Phase determination of the circadian rhythm of conidiation in heterocaryons between two out-of-phase mycelia in Neurospora crassa

Neurospora grows vegetatively as a syncytium in which multiple nuclei exist within a connected cytoplasm. Because of the ability of separate and distinct mycelia to fuse, the possibility exists of generating heterocaryotic cultures in which the nuclei and cytoplasms of two different strains are comingled into the same syncytium. We have used such heterocaryons, in which the component parts differed with respect to their circadian clock phase, to examine whether or not clock-dominant phases exist in the circadian cycle. To this end, the phase subsequent to the formation of heterocaryons by pairs of mycelial discs that are initially at different circadian phases was examined in Neurospora crassa. The resulting phase was an average of the parent phases in many cases, but was sometimes observed to correspond more closely to just one of the original parental phases. In these cases, we did not observe any dominant phases in the circadian cycle; the phase of a particular parent disc was more dominant in the heterocaryon when the proportion of the nuclei from that parent was greater in the heterocaryon. In some instances, which occurred mostly when the difference in phase of the parental discs was large, the resultant phase could not be related in a simple way to the parental phases. An interpretation based on a limit cycle model of the circadian oscillation is possible.     

Reversible steps in the reaction of aldehydes with bacterial luciferase intermediates.

Aliphatic aldehydes of different chain lengths were found to differ in their reaction at 22 °C with the B. harveyi luciferase peroxyflavin intermediate. Although similar quantum yields were obtained in the luciferase reaction with the different chain-length aldehydes, the catalytic turnover rates differed. The kinetics of a reaction utilizing two aldehydes of different chain lengths can thus indicate the degree to which the aldehyde reaction is reversible. By such criteria the reactions of octanal and decanal were found to be readily reversible, while that of dodecanal was not. This conclusion was supported both by the effects of long-chain alcohols, which are competitive inhibitors, and by the secondary addition of hydroxylamine, an aldehyde trapping agent. The results are consistent with a model in which there are many intermediates along the reaction path. Since the reactions are monitored by decay of luminescence intensity, it is difficult to determine the position of the rate-determining step. For octanal and decanal the rate-limiting step could be at an early reversible stage of the reaction, but later for dodecanal, subsequent to a less reversible step, but still prior to the final irreversible step which populates the excited state.     

The split-end model for homologous recombination at double-strand breaks and at Chi

In recent years two different styles of model for homologous recombination have been discussed, depending on whether or not the recombination event occurs in the vicinity of a double-strand break in DNA. The models of Holliday and Meselson and Radding exemplify those that do not involve a break whereas the model of Szostak et al is taken as an example of those that do. Recent advances in understanding a prototypic recombination system thought to promote exchange distant from DNA ends, at Chi sites, suggest a mechanism of initiation neither like Holliday/Meselson-Radding nor like Szostak et al. In those models, only one strand of DNA may invade a homologous DNA molecule. We propose a model for Chi in which exonuclease degrades DNA from a double-strand break to the Chi site; the exonuclease is converted into a helicase upon interaction with Chi; unwinding produces a recombinagenic split-end, and both 3'- and 5'-ending strands at the split-end are capable of invading a homologue. Different genetic consequences are proposed to result from invasion by each. We review evidence supporting the split-end model and suggest its application in at least some cases previously considered to proceed via the Meselson/Radding model and by the double-strand-break repair model of Szostak et al.     

Multiple Bacteriocin Production by Leuconostoc mesenteroidesTA33a and Other Leuconostoc/Weissella Strains

Leuconostoc (Lc.) mesenteroides TA33a produced three bacteriocins with different inhibitory activity spectra. Bacteriocins were purified by adsorption/desorption from producer cells and reverse phase high-performance liquid chromatography. Leucocin C-TA33a, a novel bacteriocin with a predicted molecular mass of 4598 Da, inhibited Listeria and other lactic acid bacteria (LAB). Leucocin B-TA33a has a predicted molecular mass of 3466 Da, with activity against Leuconostoc/Weissella (W.) strains, and appears similar to mesenterocin 52B and dextranicin 24, while leucocin A-TA33a, which also inhibited Listeria and other LAB strains, is identical to leucocin A-UAL 187. A survey of other known bacteriocin-producing Leuconostoc/Weissella strains for the presence of the three different bacteriocins revealed that production of leucocin A-, B- and C-type bacteriocins was widespread. Lc. carnosum LA54a, W. paramesenteroides LA7a, and Lc. gelidum UAL 187-22 produced all three bacteriocins, whereas W. paramesenteroides OX and Lc. carnosum TA11a produced only leucocin A- and B-type bacteriocins. Received: 11 April 1997 / Accepted: 10 June 1997     

Cycles in cannibalistic egg-larval interactions

A model of a cannibalistic larval-egg interaction such as occurs in Tribolium is developed which leads to a system of nonlinear Volterra integral equations. I determine the local stability properties of the unique equilibrium point of the model. A Hopf bifurcation analysis shows that the model always undergoes a subcritical bifurcation when stability is lost. Numerical solutions confirm the presence of multiple attractors over a range of parameter values. The form of the cycles observed in the numerical solutions is analogous to that observed in laboratory populations of Tribolium.     

Growth and luminescence of the bacterium Xenorhabdus luminescens from a human wound.

Xenorhabdus luminescens, a newly isolated luminous bacterium collected from a human wound, was characterized. The effects of ionic strength, temperature, oxygen, and iron on growth and development of the bioluminescent system were studied. The bacteria grew and emitted light best at 33 degrees C in a medium with low salt, and the medium after growth of cells to a high density was found to have antibiotic activity. The emission spectrum peaked at 482 nm in vivo and at 490 nm in vitro. Both growth and the development of luminescence in X. luminescens required oxygen and iron. The isolated luciferase itself exhibited a temperature optimum at about 40 degrees C; after purification by affinity chromatography, it showed two bands (52 and 41 kilodaltons) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicative of an alpha and beta subunit structure. Reduced flavin mononucleotide (Km of 1.4 microM) and tetradecanal (Km of 2.1 microM) were the best substrates for the luciferase, and the first-order decay constant under these conditions at 37 degrees C was 0.79 s-1.     

Identification of a ternary complex between cAMP and a trimeric form of cAMP-dependent protein kinase

One of the intermediates involved in dissociation and reassociation of the subunits of the type II cAMP-dependent protein kinase has been characterized. This intermediate can be generated when the protein kinase is prepared from the isolated catalytic subunit (C) and the isolated regulatory subunit-[3H]cAMP complex (R2-[3H]cAMP4) by dialysis for 18 h followed by gel filtration. The intermediate, which could be separated from the holoenzyme and the isolated subunits by polyacrylamide gel electrophoresis, had an apparent molecular weight of 149,000, consistent with an R2C form. Following electrophoresis, measurements of R and bound nucleotide indicated that R2C was half-saturated with [3H]cAMP. The bound [3H]cAMP exhibited biphasic dissociation kinetics indicating that both types of cAMP binding sites were occupied. These findings suggested that the intermediate is R2C-cAMP2. This intermediate was not seen when the dialysis time was increased to 5 days, but could be observed when cAMP was added to the holoenzyme or when holoenzyme was mixed with R2cAMP4 and cAMP. The presence of two occupied cAMP binding sites on this intermediate suggests that there is minimal cooperativity between the two members of the regulatory subunit dimer, i.e. one member of the dimer binds 2 molecules of cAMP while the other binds C.     

Transport by reconstituted lactose carrier from parental and mutant strains ofEscherichia coli

Summary The lactose transport carrier from parental (X71/F'W3747) and mutant cells (54/F'5441) was reconstituted into proteoliposomes. Transport by the counterflow assay showed slightly greater activity in proteoliposomes prepared from extracts of the mutant membranes compared with that for the parental cell. The mutant carrier showed a threefold lowerK _m but similarV _max compared to the parent. On the other hand proteoliposomes from the mutant showed a defect in protonmotive force-driven accumulation, compared with the parent. With a pH gradient (inside alkaline) plus a membrane potential (inside negative) the parental proteoliposomes accumulated lactose 25-fold over the medium concentration while the mutant proteoliposomes accumulated sixfold. In a series of experiments proteoliposomes were exposed to proteolytic enzymes. Chrymotrypsin treatment resulted in 30% inhibition of counterflow activity for the reconstituted carrier from both parent and mutant. Papain produced 84% inhibition of transport by the reconstituted parental carrier but only 41% of that of the mutant. Trypsin and carboxypeptidase Y treatment had no effect on counterflow activity of either parent or mutant. Exposure of purified lactose carrier in proteoliposomes to carboxypeptidase Y resulted in the release of alanine and valine, the two C-terminal amino acids predicted from the DNA sequence.     

High-frequency ventilation of ducks and geese

We studied gas exchange in anesthetized ducks and geese artificially ventilated at normal tidal volumes (VT) and respiratory frequencies (fR) with a Harvard respirator (control ventilation, CV) or at low VT-high fR using an oscillating pump across a bias flow with mean airway opening pressure regulated at 0 cmH2O (high-frequency ventilation, HFV). VT was normalized to anatomic plus instrument dead space (VT/VD) for analysis. Arterial PCO2 was maintained at or below CV levels by HFV with VT/VD less than 0.5 and fR = 9 and 12 s-1 but not at fR = 6 s-1. For 0.4 less than or equal to VT/VD less than or equal to 0.85 and 3 s-1. less than or equal to fR less than or equal to 12 s-1, increased VT/VD was twice as effective as increased fR at decreasing arterial PCO2, consistent with oscillatory dispersion in a branching network being an important gas transport mechanism in birds on HFV. Ventilation of proximal exchange units with fresh gas due to laminar flow is not the necessary mechanism supporting gas exchange in HFV, since exchange could be maintained with VT/VD less than 0.5. Interclavicular and posterior thoracic air sac ventilation measured by helium washout did not change as much as expired minute ventilation during HFV. PCO2 was equal in both air sacs during HFV. These results could be explained by alterations in aerodynamic valving and flow patterns with HFV. Ventilation-perfusion distributions measured by the multiple inert gas elimination technique show increased inhomogeneity with HFV. Elimination of soluble gases was also enhanced in HFV as reported for mammals.(ABSTRACT TRUNCATED AT 250 WORDS)