Lactose- and galactose-utilizing strains of poly(hydroxyalkanoic acid)-accumulating Alcaligenes eutrophus and Pseudomonas saccharophila obtained by recombinant DNA technology

Summary Transposon Tn 951-encoded -galactosidase was expressed in Pseudomonas saccharophila and enabled this bacterium to grow on lactose as sole carbon source. In contrast, -galactosidase was not expressed in Alcaligenes eutrophus even if the lacZ gene of Tn 951 was separated from the lacI gene. However, -galactosidase was expressed in A. eutrophus, if a DNA fragment, which was suspected to harbour the promoter of the A. eutrophus poly(3-hydroxybutyric acid)-synthetic genes, was ligated to the promoter probe vector pMC1403, which employs lac Z, Y as reporter genes. Plasmid pPL76, which harboured one of the promoter-lac fusions, enabled A. eutrophus not only to express -galactosidase but also to grow slowly on lactose (doubling time = 25–30 h). Subsequently, the promoter-lac fusion was ligated to Tn5 in pSUP5011 and was inserted into the genome of A. eutrophus H16 and of the glucose-utilizing mutant H16-G⁺1 by applying the suicide plasmid technique. Two recombinant strains, H16-cPL and H16-G⁺1-cPL, which grow with a doubling time of 16–23 h on lactose, were investigated in detail. The cells only utilized the glucose residue of lactose as a carbon source for grouth and excreted galactose into the medium. Only after the Escherichia coli gal operon had been cloned in vector pVK101 and had been mobilized to H16-cPL or H16-G⁺1-cPL, was lactose completely utilized; no galactose was detected in the medium and the growth yields increased twofold. Depending on the orientation of the gal operon in pVK101, the expression of galactokinase seems to be dependent either on the promoter of aminoglycoside phosphotransferase gene (kan) or on the promoter of the tetR gene. Offprint requests to: A. Steinbüchel     

The role of an active transport mechanism in glycerol accumulation during osmoregulation by Zygosaccharomyces rouxii

Summary At water activities (a _w) of 0.998 (no osmoticum) and 0.960 a _w(NaCl), the affinity (K _m) of glycerol transport by Zygosaccharomyces rouxii was 25.6 and 6.4 mmol/l respectively. The maximum uptake rate (V _max) was ca. 2.3 mol/g/min at both a _w's. However, at an a _wof 0.960 using polyethylene glycol (PEG) 400 the K _mand V _max for glycerol transport increased to 61.1 mmol/l and 32.2 mol/g per minute respectively. This suggests that different glycerol transport mechanisms operate during stress by the two osmotica. The addition of uncouplers (2,4-dinitrophenol or carbonylcyanide-m-chlorophenylhydrazine) resulted in the outflow of accumulated [^14C]glycerol from Z. rouxii after on osmotic upshock indicating that an active transport mechanism was operative. The transport mechanism was specific for glycerol since other polyols (mannitol, meso-erythritol and arabitol) had no effect on the uptake rate. During upshock from 0.998 to 0.960 a _w(NaCl), a transient increases in the rate of [¹⁴C]glycerol uptake was observed. However, if PEG 400 was used as osmoticum, the rate of glycerol uptake failed to increase.Offprint requests to: P. J. van Zyl     

Collection and quality of rhesus monkey semen

Electroejaculation is an accepted method of semen collection from nonhuman primates. Although both penile and rectal probe stimulation techniques have been used, there has been a general lack of consistency and detail regarding their application. This report describes the collection, processing, and evaluation of rhesus monkey semen contrasting two methods of penile electroejaculation: 1) a constant-voltage method where stimulus current is a variable and 2) a constant-current method where stimulus current is operator-controlled. The constant-current method was the more efficient procedure, requiring a lower stimulus current for successful electroejaculation. The influence on semen quality of potentially toxic agents used in the procedure, surgical glove powder and electrolyte cream, was tested; both were detrimental as measured by motility loss. No correlation was found between coagula volume and sperm numbers. The intra- and interanimal variability in semen samples from six monkeys was also evaluated. Penile electroejaculation, combined with control of stimulus current, provides a consistent, successful, and humane method for the collection of semen in the rhesus monkey.     

The role of phosphatases in signal transduction

The importance of phosphatases in regulating the phosphorylation of proteins involved in cell signaling has been demonstrated by four recent discoveries. First, a new family of receptor-like transmembrane phosphotyrosine phosphatases, highly conserved throughout evolution, was shown to be distributed in a wide variety of tissues. Extensive heterogeneity in the extracellular regions of these molecules points to the existence of a wide diversity of ligands. These ligands are thought to mediate transduction of signals to the cell interior by means of the phosphatase activity occurring within the cytoplasmic domains of the receptor-like transmembrane phosphotyrosine phosphatases. Second, cell-permeable tumor promoters, such as okadaic acid, were shown to be potent phosphatase inhibitors that have multiple effects on signaling pathways. Third, the subunits of the type 2A phosphatase were found to associate with transforming antigens encoded by DNA tumor viruses, indicating a role for phosphatases in mediating abnormal proliferative events. Fourth, several cell-cycle mutants were found to encode phosphatases. This review focuses on the significance of the transmembrane phosphotyrosine phosphatases and on the possible ways in which intracellular phosphatases function in signaling pathways.     

Effect of riparian land use on contributions of terrestrial invertebrates to streams

Since terrestrial invertebrates are often consumed by stream fishes, land-use practices that influence the input of terrestrial invertebrates to streams are predicted to have consequences for fish production. We studied the effect of riparian land-use regime on terrestrial invertebrate inputs by estimating the biomass, abundance and taxonomic richness of terrestrial invertebrate drift from 15 streams draining catchments with three different riparian land-use regimes and vegetation types: intensive grazing — exotic pasture grasses (4 streams), extensive grazing — native tussock grasses (6 streams), reserve — native forest (5 streams). Terrestrial invertebrate drift was sampled from replicated stream reaches enclosed by two 1 mm mesh drift nets that spanned the entire channel. The mean biomass of terrestrial invertebrates that entered tussock grassland (12 mg ash-free dry mass m^–2 d^–1) and forest streams (6 mg AFDM m^–2 d^–1) was not significantly different (p > 0.05). However, biomass estimated for tussock grassland and forest streams was significantly higher than biomass that entered pasture streams (1 mg AFDM m^–2 d^–1). Mean abundance and richness of drifting terrestrial invertebrates was not significantly different among land-use types. Winged insects contributed more biomass than wingless invertebrates to both pasture and tussock grassland streams. Winged and wingless invertebrates contributed equally to biomass entering forest streams. Land use was a useful variable explaining landscape-level patterns of terrestrial invertebrate input for New Zealand streams. Evidence from this study suggests that riparian land-use regime will have important influences on the availability of terrestrial invertebrates to stream fishes.     

Chloroplast DNA restriction analysis and the infrageneric grouping ofAllium (Alliaceae)

The utility of chloroplast DNA variation for checking a recently proposed infrageneric classification of the genusAllium was tested. cpDNA restriction patterns of 49 species representing the main subgenera, sections, and subsections of the existing classification were compared. 363 different fragments generated by 4 restriction enzymes were identified and analysed by UPGMA clustering. The resulting phenogram largely confirms the subgeneric classification based on an integration of morphological and other methods.     

The C-terminal KDEL sequence increases the expression level of a single-chain antibody designed to be targeted to both the cytosol and the secretory pathway in transgenic tobacco

The effects of subcellular localization on single-chain antibody (scFv) expression levels in transgenic tobacco was evaluated using an scFv construct of a model antibody possessing different targeting signals. For translocation into the secretory pathway a secretory signal sequence preceded the scFv gene (scFv-S). For cytosolic expression the scFv antibody gene lacked such a signal sequence (scFv-C). Also, both constructs were provided with the endoplasmic reticulum (ER) retention signal KDEL (scFv-SK and scFv-CK, respectively). The expression of the different scFv constructs in transgenic tobacco plants was controlled by a CaMV 35S promoter with double enhancer. The scFv-S and scFv-SK antibody genes reached expression levels of 0.01% and 1% of the total soluble protein, respectively. Surprisingly, scFv-CK transformants showed considerable expression of up to 0.2% whereas scFv-C transformants did not show any accumulation of the scFv antibody. The differences in protein expression levels could not be explained by the steady-state levels of the mRNAs. Transient expression assays with leaf protoplasts confirmed these expression levels observed in transgenic plants, although the expression level of the scFv-S construct was higher. Furthermore, these assays showed that both the secretory signal and the ER retention signal were recognized in the plant cells. The scFv-CK protein was located intracellularly, presumably in the cytosol. The increase in scFv protein stability in the presence of the KDEL retention signal is discussed.     

Transmembrane movements of artificial redox mediators in relation to electron transport and ionic currents in chloroplasts

Electrochemical data obtained with TMPD⁺-sensitive electrodes indicate that ammonium-uncoupled chloroplasts retain TMPD (N,N,N',N'-tetramethyl- p -phenylenediamine) mainly in the reduced form during illumination, whereas uncoupled DCMU-treated chloroplasts accumulate TMPD in the oxidized form (TMPD⁺). This observation indicates that the reduced plastoquinol is the preferred electron donor for photosystem I (PSI) and TMPD can only compete efficiently when plastoquinone reduction is blocked. After adding DCMU the formation of a transmembrane gradient for TMPD⁺ is reflected by a slow-down of the electrogenic electron transport and by the emerging of the overshoot of the membrane current in the light-off response. A light-dependent increase in photoelectric current generated by chloroplasts in the presence of NH₄Cl and TMPD is observed and considered to be caused by a reversible release of current limitation in the interfacial conductance barriers in the lumen.     

Biodegradation of 4-nitroanisole by two Rhodococcus spp.

Two Rhodococcus strains, R. opacus strain AS2 and R. erythropolis strain AS3, that were able to use 4-nitroanisole as the sole source of carbon and energy, were isolated from environmental samples. The first step of the degradation involved the O-demethylation of 4-nitroanisole to 4-nitrophenol which accumulated transiently in the medium during growth. Oxygen uptake experiments indicated the transformation of 4-nitrophenol to 4-nitrocatechol and 1,2,4-trihydroxybenzene prior to ring cleavage and then subsequent mineralization. The nitro group was removed as nitrite, which accumulated in the medium in stoichiometric amounts. In R. opacus strain AS2 small amounts of hydroquinone were produced by a side reaction, but were not further degraded.