Cloning of the Escherichia coli gene for the stringent starvation protein

Summary In order to clone the Escherichia coli gene for the stringent starvation protein (SSP), we determined its N-terminal sequence as well as the sequence of two peptide fragments obtained by cyanogen bromide cleavage of the protein. We then chemically synthesized four sets of oligodeoxyribonucleotide mixtures that represented possible codon combinations for parts of these amino acid sequences. The synthetic oligonucleotides were labelled with ³²P at their 5-termini and used as hybridization probes to detect DNA fragments containing the complementary sequences. Genomic Southern hybridization of E. coli chromosomal DNA gave up to ten DNA fragments hybridizing with each probe but only a few hybridized with two or more of the probes. The latter fragments were coloned in pBR322. By determining partial base sequences with a rapid method and examining proteins encoded by the DNA fragments, we were able to show that we had isolated a clone containing the complete SSP structural gene.Abbreviations SSP stringent starvation protein - PTH phenylthiohydantoin     

Isolation of an actin polymerization stimulator from bovine thyroid plasma membranes

An actin polymerization stimulator was purified from bovine thyroid plasma membranes by DNase I affinity column chromatography. Although the molecular weight of the protein was about 42,000 (42K) by sodium dodecyl sulfate polyacrylamide gel electrophoresis, it did not comigrate with actin. In the presence of 30 mM KCl, the 42K protein facilitated formation of actin filaments when analyzed by a centrifugation method, accelerated the initial phase of actin polymerization as measured in an Ostwald viscometer and increased the length of filaments as shown by electron microscopy. The 42K protein also accelerated the initial phase of actin polymerization in the presence of 100 mM KCl and 2 mM MgCl₂ but did not affect the final viscosity. The effect of the 42K protein was diminished by 5 uM cytochalasin B or 1 uM cytochalasin D. This 42K protein may anchor actin filaments onto the thyroid plasma membrane.     

Inhibition of Thiamine Pyrophosphate Utilization by Thiamine or Its Monophosphate in Escherichia coli

The growth of a thiamine pyrophosphate auxotroph of Escherichi coli was inhibited by either thiamine or thiamine monophosphate, and the growth of a thiamine monophosphate auxotroph was inhibited by thiamine. The thiamine pyrophosphate-dependent oxidation of pyruvate was inhibited by thiamine with whole cells of the thiamine pyrophosphate auxotroph but not with cell extracts prepared from the same organism. In addition, the thiamine pyrophosphate uptake of the thiamine pyrophosphate auxotroph was inhibited by either thiamine or thiamine monophosphate. Although the thiamine pyrophosphate uptake of a revertant, selected for prototrophy from the thiamine monophosphate auxotroph, was inhibited by thiamine to an extent comparable to that observed with the thiamine monophosphate auxotroph, its growth was no longer inhibited by thiamine. A possible mechanism for the inhibition by thiamine and thiamine monophosphate in the utilization of thiamine pyrophosphate is discussed.     

Localization of the ribosome-releasing factor gene in the Escherichia coli chromosome.

The ribosome-releasing factor (RRF) gene was localized at a position between 2 and 6 min on the Escherichia coli chromosome by measuring the gene-dosage-dependent production of RRF in various E. coli F' merozygotes. This position was confirmed and refined by using a nucleotide probe corresponding to a 16-amino-acid sequence in RRF. It was found that the RRF gene was contained in pLC 6-32 of the Clark-Carbon Gene Bank. Restriction enzyme mapping of E. coli genomic DNA with the above probe led us to conclude that the RRF gene is situated in the 4-min region, somewhere downstream (clockwise) of the elongation factor Ts gene, tsf. A pLC 6-32-derived DNA fragment which carries the RRF gene was found to contain a partial sequence of tsf. The exact location of the translational initiation site of the RRF gene was determined to be 1.1 kilobases downstream from the translational termination site of tsf. The RRF gene is designated frr.     

Autotrophic picoplankton in southern Lake Baikal: abundance, growth and grazing mortality during summer

Autotrophic picoplankton were highly abundant during the thermalstratification period in late July in the pelagic area (waterdepth 500–1300 m) of southern Lake Baikal; maximum numberswere 2 x 10⁶ cells ml^–1 in the euphotic zone ({small tilde}15m). Unicellular cyanobacteria generally dominated the picoplanktoncommunity, although unidentified picoplankton that fluorescedred under blue excitation were also abundant (maximum numbers4 x 10⁵ cells ml^–1) and contributed up to {small tilde}40%of the total autotrophic picoplankton on occasions. Carbon andnitrogen biomasses of autotrophic picoplankton estimated byconversion from biovolumes were 14–84 µg C l^–1and 3.6–21 µg N l^–1. These were comparableto or exceeded the biomass of heterotrophic bacteria. Autotropicpicoplankton and bacteria accounted for as much as 33% of paniculateorganic carbon and 81% of nitrogen in the euphotic zone. Measurementsof the photosynthetic uptake of [^l4C]bicarbonate and the growthof picoplankton in diluted or size-fractionated waters revealedthat 80% of total primary production was due to picoplankton,and that much of this production was consumed by grazers inthe <20 µ.m cell-size category. These results suggestthat picoplankton-protozoan trophic coupling is important inthe pelagic food web and biogeochemical cycling of Lake Baikalduring summer.     

FK506 and Cyclosporin A Enhance IL-6 Production in Monocytes: A single-Cell Assay

The effect of FK506 and cyclosporin A (CsA) on the production of interleukin 6 (IL-6) in adherent monocytes was studied at a single-cell level by the avidinbiotin- peroxidase complex methods. The percentage of IL-6-producing monocytes increased when stimulated with lipopolysaccharide (LPS) at concentrations between 10 ng/ml and 10 mug/ml, in a dose dependent manner. Both FK506 and CsA enhanced the percentage of IL-6- producing monocytes stimulated with 100 pg/ml-1 mug/ml of LPS up to values near those obtained with 10 mug/ml of LPS. The enhancement by FK506 and CsA was not seen when monocytes were stimulated with a high concentration of LPS (10 mug/ml). When monocytes were stimulated with a low concentration of LPS (10 ng/ml), FK506 and CsA enhanced IL-6 production in a dose dependent manner, at a drug concentration of 0.12 nM-1.2 muM (0.1-1 000 ng/ml) for FK506 and 0.83 nM-8.3 muM (1-10 000 ng/ml) for CsA. The optimal effect of FK506 was achieved at a concentration 7-fold lower than that of CsA. In contrast, production of turnout necrosis factor-alpha (TNFalpha and interleukin 1beta (IL-1beta) was slightly suppressed by FK506 and CsA at the concentrations tested. Moreover, pretreatment of monocytes with FK506 and CsA had a significant enhancing effect on LPS-induced IL-6 production, while treatment with FK506 or CsA after LPS stimulation had no effects on IL-6 production, suggesting that the enhancing effect of each drug is exerted before LPS stimulation or at an early stage of the post-receptor pathway after LPS stimulation. These experiments demonstrate that FK506 and CsA can selectively enhance IL-6 production in monocytes under certain conditions in vitro and, possibly, also in vivo.     

Mathematical models for the swimming pattern of a lamprey

A significant characteristic in a swimming pattern of a lamprey is the generation of a constant phase lag along its body in spite of the wide range of undulation frequencies. In this paper, we discuss a mathematical treatment for coupled oscillators with time-delayed interaction and propose a model for the central pattern generator (CPG) of a lamprey to account for the generation of a constant phase relation, with consideration of the signal conduction time. From this model, it is suggested that the desired phase relation can be produced by long ascending connections from the tail to the neck region of the CPG.     

Cytogenetical localization of Zygotic hybrid rescue (Zhr), a Drosophila melanogaster gene that rescues interspecific hybrids from embryonic lethality

Hybrid females from crosses between Drsophila melanogaster males and females of its sibling species, D. simulans, D. mauritiana, or D. sechellia die as embryos. This lethality is believed to be caused by incompatibility between the X chromosome of D. melanogaster and the maternal cytoplasm. Zygotic hybrid rescue (Zhr) prevents this embryonic lethality and has been cytogenetically mapped to a proximal region of the X chromosome of D. melanogaster, probably in the centromeric heterochromatin. We have carried out high resolution cytological mapping of Zhr using deficiencies and duplications of the X heterochromatin. Deletions of the Zhr ⁺ gene from the hybrid genome exhibit the Zhr phenotype. On the contrary, addition of the wild-type gene to the hybrid genome causes embryonic lethality, regardless of sex. The Zhr locus has been narrowed down to the region covered by Dp(1;f)1162 but not covered Dp(1;f)1205, a chromosome carrying a duplication of heterochromatin located slightly distal to the In(1)sc ⁸ heterochromatic breakpoint.     

Levels of endogenous ethylene, carbon dioxide, and soluble pectin, and activities of pectin methylesterase and polygalacturonase in ripening tomato fruits

In 4 cultivars of tomato (Lycopersicon esculentum Mill.), theearly detachment of fruits advanced ripening and considerablyreduced the threshold value of endogenous C₂H₄. This indicatesa supply from the vegetative parts of (a) labile ripening-inhibitingsubstance(s) antagonizing the action of C₂H₄. The endogenous level of CO₂ increased shortly after the risein C₂H₄, and maximum levels of C₂H₄ and CO₂ occurred almostsimultaneously. The activity of PE showed no connection with ripening, but PGactivity did not occur until the onset of ripening. However,this activity increased at considerably higher C₂H₄ concentrationsthan the rise in WSP, and was independent of the possible presenceof ripening inhibitor(s). Hence PG is considered not to be involvedin the primary events leading to fruit ripening. Exposure of fruits to different C₂H₄ concentrations in the ambientatmosphere also showed PG activity to increase only after therise in WSP had started. Other pectin degrading or synthesizingenzymes may be involved. In the non-ripening Rin mutant of cv. Rutgers, no rise occurredin C₂H₄, CO₂, WSP, and PG activity. ¹ Present address: Department of Agricultural Chemistry, Facultyof Agriculture, Kochi University, Otsu 200 Monobe, Nangoku City,Kochi Prefecture 783, Japan. (Received February 16, 1978; )     

Induction of DNA damage by dimethylarsine, a metabolite of inorganic arsenics, is for the major part likely due to its peroxyl radical

To reveal the mechanisms of previously reported lung-specific DNA strand scissions in murine after oral administration of dimethylarsinic acid (DMAA), a main metabolite of inorganic arsenics in mammals, the ultimate substance causing DNA lesion was investigated using dimethylarsine which was a further metabolite of DMAA. The alkaline elution assay using 3H-labeled DNA showed that a major portion of the strand breaks was not suppressed by SOD and catalase, suggesting an ultimate substance other than active oxygen participated in the DNA damage. By ESR analysis, a radical estimated to be (CH3)2AsOO. was detected as a reaction product of dimethylarsine and molecular oxygen. This peroxyl radical, rather than active oxygen, was assumed to play a major role in DNA damage.