The cis-acting DNA sequences required in vivo for bacteriophage Mu helper-mediated transposition and packaging

The 37,000 bp double-stranded DNA genome of bacteriophage Mu behaves as a plaque-forming transposable element of Escherichia coli. We have defined the cis-acting DNA sequences required in vivo for transposition and packaging of the viral genome by monitoring the transposition and maturation of Mu DNA-containing pSC101 and pBR322 plasmids with an induced helper Mu prophage to provide the trans-acting functions. We found that nucleotides 1 to 54 of the Mu left end define an essential domain for transposition, and that sequences between nucleotides 126 and 203, and between 203 and 1,699, define two auxiliary domains that stimulate transposition in vivo. At the right extremity, the essential sequences for transposition require not more than the first 62 base pairs (bp), although the presence of sequences between 63 and 117 bp from the right end increases the transposition frequency about 15-fold in our system. Finally, we have delineated the pac recognition site for DNA maturation to nucleotides 32 to 54 of the Mu left end which reside inside of the first transposase binding site (L1) located between nucleotides 1–30. Thus, the transposase binding site and packaging domains of bacteriophage Mu DNA can be separated into two well-defined regions which do not appear to overlap.Abbreviations attL attachment site left - attR attachment site right - bp base pairs - Kb kilobase pair - nt nucleotide - Pu Purine - Py pyrimidine - Tn transposable element State University of New York, Downstate Medical Center, Brooklyn, NY 11204 USA     

Early stimulation of ATP turnover induced by growth factors : Synergistic effect of EGF and insulin and correlation with DNA synthesis

Addition of a mixture of EGF + insulin to quiescent cell cultures synergistically stimulates the cells to reinitiate DNA synthesis and cell division. We have previously demonstrated that this mixture rapidly increases ATP turnover in quiescent cells. The present work shows that each of the two growth factors, EGF and insulin, when added separately to quiescent cells was able to stimulate the phosphorylation of the organic acid-soluble compounds (Po) pool and ATP turnover. The stimulation of ATP turnover was closely correlated with the increase in phosphorylation of the Po pool which suggests that Po labelling reflects the ATP turnover. In many experiments, the synergy between the two growth factors on the early increase in phosphorylation of the Po pool was clearly shown. Doubling the concentration of EGF (12-24 ng/ml) or insulin (50-100 ng/ml) did not increase early stimulation of phosphorylation of the Po pool, whereas simultaneous addition of the two growth factors induced a greater stimulation than that of each growth factor separately added. The augmentation in Po labelling after addition of EGF or insulin alone was transient. The synergistic effect of the two growth factors was more significant when determined 150 or 300 min after growth-factor addition. In our experimental conditions, each of the two growth factors, EGF and insulin, was able to induce a stimulation of DNA synthesis. However, the best stimulatory effect was observed with the mixture of the two which synergistically increased DNA synthesis determined between 6 and 24 h after growth-factor addition. The comparison between DNA replication and Po labelling suggests a correlation between the increase in DNA replication and in the total ATP synthesized in the first 5 h after cell stimulation by growth factors added separately or in combination.     

Thromboxane and Prostacyclin Levels in Fetal Rabbit Brain and Placenta After Intrauterine Partial Ischemic Episodes

The appearance of arachidonic acid (AA) oxidation products in fetal rabbit brain and placenta under normal or partial short-term ischemic episodes induced by placental blood vessel restriction was examined. Intracerebral administration of [3H]AA into close-to-term rabbit fetuses gave rise to radioactively labeled prostaglandin (PG) E2, thromboxane B2, and 6-keto-PGF1 alpha metabolites as detected by HPLC analysis. A significant increase of 20-30% of [3H]AA precursor into eicosanoids was detected in brain of fetuses after 2-h restriction. The thromboxane B2 and 6-keto-PGF1 alpha levels were determined by radioimmunoassay technique over a period of 48 h following ischemic episodes. Thromboxane B2 content in affected animals was higher by five- and twofold at 3 h over control fetal brain and placental tissue values, respectively, and remained significantly higher for 24 h. 6-Keto-PGF1 alpha levels reached a peak value that was greater by 2.5- and 1.5-fold at 6 h for the ischemic brain and placental tissue, respectively, compared with control fetuses. PGE2 levels were less affected, attaining a maximum of 1.9- and 1.1-fold in brain and placenta correspondingly. The thromboxane/prostacyclin ratio reached a maximum in the brain after approximately 3 h, while that in the placenta continued to rise even after 20 h. Persisting high levels of thromboxane are indicative of cerebral vasoconstriction and may suggest possible damaging effects.     

Hyperscums and the population dynamics of Microcystis aeruginosa

Conceptual models, based on 7 years of data, are constructedto simulate the annual cycle and population dynamics of Microcystisaeruginosa in hypertrophic, warm monomictic Hartbees-poort Dam,South Africa in order to assess the role of hyperscum formation.In Hartbeespoort Dam the large summer planktonic population(mean epilimnion biovolumes of 20–50 mm³ I^–1) andthe low wind speed resulted in the formation of hyperscums (thick,crusted accumulations of floating cyanobacteria at wind-protectedsites) containing up to 50% of the total standing crop for 2–3months in 4 out of 5 years. In years in which hyperscums formedthe post-maximal summer population maintained itself throughoutautumn and into late winter before declining to the annual nadir(>1000 cells ml^–1). When hyperscums did not form, orwere artificially removed, the population fell to similarlylow levels as early as May (autumn) and remained small untilthe spring growth phase began. Microcystis cells decompose inthe upper layers of a hyperscum, but this is not a major lossto the planktonic population. Hyperscums are refuges which helpmaintain large planktonic standing crops during winter whengrowth is not possible but have no effect on the long-term (perennial)survival of Microcystis.     

Ectopic expression of β-lactoglobulin/human serum albumin fusion genes in transgenic mice: hormonal regulation andin situ localization

We produced transgenic mice carrying the native sheep -lactoglobulin (BLG) or fusion genes composed of the BLG promoter and human serum albumin (HSA) minigenes. BLG was expressed exclusively in the mammary glands of the virgin and lactating transgenic mice evaluated. In contrast, transgenic females carrying the BLG/HSA fusion constructs also expressed the HSA RNA ectopically in skeletal muscle, kidney, brain, spleen, salivary gland and skin. Ectopic expression of HSA RNA was detected only in strains that express the transgene in the mammary gland. There was no obvious correlation between the level of the HSA RNA expressed in the mammary gland and that found ectopically. In three transgenic strains analysed, the expression of HSA RNA in kidney and skeletal muscle increased during pregnancy and lactation, whereas in the brain HSA expression decreased during lactation in one of the strains. HSA protein was synthesized in skeletal muscle and skin of strain #23 and its level was higher in lactating mice compared with virgin mice. Expression of HSA was also analysed in males and was found to be more stringently controlled than in females of the same strains.In situ hybridization analyses localized the expressed transgene in the skin, kidney, brain and salivary glands of various transgenic strains. Distinct strain-specific and cell-type specific HSA expression patterns were observed in the skin. This is in contrast to the exclusive expression of the HSA transgene in epithelial cells surrounding the alveoli of the mammary gland. Taken together, these results suggest that the absence of sufficient mammary-specific regulatory elements in the BLG promoter sequences and/or the juxtaposition of the BLG promoter with the HSA coding sequences leads to novel tissue- and cell-specific expression in ectopic tissues of transgenic mice.     

Long timestep dynamics of peptides by the dynamics driver approach

Previous experience with the Langevin/implicit-Euler scheme for dynamics (“LI”) on model systems (butane, water) has shown that LI is numerically stable for timesteps in the 5–20 fs range but quenches high-frequency modes. To explore applications to polypeptides, we apply LI to model systems (several dipeptides, a tetrapeptide, and a 13-residue oligoalanine) and also develop a new dynamics driver approach (“DA”). The DA scheme, based on LI, addresses the important issue of proper sampling, which is unlikely to be solved by small-time step integration methods or implicit methods with intrinsic damping at room temperature, such as LI. Equilibrium averages, time-dependent molecular properties, and sampling trends at room temperature are reported for both LI and DA dynamics simulations, which are then compared to those generated by a standard explicit discretization of the Langevin equation with a 1 fs timestep. We find that LI's quenching effects are severe on both the fast and slow (due to vibrational coupling) frequency modes of all-atom polypeptides and lead to more restricted dynamics at moderate timesteps (40 fs). The DA approach empirically counteracts these damping effects by adding random atomic perturbations to the coordinates at each step (before the minimization of a dynamics function). By restricting the energetic fluctuations and controlling the kinetic energy, we are able with a 60 fs timestep to generate continuous trajectories that sample more of the relevant conformational space and also reproduce reasonably Boltzmann statistics. Although the timescale for transition may be accelerated by the DA approach, the transitional. information obtained for the alanine dipeptide and the tetrapeptide is consistent with that obtained by several other theoretical approaches that focus specifically on the determination of pathways. While the trajectory for oligoalanine by the explicit scheme over the nanosecond timeframe remains in the vicinity of the full α_R-helix starting structure, and a high-temperature (6000°K) MD trajectory departs slowly from the a helical structure, the DA-generated trajectory for the same CPU time exhibits unfolding and refolding and reveals a range of conformations with an intermediate helix content. Significantly, this range of states is more consistent with spectroscopic experiments on small peptides, as well as the cooperative two-state model for helix–coil transition. The good, near-Boltzmann statistics reported for the smaller systems above, in combination with the interesting oligoalanine results, suggest that DA is a promising tool for efficiently exploring conformational spaces of biomolecules and exploring folding/unfolding processes of polypeptides. © 1995 Wiley-Liss, Inc.     

Catechol oxidase from green olives: Properties and partial purification

Catechol oxidase was extracted from an acetone powder prepared from green olive. The enzyme was purified 240-fold by ammonium sulphate fractionation followed by ion exchange chromatography and gel filtration. The enzyme was characterized by substrate specificity and response to inhibitors. Between 7 and 9 bands having catechol oxidase activity could be detected by gel electrophoresis and electrofocusing. The purified enzyme had an estimated MW of 42 000. The enzyme was strongly inhibited by diethyldithiocarbamate. Inhibition by chloride was strongly dependent on pH. The enzyme did not oxidise monophenols.