Open Reading Frame Of cDNA Research Articles

Molecular characterization of adenosine monophosphate deaminase 1 and the correlation analysis between its mRNA expression levels and inosine monophosphate content in large yellow croaker (Larimichthys crocea)

Declining flesh quality has drawn considerable attention in the farmed large yellow croaker (LYC; Larimichthys crocea) industry. Inosine monophosphate (IMP) is the primary flavor substance in aquatic animals. Adenosine monophosphate deaminase 1 (AMPD1) plays a critical role in IMP formation by catalyzing the deamination of AMP to IMP in the purine nucleotide cycle. To further evaluate the correlation between ampd1 mRNA expression levels and IMP content in the LYC muscle tissue, the relevant open reading frame (ORF) of L. crocea (Lcampd1) was cloned, and the IMP content and Lcampd1 mRNA expression in the muscles of LYCs of different sizes were examined. The ORF cDNA of Lcampd1 was 2211 bp in length and encoded a polypeptide of 736 amino acids (AAs). The deduced protein, LcAMPD1, possesses conserved AMPD active regions (SLSTDDP) and shows high homology with AMPD proteins of other teleost fishes. The genomic DNA sequence of Lcampd1 exhibits a high degree of evolutionary conservation in terms of structural organization among species. Phylogenetic analysis of the deduced AA sequence revealed that teleost fish and mammalian AMPD1 were separate from each other and formed a cluster with AMPD3, suggesting that AMPD1 and AMPD3 arose by duplication of a common primordial gene. In healthy LYC, Lcampd1 mRNA was expressed only in the muscle tissue. The IMP content in the muscle of LYCs with different average body weights was measured by high-performance liquid chromatography; the results showed that the IMP content in the muscle of LYCs with greater body weight was significantly higher than that in LYC with lower body weight. Moreover, a similar trend in Lcampd1 expression was observed in these muscle tissues. The Pearson correlation analysis further showed that the Lcampd1 mRNA expression was positively correlated with IMP content in the muscles of different-sized LYCs. These results suggest the potential function of Lcampd1 in determining the IMP content in LYC and provide a theoretical basis for flesh quality improvement, as well as a scientific basis for the development of the molecular breeding of LYC.

The Protective Effect of Aromatase on NSC-34 Cells with Stably Expressed hSOD1-G93A

As an adult-onset neurodegenerative disease, amyotrophic lateral sclerosis (ALS) results in progressive muscular atrophy and paralysis. However, the mechanism of ALS has not yet been elucidated, and no cure has been found. Research has revealed that a mutation of the Cu/Zn superoxide dismutase (SOD1) gene is linked to familial ALS and that potential sex discrepancies exist in ALS incidence. Here, NSC-34 cells stably expressing hSOD1-G93A (hSOD1-G93A cells) were transiently transfected with Cyp19a1 mouse open reading frame (ORF) cDNA or a short hairpin RNA (ShRNA) plasmid. Overexpression of aromatase resulting from Cyp19a1 mouse ORF cDNA plasmid transfection enhanced cell proliferation and reduced cell damage in hSOD1-G93A cells. This protective effect occurred through anti-apoptotic pathways related to estrogen receptor-alpha (ER-α) activation. Meanwhile, knockdown of aromatase with Cyp19a1 ShRNA plasmid transfection reduced cell proliferation, increased cell damage, promoted apoptosis, and decreased ER-α expression in hSOD1-G93A cells, and the induced apoptotic effects could be reversed by estradiol (E2). In brief, the results of our study suggest that aromatase plays a neuroprotective role against apoptosis in hSOD1-G93A cells by activating ER-α and may become a new intervention target for ALS treatment.

Cloning, Expression Analysis and Functional Characterization of Squalene Synthase (SQS) from Tripterygium wilfordii.

Celastrol is an active triterpenoid compound derived from Tripterygium wilfordii which is well-known as a traditional Chinese medicinal plant. Squalene synthase has a vital role in condensing two molecules of farnesyl diphosphate to form squalene, a key precursor of triterpenoid biosynthesis. In the present study, T. wilfordii squalene synthase (TwSQS) was cloned followed by prokaryotic expression and functional verification. The open reading frame cDNA of TwSQS was 1242 bp encoding 413 amino acids. Bioinformatic and phylogenetic analysis showed that TwSQS had high homology with other plant SQSs. To obtain soluble protein, the truncated TwSQS without the last 28 amino acids of the carboxy terminus was inductively expressed in Escherichia coli Transetta (DE3). The purified protein was detected by SDS-PAGE and Western blot analysis. Squalene was detected in the product of in vitro reactions by gas chromatograph-mass spectrometry, which meant that TwSQS did have catalytic activity. Organ-specific and inducible expression levels of TwSQS were detected by quantitative real-time PCR. The results indicated that TwSQS was highly expressed in roots, followed by the stems and leaves, and was significantly up-regulated upon MeJA treatment. The identification of TwSQS is important for further studies of celastrol biosynthesis in T. wilfordii.

Molecular cloning and characterization of two kind of heat-shock protein gene from Polyporus umbellatus

With RT-PCR approaches, the full-length cDNA of two heat shock protein genes were cloned from total RNA of the Polyporus umbellatus sclerotium. The full open reading frame cDNA sequence of the Hsp90 was 2 091 bp, encoding 696 amino acid residues with a predicted molecular mass of 78.9 kDa. The full open reading frame cDNA sequence of the Hsp70 was 1 944 bp, encoding 647 amino acid residues with a predicted molecular mass of 70.5 kDa. The Hsp90 and Hsp70 protein contained the conservative structure domain, respectively. Phylogenetic analysis showed that Hsp90 and Hsp90 from Trametes versicolor were clustered into one group, Hsp70 and Hsp70 from Fistulina hepatica were clustered into one group. Real-time PCR analysis showed that, the expression of Hsp90 and Hsp70 in the infected part by Amillariella mellea was upregulated. The expression profiling of Hsp90 and Hsp70 showed same patterns underbiotic stress. The results indicate that these two genes may play an important role in response to Amillariella mellea infection.

Cellular muscle growth and molecular cloning and expression of growth-related gene of Malaysian Mahseer Tor tambroides larvae fed with live and formulated feeds in indoor nursery rearing system

The influences of live and formulated feeds on growth performances, muscle fibers morphometry and muscle growth-related gene expression of Malaysian mahseer, Tor tambroides larvae were evaluated in indoor nursery rearing system. Quadruplicate groups of T. tambroides larvae (0.07±0.01g, mean±SE) were stocked in sixteen aquaria (60×30×30cm), randomly arranged in four dietary treatments viz. larvae fed artemia (LA), moina (LM), daphnia (LD) and formulated feed (FF) with stocking density of 34 larvae per aquarium. The larvae were fed to visually near satiation in two equal feedings per day, seven days per week for 75days. The growth-related parameters (mean weight gain and specific growth rate) were significantly highest in treatment FF, followed by treatment LA and the lowest in treatment LD or LM. Histological observation and muscle morphometric analysis revealed that mosaic hyperplasia was observed after 50days of the feeding trial. Hypertrophic (diameter class 50=40>d≤50μm and class 60=d>50μm), but not hyperplastic (diameter class ≤10μm), muscle fibers frequencies were significantly highest in larvae fed FF compared to those fed live feeds. The entire open reading frame cDNA sequences of two important growth-related genes, myogenin and MyoD, were successfully amplified and cloned from T. tambroides larvae. The nucleotide sequences alignment using CLUSTAL W in BioEdit program of both MyoD and myogenin genes showed the sign of existence of isoforms and highly conserved with other cyprinid fishes. Real-time PCR data demonstrated that myogenin and MyoD gene expressions were significantly upregulated in larvae fed FF. The results of the present experiment concluded that the nursery rearing of T. tambroides larvae with formulated feed showed augmented muscle growth and upregulated growth-related gene expression than feeding live feeds.

A Δ-9 Fatty Acid Desaturase Gene in the Microalga Myrmecia incisa Reisigl: Cloning and Functional Analysis.

The green alga Myrmecia incisa is one of the richest natural sources of arachidonic acid (ArA). To better understand the regulation of ArA biosynthesis in M. incisa, a novel gene putatively encoding the Δ9 fatty acid desaturase (FAD) was cloned and characterized for the first time. Rapid-amplification of cDNA ends (RACE) was employed to yield a full length cDNA designated as MiΔ9FAD, which is 2442 bp long in sequence. Comparing cDNA open reading frame (ORF) sequence to genomic sequence indicated that there are 8 introns interrupting the coding region. The deduced MiΔ9FAD protein is composed of 432 amino acids. It is soluble and localized in the chloroplast, as evidenced by the absence of transmembrane domains as well as the presence of a 61-amino acid chloroplast transit peptide. Multiple sequence alignment of amino acids revealed two conserved histidine-rich motifs, typical for Δ9 acyl-acyl carrier protein (ACP) desaturases. To determine the function of MiΔ9FAD, the gene was heterologously expressed in a Saccharomyces cerevisiae mutant strain with impaired desaturase activity. Results of GC-MS analysis indicated that MiΔ9FAD was able to restore the synthesis of monounsaturated fatty acids, generating palmitoleic acid and oleic acid through the addition of a double bond in the Δ9 position of palmitic acid and stearic acid, respectively.

Molecular cloning, expression and bioactivity of B cell activating factor (BAFF) in African ostrich

B cell activating factor (BAFF), which belongs to the tumor necrosis factor (TNF) family, is testified to play a critical role in B cell survival, proliferation, maturation and immunoglobulin secretion. In the present study, the cDNA of open reading frame (ORF) in African ostrich (Struthio camelus) BAFF (designated OsBAFF) was cloned by reverse transcription-PCR (RT-PCR). The OsBAFF gene encodes a 288-amino acid protein containing a predicted transmembrane domain and a putative furin protease cleavage site like BAFFs from chicken (cBAFF), quail (qBAFF), duck (dBAFF), goose (gBAFF) and dove (doBAFF). RT-PCR analysis showed that the OsBAFF gene is strongly expressed in the bursa of Fabricius, thymus, spleen, and bone marrow. The soluble OsBAFF had been cloned into pET28a. SDS-PAGE and Western blotting analysis confirmed that the soluble fusion protein His-OsBAFF was efficiently expressed in Escherichia coli Rosset (DE3). In vitro, purified OsBAFF was not only able to promote the survival of African ostrich bursal lymphocytes, but also able to co-stimulate proliferation of mouse splenic B cells. The expression of OsBAFF in lymphocyte cells was higher than the control after LPS stimulation. These findings indicated that OsBAFF plays an important role in survival and proliferation of African ostrich bursal lymphocytes, which may provide valuable information for research into the immune system of African ostrich and OsBAFF may serve as a potential immunologic factor for enhancing immunological efficacy in African ostrich and any other birds.

Characterization and Regulation of the Amino Acid Transporter SNAT2 in the Small Intestine of Piglets.

The sodium-dependent neutral amino acid transporter 2 (SNAT2), which has dual transport/receptor functions, is well documented in eukaryotes and some mammalian systems, but has not yet been verified in piglets. The objective of this study was to investigate the characteristics and regulation of SNAT2 in the small intestine of piglets. The 1,521-bp porcine full cDNA sequence of SNAT2 (KC769999) from the small intestine of piglets was cloned. The open reading frame of cDNA encodes 506 deduced amino acid residues with a calculated molecular mass of 56.08 kDa and an isoelectric point (pI) of 7.16. Sequence alignment and phylogenetic analysis revealed that SNAT2 is highly evolutionarily conserved in mammals. SNAT2 mRNA can be detected in the duodenum, jejunum and ileum by real-time quantitative PCR. During the suckling period from days 1 to 21, the duodenum had the highest abundance of SNAT2 mRNA among the three segments of the small intestine. There was a significant decrease in the expression of SNAT2 mRNA in the duodenal and jejunal mucosa and in the expression of SNAT2 protein in the jejunal and ileal mucosa on day 1 after weaning (P < 0.05). Studies with enterocytes in vitro showed that amino acid starvation and supplementation with glutamate, arginine or leucine enhanced, while supplementation with glutamine reduced, SNAT2 mRNA expression (P < 0.05). These results regarding the characteristics and regulation of SNAT2 should help to provide some information to further clarify its roles in the absorption of amino acids and signal transduction in the porcine small intestine.

Knockdown of the ionotropic γ-aminobutyric acid receptor (GABAR) RDL gene decreases fipronil susceptibility of the small brown planthopper, Laodelphax striatellus (Hemiptera: Delphacidae).

Insect γ-aminobutyric acid receptors (GABARs) are important molecular targets of cyclodiene and phenylpyrazole insecticides. Previously GABARs encoding rdl (resistant to dieldrin) genes responsible for dieldrin and fipronil resistance were identified in various economically important insect pests. In this study, we cloned the open reading frame cDNA sequence of rdl gene from fipronil-susceptible and fipronil-resistant strains of Laodelphax striatellus (Lsrdl). Sequence analysis confirmed the presence of a previously identified resistance-conferring mutation. Different alternative splicing variants of Lsrdl were noted. Injection of dsLsrdl reduced the mRNA abundance of Lsrdl by 27-82%, and greatly decreased fipronil-induced mortality of individuals from both susceptible and resistant strains. These data indicate that Lsrdl encodes a functional RDL subunit that mediates susceptibility to fipronil. Additionally, temporal and spatial expression analysis showed that Lsrdl was expressed at higher levels in eggs, fifth-instar nymphs, and female adults than in third-instar and fourth-instar nymphs. Lsrdl was predominantly expressed in the heads of 2-day-old female adults. All these results provide useful background knowledge for better understanding of fipronil resistance related ionotropic GABA receptor rdl gene expressed variants and potential functional differences in insects.

Molecular Identification and Expressive Characterization of an Olfactory Co-Receptor Gene in the Asian Honeybee,Apis cerana cerana

Olfaction recognition process is extraordinarily complex in insects, and the olfactory receptors play an important function in the process. In this paper, a highly conserved olfactory co-receptor gene, AcerOr2 (ortholog to the Drosophila melanogaster Or83b), cloned from the antennae of the Asian honeybee, Apis cerana cerana Fabricius (Hymenoptera: Apidae), using reverse transcriptase PCR and rapid amplification of cDNA ends. The full-length sequence of the gene was 1763 bp long, and the cDNA open reading frame encoded 478 amino acid residues, including 7 putative transmembrane domains. Alignment analysis revealed that AcerOr2 shares high homology (> 74%) with similar olfactory receptors found in other Hymenoptera species. The amino acid identity with the closely related species Apis mellifera reached 99.8%. The developmental expression analysis using quantitative real-time reverse transcriptase PCR suggested that the AcerOr2 transcript was expressed at a relatively low level in the larval stage, whereas it was expressed broadly in the pupal and adult stages, with a significantly high level on the days just before and after eclosion. In situ hybridization showed that AcerOr2 mRNA was expressed in sensilla placodea and on the basal region of the worker antennal cuticle, in accordance with the previous conclusions that the conserved genes are expressed in most olfactory receptor neurons.

Characterization of the TaMIPS gene from winter wheat (Triticum aestivum L.) and changes in its expression pattern with phytic acid accumulation in seeds during grain filling

D-myo-Inositol-3-phosphate (D-Ins(3)P1) synthase (MIPS EC 5.5.1.4) catalyzes the first step in the phytic acid biosynthetic pathway. To understand the function of the MIPS gene and phytic acid accumulation, the TaMIPS gene was characterized, and changes in gene expression and MIPS activity were analyzed. The full-length genomic DNA sequence of the TaMIPS gene, located in homologous group 4 chromosomes, was characterized. TaMIPS comprises ten exons and nine introns, with 3910 bp in total, with a cDNA open reading frame (ORF) of 1533 bp encoding a protein with 510 amino acids. The transcription of this gene was detected in roots, stems, flag leaves and immature seeds at 15 day after flowering (DAF), and high levels of gene transcripts were detected in immature seeds at 14 DAF. The enzymatic activity of MIPS was found to be high before 14 DAF, followed by a gradual reduction until seed maturity. The phytic acid concentration also gradually increased with seed maturity. A wheat cultivar with a high phytic acid content had higher enzymatic activity and higher levels of expression of MIPS than did a cultivar with low phytic acid content. The results show that the TaMIPS gene may play an important role in regulating phytate synthesis.

Glutathione Peroxidase cDNA Cloning and Expression in Soybean Roots under Heterodera glycines Infection

A gene named GmGPX1 encoding glutathione peroxidase (GPX) was cloned and sequenced from soybean roots infested Heterodera glycines by RT-PCR (Reverse Transcription PCR), which is a crucial enzyme in plant cells regulating reactive oxygen species (ROX). The cDNA length of cloned gene was 693bp, flanked by a 5'-untranslated region of 7bp and a 3'-untranslated region of 185bp, containing six exons and five introns. Genomic DNA fragment was located at Glycine max chromosome 5. The open reading frame of cDNA which encodes a polypeptide of 166 amino acid residues and protein molecular weight was 18375.8Da, theoretical isoelectric point 6.59. The deduced amino acid sequence showed about 99% and 64% homology with G max putative PHGPX (XP_003532707.1) and Arabidopsis thaliana GPX (NP_564813.1), respectively. The expression profile of the GmGPX1 in Xiaoliheidou (G. max) under H. glycines infection, which generates oxidative stress, was analyzed. Real time PCR analysis revealed that the GmGPX1 mRNA levels were increased stabilized from 1.3 to 1.47 times after exposure to H. glycines from 12h to 48h, and reduced to 1.07 times at 72h comparing with non-inoculation control. These results suggest that the GmGPX1 gene is induced by H. glycines at early stage in soybean roots and play an important role in removing oxidative damage.

Characterization of a sigma class glutathione S-transferase gene in the larvae of the honeybee (Apis cerana cerana) on exposure to mercury

Glutathione S-transferases (GSTs) are multifunctional enzymes that are mainly involved in detoxification of endogenous and xenobiotic compounds and oxidative stress resistance in insects. In this study, we identified a sigma class GST from Apis cerana cerana (AccGSTs4). The open reading frame of cDNA was 612 bp and encoded a 203 amino acid polypeptide, which exhibited the structural motif and domain organization characteristic of GST. Homology and evolutionary analysis indicated that the induced amino acid sequence of AccGSTs4 belonged to an insect sigma class group. Expression analysis indicated that AccGSTs4 was presented in all stages of development with high level in 4th instar larvae. Immunolocalization further revealed the distribution of AccGSTs4 in 4th instar larvae. RT-qPCR showed that the transcripts of AccGSTs4 from the larvae were upregulated under dietary HgCl(2). The GST activity under stress was higher than the controls fed on HgCl(2)-free diet. Disc diffusion assay provided evidence of recAccGSTs4 resistance to long-term exposure of HgCl(2) stress. Additionally, analysis of 5'-flanking region further clarified the probable expression patterns of AccGSTs4. Taken together, our findings indicate that the larvae AccGSTs4 may play a role in mercury stress response, and it will help to protect honeybees from heavy metals.

Genetic characterization and ligand specificity of the ovine Fc gamma receptor I (ovFcγRІ)

Receptors for the Fc region of IgG (FcγRs) play a critical role in the immune system and host protection against infection. In this study, we describe the cloning, mRNA expression and IgG subclass specificity of ovine Fc gamma receptor I (ovFcγRI). The ovFcγRI cDNA contains a 1047 bp open-reading frame, and is predicted to encode a 349 amino acid trans-membrane glycoprotein composed of three immunoglobulin-like domains, a trans-membrane region and a short cytoplasmic tail. The overall identity of the ovine FcγRI to its cattle, human and mouse counterparts at the level of the amino acid sequence was 92%, 61% and 54%, respectively. Rosetting analysis shows that COS-7 cells were transfected with an expressional vector carrying the cDNA open-reading frame of ovFcγRI and expressed this receptor on the surface. Identification of ovine FcγRI will aid in the understanding of molecular basis of the ovine immune system and further studies of the receptor function.

Damaged DNA-binding protein 2 (DDB2) protects against UV irradiation in human cells and Drosophila

BackgroundWe observed previously that cisplatin-resistant HeLa cells were cross-resistant to UV light due to accumulation of DDB2, a protein implicated in DNA repair. More recently, we found that cFLIP, which represents an anti-apoptotic protein whose level is induced by DDB2, was implicated in preventing apoptosis induced by death-receptor signaling. In the present study, we investigated whether DDB2 has a protective role against UV irradiation and whether cFLIP is also involved in this process.MethodsWe explored the role of DDB2 in mediating UV resistance in both human cells and Drosophila. To do so, DDB2 was overexpressed by using a full-length open reading frame cDNA. Conversely, DDB2 and cFLIP were suppressed by using antisense oligonucleotides. Cell survival was measured using a colony forming assay. Apoptosis was monitored by examination of nuclear morphology, as well as by flow cytometry and Western blot analyses. A transcription reporter assay was also used to assess transcription of cFLIP.ResultsWe first observed that the cFLIP protein was upregulated in UV-resistant HeLa cells. In addition, the cFLIP protein could be induced by stable expression of DDB2 in these cells. Notably, the anti-apoptotic effect of DDB2 against UV irradiation was largely attenuated by knockdown of cFLIP with antisense oligonucleotides in HeLa cells. Moreover, overexpression of DDB2 did not protect against UV in VA13 and XP-A cell lines which both lack cFLIP. Interestingly, ectopic expression of human DDB2 in Drosophila dramatically inhibited UV-induced fly death compared to control GFP expression. On the other hand, expression of DDB2 failed to rescue a different type of apoptosis induced by the genes Reaper or eiger.ConclusionOur results show that DDB2 protects against UV stress in a cFLIP-dependent manner. In addition, the protective role of DDB2 against UV irradiation was found to be conserved in divergent living organisms such as human and Drosophila. In addition, UV irradiation may activate a cFLIP-regulated apoptotic pathway in certain cells.

Identification of a vaccine candidate antigen, PfMAg-1, from Plasmodium falciparum with monoclonal antibody M26-32

Monoclonal antibody M26-32 has been shown to strongly inhibit the growth of Plasmodium falciparum in vitro. To identify the target antigen of M26-32, a P. falciparum Dd2 asexual stage cDNA expression library was screened with this antibody, and a full open reading frame cDNA was obtained. This gene, named pfmag-1, encodes a polypeptide of 589 amino acids. The protein PfMAg-1 was characterized as a membrane-associated protein that expressed on the surface of merozoite during erythrocytic stage. Remarkably, at the C terminus of PfMAg-1, there are 14 copies of a deca-peptide sequence of QTDEIKND (H/N) I. This tandem repeat domain was identified to harbor the epitope of the protective M26-32 monoclonal antibody, and was also recognized by sera of patients infected with P. falciparum. Rabbit antibody elicited against this deca-peptide repeat domain effectively inhibited P. falciparum invasion in vitro. Our work suggests that PfMAg-1 is a promising malaria vaccine candidate.

CONSTRUCTION OF BRAIN cDNA LIBRARIES AND MOLECULAR CLONING AND EXPRESSION ANALYSIS OF &lt;I&gt;GNRH&lt;/I&gt; GENE IN MARBLED EEL(&lt;I&gt;ANGUILLA MARMORATA&lt;/I&gt;)

Marbled eel(Anguilla marmorata) is the secondary protected animal in China.In order to save the genetic information of this rarity and clone the function genes on growth,development and reproduction,a cDNA library was constructed by CloneminerTM kit from brain of marbled eel.The titer of the amplified cDNA library was 4.3×106 cfu/mL,the total of recombinants was 5.16×107 cfu,and the percentage of recombinant efficiency was about 99.6%,the exogenous inserts of the recombinants was from 0.43kb to 3.2kb and the average size was 1532 bp.These results showed that cDNA library had excellent quality.Two types of GnRH(mGnRH and cGnRH-II) cDNA sequences were isolated from cDNA library by PCR.Sequence analysis showed that mGnRH cDNA contained an open reading frame(ORF) of 276 bp and encoded 91 amino acid residues,which consisting of a 22-amino acid signal peptide precursor(1—22 amino acid residues),mGnRH decapeptide(23—32 amino acid residues),3-amino acid signal processing site(33—35 amino acid residues),and a 56-amino acid GnRH-associated peptide(36—91 amino acid residues).cGnRH-II cDNA open reading frame(ORF) contained 264 bases encoded 87 amino acid residues,which consisting of a 24-amino acid signal peptide precursor(1—24 amino acid residues),cGnRH-II decapeptide(25—34 amino acid residues),3-amino acid signal processing site(34—36 amino acid residues),and a 50-amino acid GnRH-associated peptide(37—87 amino acid residues).The homology analysis showed that the percentage of mGnRH and cGnRH-II precursor sequence identity with Japanese eel Anguilla japonica is 98%,with fishes of Salmoniformes,Perciformes and Pleuronectiformes is 73%—78%.However,it was relative low with fishes of Cypriniformes(63%—67%).Phylogenetic tree analysis ranked the fish GnRH as three distinct groups,mGnRH and sbGnRH group,cGnRH-II group and sGnRH group,respectively.Expression analysis by RT-PCR showed that cGnRH-II and mGnRH gene expression had no obvious differences between female and male marbled eel individuals,but mGnRH gene could be expressed in more tissues than cGnRH-II.mGnRH were detected in forebrain,midbrain,hindbrain,pituitary,hypothalamus,liver,heart,spleen,kidney,intestine,gill,ovary and testis,while expression of cGnRH-II was mainly limited to forebrain,midbrain,hindbrain,ovary and testis.The present work provided evidence of two GnRH in Marbled eel reproductive system and suggested an important role of mGnRH in reproduction.

참전복(Haliotis discus hannai) Myostatin 유전자의 cDNA 동정 및 발현 분석

We cloned and sequenced the open reading frame (ORF) cDNA encoding myostatin from the muscle of abalone (Haliotis discus hannai). The ORF cDNA of the abalone myostatin is 1134 bp and encoded 377 amino acid residues that were 60-96% homologous with the amino acids of other organism myostatins. In addition, the ORF contained a conserved proteolytic cleavage site (RXRR) and nine conserved cysteine residues in the C-terminus. Semi-quantitative RT-PCR revealed the presence of myostatin mRNA in various tissues. The strongest expression was observed in the mantle of female abalone, and the gills and heart of male abalone.

Isolation and characterization of HC1: a novel human DNA repair gene

Nucleotide excision repair (NER) acts on a broad spectrum of large lesions, while base excision repair removes individual modified bases. Although both processes have been well studied in human cells, novel genes involved in these DNA repair pathways have been described. Using a heterologous complementation approach, we identified a fetal human cDNA that complemented two Escherichia coli mutants that are defective in 3-methyl adenine glycosylase and in three endonucleases, all of which are enzymes with important roles in base excision repair. The central cDNA open reading frame complemented NER mutant strains and promoted an increase in survival rate of bacteria exposed to UV light. The corresponding protein was able to restore nucleotide-excision-repair activity when added to a cell extract from Chinese hamster ovary cells deficient in the ERCC1 protein, an enzyme known to promote incision at the 5' end of the lesion during NER. In contrast, that protein was not able to complement XPG Chinese hamster ovary cells deficient in the 3' incision step of NER. These data indicate a new human repair gene, which we named HC1; it is involved in the recognition of two kinds of DNA lesions and it contributes to the 5' DNA incision step in NER.

FSCB, a Novel Protein Kinase A-phosphorylated Calcium-binding Protein, Is a CABYR-binding Partner Involved in Late Steps of Fibrous Sheath Biogenesis

We report characterization of a novel testis- and sperm-specific protein, FSCB (fibrous sheath CABYR binding), that is expressed post-meiotically and localized in mouse sperm flagella. FSCB was identified as a binding partner of CABYR, a calcium-binding protein that is tyrosine-phosphorylated during capacitation. Orthologous genes of FSCB are present in other mammals, including rat and human, and conserved motifs in FSCB include PXXP, proline-rich and extensin-like regions. FSCB is phosphorylated by protein kinase A as shown by in vitro phosphorylation assay and also by determining phosphorylation sites in native FSCB from mouse sperm. Calcium overlay assay showed that FSCB is a calcium-binding protein from sperm. FSCB is a post meiotic protein first expressed at step 11 of mouse spermatogenesis in the elongating spermatids, and it subsequently incorporates into the flagellar principal piece of the sperm. Ultrastructurally, FSCB localized to a cortical layer of intermediate electron density at the surface of the ribs and longitudinal columns of the fibrous sheath. Due to its temporal appearance during spermiogenesis and location at the cortex of the fibrous sheath, FSCB is postulated to be involved in the later stages of fibrous sheath assembly.