In vitro detection of endonuclease IV-specific DNA damage formed by bleomycin in vivo.

Endonuclease IV of Escherichia coli has been implicated by genetic studies in the repair of DNA damage caused by the antitumor drug bleomycin, but the lesion(s) recognized by this enzyme in vivo have not been identified. We used the sensitive primer activation assay, which monitors the formation of 3'-OH groups that support in vitro synthesis by E.coli DNA polymerase I, to determine whether endonuclease IV-specific damage could be detected in the chromosomal DNA of cells lacking the enzyme after in vivo treatment with bleomycin. Chromosomal DNA isolated after a 1 h bleomycin treatment from wild-type, endonuclease IV-deficient (nfo-) and endonuclease IV-overproducing (p-nfo; approximately 10-fold) strains all supported modest polymerase activity. However, in vitro treatment with purified endonuclease IV activated subsequent DNA synthesis with samples from the nfo- strain (an average of 2.6-fold), to a lesser extent for samples from wild-type cells (2.1-fold), and still less for the p-nfo samples (1.5-fold). This pattern is consistent with the presence of unrepaired damage that correlates inversely with the in vivo activity of endonuclease IV. Incubation of the DNA from bleomycin-treated nfo- cells with polymerase and dideoxynucleoside triphosphates lowered the endonuclease IV-independent priming activity, but did not affect the amount of activation seen after endonuclease IV treatment. Primer activation with DNA from the nfo- strain could also be obtained with purified E.coli exonuclease III in vitro, but a quantitative comparison demonstrated that endonuclease IV was > or = 5-fold more active in this assay. Thus, endonuclease IV-specific damage can be detected after in vivo exposure to bleomycin. These may be 2-deoxy-pentos-4-ulose residues, but other possibilities are discussed.     

POLLEN DISCOUNTING AND THE SPREAD OF A SELFING VARIANT IN TRISTYLOUS EICHHORNIA PANICULATA: EVIDENCE FROM EXPERIMENTAL POPULATIONS

Floral traits that increase self-fertilization are expected to spread unless countered by the effects of inbreeding depression, pollen discounting (reduced outcross pollen success by individuals with increased rates of self-fertilization), or both. Few studies have attempted to measure pollen discounting because to do so requires estimating the male outcrossing success of plants that differ in selfing rate. In natural populations of tristylous Eichhornia paniculata, selfing variants of the mid-styled morph are usually absent from populations containing all three style morphs but often predominate in nontrimorphic populations. We used experimental garden populations of genetically marked plants to investigate whether the effects of population morph structure on relative gamete transmission by unmodified (M) and selfing variants (M‘) of the mid-styled morph could explain their observed distribution. Transmission through ovules and self and outcross pollen by plants of the M and M’ morphs were compared under trimorphic, dimorphic (S morph absent), and monomorphic (L and S morphs absent) population structures. Neither population structure nor floral morphology affected female reproductive success, but both had strong effects on the relative transmission of male gametes. The frequency of self-fertilization in the M' morph was consistently higher than that of the M morph under all morph structures, and the frequency of self-fertilization by both morphs increased as morph diversity of experimental populations declined. In trimorphic populations, total transmission by the M and M' morphs did not differ. The small, nonsignificant increase in selfing by the M' relative to the M morph was balanced by decreased outcross siring success, particularly on the S morph. In populations lacking the S morph, male gamete transmission by the M' morph was approximately 1.5 times greater than that by the M morph because of both increased selfing and increased success through outcross pollen donation. Therefore, gamete transmission strongly favored the M' morph only in the absence of the S morph, a result consistent with the distribution of the M' morph in nature. This study indicates that floral traits that alter the selfing rate can have large and context-dependent influences on outcross pollen donation.     

Fine-scale temporal variation in recruitment of a temperate demersal fish: the importance of settlement versus post-settlement loss

In order to understand variability in recruitment to populations of benthic and demersal marine species, it is critical to distinguish between the contributions due to variations in larval settlement versus those caused by post-settlement mortality. In this study, fine-scale (1–2 days) temporal changes in recruit abundance were followed through an entire settlement season in a temperate demersal fish in order to determine 1) how dynamic the process of recruitment is on a daily scale, 2) whether settlement and post-settlement mortality are influenced by habitat structure and conspecific density, and 3) how the relationship between settlement and recruitment changes over time. Settlement is considered to be the arrival of new individuals from the pelagic habitat, and recruitment is defined as the number of individuals surviving arbitrary periods of time after settlement. Replicate standardized habitat units were placed in 2 spatial configurations (clumped and randomly dispersed) and monitored visually for cunner (Tautogolabrus adspersus) settlement and recruitment every 1–2 days throughout the settlement season. The process of recruitment in T. adspersus was highly variable at a fine temporal scale. Changes in the numbers of recruits present on habitat units were due to both settlement of new individuals and mortality of animals previously recruited. The relative importance of these two processes appeared to change from day to day. The magnitude of the change in recruit number did not differ between the clumped and random habitats. However, post-settlement loss was significantly greater on randomly dispersed than clumped habitats. During several sampling dates, the extent of the change in recruit abundance was correlated with the density of resident conspecifics; however, on other dates no such relationship appeared to exist. Despite the presence of significant relationships between the change in recruit number and density, there was no evidence of either density-dependent mortality or settlement. Initially, there was a strong relationship between settlement and recruitment; however, this relationship weakened over time. Within 2 months after the cessation of settlement, post-settlement loss was greater than 99%, and no correlation remained between recruitment and the initial pattern of settlement. The results of this study demonstrate that the spatial arrangement of the habitat affects the rate and intensity of post-settlement loss. Counter to much current thinking, this study suggests that in order to understand the population ecology of reef fishes, knowledge of what habitats new recruits use and how mortality varies with structural aspects of the habitats is essential.     

Habitat structure,conspecific presence and spatial variation in the recruitment of a temperate reef fish

Pronounced spatial variation in recruitment occurs in many marine invertebrate and fish populations and is thought to be critical to the demography of these species. In this study I examined the importance of habitat structure and the presence of conspecific residents to spatial variation in larval settlement and recruitment in a temperate fish Tautogolabrus adspersus. I define settlement as the movement of individuals from the water column to the benthic habitat, while I refer to recruitment as numbers of individuals surviving some arbitrary period of time after settlement. Experiments in which standard habitats were stocked with conspecifics showed that resident conspecifics were not an important factor contributing to small-scale variability in recruitment. Further correlative analyses demonstrated that large-scale variation in recruitment could not be explained by variability in older age classes. By contrast, manipulations of macroalgal structure within a kelp bed demonstrated that recruitment was significantly higher in habitats with a dense understory of foliose and filamentous algae than in habitats with only crustose algae. Understory algae varied in their pattern of disperison among sites, and the dispersion of fish matched that of the plants. In order to determine the effects of differences in patterns of algal dispersion on the demography of associated T. adspersus populations, I used experimental habitat units to manipulate patterns of dispersion. Settlement was significantly greater to randomly placed versus clumped habitats; however, no differences in recruitment between random and clumped habitats were detected. Because recruitment is a function of the numbers of settlers minus the subsequent loss of settlers, rates of mortality or migration must have been higher in the randomly placed habitats. These results are counter to the current paradigm for reef fishes which suggests that larval settlement is the crucial demographic process producing variability in population abundance. In this experiment patterns of settlement were modified by varying the patch structure of the habitat.Contribution number 278 from the Center for Marine Biology, University of New Hampshire     

Polyamines and cytokinins in celery embryogenic cell cultures

The effect of inhibitors of polyamine biosynthesis on the development of embryogenic cell cultures of celery (Apium graveolus L.) was studied. Several developmental stages of somatic embryos were compared for differences in the content and biosynthesis of free polyamines and for cytokinin content. Cyclohexylamine and particularly methylglyoxal bis(guanylhydrazone), inhibited both cell division and the organization of polar embryos from globular embryos. Difluoromethylornithine slightly promoted embryo development, especially cell division.The free putrescine content of globular embryos was 6-fold that of fully differentiated plantlets, and that of spermidine 2-fold. Only a slight increase in the spermine content was found with embryo development. These differences were confirmed by data from polyamine biosynthesis. Incorporation of ¹⁴C-arginine into polyamines was slightly higher than that of ¹⁴C-ornithine. Over 96% of this incorporation was detected in the putrescine fraction. Incorporation of ¹⁴C into putrescine in globular embryos was 3 to 4-fold that in fully-differentiated plantlets. Incorporation into spermidine and spermine was, however, higher in plantlets than in globular embryos.Cytokinin analysis revealed considerable differences in the biological activity between the developmental stages of embryogenesis. This could be due to endogenous cytokinins and/or BA taken up from the maintenance medium. Cytokinin levels decreased with increased embryo development. Most of the detected cytokinin-like activity co-chromatographed with BA and its metabolites. Some as yet unidentified peaks of activity were recorded in the globular embryos.The results are considered with respect to the possible participation of polyamines and cytokinins in the development of embryogenic cell cultures of celery. It is suggested that the onset of embryogenesis is characterized by a high content of putrescine and cytokinins, while a decrease in putrescine synthesis and cytokinin content, and an increase in spermidine and spermine content, accompany further embryo development and plantlet formation.Abbreviation ADC arginine decarboxylase - ODC ornithine decarboxylase - 2,4-D dichlorophenoxyacetic acid - DFMA difluoromethylarginine - DFMO difluoromethylornithine - MGBG methylglyoxal bis(guanylhydrazone) - CHA cyclohexylamine - BA benzyladenine - BAR benzyladenine riboside     

A Julia set model of field-directed morphogenesis: developmental biology and artificial life

One paradigm used in understanding the control of morpho-geneticevents is the concept of positional information, where sub-organismiccomponents (such as cells) act in response to positional cues.It is important to determine what kinds of spatiotemporal patternsmay be obtained by such a method, and what the characteristicsof such a morphogenetic process might be. This paper presentsa computer model of morphogenesis based on gene activity drivenby interpreting a positional information field. In this model,the interactions of mutually regulating developmental genesare viewed as a map from R² to R², and are modeled by the complexnumber algebra. Functions in complex variables are used to simulategenetic interactions resulting in position-dependent differentiation.This is shown to be equivalent to computing modified Julia sets,and is seen to be sufficient to produce a very rich set of morphologieswhich are similar in appearance and several important characteristicsto those of real organisms. The properties of this model canbe used to study the potential role of fields and positionalinformation as guiding factors in morphogenesis, as the modelfacilitates the study of static images, time-series (movies)and experimental alterations of the developmental process. Itis thus shown that gene interactions can be modeled as a multi-dimensionalalgebra, and that only two interacting genes are sufficientfor (i) complex pattern formation, (ii) chaotic differentiationbehavior, and (iii) production of sharp edges from a continuouspositional information field. This model is meant to elucidatethe properties of the process of positional information-guidedbiomorphogenesis, not to serve as a simulation of any particularorganism's development. Good quantitative data are not currentlyavailable on the interplay of gene products in morphogenesis.Thus, no attempt is made to link the images produced with actualpictures of any particular real organism. A brief introductionto top-down models and positional information is followed bythe formal definition of the model. Then, the implications ofthe resulting morphologies to biological development are discussed,in terms of static shapes, parametrization studies, time series(movies made from individual frames), and behavior of the modelin light of experimental perturbations. All figures (in grayscale),formulas and parameter values needed to re-create the figuresand movies are included.     

Characterization of de novo duplications in eight patients by using fluorescence in situ hybridization with chromosome-specific DNA libraries.

Fluorescence in situ hybridization (FISH) with chromosome-specific DNA libraries was performed on samples from eight patients with de novo chromosomal duplications. In five cases, the clinical phenotype and/or cytogenetic evaluations suggested a likely origin of the duplicated material. In the remaining three cases, careful examination of the GTG-banding pattern indicated multiple possible origins; hybridization with more than one chromosome-specific library was performed on two of these cases. In all cases, FISH conclusively identified the chromosomal origin of the duplicated material. In addition, the hybridization pattern was useful in quantitatively delineating the duplication in two cases.     

MKK1 and MKK2, which encode Saccharomyces cerevisiae mitogen-activated protein kinase-kinase homologs, function in the pathway mediated by protein kinase C.

The PKC1 gene of Saccharomyces cerevisiae encodes a homolog of mammalian protein kinase C that is required for normal growth and division of yeast cells. We report here the isolation of the yeast MKK1 and MKK2 (for mitogen-activated protein [MAP] kinase-kinase) genes which, when overexpressed, suppress the cell lysis defect of a temperature-sensitive pkc1 mutant. The MKK genes encode protein kinases most similar to the STE7 product of S. cerevisiae, the byr1 product of Schizosaccharomyces pombe, and vertebrate MAP kinase-kinases. Deletion of either MKK gene alone did not cause any apparent phenotypic defects, but deletion of both MKK1 and MKK2 resulted in a temperature-sensitive cell lysis defect that was suppressed by osmotic stabilizers. This phenotypic defect is similar to that associated with deletion of the BCK1 gene, which is thought to function in the pathway mediated by PCK1. The BCK1 gene also encodes a predicted protein kinase. Overexpression of MKK1 suppressed the growth defect caused by deletion of BCK1, whereas an activated allele of BCK1 (BCK1-20) did not suppress the defect of the mkk1 mkk2 double disruption. Furthermore, overexpression of MPK1, which encodes a protein kinase closely related to vertebrate MAP kinases, suppressed the defect of the mkk1 mkk2 double mutant. These results suggest that MKK1 and MKK2 function in a signal transduction pathway involving the protein kinases encoded by PKC1, BCK1, and MPK1. Genetic epistasis experiments indicated that the site of action for MKK1 and MKK2 is between BCK1 and MPK1.     

The stimulatory effect of chronic lithium treatment on basal thyrotropin secretion in rats: In vivo antagonism by methylparaben

Chronic treatment of rats with lithium chloride was examined in order to determine its effect on hypothalamic monoamine and metabolite content, basal thyrotropin (TSH) secretion and thyroid function. The hypothalamic concentrations of noradrenaline (NA), dopamine (DA) and its metabolites, dihydroxyphenylacetic acid. (DOPAC) and homovanillic acid (HVA) in the lithium treated rats remained unaltered when compared to control levels. NA turnover and the NA metabolite, 3-methoxy-4-hydroxyphenylglycol (total MHPG), were significantly lower (p<0.01), whereas both serotonin (5-HT) and its metabolite, 5-hydroxyindole-3-acetic acid (5-HIAA), were significantly higher (p<0.01 and p<0.02, respectively) in the lithium treated rat hypothalami than in controls. Chronic lithium treatment significantly elevated basal TSH levels (p<0.05). This effect was antagonized by methylp-hydroxybenzoate (methylparaben, p<0.01), which did not itself affect basal TSH levels. Free serum T₃ and T₄ levels were not significantly affected by chronic lithium treatment, although T₄ tended to be slightly lower than control levels. The monoamine changes observed in the hypothalamus of lithium treated rats did not appear to account for the elevated TSH levels observed in these rats since NA activity which is generally regarded as stimulatory was decreased and 5-HT which has an inhibitory effect on TSH secretion, was increased. The elevated TSH levels may have been due to a reduced negative feedback inhibition of TSH release by the mildly reduced circulating T₄ levels caused by chronic lithium treatment. A further possibility is that the pituitary cGMP (and hence TSH) response to TRH may have been enhanced by chronic lithium treatment and methylparaben may have antagonized this effect.