Significant fluctuations in ecdysteroid receptor gene (EcR) expression in relation to seasons of molt and reproduction in the grapsid crab, Metopograpsus messor (Brachyura: Decapoda).

Metopograpsus messor, a brachyuran crab inhabiting the estuaries of North Kerala (India), is a prolific breeder releasing approximately 14-16 broods a year. The present paper reports the sequence information on the DNA binding domain (C domain, DBD), linker (D domain) and ligand binding domain (E domain, LBD) of M. messor ecdysteroid receptor (MmEcR) gene, the first grapsid brachyuran crab EcR examined. We have also measured MmEcR transcript levels in the ovary and the hepatopancreas throughout the annual cycle, with special reference to seasons of molt and reproduction. MmEcR expression in both the tissues is found to be at its peak (P<0.05) in late premolt crabs (January/May, molt/reproduction season); the expression levels are lowest (P<0.05) during June/July, when the females would neither molt nor reproduce (season for molt/reproduction repose). Intermediate levels of expression were found during the breeding season (August/December). Interestingly, this pattern of gene expression is in concordance with the fluctuating ecdysteroid levels of the hemolymph and Y organ secretory activity. The significant levels of fluctuation in the ovarian expression of MmEcR strongly suggest the ovary as a potential target for ecdysteroid action. A season-wise comparison of the gene expression reveals that ovarian MmEcR transcript levels are higher in breeding crabs (August/December) than the non-breeding animals (June/July), implicating a possible ecdysteroid role in reproduction in M. messor.

Transcriptomic analysis of crustacean molting gland (Y-organ) regulation via the mTOR signaling pathway.

The intermolt crustacean Y-organ (YO) maintains a basal state mediated by pulsatile release of molt inhibiting hormone (MIH), a neuropeptide produced in the eyestalk ganglia, inhibiting YO ecdysteroidogenesis. Reduction of MIH results in YO activation and the animal enters premolt. In the crab, Gecarcinus lateralis, molting was induced by eyestalk ablation (ESA). ESA animals were injected with either rapamycin, an mTOR inhibitor, or DMSO vehicle at Day 0. YOs were harvested at 1, 3, and 7 days post-ESA and processed for high throughput RNA sequencing. ESA-induced increases in mRNA levels of mTOR signaling genes (e.g., mTOR, Rheb, TSC1/2, Raptor, Akt, and S6 kinase) declined following rapamycin treatment. In concert with mTOR inhibition, mRNA levels of ecdysteroid biosynthesis genes (e.g., Nvd, Spo, Sad, Dib, and Phm) were decreased and accompanied by a decrease in hemolymph ecdysteroid titer. By contrast, rapamycin increased the mRNA level of FKBP12, the rapamycin-binding protein, as well as the mRNA levels of genes associated with Wnt and insulin-like growth factor signaling pathways. Many MIH and transforming growth factor-ß signaling genes were down regulated in ESA animals. These results indicate that mTOR activity either directly or indirectly controls transcription of genes that drive activation of the YO.

Actin gene expression in developing sea urchin embryos.

We show that the synthesis of actin is regulated developmentally during early sea urchin embryogenesis and that the level of synthesis of this protein parallels the steady-state amounts of the actin messenger ribonucleic acids (RNA). An in vitro translation and RNA blotting analysis of embryo RNA from several stages of early development indicated that during the first 8 h after fertilization there was a low and relatively constant level of actin messenger RNA in the embryo. Between 8 and 13 h of development, the amount of actin messenger RNA began to increase both in the cytoplasm and on polysomes, and by 18 h the amounts of actin message per embryo had risen between approximately 10- and 25-fold in the cytoplasm and between 15- and 40-fold on polysomes. Two size classes of actin messenger RNA (2.2 and 1.8 kilobases) were identified in unfertilized eggs and in all of the developmental stages examined. The amount of each actin message class increased over a similar time interval during early development. However, the amounts of these size classes in the cytoplasm relative to each other shifted between the earliest stages examined (2 to 5 h) and the hatching blastula stage (18 h), with the ratio of the 1.8-kilobase actin messenger RNA to the 2.2-kilobase actin messenger RNA increasing almost threefold during this period.

Organization and expression of multiple actin genes in the sea urchin.

A set of at least 11 actin genes has been isolated from genomic recombinant deoxyribonucleic acid libraries of the sea urchin Strongylocentrotus purpuratus. Most of the isolates derive from a library which represents the genome of a single animal. There are at least five distinct types of sea urchin actin gene, some of which are represented by multiple copies in the genome. The actin gene types are distinguished by nonhomologous flanking sequences and intervening sequences, though the protein coding sequences appear in most cases to be quite similar. Eight of the 11 genes isolated have been recovered in lambda recombinants that contain two actin genes, linked at 5- to 9-kilobase distances. Restriction map overlaps suggest that the genome contains an array of at least three of these genes spaced over about 30 kilobases of deoxyribonucleic acid. In the linkage patterns observed, actin genes of diverse types were linked to each other. In early embryos, actin messenger ribonucleic acid (RNA) transcripts of 1.8 and 2.2 kilobases were found, and the longer of these transcripts was more prevalent in the maternal RNA of the egg. From RNA gel blot experiments, we conclude that the two transcripts derive from different actin gene types. Different repetitive sequences were located to either side of most of the actin genes, and in most observed cases the repeat sequences which were adjacent to actin genes of a given type were similar. The repeat sequences flanking the actin genes belonged to families which were transcribed, but those repeats in the neighborhood of the actin genes which have been investigated were not themselves represented in the stable RNAs of eggs or early embryos.

Studies on the Ribosomal RNA Cistrons in Interspecific Drosophila Hybrids. II. Heterochromatic Regions Mediating Nucleolar Dominance.

Interspecific hybrids of D. melanogaster and D. simulans normally exhibit a secondary constriction only at the D. melanogaster nucleolus organizer (NO). This phenomenon, termed nucleolar dominance, occurs only when the NO-bearing sex chromosomes of both species are present in conjunction. Experiments were initiated to localize regions on the sex chromosomes of D. melanogaster involved in mediating this suppression. Sex chromosome heterochromatic rearrangements and deficiencies were introduced into F(1) hybrids and their corresponding effect on simulans NO constriction formation was examined in hybrid mitotic neuroblast tissue. Sex chromosomes deficient for both the D. melanogaster NO and adjacent heterochromatin were unable to restrict the formation of a constriction at the D. simulans NO. The presence of a D. melanogaster NO, however, was not sufficient for the establishment of nucleolar dominance. Results from an array of NO-bearing X and Y chromosome rearrangements and deficiencies indicate that at least one heterochromatic region, proximal to the NO on the D. melanogaster X and distal to the NO on the D. melanogaster Y, affects the induction of this interchromosomal phenomenon.

Sequence analysis of the indirect flight muscle actin-encoding gene of Drosophila simulans.

A genomic clone of the Drosophila simulans indirect flight muscle actin-encoding gene (Act88F) has been isolated, and the sequence of a 3358-nucleotide segment corresponding to the Drosophila melanogaster Act88F transcription unit is presented. The ACt88F homologs in these two sibling species encode identical proteins and the general genomic organization of the Act88F locus is highly conserved, including the location of the transcription start point, and the size and position of intron/exon junctions. Substitutions within the 5' flanking region, however, are clearly nonuniform and the regions of lowest divergence coincide with regions that have been implicated in transcript accumulation and the regulation of tissue-specific expression. Silent substitutions within the coding regions have been compared to five other gene homologs in these sibling species. The rate of silent substitution at these loci varies more than threefold, suggesting selection at the codon level.

Expression of the genes encoding the ecdysteroid and retinoid receptors in regenerating limb tissues from the fiddler crab, Uca pugilator.

Using sequence information derived from the Drosophila melanogaster (Dm) ecdysteroid receptor (EcR)- and retinoid X receptor (RXR)-encoding gene homologs, we have isolated cDNA clones corresponding to the DNA-binding domains (DBD) for these two nuclear receptors from the fiddler crab, Uca pugilator (Up). Both genes appear to be represented in 1-2 copies in the Up genome, and unlike Dm, contain an intron within the DBD-encoding region. Sequence comparisons to the Dm EcR and RXR homologs indicate 76 and 82% nucleotide identity, respectively, corresponding to 6 and 4 single-amino acid substitutions which primarily cluster in the region of the molecule involved in dimerization. RT-PCR analysis indicates that both the EcR and RXR homologs are expressed during the initial stages of limb regeneration, temporally concomitant with early blastema formation and the secretion of a flexible sac cuticle at the site of limb loss.

Gene fusion in vivo using transductional cointegrates.

A method is described which allows the efficient construction of hybrids between homologous genes. The technique is based on the phenomenon of cointegrate transduction in which the homology between cloned sequences present on a bacteriophage lambda vector and a plasmid vector is exploited to allow the packaging of a plasmid-phage recombinant. The size of the cointegrate molecule can be far beyond the normal packaging limit of lambda and still allow the transduction of plasmid-borne drug-resistance markers. This method allows the exchange of the 5' and 3' ends of the participating genes as well as the exchange of sequences residing between the end-points of homology between the two genes. Hybrids of either type were constructed between a sea urchin and a Drosophila actin gene using the transductional cointegrate method in vivo. This approach does not require the use of specialized phage or plasmid vectors and can also be used to screen plasmid libraries with a bacteriophage lambda probe.

Isolation and characterization of abl gene sequences in Calliphora erythrocephala.

Screening of genomic DNA libraries with hybridization probes derived from a Drosophila melanogaster c-abl proto-oncogene homologue resulted in the isolation of a set of related sequences from the dipteran Calliphora erythrocephala. Although the region encompassing the c-abl protein kinase domain encodes a polypeptide extremely similar to the Drosophila gene, considerable inter- and intraspecific divergence is found adjacent to this region. Restriction-site heterogeneity and cross-hybridization studies between individual cloned isolates suggest that abl homologues represent a small gene family in the Calliphora genome. As is the case in Drosophila, abl-related transcripts appear to be low in abundance, are synthesized during oogenesis and stored as a maternal mRNA.

Cloning of crustacean ecdysteroid receptor and retinoid-X receptor gene homologs and elevation of retinoid-X receptor mRNA by retinoic acid.

We report the cloning and analysis of ecdysteroid receptor (bpEcR) and retinoid-X receptor (UpRXR) cDNA homologs from the fiddler crab Uca pugilator. The deduced amino acid sequence of this crustacean EcR most closely resembles the insect EcRs within the DNA binding and ligand binding domains (LBDs). For UpRXR, the DNA binding domain (DBD) shares greatest identity to the insect USPs. The ligand binding domain, however, is closer to vertebrate RXRs but may have a nonfunctional AF-2 domain. Probes derived from these clones were used to examine transcript levels in blastemas during early limb regeneration. Both UpEcR and UpRXR transcripts were detected in low amounts 1 day after limb loss, but increased during the next 4 days. Immersion of crabs in sea water containing all-trans retinoic acid increased the steady state concentrations of UpRXR transcript and altered the pattern of circulating ecdysteroids. These effects correlate with the disruptive effects of retinoic acid on blastemal differentiation observed in earlier studies.

Differential recovery of mRNA transcripts using acid organic extraction techniques: quantification of abl mRNA abundance in the blowfly, Calliphora erythrocephala.

A commonly used RNA isolation technique that employs a single-step acid phenol-chloroform extraction (Chomczynski and Sacchi, 1987) produces a different distribution pattern of C. erythrocephala c-abl (Ceabl) transcripts following Northern blot analysis than methods that rely on phenol-chloroform extraction at basic pH (Jowett, 1986). Only Ceabl transcripts of 10.5 kb and 6.5 kb are recovered, while Ceabl transcripts of 9 kb and 4.4 kb are lost, when acidic phenol-chloroform methods are used. All tested extraction methods which do not rely on phenol-chloroform extraction under acidic conditions can recover Ceabl transcripts of 10.5 kb, 9 kb, 6.5 kb and 4.4 kb. The reason for this discrepancy is apparently due to differential loss of mRNA at the phenol phase and/or interphase boundary during the acidic extractions.

Organization of actin gene sequences in the sea urchin: molecular cloning of an intron-containing DNA sequence coding for a cytoplasmic actin.

Southern transfer and solution hybridization experiments, using as probe a DNA fragment that encodes for Drosophila actin, demonstrate cross hybridization to DNA from the sea urchin Strongylocentrotus purpuratus. Recombinant DNA clones that contained sea urchin genomic DNA fragments were constructed and screened for the presence of actin-encoding DNA sequences by colony hybridization with the Drosophila actin sequence. Two different putative actin-encoding clones were identified and were shown to specifically hybridize actin-encoding mRNA from a complex mRNA population. Southern blot hybridization experiments with both the Drosophila actin sequence and one of the cloned sea urchin sequences, in conjunction with solution hybridization data, suggest an actin gene copy number of 5-20 per haploid genome. DNA sequence analysis of one of the cloned sequences indicates that this fragment codes for a cytoplasmic form of actin and contains an intervening sequence of at least 200 nucleotides beginning immediately after amino acid 121 in the protein sequence.

DNA sequence analysis of an abl-related gene in the blow fly, Calliphora erythrocephala.

The Ceabl locus is a member of a small family of abl-related sequences in C. erythrocephala. The catalytic, SH2 and SH3 domains of the Ceabl-encoded protein show greater than 75% sequence identity to vertebrate abl protein tyrosine kinases and greater than 95% identity to the D. melanogaster abl polypeptide. Ceabl diverges from the vertebrate proteins, however, at the extreme amino terminus, near the beginning of the vertebrate abl exon involved in differential splicing. The carboxyl region shows no detectable similarity to the vertebrate abl proteins, while identity to Drosophila abl falls to 55%. Regions conserved between the two dipteran genes revealed no strong similarities to other proteins in the Genbank and EMBL databases.

Tissue-specific patterns and steady-state concentrations of ecdysteroid receptor and retinoid-X-receptor mRNA during the molt cycle of the fiddler crab, Uca pugilator.

In the fiddler crab, Uca pugilator, we have investigated the temporal expression of receptors in various tissues using probes that encode Uca ecdysteroid receptor (UpEcR) and retinoid-X-receptor (UpRXR) gene homologs. During molt stages C4 through D1-4, UpEcR and UpRXR transcripts are expressed in regenerating limb buds, gills, eyestalks, hypodermis, hepatopancreas, muscle from nonregenerating walking legs, and the large cheliped. Some of these tissues have not previously been recognized as ecdysteroid-target tissues. Levels of ecdysteroids in the hemolymph fluctuate significantly during the molt cycle of U. pugilator. The variation in steady-state concentrations of UpEcR transcripts in tissues from C4 to D1-4 implies molt cycle-related differences in the potential of these tissues to respond to changing titers of ecdysteroids in the hemolymph. In singly autotomized crabs, highest concentrations of UpEcR transcript in some tissues did not coincide with the highest levels of circulating ecdysteroids, suggesting that UpEcR expression in these tissues is not dependent on high ecdysteroid titers and may be induced by low or rising concentrations of ecdysteroids. UpEcR and UpRXR genes were expressed simultaneously in tissues, supporting the possibility of heterodimerization for EcR and RXR in vivo. In some tissues, however, levels of transcripts differed, suggesting other possible receptor interactions. Moreover, UpEcR expression in tissues from multiply autotomized crabs differed from the expression patterns in tissues from singly autotomized crabs.

Invariant temporal order of replication of the four actin gene loci during the naturally synchronous mitotic cycles of Physarum polycephalum.

The chronological sequence of replication for the four unlinked actin gene loci of Physarum has been established. Southern hybridization analysis of density-labeled, bromodeoxyuridine-substituted DNA isolated from defined periods of S phase demonstrates that three actin loci (ardB, ardC, ardD) are duplicated early, corresponding to the first 10% of the genome. The fourth locus (ardA) replicates later, between 80 and 100 min into S phase and after 75% of DNA synthesis is completed. Gene-dosage determinations, based on the quantitation of hybridization signals from DNAs isolated from various times during S phase, confirm the results obtained with bromodeoxyuridine-substituted DNA and increase the temporal resolution. The chronological order of replication in the macroplasmodium appears constant through two consecutive cell cycles and after prolonged growth in suspension culture. The precise chronology of DNA synthesis at the gene level extends to the coordinate replication of allele pairs.

DNA sequence analysis and structural relationships among the cytoskeletal actin genes of the sea urchin Strongylocentrotus purpuratus.

The general organization and primary amino acid sequences of the S. purpuratus cytoskeletal actin genes CyIIb and CyIIIb have been determined from restriction enzyme analysis, DNA sequencing, and RNA mapping studies. As is the case with the other sea urchin cytoskeletal actin genes previously studied, the CyIIb and CyIIIb genes contain two introns that interrupt the coding DNA following codon 121 and within codon 204. An intron ending 26-27 nucleotides (nt) upstream of the initiation codon has also been localized in the 5'-flanking region of both genes. The CyIIb gene, which is part of a cluster of three genes linked in the order CyI-CyIIa-CyIIb, encodes a protein that differs from CyI by a single residue and from CyIIa by three residues. The substitutions observed within this linkage group are relatively conservative changes, and pairwise comparisons between genes indicate less than 5% mismatch in nucleotide sequence within the coding region. Nucleotide sequence comparisons of 5'-flanking region and intron DNA, however, indicate greater similarity between the CyI and CyIIb genes than the CyIIa gene that separates them, suggestive of a potential gene conversion event between the flanking genes in the CyI-CyIIa-CyIIb linkage. The CyIIIb gene, part of a separate cluster of two functional genes ordered CyIIIa-CyIIIb, shares little similarity outside of coding DNA with genes of the other linkage group. Although CyIIIb exhibits strong nucleotide sequence similarity outside of coding DNA with the neighboring CyIIIa gene, it differs from that gene at six codons. The CyIIIb gene encodes a protein considerably different from all cytoskeletal actins previously reported, with changes clustered in the latter 40% of the coding sequence. An 81-nt tandem duplication of the C-terminal coding region is located adjacent to the termination codon of the CyIIIb gene, a potential relic of a slipped mispairing and replication event.

The sequence of a sea urchin muscle actin gene suggests a gene conversion with a cytoskeletal actin gene.

We report the nucleotide sequence of the single muscle actin gene of the sea urchin Strongylocentrotus purpuratus. Comparison of the protein-coding sequence of this muscle actin gene (pSpG28) with that of two linked sea urchin cytoskeletal actin genes (pSpG17 and CyIIa) reveals a region of exceptional sequence conservation from codon 61 through codon 120. Furthermore, when silent nucleotide changes are compared, the conservation of this region is still evident (7.9% silent site differences in the conserved region vs 43.3% silent site differences in the rest of the gene when pSpG28 and CyIIa are compared), indicating that the conservation is not due to particularly stringent selection on the portion of the protein encoded by this region of the genes. These observations suggest that a gene conversion has occurred between the muscle actin gene and a cytoskeletal actin gene recently in the evolution of the sea urchin genome. Gene conversion between nonallelic actin genes may thus play a role in maintaining the homogeneity of this highly conserved gene family.