Dichogamy,gender variation and bet-hedging inPseudowintera colorata

Summary Temporal patterns of variability in the longevity of the male and female phases of individual flowers and in the gender expression of plants of a dichogamous New Zealand tree,Pseudowintera colorata (Winteraceae), were documented in field studies. Two measures for the duration of phases in a dichogamous flower are distinguished; the nominal phases based on morphological features of the flower, and the effective phases reflecting the duration of their functions. Flower and phase longevity and phenotypic gender varied considerably throughout the season and among individuals. Temporal variability in phenotypic gender was loosely synchronized among the 12 plants sampled. Three effects of an environmental factor (temperature) were noted. First, increased temperatures shortened the duration of the female phase but had no effect on the duration of the male phase. Second, pollination frequency was positively correlated with temperature. These results indirectly suggest that increased pollination may shorten the duration of the female phase. Third, average population maleness, measured as the proportion of open flowers in the population on a given day which were in the male phase, was positively correlated with temperature. It is postulated that temperature indirectly influences temporal patterns of gender expression in the population through its differential effects on the longevity of the male and female phases in individual flowers. A theoretical model of bet-hedging shows that, if the direction of an environmental effect on the proportions of the sexual phases is irreversible, selection favours asynchronous dichogamy and reduces the temporal variability as much as possible. If the direction of the response is reversible, heterodichogamy is favoured.     

Fruiting at High Temperature and Its Genetic Control in the Basidiomycete Flammulina velutipes

Of 10 geographic strains of Flammulina velutipes, 4 were found capable of fruiting at 22°C (Fr_H) rather than at the typical 15°C (Fr_L). Crosses made between Fr_H and Fr_L monokaryons were never observed to fruit at 22°C. However, some hybrids did fruit at the intermediate temperature of 18°C when grown on appropriate substrates, indicating incomplete dominance of the low-temperature requirement. Analysis of progeny of five Fr_H × Fr_L crosses indicated that a minimum of two genes appears to control the requirement for fruiting at ≤15°C. The genes are not closely linked to either incompatibility locus.     

A comparison of confidence interval methods for the intraclass correlation coefficient

Different methods of obtaining confidence intervals for the intraclass correlation coefficient rho in the unbalanced one-way random-effects model are investigated, focusing on applications to family studies. Methods based on simple modifications of formulas for the case of equal group sizes are found to provide adequate coverage at small to moderate values of rho. A method based on the large-sample standard error of the sample intraclass correlation, as derived by Smith (1956, Annals of Human Genetics 21, 363-373), is shown to provide consistently good coverage at all values of rho. A method proposed by Thomas and Hultquist (1978, Annals of Statistics 6, 582-587) also provides consistently good coverage, but generates mean interval widths substantially greater than those generated by Smith's method at values of rho likely to arise in practice.     

Assessment of mechanistic proposals for the binding of agonists to cardiac muscarinic receptors

N-[3H]Methylscopolamine has been used to characterize muscarinic receptors in crude homogenates prepared from hearts of Syrian golden hamsters. The Hill coefficient is one for specific binding of the radioligand itself and for its inhibition by muscarinic antagonists; markedly lower values are obtained for its inhibition by muscarinic agonists. The binding patterns of agonists have been analyzed in terms of a mixture of sites differing in affinity for the drug and reveal the following. All agonists discern at least two classes of receptor in atrial and ventricular homogenates. The number of classes and the relative size of each differ for different agonists in the same region and for the same agonist in different regions. Atrial and ventricular affinities are in good agreement for some agonists but differ for others. Guanylyl imidodiphosphate (GMP-PNP) is without effect on the specific binding of the radioligand but alters the binding of carbachol via an apparent redistribution of receptors from one class to another; the apparent affinity at either class remains unchanged. Carbachol reveals two classes of sites in ventricular preparations, and the nucleotide mediates an interconversion from higher to lower affinity; three classes are revealed in atrial preparations, and the nucleotide eliminates the sites of highest affinity with a concomitant increase in the number of sites of lowest affinity. Taken together, the data are incompatible with the notion of different, noninterconverting sites; rather, there appear to be several possible states of affinity such that the equilibrium distribution of receptors among the various states is determined by the tissue, by the agonist, and by neurohumoral modulators such as guanylyl nucleotides. The effects of agonists and GMP-PNP cannot be rationalized in terms of a ternary complex model in which the low Hill coefficients arise from a spontaneous equilibrium between receptor (R) and G protein (G) and in which agonists bind preferentially to the RG complex.     

DNA requirements at the bacteriophage G4 origin of complementary-strand DNA synthesis.

An in vivo assay was used to define the DNA requirements at the bacteriophage G4 origin of complementary-strand DNA synthesis (G4 origin). This assay made use of an origin-cloning vector, mRZ1000, a defective M13 recombinant phage deleted for its natural origin of complementary-strand DNA synthesis. The minimal DNA sequence of the G4 genome sufficient for the restoration of normal M13 growth parameters was determined to be 139 bases long, located between positions 3868 and 4007. This G4-M13 construct was also found to give rise to proper initiation of complementary-strand synthesis in vitro. The cloned DNA sequence contains all the regions of potential secondary structure which have been implicated in primase-dependent replication initiation as well as additional sequence information. To address the role of one region which potentially forms a DNA secondary structure, the DNA sequence internal to the G4 origin was altered by site-directed mutagenesis. A 3-base insertion at the AvaII site as well as a 17-base deletion between the AvaI and AvaII sites both resulted in loss of origin function. The 17-base deletion was also generated within the G4 genome and found to dramatically reduce the infectious growth rate of the resulting phage. These results are discussed with respect to the role of the G4 origin as the recognition site for primase-dependent replication initiation and its possible role in stage II replication.     

Canopy photosynthesis and its relationship to plant productivity in near-isogenic cotton lines differing in leaf morphology

A 2-year study was conducted to determine the relationships between plant canopy photosynthesis, canopy light interception, and plant productivity of cotton (Gossypium hirsutum L.) exhibiting differing leaf morphologies. The near-isogenic lines were from a single background (MD 65-11) and represented the leaf shapes Normal (small leaf lobing), Sub-Okra (intermediate leaf lobing), Okra (large leaf lobing), and Super Okra (severe leaf lobing). The F₁ of a cross Normal × Okra (intermediate leaf lobing) and the F₂ (segregating 1:2:1 for Normal Sub-Okra, and Okra, respectively) were also grown. Reduced plant canopies were produced by Okra and Super Okra lines, which translated into increased light penetration to the ground, and hence, in reduced canopy photosynthesis. Integrated canopy photosynthesis (ICAP) was significantly associated with light interception by the plant canopy. Part of the remaining variability in ICAP was associated with confounding factors associated with plant maturity and other unmeasured genotypic factors. Intermediate (F₁ and Sub-Okra) and normal leaf types displayed the largest ICAP values in both years. Lint production was positively related to ICAP (R² = 0.53). The combination of high ICAP values and competitive lint yields indicate that intermediate lobed leaf morphologies offer promise as productive sources of physiological variation for cotton germplasm development.     

Changes in lipoprotein composition during larval-pupal metamorphosis of an insect, Manduca sexta

During the transition from the last feeding larval stage to the pupal stage of the tobacco hornworm, Manduca sexta, significant changes occur in the properties of lipophorin, the major hemolymph lipoprotein. Within the first 24 h after cessation of feeding, the larval lipophorin (HDLp-L) is first converted to a higher density form (HDLp-W2) and then HDLp-W2 is converted to a lower density form (HDLp-W1). HDLp-W1 remains in the hemolymph until pupation, when another form, HDLp-P, with a density between HDLp-W1 and HDLp-L, is present. Although all the lipophorins contain identical apoproteins, they differ in lipid content and composition; the differences in density being primarily related to diacylglycerol content. The conversion of HDLp-L to HDLp-W1 is accompanied by a loss of hydrocarbon and uptake of carotenes. These latter changes in lipophorin composition reflect alterations in cuticular lipid composition. HDLp-L was radiolabeled in the apoproteins by injecting animals with 3H-amino acids early in the last larval stage. Subsequently HDLp-L was isolated at the end of the larval stage, HDLp-W2 and HDLp-W1 were isolated during the wandering stage, and HDLp-P was isolated after pupation. The specific activity of the apoproteins in the four lipophorins was not significantly different, suggesting that the observed alterations in lipophorin properties do not require synthesis of new apoproteins but result from retailoring the lipid composition of preexisting molecules. Examination of the hemolymph of individual animals during these transitions showed that only one species of lipoprotein was present, never a mixture of two or more species. These observations suggest that the lipoprotein conversions are precisely timed and that lipoprotein metabolism during larval development and pupation cannot be considered a static process. The unique finding of these studies was that synthesis of lipophorin apoproteins proceeds actively during the first part of the fifth instar but then ceases and does not recommence during the wandering or early pupal stages.     

Lipoprotein biosynthesis in the larvae of the tobacco hornworm, Manduca sexta

Lipoprotein biosynthesis in larvae of the tobacco hornworm (Manduca sexta) was investigated. By immunoblotting, it was shown that the apoproteins are present in the fat body, but not in the midgut. Fat body incubated in vitro with [35S]methionine secreted labeled apoproteins. However, when the density of the secreted particle was determined, it was found at 1.24-1.28 g/ml instead of 1.15 g/ml, which is the density of the circulating lipoprotein. Lipid analysis of immunoprecipitated lipoprotein secreted by the fat body showed a phospholipid/diacylglycerol ratio of 8.3 rather than 0.9, the ratio found in the circulating lipoprotein. When labeled oleic acid or triolein was fed to larvae, it was found that greater than 98% of the label in the circulating lipoprotein was in diacylglycerol. In studies using animals raised on a fat-free diet, it was shown that the circulating lipoprotein has properties comparable to those of the material secreted in vitro by the fat body and that this diacylglycerol-poor particle can be converted to the normal lipoprotein by feeding a bolus of triolein. These data support the hypothesis that the fat body makes and secretes a "nascent" lipoprotein which contains apoproteins and phospholipid, but is devoid of diacylglycerol. The diacylglycerol is then picked up from the midgut to complete assembly of the mature circulating lipoprotein.     

Lipoprotein interconversions in an insect, Manduca sexta. Evidence for a lipid transfer factor in the hemolymph

Hemolymph lipoproteins (lipophorins) of adult Manduca sexta are disinct from larval forms in density, lipid content, composition, and the presence of a third, low molecular weight apoprotein. Generally, only one lipoprotein species exists in M. sexta hemolymph during any given life stage. Progression through the life cycle results in alterations of existing lipoproteins to produce new forms, without new protein synthesis. The observed alterations in lipoprotein density could result from facilitated lipid transfer in insect hemolymph. An in vitro assay of facilitated lipid transfer was developed which employs a high density lipophorin from the wandering larva (density = 1.18 g/ml) as acceptor and adult low density lipophorin (density = 1.03 g/ml) as donor. Adult lipophorin-deficient hemolymph was shown to catalyze a time-dependent equilibration of the starting lipoproteins to produce a new intermediate lipophorin, Lp-I. Hydrodynamic experiments on the donor, acceptor, and product lipoproteins excluded fusion as the mechanism whereby Lp-I is produced. Thus, it is concluded that Lp-I results from facilitated net lipid transfer from low to high density lipoprotein. Furthermore, experiments conducted with radioiodinated donor and radioiodinated acceptor lipoproteins demonstrated that apoprotein exchange does not occur during the lipid transfer reaction. When donor lipoprotein was labeled in the lipid moiety with carbon-14, evidence of diacylglycerol and phospholipid exchange was obtained. Partial characterization of the lipid transfer factor revealed a relationship between incubation time, donor concentration, acceptor concentration, lipophorin-deficient hemolymph concentration, and transfer activity, as measured by Lp-I production. It is concluded that lipophorin-deficient hemolymph contains one or more factor(s) that catalyze net lipid transfer as well as diacylglycerol and phospholipid exchange between lipophorins to produce a single form at equilibrium.     

In vivo formation and stability of engineered disulfide bonds in subtilisin

Computer modeling suggested that a disulfide bond could be built into Bacillus amyloliquefaciens subtilisin between positions 22 (wild-type, Thr) and 87 (Ser) or between positions 24 (Ser) and 87 (Ser). Single cysteines were introduced into this cysteine-free protease at positions 22, 24, or 87 by site-directed mutagenesis of the cloned subtilisin gene. The corresponding double-cysteine mutants were constructed, and recombinant plasmids were expressed in Bacillus subtilis. Double-cysteine mutant enzymes were secreted as efficiently as wild-type, and disulfide bonds were formed quantitatively in vivo. These disulfide bonds were introduced approximately 24 A away from the catalytic site and had no detectable effect on either the specific activities or the pH optima of the mutant enzymes. The equilibrium constants for the reduction of the mutant disulfide bonds by dithiothreitol were determined to be 82 +/- 22 and 20 +/- 5 for Cys22/Cys87 and Cys24/Cys87, respectively. Studies of autoproteolytic inactivation of wild-type subtilisin support a relationship between autolytic stability and conformational stability of the protein. The stabilities of Cys24/Cys87 and wild-type enzymes to autolysis were essentially the same; however, Cys22/Cys87 was actually less stable to autolysis. Reduction of the disulfide cross-bridge lowered the autolytic stability of both double-cysteine mutants relative to their disulfide forms. This correlates with a lowered autolytic stability for the Cys22 and Cys87 single-cysteine mutants, and the fact that an intramolecular hydrogen bond between the hydroxyl groups of Thr22 and Ser87 is likely to be disrupted in the Cys22 and Cys87 single-cysteine mutant proteins.