A restriction endonuclease map of Streptomyces phage VWB

Summary A physical map of the actinophage VWB has been constructed using the restriction endonucleases BglII, ClaI, EcoRI, EcoRV, HindIII, KpnI and SphI. Phage VWB, genome size 47.3 kb, propagates on Streptomyces venezuelae, and it can also lysogenise this species. The three BglII-generated fragments of VWB DNA were cloned in pBR322, and subsequently mapped. In this manner the restriction map of the VWB phage genome was constructed.Abbreviations dam DNA adenine methylase activity - kb kilobase pairs - :: novel joint     

Frequencies of simultaneous transformation with different T-DNAs and their relevance to the Agrobacterium/plant cell interaction

Summary We investigated whether the efficiency of transformation of plant cells by Agrobacterium tumefaciens during cocultivation is limited by the properties of the plant cells or by the infecting bacteria.Therefore, tobacco protoplasts were infected by cocultivation with two different agrobacteria strains carrying Ti plasmids with distinguishable T-DNAs. These T-DNAs cotransform plant cells at a frequency equal to the product of their independent transformation frequencies, which indicates that all plant cells are equally competent. On the other hand, when these T-DNAs are located on the same Ti plasmid vector within one bacterial strain, the cotransformation frequency is significantly higher than the product of the single transformation frequencies. We interpret these results to indicate that transformation is limited more by the establishment of effective bacteria/plant cell interaction than by (i) the process of DNA integration and (ii) by the number of plant cells capable of being transformed by Agrobacterium. We found that most plant cells are transformed by only one or a few agrobacteria. Analysis of the number of T-DNA copies in these clonally transformed lines indicates amplification of the original, infecting T-region copy.     

Primary production of the periphyton in the littoral of the Danube

Summary The gross primary production of periphyton, grown on artificial substrata in the littoral of the Danube, was measured by the light and dark bottle method from April 1970 to March 1971. The periphyton used for the measurements was sampled from various depths, in order to cover the production of the whole littoral. In connection with this, the littoral area was divided into four zones limited by the heights 600 to 200 cm, according to the local water level gauge. The highest localized zone A (500–600 cm) was inundated only at maximum water levels for rather short period of time, the deepest zone D (200–300 cm) was permanently flooded.The zone A showed an average periphyton primary production of 22.5, the zone B of 54.8, the zone C of 28.9 and the zone D of 9.1 mg O₂/dm²/day. When recalculated to periods when the individual zones were inundated, the annual production was as follows: in zone A: 0.94, zone B: 9.20, zone C: 7.29 and zone D: 3.06g O₂/dm². The highest primary production was always found in the zone just below the water level. Exceptions occurred only when this zone was inundated for a short time as a result of a temporary rise of the water level and the periphyton was insufficiently developed.In order to compare the values of primary production of periphyton obtained from shallower rivers, where the whole bottom is well illuminated, or from rivers that do not exhibit such frequent and extensive level oscillations as the Danube, average value calculated from results obtained from the zone closest to the water level at the time of measurements, were always used. These results represent a special zone following the water level changes which is called surface zone. Primary periphytic production in the surface zone was 43.8 mg O₂/dm²/day. For the annual period it corresponds to the value of 14.74 g O₂/dm². Efficiently of gross photosynthesis in this zone was on the average 1.72%.The height of the water level and the water temperature were higly correlated with gross periphytic production. Close relationships between chlorophyll a concent, biomass and gross primary production of periphyton were found.

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Zusammenfassung Die Bruttoprimärproduktion des auf künstlichem Substrat im litoralen Bereich der Donau wachsenden Periphytons, wurde vom April 1970 bis zum März 1971, mit Hilfe der Hell-Dunkelflaschenmethode, gemessen. Das für die Messung herangezogene Periphyton wurde aus verschiedenen Tiefenzonen entnommen, um die Produktion der gesamten Uferzone festzuhalten. Im Zusammenhang damit wurde der litorale Bereich der Donau in 4 Zonen gegliedert, welche ihre Grenzen bei einem Pegelstand von 600–200 cm des Ortspegels hatten. Die höchste Zone A (500–600 cm) wurde nur bei den höchsten Wasserstanden und nur für kurze Zeit vom Wasser überflutet, die tiefste Zone D (200–300 cm) war dauernd unter Wasser.In der Zone A war die durchschnittliche Produktion des Periphytons 22,5 mg O₂/dm²/Tag, in Zone B 54,8 mg, in Zone C 28,9 mg und in Zone D 9.1 mg. Nach Berechnung für die Zeit, in der die einzelnen Zonen vom Wasser bedeckt waren war folgende Produktion in den einzelnen Zonen festzustellen: in Zone A 0,94 g O₂/dm², in Zone B 9,20 g, in Zone C 7,29 g und in Zone D 3,06 g. Die höchste Periphytonproduktion wurde jeweils in der Zone festgestellt, welche dem Wasserspiegel am nächsten war. Eine Ausnahme bildeten nur jene Fälle, in denen these Zone nach vorübergehender Erhöhung des Wasserspiegels erst kurze Zeit vom Wasser bedeckt war und das Periphyton ungenügend entwickelt war.Zum Vergleich mit den Werten der Primärproduktion des Aufwuchses aus seichteren Gewässern mit gut belichteter Sohle, oder aus Flüssen welche nicht so häufige und weitreichende Spiegelschwankungen aufweisen wie die Donau, wird in der vorgelegten Arbeit der Durchschnittswert aus den Ergebnissen der Zone verwendet, welche zur Zeit der Messungen dem Wasserspiegel am nächsten war. Diese Ergebnisse repräsentieren eine besondere Zone, welche den Schwankungen des Wasserspiegels folgt und von den Autoren durch den Begriff Oberflachenzone bezeichnet wird. Die Primärproduktion des Periphytons in dieser Oberflächenzone war 43,8 mg O₂/dm²/Tag. Dem entspricht im Jahresverlauf ein Wert von 14,74 g O₂/dm². Die Ausnützung der Sonnenenergie war in dieser Zone im Durchschnitt 1,72%.Von den Umweltfaktoren zeigt die Höhe des Wasserspiegels und die Wassertemperatur den engsten Zusammenhang mit der Höhe der Bruttoprimärproduktion des Periphytons. In kürzeren Zeitabschnitten, in denen es zu keinen raschen ausgeprägten Schwankungen des Wasserspiegels des Flusses kommt, wurde eine enge Abhängigkeit zwischen den Werten der Primärproduktion und dem a-Chlorophylgehalt und auch der Biomasse festgestellt.

     

Isoforms of Na,K-ATPase inArtemia salina: II. Tissue distribution and kinetic characterization

Summary To characterize the molecular properties conveyed by the isoforms of the subunit of Na,K-ATPase, the two major transepithelial transporting organs in the brine shrimp (Artemia salina), the salt glands and intestines, were isolated in pure form. The isoforms were quantified by ATP-sensitive fluorescein isothiocyanate (FITC) labeling. The salt gland enzyme exhibits only the 1 isoform, whereas the intestinal enzyme exhibits both the 1 and the 2 isoforms. After 32 hours of development, Na,K-ATPase activity [in mol P_i/mg protein/hr (1u)] in whole homogenates was 32±6 in the salt glands and 12±3 in the intestinal preparations (mean±sem). The apparent half-maximal activation constants (K _1/2) of the salt gland enzyme as compared to the intestinal enzyme were 3.7±0.6mm vs. 23.5±4mm (P<0.01) for Na⁺, 16.6±2.2mm vs. 8.29±1.5mm for K⁺ (P<0.01), and 0.87±0.8mm vs. 0.79±1.1mm for ATP (NS). The apparentK _i's for ouabain inhibition were 1.1×10^–4 m vs. 2×10^–5 m, respectively. Treatment of whole homogenates with deoxycholic acid (DOC) produced a maximal Na,K-ATPase activation of 46% in the salt gland as compared to 23% in the intestinal enzyme. Similar differences were found with sodium dodecyl sulfate (SDS). The two distinct forms of Na,K-ATPase isolated from the brine shrimp differed markedly in three kinetic parameters as well as in detergent sensitivity. The differences inK _1/2 for Na⁺ and K⁺ are more marked than those reported for the mammalian Na,K-ATPase isoforms. These differences may be attributed to the relative abundances of the subunit isoforms; other potential determinants (e.g. differences in membrane lipids), however, have not been investigated.During the tenure of an Educational Commission For Foreign Medical Graduates Visiting Associate Professorship.     

Photosynthetic Nitrite Reduction as Influenced by the Internal Inorganic Carbon Pool in Air-Grown Cells of Synechococcus UTEX 625

Photosynthetic reduction of NO2- was studied in air-grown cells of a cyanobacterium, Synechococcus UTEX 625. Addition of NO2- resulted in significant amounts of chlorophyll a fluorescence quenching both in the absence and presence of CO2, fixation inhibitors, glycolaldehyde or iodoacetamide. The degree of NO2- quenching was insensitive to the O2 concentration in the medium. Addition of 100 [mu]M inorganic carbon in the presence of glycolaldehyde and O2, leading to formation of the carbon pool within the cells, resulted in pronounced fluorescence quenching. Removal of O2 from the medium restored the fluorescence yield completely, and the subsequent addition of NO2- quenched 36% of the variable fluorescence. From the response to added 3-(3,4-dichlorophenyl)-1,1-dimethylurea, the quenching by NO2- appeared to be photochemical quenching, and nonphotochemical quenching did not seem to be present. The reduction of NO2- observed on its addition to inorganic carbon-depleted cells remained uninfluenced by O2 or glycolaldehyde. The internal inorganic carbon pool in the cells stimulated NO2- reduction, both in the presence and absence of O2, by 4.8-fold. An increase in NO2- reduction by 0.5-fold was also observed in the presence of O2 during simultaneous assimilation of carbon and nitrogen in inorganic carbon-depleted cells. Contrary to this, under anaerobiosis, NO2- reduction was suppressed when carbon and nitrogen assimilation occurred together.     

Analysis of methionine enkephalin in human pituitary by multi-dimensional reversed-phase high-performance liquid chromatography, radioreceptor assay, radioimmunoassay, fast atom bombardment mass spectrometry, and mass spectrometry—mass spectrometry

Methionine enkephalin (ME = YGGFM) was measured in five individual human post-mortem pituitaries using four different analytical methods, with the objective of comparing the molecular specificities of the methods. Radioreceptor assay (RRA) used a receptor-rich preparation from brain and [³H]etorphine as radioligand to determine ME-like receptoractivity (ME-LR). Radioimmunoassay (RIA) measured ME-like immunoreactivity (ME-LI). Pituitary samples analyzed by RRA and RIA were purified first with a high-performance liquid chromatography (HPLC) gradient on a polymer analytical column. Fast atom bombardment mass spectrometry (FAB-MS) in two different detection modes quantified ME using the protonated molecular ion MH⁺ of ME at 574 a.m.u. and B/E linked-field selected reaction monitoring (SRM) to monitor the specific unimolecular metastable transition that produced the unique amino acid sequence-determining tetrapeptide fragment ion YGGF⁺ from the MH⁺ precursor ion. Both FAB-MS methods used the deuterated internal standard YGG[²H₅-F]M. Samples analyzed with FAB-MS were purified first with multi-dimensional reversed-phase HPLC. The first dimension was an ODS gradient, and the second dimension was a polymer isocratic elution. The following ME amounts were measured (mean ± standard error of the mean): ME-LR, 7.0 ± 1.9 μg g⁻¹ tissue; ME-LI, 1.8 ± 0.7 μg g⁻¹ tissue; MH⁺, 2.7 ± 0.6 μg g⁻¹ tissue; SRM, 3.0 ± 0.8 μg g⁻¹ tissue. The FAB SRM method provided the highest level of molecular specificity amount these four analytical methods used to measure picomole amounts of endogenous ME in a human pituitary.