The temporal pattern of vitellogenin synthesis in Drosophila grimshawi

The temporal pattern of protein production and, in particular, vitellogenin protein synthesis during the sexual maturation of Drosophila grimshawi females has been studied in vivo by briefly feeding the flies with 35S-methionine and 3H-amino acids. The overall level of incorporation was very low in young flies; it then progressively increased to reach a maximum with the onset of sexual maturity at 13-15 days. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses revealed three classes of proteins: those synthesized throughout the age spectrum, which constitute the majority of protein species; proteins synthesized primarily or only in young flies; and proteins synthesized only by the older flies. In this Drosophila species, the three vitellogenins (V1, V2, and V3) appeared to be synthesized in a two-phase pattern. In the first phase, small quantities of V1 and V2 were detected immunologically in the fat body and hemolymph of newly emerged and 1 day-old flies. These proteins did not accumulate in the hemolymph or the ovaries, apparently being unstable proteins. The second phase commenced in early vitellogenesis (7-9 days of age) with synthesis in the fat body of small quantities of V1 and V2, followed by V3 proteins. These proteins were secreted and accumulated in the hemolymph and 24 h later were found in the ovaries. Their quantities increased rapidly and a steady state of synthesis, release into the hemolymph, and uptake by the ovaries was reached by days 13-15. We have estimated that during the steady state of vitellogenin synthesis, a fly can synthesize in 24 h at least 152 micrograms of vitellogenins, which is more than 2% of its body weight, at an average rate of about 6.3 micrograms vitellogenins/h. About 2 micrograms of this are synthesized in the fat body, and about 4 micrograms in the ovaries. These findings are discussed in terms of their physiological implications and contrasted with the available data on Drosophila melanogaster.     

Dynamic filtration of the expression pattern variability of Drosophila zygotic segmentation genes

Analysis of the quantitative data obtained by processing the confocal images showed that the initial variability of the expression pattern of Drosophila zygotic segmentation genes was strongly reduced by the onset of gastrulation. The following variability components were studied: the range of gene expression intensity in different embryos, the time and succession of the formation of expression domain, types of formation, and domain positioning. At the level of zygotic genes, the positioning error proved to be dynamically filtered with time.     

Noncoordinate synthesis of the vitellogenin proteins in tissues of Drosophila grimshawi

In analyzing the in vitro pattern of protein synthesis by the fat body and ovaries of the Hawaiian species Drosophila grimshawi, we have found that the ovaries synthesize much more protein than the female fat body and that the majority of the synthesized proteins are retained by the ovarian tissues. In contrast, the fat body secrets most of the proteins into the culture medium. Vitellogenins are the major class of proteins synthesized and released into the medium by both tissues. The synthesis of the three vitellogenin proteins (V1, V2, V3) is noncoordinate in the two tissues. Ovaries synthesize much more of the V2 protein, less V1 and very little V3, whereas fat body synthesizes more V1 protein with lesser quantities of the other two. The follicle cells were identified as the site of ovarian vitellogenin synthesis in D. grimshawi, confirming the findings in D. melanogaster. In D. grimshawi, the three vitellogenins are synthesized by the follicle cells in a noncoordinate and developmentally regulated manner. V2 and V1 are the predominant proteins at the onset of vitellogenesis (S8-9); their production peaks together with that of V3 a few hours later (S10) and then decreases to quantities equal to that of V3 during early choriogenesis (S11). During active choriogenesis (S12), V2 and V1 cease to be synthesized, but V3 synthesis continues. The vitellogenins synthesized by the follicles in vitro are released into the medium and not incorporated into the oocyte.     

Hox genes and the regulation of movement in Drosophila

Many animals show regionally specialized patterns of movement along the body axis. In vertebrates, spinal networks regulate locomotion, while the brainstem controls movements of respiration and feeding. Similarly, amongst invertebrates diversification of appendages along the body axis is tied to the performance of characteristically different movements such as those required for feeding, locomotion, and respiration. Such movements require locally specialized networks of nerves and muscles. Here we use the regionally differentiated movements of larval crawling in Drosophila to investigate how the formation of a locally specialized locomotor network is genetically determined. By loss and gain of function experiments we show that particular Hox gene functions are necessary and sufficient to dictate the formation of a neuromuscular network that orchestrates the movements of peristaltic locomotion.     

The distribution of the dinucleotide CpG and cytosine methylation in the vitellogenin gene family

Summary Sequence data from regions of five vertebrate vitellogenin genes were used to examine the frequency, distribution, and mutability of the dinucleotide CpG, the preferred modification site for eukaryotic DNA methyltransferases. The observed level of the CpG dinucleotide in all five genes was markedly lower than that expected from the known mononucleotide frequencies. CpG suppression was greater in introns than in exons. CpG-containing codons were found to be avoided in the vitellogenin genes, but not completely despite the redundancy of the genetic code. Frequency and distribution patterns of this dinucleotide varied dramatically among these otherwise closely related genes. Dense clusters of CpG dinucleotides tended to appear in regions of either functional or structural interest (e.g., in the transposon-like Vi-element ofXenopus) and these clusters contained 5-methylcytosine (5 mC). 5 mC is known to undergo deamination to form thymidine, but the extent to which this transition occurs in the heavily methylated genomes of vertebrates and its contribution to CpG suppression are still unclear. Sequence comparison of the methylated vitellogenin gene regions identified CT and GA substitutions that were found to occur at relatively high frequencies. The predicted products of CpG deamination, TpG and CpA, were elevated. These findings are consistent with the view that CpG distribution and methylation are interdependent and that deamination of 5 mC plays an important role in promoting evolutionary change at the nucleotide sequence level.     

果蝇基因组与功能基因研究进展

黑腹果蝇Drosophila melanogaster是生物科学研究中重要的模式动物之一。2000年,黑腹果蝇全基因组测序完成,随后基因组序列质量不断完善,对其功能基因进行深入研究,为其他高等动物基因组和功能基因的研究提供了巨大帮助。本文综述了近年来基因组功能元件、比较基因组学等方面的最新研究成果,着重介绍了功能基因在Hh信号通路、细胞凋亡方面的研究进展,并对最新的功能基因研究技术进行了简要概述。     

Involvement of polycomb-group genes in establishing HoxD temporal colinearity

Temporal colinearity in mouse HoxD is dependent on repressive activity of sequences within the 5' end of the complex. We show that a 5-kb DNA fragment from this region represses transgenes when combined in mouse as well as in Drosophila melanogaster. Moreover, repressive activity in Drosophila depends on some members of the Polycomb-group (PcG) genes, for example, extra sex combs. We also showed direct association of these factors with the repressive fragment, both in transgenic flies and in the context of the native mouse HoxD complex. These results suggest that the global repressive region of the HoxD complex functions in two very different species and that some PcG genes are involved in establishing the early repressive state of the HoxD complex, thus contributing to temporal colinearity.     

Survival and reproduction when food is scarce: implications for a lekking Hawaiian Drosophila

Abstract.  1. In insects, larval diet can have a major impact on development, survival, and reproductive success. However, resource availability at the adult phase of the life cycle is also likely to have strong effects in species where there is an extended period of sexual maturation following adult eclosion.
2. The effect of diet on the survival and reproductive success of the lekking Hawaiian fruit fly, Drosophila grimshawi , was explored. Two generations of emerging adults were exposed to one of two feeding regimes: 'constant' and 'varied' (corresponding to food 'each day' or 'every other day' respectively). The impact of resource availability on survival and reproductive success in each generation was then investigated.
3. The probability of survival to 5 weeks old was higher for individuals fed a constant diet than individuals fed a varied diet, but was comparable for males and females.
4. There was a significant maternal effect on offspring survival. Offspring whose mothers were reared on a constant feeding regime had higher survival than offspring whose mothers were reared on a varied diet.
5. There was no relationship between feeding regime and the quantity of pheromones deposited by males (a measure of male reproductive investment); however F₂ sons were more likely to deposit pheromones and deposited a larger quantity of pheromone than their F₁ sires. The number and sex ratio of offspring (a measure of female reproductive effort) emerging from the F₁ generation was unrelated to maternal or paternal feeding regime.
6. The implications of variation in the foraging environment for mate choice in D. grimshawi are discussed.     

Genetic control of the developmental program of L-glycerol-3-phosphate dehydrogenase isozymes in Drosophila melanogaster: Identification of a cis-acting temporal element affecting GPDH-3 expression

The complete developmental program of glycerol-3-phosphate dehydrogenase in wild type Drosophila is described with respect to activity, isozyme expression, and GPDH-specific CRM. Variants of this developmental program have been isolated from natural populations which affect the rate of accumulation of only the GPDH-3 isozyme in both the larval and adult stages of development. This activity variation segregates as a single gene which is tightly linked to the structural element on Chromosome II, exhibits cis-control, and is tissue specific in expression. This gene meets all the criteria for temporal regulatory genes.     

Cloning and expression in Escherichia coli of tryptophan genes from Streptomyces griseus IMRU 3570

Two Sau3A fragments of Streptomyces grisues IMRU 3570 were cloned in pBR322 as a vector. One of these clones contained the genetic information needed to complement trpA and trpB mutations in Escherichia coli. The other complements trpA, trpB and trpC mutations in E. coli. Both fragments originated in the same region of the chromosome but the latter is 1 kilobase (kb) longer in the region nearest the tetracycline promoter.     

Complex cis-acting regulatory genes demonstrated in Drosophila hybrids

Drosophila heteroneura and D differens are closely related, interfertile species of the Hawaiian picture-winged group. They display marked qualitative and quantitative differences in the pattern of expression of alcohol dehydrogenase (ADH) and an aldehyde oxidase (AO-1). These presumptive regulatory differences are revealed by comparisons of the relative levels of these enzymes in various tissues in larvae and adults. In hybrids produced between parents carrying different electrophoretic alleles at the structural loci for these two enzymes, each allele is expressed according to the developmental program characteristic of the parent from which it was derived. This result indicates control of the differences in pattern of expression by one or more cis-acting sites associated with each structural locus. The distribution of activity among all the three forms of these dimeric enzymes produced in hybrids indicates that the pattern differences reflect differential accumulation of enzyme molecules, not altered catalytic properties. As expected, the regulatory differences segregate with the electrophoretic markers in backcrosses.     

Structural and expression analyses of two vitellogenin genes in the carp, Cyprinus carpio

We cloned and sequenced two vitellogenin (vg) cDNAs of the carp, Cyprinus carpio, using a cDNA library constructed from estradiol-17β (E₂)-treated livers. One was a novel, longer 5000 bp-long cDNA termed vg-B2 encoding 1624 amino acids in a single open reading frame. The other was a shorter cDNA (vg-B1), identical to that registered previously as carp vg cDNA in the international nucleotide sequence database. The deduced amino acid sequences of these two molecules were well-aligned with known vertebrate Vgs sharing common characteristics such as N-terminal lipovitellin I (LVI), phosvitin (PV) and C-terminal lipovitellin II (LVII). The novel Vg-B2 bore a highly conserved GL/ICG motif within the LVII region, in contrast to the shorter Vg-B1 that has a truncated C-terminal and lacks the β-component within the LVII region including the GL/ICG motif. Both vg-B2 and vg-B1 genes were expressed in the livers of females and E₂-injected males. Western blot analysis using anti-Vg and anti-vitellin (Vn) antisera demonstrated that both Vg-B2 and Vg-B1 were detected as polypeptides with an estimated molecular mass of 180 kDa and 160 kDa, respectively, in the blood of females and E₂-injected males. The results suggest the potential utilization of these genes as sensitive xenoestrogenic markers.     

Genetic interaction between homoeotic Sex combs reduced and Regulator of bithorax (or trithorax) genes of Drosophila melanogaster

Summary Regulator of bithorax (Rg-bx)^–, or trithorax (trx)^– lethal larvae occasionally show a homoeotic transformation of the dorsal prothorax to mesothoracic structures. This transformation suggests a reduced activity of the Sex combs reduced (Scr) gene on the basis of gene dosage studies, as well as enhanced expression of the phenotypes of the weak Scr^– alleles in Rg-bx^– larvae. Morphological observations of adult flies doubly heterozygous for Rg-bx and Scr mutations also suggest the enhancement of an aspect of Scr adult phenotypes. I conclude that the Rg-bx⁺ gene function is required for the optimal expression of the Scr gene in larval and imaginai cells.     

Genomic background drives the divergence of duplicated amylase genes at synonymous sites in Drosophila

In some Drosophila species, there are two types of greatly diverged amylase (Amy) genes (Amy clusters 1 and 2), each encoding active amylase isozymes. Cluster 1 is located at the middle of its chromosomal arm, and the region has a normal local recombination rate. However, cluster 2 is near the centromere, and this region is known to have a reduced recombination rate. Although nonsynonymous substitutions follow a molecular clock, synonymous substitutions were accelerated in cluster 2 after gene duplications. This resulted in a higher GC content at the third codon position (GC3) and codon usage bias in cluster 1, and lower GC3 content and codon usage bias in the cluster 2. However, no systematic difference in GC content was observed in the first and second codon positions or the 3'-flanking regions. Therefore, differences in local recombination rate rather than mutation bias might explain the divergence at synonymous sites between the two Amy clusters within species (Hill-Robertson effect). Alternatively, the different patterns and levels of expression between the two clusters may imply that the reduced expression level in cluster 2 caused by chromatin potentiation decreased the codon bias. Both of these hypotheses imply the importance of the genomic background as a driving force of divergence between non-tandemly duplicated genes.     

Isolation of developmentally regulated genes in Drosophila melanogaster

We used a molecular approach to search for maternally expressed genes in Drosophila melanogaster. The relative merits of differential and competition screens were analyzed in a series of reconstruction experiments using either purified phage plaques or derivative DNA sequences. In the course of this study, we isolated 5 clones whose RNA level varies during early embryogenesis. Three gastrula differential clones, b4, b8 and d3, are present in numerous copies in the genome; clone b4 hybridizes with the copia-like B104 repetitive sequence described by Scherer et al. We also isolated 2 maternally-expressed genes, not previously identified in either classical genetic or similarly molecular-based screens. These clones, b11 and d6, map at cytogenetic positions 98F and 4F respectively, on the polytene chromosome map.