Differential expression of boule and dazl in adult germ cells of the Asian seabass

Fertility genes boule and dazl constitute the evolutionarily conserved DAZ (Deleted in AZoospermia) family of RNA binding proteins essential for germline development across animal phyla. Here we report the cloning and expression analysis of boule and dazl from the Asian seabass (Lates calcarifer), a marine fish that undergoes sequential male-to-female sex reversal. Molecular cloning and sequence comparison led to the identification of boule and dazl cDNAs. RT-PCR analysis showed that both boule and dazl RNAs were restricted to the gonads among adult organs examined. Chromogenic in situ hybridization revealed germ cell-specific expression for both boule and dazl in female and male adults. Importantly, distinct differences were found between boule and dazl in terms of temporospatial expression and subcellular distribution. The boule RNA was abundant in late gametogenic cells except sperm. Interestingly, dazl expression increases in early oocytes and concentrates in a perinuclear speckle that appears to develop ultimately into the Balbiani body in advanced oocytes. The dazl RNA was found to be abundant in spermatocytes but hardly detectable in sperm. These data demonstrate that boule and dazl are germ cell markers in the adult Asian seabass, and that bisexual germline-specific expression has been conserved for boule and dazl in fish.     

Conserved function of Rho-related Rop/RAC GTPase signaling in regulation of cell polarity in Physcomitrella patens

Cell polarity is fundamentally important to growth and development in higher plants, from pollen tubes to root hairs. Basal land plants (mosses and ferns) also have cell polarity, developing protonemal apical cells that show polar tip growth. Flowering plants have a distinct group of Rho GTPases that regulate polarity in polarized cell growth. Rop/RAC signaling module components have been identified in non-flowering plants, but their roles remain unclear. To understand the importance and evolution of Rop/RAC signaling in polarity regulation in land plants, we examined the functions of PpRop and PpRopGEF in protonemal apical cells of the moss Physcomitrella patens. Inducible overexpression of PpRop2 or PpRopGEF3 caused depolarized growth of tip-growing apical cells. PpRop2 overexpression also caused aberrant cross wall formation. Fluorescent protein-tagged PpRop2 localized to the plasma membrane, including the cross wall membrane, and fluorescent-tagged PpRopGEF3 showed polarized localization to the tip region in apical cells. Thus, our results suggest common functions of PpRop and PpRopGEF in the tip-growing apical cells and the importance of a conserved Rop/RAC signaling module in the control of cell polarity in land plants.     

Molecular cloning and sequencing of metallothionein in squamates: New insights into the evolution of the metallothionein genes in vertebrates

Metallothioneins are cysteine-rich, metal-binding proteins ubiquitously expressed in living organisms. In the last past years, a plethora of vertebrate metallothionein sequences have become available, but so far there has been an almost absolute lack of data about sequences of metallothionein of non-avian diapsida. In the framework of the investigations on structural and functional properties of non-mammalian metallothioneins, we have cloned and sequenced the cDNAs encoding for metallothioneins of 10 squamate reptiles, belonging to 5 different infraorders. These sequences have been used to gain insight into the evolutionary history of metallothioneins in reptiles. Phylogenetic analysis shows that reptilian metallothionein phylogeny is inconsistent with the species phylogeny. Such findings allow us to hypothesize that the identified metallothionein in each squamate species used for this study might be considered a paralogous gene derived from more events of gene duplication and losses occurred during the diversification of the squamate species. Finally, through vertebrate metallothionein comparisons and phylogenetic analysis, we also add a novel contribution to the understanding of the evolution of metallothionein genes along the major vertebrate lineages.     

Cloning of two members of the calcitonin-family receptors from stingray, Dasyatis akajei: possible physiological roles of the calcitonin family in osmoregulation

In cartilaginous fish, two cDNAs encoding calcitonin-family receptors were isolated for the first time from the stingray brain. The open reading frame of one receptor cDNA coded a 525-amino acid protein. The amino acid identity of this receptor to human calcitonin-receptor-like receptor (CRLR) is 64.5%, frog CRLR is 64.7%, and flounder CRLR is 61.2% and this was higher than to human calcitonin receptor (CTR) (46.1%), frog CTR (54.7%), and flounder CTR (48.9%). We strongly suggested that this receptor is a ray CRLR based on phylogenetic analysis. In case of the second receptor, amino acid identity among CRLRs (human 50.5%, frog 50.7%, flounder 48.0%) and CTRs (human 43.2%, frog 49.1%, flounder 41.8%) was similar. From phylogenetic analysis of both CRLRs and CTRs, we believe that this receptor is ray CTR. The expression of ray CRLR mRNA was predominantly detected in the nervous system (brain) and vascular system (atrium, ventricle, and gill), which reflects the similar localization of CGRP in the nervous and vascular systems as mammals. It was observed that the second receptor was expressed in several tissues, namely cartilage, brain, pituitary gland, gill, atrium, ventricle, pancreas, spleen, liver, gall bladder, intestine, rectal gland, kidney, testis and ovary. This localization pattern was very similar to flounder CTR. Both receptor mRNAs were strongly expressed in the gill. This suggests that the calcitonin-family members are involved in the osmoregulation of stingray as this fish is known to be euryhaline. When a stingray was transferred to diluted seawater (20% seawater), the expression of both receptors significantly decreased in the gill. Similar results were obtained in the kidney of the stingray. Thus, our cloning and isolation of both receptors in the stingray will be helpful for elucidation of their physiological role(s) such as osmoregulation including calcium metabolism of cartilaginous fish.     

Segregation of mutant ovalbumins and ovalbumin-globin fusion proteins in Xenopus oocytes: Identification of an ovalbumin signal sequence

The intramolecular signals for chicken ovalbumin secretion were examined by producing mutant proteins in Xenopus oocytes. An ovalbumin complementary DNA clone was manipulated in vitro, and constructs containing altered protein-coding sequences and either the simian virus 40 (SV40) early promoter or Herpes simplex thymidine kinase promoter, were microinjected into Xenopus laevis oocytes. The removal of the eight extreme N-terminal amino acids of ovalbumin had no effect on the segregation of ovalbumin with oocyte membranes nor on its secretion. A protein lacking amino acids 2 to 21 was sequestered in the endoplasmic reticulum but remained strongly associated with the oocyte membranes rather than being secreted. Removal of amino acids 231 to 279, a region previously reported to have membrane-insertion function, resulted in a protein that also entered the endoplasmic reticulum but was not secreted. Hybrid proteins containing at their N terminus amino acids 9 to 41 or 22 to 41 of ovalbumin fused to the complete chimpanzee α-globin polypeptide were also sequestered by oocyte membranes. We conclude that the ovalbumin “signal” seque?ce is internally located within amino acids 22 to 41, and we speculate that amino acids 9 to 21 could be important for the completion of ovalbumin translocation through membranes.     

Population genetic structure of Penaeus monodon, in relation to monsoon current patterns in Southwest, East and Andaman coastal waters of India

The black tiger shrimp (Penaeus monodon), a commercially important penaeid species, is widely distributed across the Indo-Pacific region. Genetic diversity in P. monodon collected from eight geographical regions in Southwest, East and Andaman coastal waters of India (N = 418) was investigated using 10 polymorphic microsatellite loci. Average observed heterozygosity at sampled loci were high, ranging from 0.643 (Coromandel Coast) to 0.753 (South Andaman). Pairwise F_ST (ranged from 0.005 to 0.078) and R_ST (ranged from 0.005 to 0.171) estimates revealed surprisingly strong and statistically significant genetic structure among tiger shrimp populations. A synthetic map generated by multidimensional scaling shows an apparent cline in allele frequencies paralleling the roughly circular flow of surface currents in the Bay of Bengal. Significant heterozygote deficiencies were noted in most population samples at most loci. Andaman Island sites showed the highest diversity. Recognition of high genetic diversity and distinct population structuring of P. monodon in Indian seas has important implications for future domestication of this species in India, for two reasons: identification of the best wild founding stocks for aquaculture and, subsequently, the potential impacts of release of domesticates to the wild, either accidentally or deliberately (i.e. for stock enhancement).     

Identification of a mutation that relieves gamma-glutamyl kinase from allosteric feedback inhibition by proline

A 1.75-kb DNA fragment containing the entire Escherichia coli proB+ gene has been sequenced. The proB locus encodes the structural gene for gamma-glutamyl kinase (GK), the enzyme responsible for the first step in proline biosynthesis, and the primary regulatory point of the pathway. We have previously reported the nucleotide (nt) sequence of a mutant proB gene isolated from an E. coli strain resistant to the toxic analog of proline, 3,4-dehydro-DL-proline (DHP). This mutant gene encodes a GK which is refractory to allosteric feedback inhibition by proline (DHPR). Comparison of the proB+ and DHPR proB sequences revealed a single base difference, an A-T to C-G transversion localized at nt position 428 within the amino acid (aa) coding region of proB. This mutation predicts an aa change from glutamic acid in the wild-type (wt) enzyme to alanine in the DHPR enzyme.     

A conserved DYW domain of the pentatricopeptide repeat protein possesses a novel endoribonuclease activity

Many plant pentatricopeptide repeat (PPR) proteins are known to contain a highly conserved C-terminal DYW domain whose function is unknown. Recently, the DYW domain has been proposed to play a role in RNA editing in plant organelles. To address this possibility, we prepared recombinant DYW proteins and tested their cytidine deaminase activity. However, we could not detect any activity in the assays we used. Instead, we found that the recombinant DYW domains possessed endoribonuclease activity and cleaved before adenosine residues in the RNA molecule. Some DYW-containing PPR proteins may catalyze site-specific cleavage of target RNA species.     

Small inverted repeats drive mitochondrial genome evolution in Lake Baikal sponges

Demosponges, the largest and most diverse class in the phylum Porifera, possess mitochondrial DNA (mtDNA) markedly different from that in other animals. Although several studies investigated evolution of demosponge mtDNA among major lineages of the group, the changes within these groups remain largely unexplored. Recently we determined mitochondrial genomic sequence of the Lake Baikal sponge Lubomirskia baicalensis and described proliferation of small inverted repeats (hairpins) that occurred in it since the divergence between L. baicalensis and the most closely related cosmopolitan freshwater sponge Ephydatia muelleri. Here we report mitochondrial genomes of three additional species of Lake Baikal sponges: Swartschewskia papyracea, Rezinkovia echinata and Baikalospongia intermedia morpha profundalis (Demospongiae, Haplosclerida, Lubomirskiidae) and from a more distantly related freshwater sponge Corvomeyenia sp. (Demospongiae, Haplosclerida, Metaniidae). We use these additional sequences to explore mtDNA evolution in Baikalian sponges, paying particular attention to the variation in the rates of nucleotide substitutions and the distribution of hairpins, abundant in these genomes. We show that most of the changes in Lubomirskiidae mitochondrial genomes are due to insertion/deletion/duplication of these elements rather than single nucleotide substitutions. Thus inverted repeats can act as an important force in evolution of mitochondrial genome architecture and be a valuable marker for population- and species-level studies in this group. In addition, we infer (((Rezinkovia+Lubomirskia)+Swartschewskia)+Baikalospongia) phylogeny for the family Lubomirskiidae based on the analysis of mitochondrial coding sequences from freshwater sponges.     

The co-existence of two growth hormone receptors and their differential expression profiles between female and male tongue sole (Cynoglossus semilaevis)

Growth hormone receptor (Ghr) is a single-transmembrane pass protein which is important in initiating the ability of growth hormone (Gh) to regulate development and somatic growth in vertebrates. In this study, molecular cloning, expression analysis of two different ghr genes (ghr1 and ghr2) in the tongue sole (Cynoglossus semilaevis) was conducted. As a result, the ghr1 and ghr2 cDNA sequences are 2364 bp and 3125 bp, each of which encodes a transmembrane protein of 633 and 561 amino acids (aa), respectively. Besides, the ghr1 gene includes nine exons and eight introns. The sex-specific tissue expression was analyzed by using 14 tissues from females, normal males and extra-large male adults. Both the ghr1 and ghr2 were predominantly expressed in the liver, and the ghr1 expression level in normal males was 1.6 and 1.4 times as much as those in females and extra-large males, while the ghr2 mRNA expression level in normal males was 1.1 and 1.2 times as much as those in females and extra-large males, respectively. Ontogenetic expression analysis at early life stages indicated that the ghr1 and ghr2 mRNAs were detected at all of the 35 sampling points (from oosphere to 410 days-old). Furthermore, the sex differences in ghr mRNA expressions were also examined by using a full-sib family of C. semilaevis. Significantly higher levels of ghr1 mRNA were observed in males than in females at most stages of the sampling period (P < 0.01). The ghr2 mRNA expression at most stages exhibited a significant sexual difference at each sampling point (P < 0.01) without any variation trend related with the sexes during the whole sampling period.     

Molecular cloning,characterization and expression analysis of ATG1 in the silkworm, Bombyx mori

Atg1 is a Serine/Threonine protein kinase that plays a pivotal role in autophagy. A complete coding sequence of ATG1 is not available for the silkworm, Bombyx mori which is a good model for studying the autophagic process.     

Molecular cloning and expression in vitro of a carboxylesterase gene from the Glanville fritillary butterfly (Melitaea cinxia)

Carboxylesterase (EC 3.1.1.1) is a member of the carboxyl/cholinesterase (CCE) superfamily, which is widely distributed in animals, plants and microorganisms. This enzyme has been known to be associated with insecticide resistance and detoxification. Although CCEs have been extensively studied in insects, including lepidopterans, the research on butterflies, a major subgroup in Lepidoptera, is still poor. In the present study, we cloned a CCE gene (McCCE1) from the Glanville fritillary butterfly (Melitaea cinxia, Lepidoptera: Nymphalidae). The full-length cDNA encoding McCCE1 was 1786 bp, containing a 1641 bp open reading frame encoding 546 amino acids, a 38 bp 5′-untranslated region (5′-UTR), and a 107 bp 3′-UTR with a poly(A) tail. The functionally conserved amino acids in McCCE1 shared the 55% identity with the cytoplasmic esterase CCE017a in Helicoverpa armigera (Lepidoptera: Noctuidae), which has been associated with detoxification. Assays in vitro showed that the recombinant McCCE1 could hydrolyze α- and β-naphthyl acetate. Thus, the present study adds to the body of knowledge concerning the detoxification of pesticides by lepidopterans.