Synergistic interaction between particular X-chromosome deletions andSex-lethal causes female lethality inDrosophila melanogaster

We studied the effect on female viability of trans-heterozygous combinations of X-chromosome deficiencies and Sxt-(fl), a null allele of Sex-lethal. Twentyfive deficiencies, which together covered 80% of the X chromosome, were tested. Seven of these trans-heterozygous combinations caused significant levels of female lethality. Two of the seven interacting deficiencies include the previously known sex determination genes sans fille and sisterless-a. Four of the remaining uncover X-chromosomal regions that were not hitherto known to contain sex determination genes. These newly identified regions are defined by deficiencies Df(1)RA2 (7D10; 8A4-5), Df(1)KA14 (7F1-2; 8C6), Df(1)C52 (8E; 9C-D) and Df(1)N19 (17A1; 18A2). These four deficiencies were characterized further to determine whether it was the maternal or zygotic dosage that was primarily responsible for the observed lethality of female embryos, daughterless and extra macrochaetae, two known regulators of Sxl, influence the interaction of these deficiencies with Sxl.

Synthesis of Trilaurin by Developing Pisa Seeds (Actinodaphne hookeri)

The developing seeds of Actinodaphne hookeri were investigated to delineate their ability to synthesize large amounts of trilaurin. Until 88 days after flowering the embryos contained 71% neutral lipids (NL) and 29% phospholipids (PL) and both these components contained C-16:0, C-18:0, C-18:2, and C-18:3 as the major fatty acids (FA). At 102 days after flowering the seeds began to accumulate triacylglycerols (TAG) and to synthesize lauric acid (C-12:0). By 165 days after flowering, when the seeds were mature, they contained about 99% NL and 1% FL. At this stage the TAG contained exclusively C-12:0, while the PL consisted of long-chain fatty acids (LCFA) only. Leaf lipids in contrast did not contain any C-12:0. Experiments on [1-C-14]acetate incorporation into developing seed slices showed that at 88 days after flowering only 4% of the label was in TAG, 1% in diacylglycerols (DAG), and 87% in FL. One hundred two days after flowering seeds incorporated only 2% of the label into TAG, 30% into DAG, and 64% into FL. In contrast at 114 days after flowering 71% of the label was incorporated into TAG, 25% into DAG, and only 2% into FL. Analysis of labeled FA revealed that up to 102 days after flowering it was incorporated only into LCFA, whereas at 114 days after flowering it was incorporated exclusively into C-12:0. Furthermore, 67% of the label in PL at 114 days after flowering was found to be dilaurylglycerophosphate. Analysis of the label in DAG at this stage showed that it was essentially in dilaurin species. These observations indicate the induction of enzymes of Kennedy pathway for the specific synthesis of trilaurin at about 114 days after flowering, Homogenates of seeds (114 days after flowering) incubated with labeled FA in the presence of glycerol-3-phosphate and coenzymes A and ATP incorporated 84% of C-12:0 and 61% of C-14:0, but not C-16:0, C-18:2, and C-18:3, into TAG. In contrast the LCFA were incorporated preferentially into FL. It is concluded that, between 102 and 114 days after flowering, a switch occurs in A. hookeri for the synthesis of C-12:0 and trilaurin which is tissue specific. Since the seed synthesizes exclusively C-12:0 at 114 days after flowering onwards and incorporates specifically into TAG, this system appears to be ideal for identifying the enzymes responsible for medium-chain fatty acid as well as trilaurin synthesis and for exploiting them for genetic engineering. (C) 1994 Academic Press, Inc.

Social selection and the evolution of cooperative groups: The example of the cellular slime moulds

In social selection the phenotype of an individual depends on its own genotype as well as on the phenotypes, and so genotypes, of other individuals. This makes it impossible to associate an invariant phenotype with a genotype: the social context is crucial. Descriptions of metazoan development, which often is viewed as the acme of cooperative social behaviour, ignore or downplay this fact. The implicit justification for doing so is based on a group-selectionist point of view. Namely, embryos are clones, therefore all cells have the same evolutionary interest, and the visible differences between cells result from a common strategy. The reasoning is flawed, because phenotypic heterogeneity within groups can result from contingent choices made by cells from a flexible repertoire as in multicellular development. What makes that possible is phenotypic plasticity, namely the ability of a genotype to exhibit different phenotypes. However, co-operative social behaviour with division of labour requires that different phenotypes interact appropriately, not that they belong to the same genotype, or have overlapping genetic interests. We sketch a possible route to the evolution of social groups that involves many steps: (a) individuals that happen to be in spatial proximity benefit simply by virtue of their number; (b) traits that are already present act as preadaptations and improve the efficiency of the group; and (c) new adaptations evolve under selection in the social context-that is, via interactions between individuals-and further strengthen group behaviour. The Dictyostelid or cellular slime mould amoebae (CSMs) become multicellular in an unusual way, by the aggregation of free-living cells. In nature the resulting group can be genetically homogeneous (clonal) or heterogeneous (polyclonal); in either case its development, which displays strong cooperation between cells (to the extent of so-called altruism) is not affected. This makes the CSMs exemplars for the study of social behaviour.

Developmental analysis of a female-specific 16S rRNA gene from mycetome-associated endosymbionts of a mealybug, Planococcus lilacinus

A clone showing female-specific expression was identified from an embryonic cDNA library of a mealybug, Planococcus lilacinus, In Southern blots this clone (P7) showed hybridization to genomic DNA of females, but not to that of males, However, P7 showed no hybridization to nuclei of either sex, raising the possibility that it was extrachromosomal in origin, In sectioned adult females P7 hybridized to an abdominal organ called the mycetome. The mycetome is formed by mycetocytes, which are polyploid cells originating from the polar bodies and cleavage nuclei that harbour maternally transmitted, intracellular symbionts. Electron microscopy confirmed the presence of symbionts within the mycetocytes, Sequence analysis showed that P7 is a 16S rRNA gene, confirming its prokaryotic origin, P7 transcripts are localized to one pole in young embryos but are found in the pole as well as in the germ band during later stages of development, P7 expression is detectable in young embryos of both sexes but the absence of P7 in third instar and adult males suggests that this gene, and hence the endosymbionts, are subject to sex-specific elimination. Copyright (C) 1997 Elsevier Science Ltd.

The determination of spatial pattern in Dictyostelium discoideum

Free-living amoebae of the cellular slime mould Dictyostelium discoideum aggregate when starved and give rise to a long and thin multicellular structure, the slug. The slug resembles a metazoan embryo, and as with other embryos it is possible to specify a fate map. In the case of Dictyostelium discoideum the map is especially simple: cells in the anterior fifth of the slug die and form a stalk while the majority of those in the posterior differentiate into spores. The genesis of this anterior-posterior distinction is the subject of our review. In particular, we ask: what are the relative roles of individual pre-aggregative predispositions and post-aggregative position in determining cell fate? We review the literature on the subject and conclude that both factors are important. Variations in nutritional status, or in cell cycle phase at starvation, can bias the probability that an amoeba differentiates into a stalk cell or a spore. On the other hand, isolates, or slug fragments, consisting of only prestalk cells or only prespore cells can regulate so as to result in a normal range of both cell types. We identify three levels of control, each being responsible for guiding patterning in normal development: (i) 'coin tossing', whereby a cell autonomously exhibits a preference for developing along either the stalk or the spore pathway with relative probabilities that can be influenced by the environment; (ii) 'chemical kinetics', whereby prestalk and prespore cells originate from undifferentiated amoebae on a probabilistic basis but, having originated, interact (e.g. via positive and negative feedbacks), and the interaction influences the possibility of conversion of one cell type into the other, and (iii) 'positional information', in which the spatial distribution of morphogens in the slug influences the pathway of differentiation. In the case of possibilities (i) and (ii), sorting out of like cell types leads to the final spatial pattern. In the case of possibility (iii), the pattern arises in situ.

Secretion of endocrine signals by the primate embryo during the peri-implantation period

Development of preimplantation embryos and blastocyst implantation are critical early events in the establishment of pregnancy. In primates, embryonic signals, secreted during the peri-implantation period, are believed to play a major role in the regulation of embryonic differentiation and implantation. However, only limited progress has been made in the molecular and functional characterization of embryonic signals, partly due to severe paucity of primate embryos and the lack of optimal culture conditions to obtain viable embryo development. Two embryonic (endocrine) secretions, i.e. chorionic gonadotrophin (CG) and gonadotrophin releasing hormone (GnRH) are being studied. This article reviews the current status of knowledge on the recovery and culture of embryos, their secretion of CG, GnRH and other potential endocrine signals and their regulation and physiological role(s) during the peri-implantation period in primates, including humans.

Structure and activity cyclase activated of OK-GC: A kidney receptor-guanylate cyclase activated by guanylin peptides

Uroguanylin, guanylin, and lymphoguanylin are small peptides that activate renal and intestinal receptor guanylate cyclases (GC). They are structurally similar to bacterial heat-stable enterotoxins (ST) that cause secretory diarrhea. Uroguanylin, guanylin, and ST elicit natriuresis, kaliuresis, and diuresis by direct actions on kidney GC receptors. A 3,762-bp cDNA characterizing a uroguanylin/guanylin/ST receptor was isolated from opossum kidney (OK) cell RNA/cDNA. This kidney cDNA (OK-GC) encodes a mature protein containing 1,049 residues sharing 72.4�75.8% identity with rat, human, and porcine forms of intestinal GC-C receptors. COS or HEK-293 cells expressing OK-GC receptor protein were activated by uroguanylin, guanylin, or ST13 peptides. The 3.8-kb OK-GC mRNA transcript is most abundant in the kidney cortex and intestinal mucosa, with lower mRNA levels observed in urinary bladder, adrenal gland, and myocardium and with no detectable transcripts in skin or stomach mucosa. We propose that OK-GC receptor GC participates in a renal mechanism of action for uroguanylin and/or guanylin in the physiological regulation of urinary sodium, potassium, and water excretion. This renal tubular receptor GC may be a target for circulating uroguanylin in an endocrine link between the intestine and kidney and/or participate in an intrarenal paracrine mechanism for regulation of kidney function via the intracellular second messenger, cGMP.

Structure and activity of OK-GC: a kidney receptor guanylate cyclase activated by guanylin peptides

Uroguanylin, guanylin, and lymphoguanylin are small peptides that activate renal and intestinal receptor guanylate cyclases (GC). They are structurally similar to bacterial heat-stable enterotoxins (ST) that cause secretory diarrhea. Uroguanylin, guanylin, and ST elicit natriuresis, kaliuresis, and diuresis by direct actions on kidney GC receptors. A 3,762-bp cDNA characterizing a uroguanylin/guanylin/ST receptor was isolated from opossum kidney (OK) cell RNA/cDNA. This kidney cDNA (OK-GC) encodes a mature protein containing 1,049 residues sharing 72.4-75.8% identity with rat, human, and porcine forms of intestinal GC-C receptors. COS or HEK-293 cells expressing OK-GC receptor protein were activated by uroguanylin, guanylin, or ST13 peptides. The 3.8-kb OK-GC mRNA transcript is most abundant in the kidney cortex and intestinal mucosa, with lower mRNA levels observed in urinary bladder, adrenal gland, and myocardium and with no detectable transcripts in skin or stomach mucosa. We propose that OK-GC receptor GC participates in a renal mechanism of action for uroguanylin and/or guanylin in the physiological regulation of urinary sodium, potassium, and water excretion. This renal tubular receptor GC may be a target for circulating uroguanylin in an endocrine link between the intestine and kidney and/or participate in an intrarenal paracrine mechanism for regulation of kidney function via the intracellular second messenger, cGMP.

Molecular cloning and expression pattern of a Cubitus interruptus homologue from the mulberry silkworm Bombyx mori

A homologue of the segment polarity gene Cubitus interruptus from Bombyx Mori, (BmCi) has been cloned and characterized. This region harbouring Zn2+ finger motif is highly conserved across species. In B. Mori, BmCi RNA expression was first detected at stage 6 of embryogenesis, which reached maximum levels at stage 21C and was maintained until larval hatching. The segmentally reiterated striped pattern of transcript distribution in stage 21C embryos was in conformity with its predicted segment polarity nature. BmCi was expressed in the fore- and hind-wing discs, ovaries, testes and gut during fifth larval intermolt, reminiscent of its expression domains in Drosophila. Besides, BmCi expression was seen in the. anterior part of the middle silkglands in late embryonic stages, and this pattern was maintained during larval development. The transition from third to fourth and fifth larval intermolts was accompanied by an increase in the transcript levels in the middle silkglands. Our results demonstrate the presence of a novel expression domain for Ci in Bombyx. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.

Prevalence of, and antigenic variation in, serotype G10 rotaviruses and detection of serotype G3 strains in diarrheic calves: Implications for the origin of G10P11 or P11 type reassortant asymptomatic strains in newborn children in India

Previous studies have shown predominant association of G10P11 type bovine rotavirus-derived reassortant strains with asymptomatic infections in newborn children in India. To understand the epidemiological and genetic basis for the origin of these strains in humans, the relative frequencies of different serotypes among bovine rotaviruses (BRVs) isolated from southern, western and central regions of the country were determined by subgroup and serotype analysis as well as nucleotide (nt) sequence analysis of the genes encoding the outer capsid proteins VP4 and VP7. Since the human G10P11 asymptomatic neonatal strain I321 possessed NSP1 from a human rotavirus, to determine its genetic origin in the bovine strains, comparative analysis of partial gene sequences from representative G10P11 strains was also carried out. The following observations were of great epidemiological significance, (i) G10P11 strains predominated in all the three regions with frequencies ranging between 55.6% and 85.2%. In contrast to the high prevalence of G6 strains in other countries, only one G6 strain was detected in this study and G8 strains represented 5.8% of the isolates, (ii) among the G10 strains, in serotyping ELISA, four patterns of reactivity were observed that appeared to correlate with the differences in electropherotypic patterns and amino acid (aa) sequence of the VP7, (iii) surprisingly, strains belonging to serotype G3 were detected more frequently (10.7%) than those of serotypes G6 and G8 combined, while strains representing the new serotype (G15) were observed in a single farm in Bangalore, and (iv) about 3.9% of the isolates were nontypeable as they exhibited high cross-reactivity to the serotyping MAbs used in the study. Comparative analysis of the VP7 gene sequence from the prototype G3 MAb-reactive bovine strain J63 revealed greatest sequence relatedness (87.6% nt and 96.0% aa) with that of serotype G3 rhesus-monkey strain RRV. It also exhibited high sequence homology with the VP7 from several animal and animal rotavirus-related human G3 strains (Simian SA11; equine ERV316 and FI-14. canine CU-1 and K9; porcine 4F; Feline Cat2 and human HCR3, YO and AU1). Partial nucleotide sequence analysis of the NSP1 gene of J63 showed greatest nt sequence homology (95.9%) to the NSP1 gene allele of the Indian G8 strain, isolated from a diarrheic child, which is likely to have been transmitted directly from cattle and 92.6% homology to that of the bovine G8 strain A5-10 suggesting the likely origin of J63 by gene reassortment between a bovine G8 strain and a G3 animal strain. Prevalence of G10P11 strains in cattle and G10P11 or P11 type reassortant strains in asymptomatic neonates as well as detection of G8P[1] strains in diarrheic children support our hypothesis for bidirectional transmission of rotaviruses between humans and cattle and origin of novel strains catalyzed by the age-old traditions and socio-economic conditions in India.

Reconstructing Solid Model from 2D Scanned Images of Biological Organs for Finite Element Simulation

This work presents a methodology to reconstruct 3D biological organs from image sequences or other scan data using readily available free softwares with the final goal of using the organs (3D solids) for finite element analysis. The methodology deals with issues such as segmentation, conversion to polygonal surface meshes, and finally conversion of these meshes to 3D solids. The user is able to control the detail or the level of complexity of the solid constructed. The methodology is illustrated using 3D reconstruction of a porcine liver as an example. Finally, the reconstructed liver is imported into the commercial software ANSYS, and together with a cyst inside the liver, a nonlinear analysis performed. The results confirm that the methodology can be used for obtaining 3D geometry of biological organs. The results also demonstrate that the geometry obtained by following this methodology can be used for the nonlinear finite element analysis of organs. The methodology (or the procedure) would be of use in surgery planning and surgery simulation since both of these extensively use finite elements for numerical simulations and it is better if these simulations are carried out on patient specific organ geometries. Instead of following the present methodology, it would cost a lot to buy a commercial software which can reconstruct 3D biological organs from scanned image sequences.

On the dynamics of activation of mammalian X chromosomes

We have investigated a mathematical model of the process of activation of the X chromosomes in eutherian mammals. The model assumes that the activation is brought about over some definite time interval T by the complete saturation of N receptor sites on an X chromosome by M activating molecules (or multiples of M). The probability λ of a first hit on the receptor site is considered to be very much lower than that of subsequent hits; that is, we assume strong co-operative binding. Assuming further that an incomplete saturation of receptor sites is malfunctional, we can show that for proper activation of X chromosomes in normal diploid males and females, we must have λMT ≥ 3 and 0·96 ≤ N/M ≤ 1. An extension of this analysis for the triploid cases shows that under these conditions, we cannot explain the activation of two X's if the number of activating molecules is fixed at M. This suggests that there must be two classes of triploid embryos differing from each other in a step-wise manner in the number of activating molecules. In other words, triploids with two active X chromosomes would require 2M activating molecules as opposed to M molecules in triploids with a single active X. This interpretation of the two classes of triploids would be consistent with differing imprinting histories of the parental contributions to the triploid zygote.

Time-Dependent Predominance of Nonhomologous DNA End-Joining Pathways during Embryonic Development in Mice

Repair of DNA double-strand breaks (DSBs) is crucial for maintaining genomic integrity during the successful development of a fertilized egg into a whole organism. To date, the mechanism of DSB repair in postimplantation embryos has been largely unknown. In the present study, using a cell-free repair system derived from the different embryonic stages of mice, we find that canonical nonhomologous end joining (NHEJ), one of the major DSB repair pathways in mammals, is predominant at 14.5 day of embryonic development. Interestingly, all four types of DSBs tested were repaired by ligase IV/XRCC4 and Ku-dependent classical NHEJ. Characterization of end-joined junctions and expression studies further showed evidences for canonical NHEJ. Strikingly, in contrast to the above, we observed noncanonical end joining accompanied by DSB resection, dependent on microhomology and ligase III in 18.5-day embryos. Interestingly, we observed an elevated expression of CtIP, MRE11, and NBS1 at this stage, suggesting that it could act as a switch between classical end joining and microhomology-mediated end joining at later stages of embryonic development. Thus, our results establish for the first time the existence of both canonical and alternative NHEJ pathways during the postimplantation stages of mammalian embryonic development. (C) 2012 Elsevier Ltd. All rights reserved.

NMR studies of preimplantation embryo metabolism in human assisted reproductive techniques: a new biomarker for assessment of embryo implantation potential

There has been growing interest in understanding energy metabolism in human embryos generated using assisted reproductive techniques (ART) for improving the overall success rate of the method. Using NMR spectroscopy as a noninvasive tool, we studied human embryo metabolism to identify specific biomarkers to assess the quality of embryos for their implantation potential. The study was based on estimation of pyruvate, lactate and alanine levels in the growth medium, ISM1, used in the culture of embryos. An NMR study involving 127 embryos from 48 couples revealed that embryos transferred on Day 3 (after 72 h in vitro culture) with successful implantation (pregnancy) exhibited significantly (p < 10(-5)) lower pyruvate/alanine ratios compared to those that failed to implant. Lactate levels in media were similar for all embryos. This implies that in addition to lactate production, successfully implanted embryos use pyruvate to produce alanine and other cellular functions. While pyruvate and alanine individually have been used as biomarkers, the present study highlights the potential of combining them to provide a single parameter that correlates strongly with implantation potential. Copyright (C) 2012 John Wiley & Sons, Ltd.

Expression and function of cyclooxygenase-2 is necessary for hamster blastocyst hatching

Blastocyst hatching is critical for successful implantation leading to pregnancy. Its failure causes infertility. The phenomenon of blastocyst hatching in humans is poorly understood and the available information on this stems from studies of rodents such as mice and hamsters. We and others showed that hamster blastocyst hatching is characterized by firstly blastocyst deflation followed by a dissolution of the zona pellucida (zona) and accompanied by trophectodermal projections (TEPs). We also showed that embryo-derived cathepsins (Cat) proteases, specifically Cat-L, -B and -P act as zonalysins and are responsible for hatching. In this study, we show the expression and function of one of the potential regulators of embryogenesis, cyclooxygenase (COX)-2 during blastocyst development and hatching. The expression of COX-2 mRNA and protein was observed in 8-cell through hatched blastocyst stages and it was also localized to blastocysts TEPs. Specific COX-2 inhibitors, NS-398 and CAY-10404, inhibited blastocyst hatching; percentages achieved were only 28.4 5.3 and 32.3 5.4, respectively, compared with 90 with untreated embryos. Interestingly, inhibitor-treated blastocysts failed to deflate, normally observed during hatching. Supplementation of prostaglandins (PGs)-E-2 or -I-2 to cultured embryos reversed the inhibitors effect on hatching and also the deflation behavior. Importantly, the levels of mRNA and protein of Cat-L, -B and -P showed a significant reduction in the inhibitor-treated embryos compared with untreated embryos, although its mechanism remains to be examined. These data provide the first evidence that COX-2 is critical for blastocyst hatching in the golden hamster.