Drosophila “enhancer-trap” transposants: Gene expression in chemosensory and motor pathways and identification of mutants affected in smell and taste ability

We have isolated about a thousandDrosophila P-element transposants that allow thein situ detection of genomic enhancer elements by a histochemical assay for β-galactosidase activity. We summarize the β-galactosidase staining patterns of over 200 such transposants in the adult. Our aim was to identify genes that are likely to be involved in the chemosensory and motor pathways ofDrosophila. Based on β-galactosidase expression patterns in the tissues of our interest, we have chosen some strains for further analysis. Behavioral tests on a subset of the transposants have, in addition, identified several strains defective in their chemosensory responses.     

Does genetic conflict drive rapid molecular evolution of nuclear transport genes in Drosophila?

The Segregation Distorter (SD) system of Drosophila melanogaster is one the best-characterized meiotic drive complexes known. SD gains an unfair transmission advantage through heterozygous SD/SD(+) males by incapacitating SD(+)-bearing spermatids so that virtually all progeny inherit SD. Segregation distorter (Sd), the primary distorting locus in the SD complex, is a truncated duplication of the RanGAP gene, a major regulator of the small GTPase Ran, which has several functions including the maintenance of the nucleocytoplasmic RanGTP concentration gradient that mediates nuclear transport. The truncated Sd-RanGAP protein is enzymatically active but mislocalizes to the nucleus where it somehow causes distortion. Here I present data consistent with the idea that wild-type RanGAP, and possibly other loci able to influence the RanGTP gradient, has been caught up in an ancient genetic conflict that predates the SD complex. The legacy of this conflict could include the unexpectedly rapid evolution of nuclear transport-related proteins, the accumulation of chromosomal inversions, the recruitment of gene duplications, and the turnover of repetitive sequences in the centric heterochromatin.     

Metabonomic phenotype and identification of “heart blood stasis obstruction pattern” and “qi and yin deficiency pattern” of myocardial ischemia rat models

The traditional Chinese medicine concepts of “Xinxueyuzuzheng (heart blood stasis obstruction pattern)” and “Qiyinliangxuzheng (qi and yin deficiency pattern)” for myocardial ischemia rat models were constructed in the present study. Endogenous metabolites in rat plasma were analyzed using the GC/TOF-MS-based metabonomic method. Significant metabolic differences were observed between the control and two model groups, and the three groups were distinguished clearly by pattern recognition. Compared with those of the control, the levels of hydroxyproline, threonic acid, glutamine and citric acid were strikingly up- or down-regulated in model rats. The metabolites contributing most to the classification between the two “pattern” rats were identified, such as valine, serine, threonine, ornithine, hydroxyproline, lysine, 2-hydroxybutanoic acid, 3-hydroxybutanoic acid, galactofuranose and inositol. These compounds were indicated as the potential biomarkers. The results suggested that the two “patterns” are involved in dysfunction in oxidative stress, energy metabolism and amino acid metabolism. These findings also provided the substantial foundation for exploring the scientific connotation of these two “Zhengxing (pattern types)” of myocardial ischemia, and “Bianzheng (pattern identification)”.     

Expression and regulation of Spätzle-processing enzyme in Drosophila

The Drosophila melanogaster Toll receptor controls embryonic dorsal-ventral axis formation and is crucial for the innate immune response. In both cases, Toll is activated by the enzymatically cleaved form of its ligand Sp?tzle (Spz). During axis formation, Spz is cleaved by the maternally provided serine protease Easter while the Sp?tzle-processing enzyme (SPE) activates Spz after infection. We confirm the role of SPE in immunity and show that it is a zygotic gene specifically expressed in immune tissues implying that the dual activation of Spz is achieved by differential spatiotemporal expression of two similar but distinct serine proteases.     

Uptake and binding of β-ecdysone in imaginal discs of Drosophila melanogaster

The kinetics of uptake and retention of β-ecdysone by imaginal discs from late third instar larvae of Drosophila melanogaster correspond well with those of the first synthetic response of discs to hormone, an increase in RNA synthesis.Competition studies indicate the presence of two types of hormone binding sites, specific and non-specific. The specific sites are saturated at hormone concentrations which fully induce morphogenesis. Results are consistent with the hypothesis that analogs which induce morphogenesis at differing concentrations bind to the same sites. Experiments with the inhibitors N-ethylmaleimide, actinomycin d, and cycloheximide suggest that the binding sites are pre-existing in the cell and require functional sulfhydryl groups for binding.Specific binding, binding that is competed by excess unlabeled β-ecdysone, is saturable (70–80 nM). Kinetic rate constants for this specific binding were estimated to be k_a = 1.5 × 10⁵M^?1 min^?1, k_d = 3 × 10^?2 min^?1. The equilibrium dissociation constant calculated from the kinetic rate constants was K_eq = 2 × 10^?7M compared to 1.7 × 10^?7M β-ecdysone required to induce morphogenesis in vitro and 2.5 × 10^?7M determined to be the in vivo concentration at the time of induction of morphogenesis.     

Production of Anti-glucagon Antibodies in Poly-L-lysine “Responder” Guinea-pigs

THE availability of anti-glucagon antiserum would greatly facilitate studies on the action of the pancreatic peptide hormone glucagon (molecular weight 3,48s)¹; this peptide is poorly immunogenic^2–8. Grey et al.⁹ report that immunization of rabbits with a glucagon-haemocyanin conjugate yielded antibodies of high affinity. We now describe a method of producing and assaying antibodies to glucagon in which glucagon is coupled to poly-L-lysine (PLL). Immunization of guinea-pigs with glucagon-PLL conjugate yields large amounts of high affinity anti-glucagon antibody; the immune response to PLL and hapten-PLL conjugates is determined by a dominant autosomal genetic factor¹⁰.     

The “survivor effect” and the evolution of parthenogenesis and self-fertilization

Parthenogenetic or self-fertilizing species are often reduced to small numbers of survivors or colonists. These types of reproduction may therefore evolve because they eliminate problems of finding a mate. It is argued here that outbreeding populations which are often reduced to a few survivors will experience heavy offspring losses when population expansion occurs. This ‘survivor effect’ results from the production of many individuals homozygous for deleterious mutations (i.e. inbreeding depression). Parthenogenetic and selfing species do not experience a “survivor effect”. This effect together with mate-finding problems will tend to cause the evolution of parthenogenesis and selfing in weedy or colonizing species.     

Production and degradation of β-Poly-L-malate in cultures of Physarum polycephalum

β-Poly-L-malate (PMA) is synthesized by plasmodia of Physarum polycephalum during growth and secreted into the culture medium. There it is degraded to L-malate after growth has ceased. Its concentration is highest in cell nuclei, where it probably performs a plasmodium-specific function.     

“Fitness Fingerprints” Mediate Physiological Culling of Unwanted Neurons in Drosophila

1. Download : Download high-res image (234KB)
2. Download : Download full-size image

     

Enzyme specificity: “pseudopolymorphism” of lactate dehydrogenase in Drosophila melanogaster

An electrophoretic variant in the LDH (l-lactate:NAD oxidoreductase, E.C.1.1.1.27) of Drosophila melanogaster was observed on starch (or polyacrylamide) gels. This variant was found to exhibit an identical isozymic pattern (three isozymes with a decreasing staining density) on starch gel and map position as the Adh locus. On the other hand, anodal polyacrylamide gel electrophoresis in crude extracts has shown LDH to consist of nine bands and ADH of four bands. We have shown that ADH (Alcohol:NAD oxidoreductase, E.C.1.1.1.1) also oxidizes l(+)-lactate or d(–)-lactate with the NAD, while LDH oxidizes ethanol. By using various genetic and biochemical techniques, we have shown that the observed Ldh electrophoretic variant was not a real one and could be attributed to the presence of ADH. We have called this phenomenon pseudopolymorphism, and the problem of enzyme specificity has been examined. The appearance of a band in an assay using lactic acid as a substrate is not sufficient evidence for the presence of LDH. Hence, caution is called for before characterizing an electrophoretic band on a gel as being equivalent to the presence of a genetic locus. Out of the nine electrophoretic zones of activity observed on polyacrylamide gel (or out of the six previously observed) using crude extract, only two (one major and one minor) belong to LDH, as revealed by purified enzyme preparations. Furthermore, purified LDH exhibits activity in two bands on starch gel (out of three observed in crude extracts), which appear in different positions as compared with those of ADH. Finally, one band which responds to the presence of d(–)-lactate but not to l(+)-lactate has been revealed.     

Quantitative variation of “Mus musculus-like” constitutive heterochromatin and satellite DNA-sequences in the genus Mus

The extent of conservation of constitutive heterochromatin in three species of Mus viz. M. musculus, M. booduga and M. dunni, with shared cytological properties and homologous DNA sequences has been studied. The cytological properties were investigated by doing fluorescence staining and condensation inhibition of their chromosomes with Hoechst 33258. Both the parameters indicate the occurrence of a reduced quantum of M. musculus like heterochromatin at specific sites in the other two genomes. In situ hybridization of the nick translated ³H-labelled M. musculus satellite DNA with M. booduga and M. dunni chromosomes, also corroborates our Hoechst 33258 findings and comparable variation in the amount and site of occurrence of sequences homologous to M. musculus satellite DNA in these species are noticed. The study thus provides a good example of a gradual quantitative variation of a particular type of heterochromatin and in turn of the repetitive DNA constituting it in different related species. Further since the heterochromatin in M. booduga and M. dunni is expected to contain different repetitive DNA sequences in addition to those homologous to M. musculus satellite DNA, it is proposed that a change in the balance between two or more repetitive sequences in heterochromatin may be more crucial in its evolutionary consequences rather than a mere increase or decrease of a homogeneous repetitive sequence.     

Genetics and ontogeny of α-glycerophosphate dehydrogenase isozymes in Drosophila melanogaster

-Glycerophosphate dehydrogenase (GPDH) occurs in Drosophila melanogaster in three isozymic forms. These are separable by starch gel electrophoresis and have been tentatively numbered 1, 2, and 3. GPDH-1 is most concentrated in the adult thorax and GPDH-3 in the abdomen; 1 and 3 are in approximately equal amounts in the head. GPDH-2 is relatively weak in all preparations. In larvae, only GPDH-3 is present. Purified GPDH-1 has optimal activity at pH 6.7–7.0. GPDH-3 at pH 7.5, and GPDH-2 is intermediate. Changes in total GPDH activity parallel larval growth, pupal histolysis, and differentiation of adult tissues. In the latter period the ratio of activity at pH 6.7 to pH 7.6 increases, reflecting the shift from GPDH-3 to GPDH-1. Two types of homozygous GPDH patterns which differ in the electrophoretic mobilities of all three isozymes have been found in inbred strains. In heterozygous adults six bands, the parental forms of GPDH-1 and GPDH-3 and hybrid forms of each, can be resolved. Analysis of F₂ and backcross progeny suggests that a single genetic locus affects all three isozymes. Heterozygous embryos have only the maternal form of GPDH-3 until just before they hatch as first instar larvae. At this stage they have maternal and paternal GPDH-3 plus an intermediate band.This project was supported in part by National Institutes of Health research grant GM-15597.     

Genetics and physiological expression of β-hydroxy acid dehydrogenase in Drosophila

A mutant Had ^nl was induced in Drosophila melanogaster and found to be deficient in -hydroxy acid dehydrogenase. This mutation was utilized to study the genetics and physiological expression of Had ⁺. Had⁺ was mapped to the X chromosome at 54.4 and seems to be the structural gene for the enzyme. Enzyme activity in male and female flies indicates that the gene shows both dosage compensation independent from dose effect and differential activity during ontogeny. Electrophoretic mobility data indicate that the enzyme is a dimer which forms by random association of subunits. The fact that the mutant shows no detrimental effect implies that the enzyme is dispensable, at least under laboratory conditions. The biological and technical implications of this gene-enzyme system are discussed.This research was sponsored by the Energy Research and Development Administration under contract with the Union Carbide Corporation. J. E. T. was a postdoctoral investigator supported by USPHS Fellowship No. 1-F02-GM53673-01 during a portion of this work.     

Hybridization and recurrent evolution of left–right reversal in the land snail genus Schileykula (Orculidae,Pulmonata)

The land snail genus Schileykula Gittenberger, 1983 is distributed in arid limestone areas from western Turkey to north-western Iran. It comprises eight species, which display high variation in shell size and morphology. The cylindrical shells are 5–12 mm in height and the last shell whorls bear several inner lamellae and plicae. Two taxa differ in their chirality having sinistral shells, while all the others are dextrals such as the vast majority of orculids. The aim of this study was to establish a molecular genetic phylogeny of Schileykula and to test whether it conforms to the current morphology-based classification. Furthermore, we were interested in the phylogenetic position of the two sinistral forms in order to assess whether one or two reversals happened in the evolution of the genus. Nine out of ten species, including all four subspecies of Schileykula trapezensis and three of six subspecies of Schileykula scyphus, were investigated. A section of the mitochondrial cytochrome c oxidase subunit I gene was analyzed in 54 specimens of Schileykula and from a subsample, partial sequences of the mitochondrial genes for the 12S rRNA and the 16S rRNA, and a section of the nuclear H4/H3 histone gene cluster were obtained. The phylogenetic trees based on the mitochondrial sequences feature high support values for most nodes, and the species appear well differentiated from each other. The two chiral forms evolved independently and are not sister lineages. However, some groupings disagree with the present morphology-based classification and taxonomical conclusions are drawn. Schileykula trapezensis is polyphyletic in the molecular genetic trees; therefore, three of its subspecies are elevated to species level: Schileykula acampsis Hausdorf, 1996 comb. nov., Schileykula neuberti Hausdorf, 1996 comb. nov., and Schileykula contraria Neubert, 1993 comb. nov. Furthermore, Schileykula sigma is grouped within S. scyphus in the mitochondrial and nuclear trees and consequently treated as a subspecies of the latter (Schileykula scyphus sigma Hausdorf, 1996 comb. nov.). Schileykula nordsiecki, whose shell morphology is indistinguishable from that of the neighboring Schileykula scyphus lycaonica, but who differs in its genital anatomy, was confirmed to represent a distinct lineage. The phylogenies produced by the mitochondrial and nuclear data sets are to some extent conflicting. The patterns differ concerning the grouping of some specimens, suggesting at least two independent hybridization events involving S. contraria, S. scyphus and S. trapezensis. The results exemplify the importance of integrating both mitochondrial and nuclear sequence data in order to complement morphology-based taxonomy, and they provide further evidence for hybridization across distantly related lineages in land snails.     

Production of α-Glucosidase and Its Role in Regulation of Amylase Production by Bacillus circulons F-2

Bacillus circulans F-2 requires a special carbon source or cultural conditions for amylase production. The α-glucosidase production of this bacterium was studied in various cultural conditions with measured glucose concèntrations. High amylase production was always accompanied by low α-glucosidase production and the absence of glucose in culture broth. Usually higher α-glucosidase production was observed in cultural conditions where little amylase was produced. In the presence of 1-deoxynojirimycin, an inhibitor of α-glucosidase activity, the bacterium produced significant amounts of amylase even in conditions giving high α-glucosidase production. It was concluded that the special requirement of this bacterium to produce amylase is effected by its high sensitivity to glucose repression and by the production of α-glucosidase which leads to the formation of glucose. Production of α-glucosidase was, like that of amylase, induced by maltooligosaccharides and repressed by glucose, but both its induction and repression are less sensitive to glucose than those of amylase.     

“Neutral theory” and the dynamics of the evolution of “Modern” human morphology

There is a widespread assumption, even among those who reject the Synthetic Theory of Evolution, that the form of “modern”Homo sapiens is somehow superior to that of archaicHomo sapiens (Tattersall 2000). Those who accept the general outlook of evolutionary biology also tend to assume that “modern” form emerged because it was selected for, which also implies that it was better than that which preceded it. However, after years of using craniofacial measurements to compare human populations, I finally came to realize that, with only a few exceptions, the dimensions measured have no relation to differences in adaptation (Brace 1989, 1996, 2000; Brace et al., 1993). Elsewhere the conclusion has been supported that what is shown by craniometrics is selectively neutral on the average (Relethford 2002). With the documentation that approximately 95% of the genome is not functional, molecular genetics has proved to be useful in documenting the length of time of separation of related human populations by investigating the differences that have accumulated in the neutral parts of the genome. Not surprisingly, the picture revealed by the study of genetic differences is very similar to the one revealed by the study of craniometric differences (Brace et al., 2001). For this reason, the logic behind the “neutral theory” in molecular genetics is very similar to that applied to what happens to morphological characteristics when selection ceases (Brace 1963; Kimura 1968). The difference is that random changes in the neutral part of the genome have no other consequences. However, random changes in the genes that produce specific aspects of morphology will be visible even when selection is no longer controlling the particular trait in question. From an assessment of what random changes in genes controlling morphological traits are likely to do, it follows that the most likely change will probably be a reduction in the trait in question, i.e. the Probable Mutation Effect will produce structural reduction. When survival in the temperate zone during the last glaciation dependend on “obligatory cooking”, one of the unintended consequences was a reduction in the selective pressures maintaining a Middle Pleistocene-sized dentition. The result was a gradual reduction in tooth size and a conversion, of a Neanderthal-sized face into one of “modern” dimensions. The manufacture and use of string for snares and nets similarly reduced the selective pressures maintaining post-cranial levels of robustness and muscularity. The reduction in the latter resulted in the emergence of moderm post-cranial robustness out of what had been a Neanderthal level,in situ wherever the technology can be documented and without any need for invasions and replacements.     

Diversity and evolution of samara in angiosperm北大核心CSCD

Samara (winged fruit) can be dispersed easily by wind and may be a crucial factor for angiosperm spread and diversification. In a narrow sense, a samara is an indehiscent dry fruit with wing(s) developed from fruit pericarp, while in a broad sense samaras also include all winged fruits with wings developed from both pericarp and peri-anth or bracts. According to the wing shape and growth patterns of samaras, we divided samaras into six types, i.e. single-winged, lanceolate-winged, rib-winged, sepal-winged, bract-winged, and perigynous samaras. Perigynous samaras can be further classified into two forms, i.e. round-winged and butterfly-winged samaras. Accordingly, the aerodynamic behavior of samaras can be classified into five types, autogyro, rolling autogyro, undulator, helicopter, and tumbler. The rib-winged and round-winged samaras can be found in Laurales, a basal angiosperm, and may represent the primitive type of early samaras. In the derived clades, samaras evolved enlarged but unequal wings and decreased wing loading (the ratio of fruit weight to wing size), which is likely an adaptation to gentle wind and secondary dispersal through water or ground wind. The wings of some samaras (such as sepal-winged and bract-winged samaras) may have multiple functions including wind dispersal, physical defense for the seeds, and adjust seed germination strategy. The pantropical family Malpighiaceae is extraordinarily rich in samara types, which is likely related to its multiple inter-continent dispersal in history, which is known as “Malpighiaceae Route”. Therefore, Malpighiaceae can be used as a model system for the studies on samara adaptation and evolution. We identified the following issues that deserve further examination in future studies using both ecological and evo-devo methods: 1) the adaption of different types of samaras in dispersal processes, 2) the molecular and developmental mechanism of sepal- and bract-wings, and 3) the evolution of samara types and their effects on angiosperm diversification. © 2018 Editorial Office of Chinese Journal of Plant Ecology. All rights reserved.     

“Hybrid” X-Y translocation chromosomes of Drosophila hydei and D. neohydei

The Y chromosome of Drosophila carries fertility genes which, in part, develop lampbrush loops during the meiotic prophase. Hybrid males from crosses between D. hydei and D. neohydei are fertile although the morphology of the lampbrush loops differs between both species. With the aid of X ray induced hybrid X – Y translocation chromosomes the question has been studied whether Y chromosomal genes of D. neohydei can substitute deletions in the Y chromosome of D. hydei. Although the induction of translocation chromosomes almost regularly results in an inactivation of the translocated Y fragment within a few generations, one case of successful complementation has been demonstrated. Furthermore, a new lampbrush loop pair has been detected in D. neohydei which is morphologically similar to the nooses of D. hydei. Preliminary evidence for the location of the lampbrush loops on the Y chromosome of D. neohydei is discussed.