefore, carthlicum is likely to be an ancestor or a derivate of some forms of bread wheat. There were also chromosome 1A and 6A-, but not 1B-encoded blocks which were identical in durum and carthlicum wheats. The results confirm that all three wheats share the same genome A, but emphasize the heterogeneity of genotypes among donors of this genome. Discovery of identical blocks in tetraploids and hexaploids indicates polyphyletic [from different genotypes of donor (s)] origin of these wheats.     

Proteoglycan synthesis by normal and neoplastic human transitional epithelial cells

Metabolically 35S-labeled proteoglycans were isolated from cell-associated matrices and media of confluent cultures of human normal transitional epithelial cells and HCV-29T transitional carcinoma cells. On Sepharose CL-4B columns, the cell-associated proteoglycans synthesized from both cell types separated into three identical size classes, termed CI, CII, and CIII. Normal epithelial cell C-fractions eluted in a 22:34:45 proportion and contained 64%, 64%, and 72% heparan sulfate, whereas corresponding HCV-29T fractions eluted in a 29:11:60 proportion, and contained 91%, 77%, and 70% heparan sulfate, respectively. Medium proteoglycans from normal cells separated into two size classes in a proportion of 6:94 and were composed of 35% and 50% heparan sulfate. HCV-29T medium contained only one size class of proteoglycans consisting of 23% heparan sulfate. The remaining percentages were accounted for by chondroitin/dermatan sulfate. On isopycnic CsCl gradients, proteoglycan fractions from normal cells had buoyant densities that were higher than the corresponding fractions from HCV-29T cells. DEAE-Sephacel chromatography showed that cell and medium associated heparan sulfate from HCV-29T cells was consistently of lower charge density (undersulfated) than that from normal epithelial cells. In contrast, the chondroitin/dermatan sulfate of HCV-29T was of a charge density similar to that of normal cells. These as well as other structural and compositional differences in the proteoglycan may account, at least in part, for the altered behavioral traits of highly invasive carcinoma cells.     

Functional and phenotypic analyses of interleukin 2-activated tumor-infiltrating lymphocytes

The tumor-infiltrating lymphocytes (TILs) were cultured with interleukin 2 (IL-2) to induce the activated killer cells possessing autologous tumor-killing activity, and analysed their cell surface phenotypes and assessed anti-tumor killing activity. Furthermore, the activated TILs were transferred into 7 patients adoptively resulting in complete remission in a patient with pancreatic cancer and partial remission in another patient with gastric cancer.The cytotoxic activities of activated TILs at 3 weeks-incubation was 72 ± 15, 42 ± 26, 27 ± 21 and 25 ± 15% against K562, Daudi, KATO-III and autologous tumor, respectively. The negative selection method, indicated that the killer cells recognizing autologous tumor cells consisted of CD4- or CD8-positive T lymphocytes and CD16- or CD56-positive natural killer cells. The activated TILs could not only lyse cultured tumor cell lines, but also autologous tumor cells.     

Utilization of organic compounds as the sole source of nitrogen by Thiobacillus thiooxidans

Thiobacillus thiooxidans DSM 504 was shown to grow with adenine, hypoxanthine, xanthine and uric acid as sole sources of nitrogen. Growth with these compounds was observed after lag periods of varying lengths, unless the cells had been previously grown with the same purine base. The disappearance of adenine was accompanied by a temporary accumulation of hypoxanthine in the medium. The utilization of purines was inhibited by ammonia (1 mM). Guanine, pyrimidines and some other organic compounds were not utilized.Non-standard abbreviation U-¹⁴C uniformly labeled by ¹⁴C     

Differences in the Biophysical Properties of the Benzodiazepine/γ-Aminobutyric Acid Receptor Chloride Channel Complex in the Long-Sleep and Short-Sleep Mouse Lines

Significant differences in the thermal stability of benzodiazepine receptors were found in cerebral cortical membranes prepared from the long-sleep (LS) and short-sleep (SS) selected mouse lines. Thus, benzodiazepine receptors from LS mice were heat inactivated (55 degrees C) at a significantly faster rate than those from SS mice. Although gamma-aminobutyric acid (GABA) reduced the rate of heat inactivation in both lines, the more rapid rate of inactivation in the LS line was maintained. Furthermore, the potency of GABA to enhance [3H]flunitrazepam binding decreased threefold in membranes from LS mice as the incubation temperature was increased from 0 degrees to 37 degrees C, but was unaltered in membranes from SS mice. These differences in the biophysical properties of the benzodiazepine/GABA receptor chloride channel complex ("supramolecular complex"), together with a higher KD for t-[35S]butylbicyclophosphorothionate in membranes from LS compared to SS mice, suggest that the supramolecular complex may modulate the differential sensitivity to some depressants and convulsants in these lines.     

Correlation of immunomodulatory and therapeutic activities of interferon and interferon inducers in metastatic disease

The mechanism of therapeutic activity of recombinant murine interferon-gamma (rMu IFN-gamma) and the IFN inducer polyinosinic-polycytidylic acid solubilized with poly-L-lysine in carboxy methyl cellulose (pICLC) in treating metastatic disease was investigated by comparing effector cell augmentation with therapeutic activity in mice bearing experimental lung metastases (B16-BL6 melanoma). Effector cell functions in spleen, peripheral blood, and lung (the organ with tumor) were tested after 1 and 3 weeks of rMu IFN-gamma or pICLC administration (intravenous, three times a week). In these studies, natural killer (NK), lymphokine-activated killer (LAK), cytolytic T lymphocytes (CTL) (against specific and nonspecific targets), and macrophage tumoricidal and tumoristatic activities were measured. rM IFN-gamma and pICLC had therapeutic activity and immunomodulatory activity in most assays of immune function examined. Specific CTL activity of pulmonary parenchymal mononuclear cells (PPMC), but not in splenocytes or peripheral blood lymphocytes (PBL), during week 3 and not during week 1, correlated with the therapeutic activity of rMu IFN-gamma and of pICLC. Macrophage tumoricidal activity in PPMC, but not in alveolar macrophages, also correlated with the therapeutic activity of rMu IFN-gamma, but the opposite was true for the therapeutic activity of pICLC. NK activity of PPMC, but not of splenocytes or PBL, during week 1 correlated with the therapeutic activity of pICLC; in contrast, NK activity at any site did not correlate with the therapeutic activity of rMu IFN-gamma. LAK activity at any site did not correlate with the therapeutic activity of either agent.     

Transforming activities of sodium fluoride in cultured Syrian hamster embryo and BALB/3T3 cells

The transforming activity of sodium fluoride was studied in the SHE and the BALBl3T3 cell culture systems. Initiating and promoting activities were then investigated by means of the orthogonal methodology. Sodium fluoride was found to induce morphological transformation of SHE cells seeded on a feeder layer of X-irradiated cells at high concentrations (75–125 g/ ml). When the cells were seeded in the absence of a feeder-layer, the transformation frequencies increased in a dose-dependent manner with the concentrations of sodium fluoride ranging from 0 to the highly toxic concentration of 200 g/ml. In the BALBl3T3 cell system, sodium fluoride was negative in the standard Kakunaga procedure, while through the experiment designed by table L₈ (2⁷) of the orthogonal method, an initiating-like effect and a weak promoting activity were detected within the concentrations ranging from a 25 g/ ml to a 50 g/ ml concentration which is highly toxic for BALBl3T3 cells. From these results, it is suggested that, besides a genetic mode of action, sodium fluoride could possibly act through a non-genotoxic mechanism.Abbreviations CE cloning efficiency - NaF sodium fluoride - SHE Syrian hamster embryo - TF transformation frequency