Cloning and expression of the Erwinia chrysanthemi asparaginase gene in Escherichia coli and Erwinia carotovora

A genomic library of Erwinia chrysanthemi DNA was constructed in bacteriophage lambda 1059 and recombinants expressing Er. chrysanthemi asparaginase detected using purified anti-asparaginase IgG. The gene was subcloned on a 4.7 kb EcoRI DNA restriction fragment into pUC9 to generate the recombinant plasmid pASN30. The position and orientation of the asparaginase structural gene was determined by subcloning. The enzyme was produced at high levels in Escherichia coli (5% of soluble protein) and was shown to be exported to the periplasmic space. Purified asparaginase from E. coli cells carrying pASN30 was indistinguishable from the Erwinia enzyme on the basis of specific activity [660-700 units (mg protein)-1], pI value (8.5), and subunit molecular weight (32 X 10(3]. Expression of the cloned gene was subject to glucose repression in E. coli but was not significantly repressed by glycerol. Recombinant plasmids, containing the asparaginase gene, when introduced into Erwinia carotovora, caused increased synthesis of the enzyme (2-4 fold higher than the current production strain).     

Endocrine changes before and after weaning in response to boar exposure and altered suckling in sows

Eighteen sows (6 primiparous and 12 multiparous) were allotted randomly within parity to two lactational treatments: litter separation (LS; 6 h/day) plus boar exposure (BE; 1 h/day; N = 14) beginning 8 days before weaning (4 weeks) and no LS + no BE (controls; N = 4). Blood was collected from all sows via indwelling venous catheters at 20-min intervals for 5 h on Days -1, 0, 1, 2 and 3 from start of treatment. Control sows and those exposed to LS + BE not exhibiting oestrus during lactation were resampled on Days -1, 0, 1 and 2 from weaning. All 10 multiparous sows receiving LS + BE exhibited oestrus during lactation, whereas none of the 4 primiparous sows exposed to LS + BE or the 2 control multiparous and 2 control primiparous sows exhibited lactational oestrus. Overall concentrations of LH in serum were higher (P less than 0.05) in sows receiving LS + BE than in control sows during lactation, whereas overall FSH was higher (P less than 0.05) in primiparous than multiparous sows. Number and amplitude of pulses of LH were greater (P less than 0.05) for treated primiparous than multiparous sows during lactation. Oestradiol-17 beta increased (P less than 0.05) in sows during LS + BE and was higher (P less than 0.01) in multiparous sows of this group than control multiparous or treated primiparous sows. Preweaning concentrations of cortisol and progesterone in serum were higher (P less than 0.05) in treated than control sows for multiparous and primiparous animals. In sows resampled at weaning, the number of pulses of LH was greater (P less than 0.05) in treated primiparous than in control sows. Postweaning concentrations of FSH in serum were unaffected by preweaning treatments. It was concluded that (1) litter separation and boar exposure increased basal and pulsatile secretion of LH in multiparous and primiparous sows; (2) lack of ovarian follicular development and oestradiol secretion may preclude expression of oestrus in primiparous sows during lactation, despite elevated concentrations of FSH and LH in serum; and (3) if elevated concentrations of cortisol and progesterone inhibit the onset of oestrous cycles, in response to litter separation and boar exposure during lactation, the effect is limited to primiparous sows.     

Lateral diffusion of membrane proteins in protein-rich membranes. A simple hard particle model for concentration dependence of the two-dimensional diffusion coefficient.

A model for the effect of protein concentration on the rate of lateral diffusion of integral membrane proteins is presented, in which the proteins are represented by equivalent hard circular particles on a surface. As the density of particles increases, the probability of finding a vacancy immediately adjacent to a tracer particle into which it may diffuse decreases, resulting in a concomitant reduction of the tracer diffusion coefficient. Using scaled particle theory to calculate the concentration-dependent probabilities, a simple approximate result is obtained in closed form, that is compared with the results of previously published Monte Carlo lattice simulations and experimental observations.     

Hydrolysis and protection from hydrolysis of enkephalins in human plasma

Seven groups of enkephalin-degrading enzymes and three groups of inhibitors active on these enzymes were separated from human plasma. The activity of the enzymes in hydrolyzing enkephalins and of the inhibitors in protecting enkephalins from proteolysis was measured. Results obtained with the endogenous inhibitors were compared to those relative to synthetic inhibitors. Data obtained indicate that all enkephalin-degrading enzymes found in plasma are significantly inhibited by the endogenous substances present in this tissue. The inhibition of the different classes of plasma enzymes by two of the three groups of endogenous substances is quite uniform, while one group of inhibitors appears specific to dipeptidylpeptidases. Results obtained are discussed in terms of the functional role of the inhibitory substances and of the possible pharmacological implication of their presence in human plasma.     

The pMTL nic- cloning vectors. I. Improved pUC polylinker regions to facilitate the use of sonicated DNA for nucleotide sequencing

A series of nic- cloning vectors have been constructed analogous to the pUC plasmids but which are smaller in size and carry more extensive polylinker regions within the lacZ' gene. The vectors pMTL20 and pMTL21 carry six additional sites (AatII, MluI, NcoI, BglII, XhoI and StuI) to those present in pUC18 and pUC19, while pMTL22 and -23 possess eleven new cloning sites (ActII, MluI, NcoI, BglII, XhoI, StuI, NaeI, EcoRV, ClaI, NdeI and NruI). More importantly, the relative order of the restriction sites within the polylinker of these latter vectors has been totally rearranged, relative to pUC18 and pUC19, to facilitate the conversion of DNA fragments with incompatible ends to fragments with compatible termini. The availability of such DNA fragments is a crucial requirement when M13 templates are generated for dideoxy sequencing by the sonication procedure. Derivatives of these vectors have also been constructed which demonstrate improved segregational stability by incorporation of the pSC101 par locus. During the construction of these new vectors data were obtained which demonstrated that the pUC and pMTL plasmids contain a previously unreported single base pair difference within the RNA I/RNA II region (compared to pBR322) responsible for a three-fold increase in plasmid copy number. The pUC and pMTL plasmids were also shown to be functionally nic-, thus affording the lowest categorisation in genetic manipulation experiments.     

UV-induced interstrand cross-linking of d(GT)n.d(CA)n is facilitated by a structural transition

Photochemical alterations following ultraviolet irradiation of the alternating copolymer d(GT)n.d(CA)n were studied. We found that in solution conditions which produced circular dichroism spectra compatible with B-form or A-form DNA, no interstrand cross-linking or photoproduct formation could be demonstrated. Zimmer et al. (Zimmer, C., Tymen, S., Marck, C., and Guschlbaumer, W. (1982) Nucleic Acids Res. 10, 1081-1091) and Vorlickova et al. (Vorlickova, M., Kypr, J., Sotkrova, S., Sponar, J. (1982) Nucleic Acids Res. 10, 1071-1080) have reported a number of solution conditions which produce a structural transition of this polymer characterized by a negative deviation of the circular dichroism spectrum in the region of 280 nm. The nature of this transition has not yet been elucidated. Following ultraviolet irradiation of d(GT)n.d(CA)n under two conditions which produce this transition (manganese solution or ethanol plus trace salts solution) we found ultraviolet dose-dependent interstrand cross-linking as well as dose-dependent formation of thymine-containing photoproduct. Interstrand cross-linking is demonstrated by two criteria: increase in polymer size as detected by alkaline agarose gel electrophoresis, and generation of intermediate density material in alkaline cesium sulfate isopycnic gradients. The thymine-containing photo-product was demonstrated by thin layer chromatography of acid hydrolysates of the polymer. The photo-product is at least partially photoreversible. These findings suggest that the geometry of the alternative conformation is such that pyrimidines from different strands are closely approximated, allowing for photodimerization.