A rapid procedure for DNA sequencing using transposon-promoted deletions in Escherichia coli

A simple procedure has been developed for sequencing long fragments of DNA. The fragment (which can be several kb in length) is cloned in pAA3.7X, and subdivided into many overlapping segments by Tn9-promoted deletions. The deletions are isolated by positive selection for galactose resistance. A rapid plasmid preparation from several hundred galactose-resistant colonies is fractionated by agarose gel electrophoresis to pick a series of deletions terminating at approx. 200-bp intervals across the entire length of the fragment. Selected plasmids are purified by rapid alkaline extraction, and used directly for supercoil sequencing with a primer derived from IS1. Sequences of adjacent deletions contain overlaps which are used to connect individual sequences to give the complete sequence.     

Isolation of protoplasts and regeneration of callus from suspension cultures of cultivated beets

Conditions necessary for the isolation and culture of protoplasts from suspension cultures of sugar, fodder and garden beets were investigated. Good yields of protoplasts were obtained by treating cells with a mixture of cellulase, Macerozyme and Driselase enzymes. Nutritional requirements of beet protoplasts were found to be quite simple: protoplasts could be cultured in MS, B₅ or PGo based media with 0.4 M glucose with the optimum result being produced on KM8p medium. Plating efficiency (P.E) was genotype-dependent with the sugar beet giving better P.E. than the fodder or garden beets used, and higher values being achieved with the use of desalted Driselase for isolation followed by culture on KMBp medium.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BAP N⁶ benzylaminopurine - NAA naphthaleneacetic acid - P.E. plating efficiency - * University of Birmingham beet germplasm accession number     

Phosphoglucose isomerase isozymes in teleostean fishes from the Arabian Gulf

Two phosphoglucose isomerases (PGI) with different electrophoretic mobilities have been found in all groups of teleostean fishes studied, with the exception of the Clupeomorpha. PGI proved to be a good taxonomic criterion to differentiate members of the Nemipteridae, Sciaenidae, Platycephalidae and Stromateidae from the other teleost families.     

Flavonoids of the Hydrangeaceae Dumortier

Fourteen species representing nine genera of the Hydrangeaceae Dumortier were surveyed for their flavonoid pigments. All taxa exhibited profiles based upon common flavonols. Myricetin was seen in two genera: Jamesia and Decumaria. Jamesia was further distinguished by the absence of kaempferol or its glycosides. A complex array of 3-O-mono-, 3-O-di- and 3-O-triglycosides was observed, although not all species had all levels of glycosylation. Decumaria barbara was unique within the species studied in its possession of 3,7-di- and 3,7-triglycosides. The overall pattern of flavonol glycosides observed for the Hydrangeaceae closely resembles that found in herbaceous genera of Saxifragaceae. The comparatively low frequency of myricetin contrasts with its high occurrence in herbaceous genera.     

The invasion of Biomphalaria pfeifferi by Schistosoma mansoni miracidia and the development of daughter sporocysts

Laboratory experiments have been carried out to determine the susceptibility of Gezira Biomphalaria pfeifferi snails to S. mansoni miracidia and the relationship between miracidia and daughter sporocyst production at the 10–17 day development stage. The relationship between snail numbers, miracidia numbers and water volume has also been studied. Two non susceptible snails, Bulinus truncatus and Cleopatra bulimoides, both of which occur naturally in Gezira canals, were tested to see if they act as decoys for S. mansoni miracidia.The results showed that the B. pfeifferi are 100% susceptible to S. mansoni invasion, at least to the daughter sporocyst development stage. The more miracidia that penetrated the more daughter sporocysts were produced, however individual variation and overlap were great. When one miracidium was released to find one snail it succeeded in low water volumes (5 m, 50 ml), but failed in 5 litres. When 100 miracidia were released mortality of snails was high suggesting superinfection particularly when only one or five snails were available. Among survivors daughter sporocyst counts were very high. Cleopatra and Bulinus snails do have a decoy effect when present in large numbers. In their presence the number of infected snails was marginally reduced and the number of daughter sporocysts greatly reduced. However, if superinfection is reduced by decoy effect, it is conceivable that Biomphalaria may be protected by decoy snails in circumstances where miracidia counts are high.     

Spectral shifting by dyes to enhance algae growth

The photosynthetic growth action spectrum of a green alga at three bands of visible light (blue, orange, and red) at fixed quanta input and under light-limiting conditions was measured in a batch cultivation system. Quantum efficiencies (biomass dry weight increment per quanta absorbed) were better in the yellow-red region than in the blue region. Results served as a basis for the design and optimization of a dye system that would shift the energy of solar radiation to the required wavelength range by absorbing ultraviolet to blue radiation and emitting in the yellow-red, thus enhancing algae growth. Direct incorporation of dyes into the growth medium, although theoretically expected to enhance growth, in fact resulted in dye decomposition, toxicity to algae and consequently in growth inhibition. Indirect application of dyes in a double tubular reactor (algae inside and dye solution outside) demonstrated growth enhancement for certain dyes with high quantum yields and stability, which had suitable absorption/emission spectra for artificial light sources used. The maximum indirect growth enhancement was obtained using rhodamine 6G at a concentration of 3x10(-5)M with tungsten filament lamp sources.     

The effect of short-term fluctuations in pH on NO−3 uptake and intracellular constituents in Skeletonemacostatum (Grev.) Cleve

Measurements of uptake rates, intracellular nitrogen pools, and other key intracellular constituents were made during exponential growth in Skeletonema costatum (Grev.) Cleve under varying pH levels. An understanding of the overall effects of extracellular pH on the above mentioned cellular parameters is crucial in order to ascertain the degree to which pH must be regulated and monitored in laboratory experiments with marine phytoplankton.It was found that uptake rates and intracellular pool sizes of NO^?₃ were directly influenced by the extracellular pH level, whereas, other cellular compounds remained relatively unchanged. Therefore, nitrogen uptake and intracellular nitrogen storage are dependent on key H⁺ and OH_? ion transport mechanisms that are associated with phytoplankton metabolism. These findings reiterate the fact that investigators examining nitrogen uptake and assimilatory mechanisms in marine phytoplankton must be conscious of cellular H ⁺ and OH^? fluxes that contribute to intracellular pH regulation and changes in extracellular pH levels, both of which interact to affect phytoplankton metabolic processes.     

Recognition and response to alloantigens in vivo. I. Negative and positive selection of MLR reactivity in murine peripheral blood lymphocytes to major histocompatibility complex and Mls antigens

Specific mixed lymphocyte reaction (MLR) responsiveness to allogeneic major histocompatibility complex (MHC), or minor lymphocyte-stimulating (Mls) determinants, was depleted in the peripheral blood lymphocytes (PBL) obtained from mice 24 to 48 hr after i.v. injection of 5 to 7.5 X 10(7) MHC or Mlsa-incompatible spleen cells, respectively. Results of cell mixture experiments suggest that the generation of suppressor cells was not the explanation for this specific reduction in MLR proliferation occurring with these PBL responder cells. To gain additional insight into parameters involved in the recognition of allodeterminants in vivo, experimental manipulations of the host environment and donor cell inoculum utilized in the negative selection procedure were employed. For example, removal of the spleen in the recipient animal, an anatomic site in which injected allogeneic cells and corresponding host antigen-reactive cells (ARC) are trapped, still permitted the specific depletion in murine PBL of host ARC for donor foreign MHC antigens. This finding may implicate other sites such as the liver where unprimed host alloreactive clones are trapped. In addition, irradiation of allogeneic donor cells significantly reduced their capacity to trap alloreactive T cell clones in vivo, whereas heat treatment of the donor cells completely eliminated this ability, even though the Ia determinants were still expressed, measured by flow cytometry. After the negative selection period, kinetic analysis of proliferation showed that 3, 4, or 5 days after injection of MHC-incompatible allogeneic spleen cells, the PBL of the recipient showed specific hyperresponsiveness to the MHC-haplotype of the donor cells. Interestingly, these primed PBL responder cells had the volume distribution of small resting cells; thoracic duct lymphocytes (TDL), positively selected by adoptive transfer of T cells to irradiated semiallogeneic recipients, are reported to be mainly blast cells. In contrast to the MLR hyperresponsiveness that results from priming with MHC-incompatible splenocytes, PBL, obtained at these later time points from mice primed with Mlsa-incompatible, H-2-compatible splenocytes, showed complete unresponsiveness in MLR to these Mlsa-bearing stimulator cells, as well as some nonspecific reduction in proliferation to MHC-incompatible stimulator cells regardless of their Mls genotype.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ammonium-nitrogen metabolism and nitrogen fixation in azotobacter vinelandii

The probable effect of increasing levels of ammonium nitrogen on the growth, efficiency of nitrogen fixation, and main cellular constituents of Azotobacter vinelandii was studied under shaking and static culture conditions. The presence of NH₄⁺-N up to 50 mgl^-1 level has no harmful effect on the multiplication as well as the yield efficiency ratio of the tested organism. A. vinelandii was able to fix dinitrogen in the presence of NH₄⁺-N when both nitrogen sources were available in the culturing medium. The efficiency of nitrogen fixation was affected by the initial presence of NH₄⁺-N in the medium, it was quite low at the highest level. The crude protein efficiency ratio was correlated inversely with the initial NH₄⁺-N concentration, whereas the total carbohydrate efficiency ratio as well as the total lipid efficiency ratio were positively correlated with the NH₄⁺-N concentration. The presence of NH₄⁺-N in the culturing medium has no essential influence on the qualitative composition of the amino acids in the Azotobacter cells.