Sequence Of cDNA Clone Research Articles

Creatine in giant grouper (Epinephelus lanceolatus): Insights into biosynthesis and transporter genes molecular characterization, regulation and impact on muscle metabolism

Creatine is synthesized from glycine, arginine, and methionine with the catalysis of glycine amidinotransferase (GATM) and guanidinoacetate N-methyltransferase (GAMT), playing a crucial role in regulating the cellular metabolism and whole-body energy balance. However, information regarding the molecular characteristics of GATM and GAMT is limited in fish species. In this study, GATM and GAMT cDNA were cloned from giant grouper (Epinephelus lanceolatus). The cloned cDNA sequence of GATM was 1503 bp encoding a 421 aa containing conserved regions of amidinotransferase superfamily. GAMT cDNA was 892 bp encoding a 235 aa containing conserved regions of S-adenosylmethionine-dependent methyltransferases superfamily. The phylogenetic tree indicated that GATM and GAMT were clustered distinctly into teleost and featured common function domains and homology with teleost proteins, but they were distinct from the terrestrial clusters. The mRNA transcripts of GATM and GAMT were detectable mainly in muscle tissue. Additionally, as the creatine synthesis limited enzyme, GATM gene and protein expression were both significantly regulated by supplement of dietary creatine (P < 0.05). Metabolomics analysis showed that dietary creatine significantly affected skeletal muscle nutrients and energy metabolism. Meanwhile, inclusion of dietary creatine led to a significant inhibition of AMPK phosphorylation. What is more, we also recorded that cytochrome oxidase (cox) and mitochondrial fission factor (mff) gene expression were remarkable up-regulated by dietary creatine supplementation. In summary, the results obtained from the present study would help us better understand the creatine synthesis pathway in fish species. We also demonstrated that creatine plays a significant role in fish muscle metabolism.

The responsive characteristics of phytochrome genes to photoperiod, abiotic stresses and identification of their key natural variation sites in foxtail millet (Setaria italica L.)

The responsive patterns of phytochrome gene family members to photoperiod and abiotic stresses were comparatively analyzed and the favorable natural variation sites of these genes were identified. This would help understand the mechanism of phytochrome gene family in photoperiod-regulated growth and development and abiotic stress response. In addition, it may facilitate the molecular marker assisted selection of key traits in foxtail millet. In this study, we used RT-PCR to clone three phytochrome genes SiPHYA, SiPHYB and SiPHYC from ultra-late maturity millet landrace variety 'Maosu'. After primary bioinformatics analysis, we studied the photoperiod control mode and the characteristics of these genes in responding to five abiotic stresses including polyethylene glycol (PEG)-simulated drought, natural drought, abscisic acid (ABA), high temperature and NaCl by fluorescence quantitative PCR. Finally, we detected the mutation sites of the three genes among 160 foxtail millet materials and performed haplotype analysis to determine the genes' functional effect. We found that the cloned cDNA sequences of gene SiPHYA, SiPHYB and SiPHYC were 3 981, 3 953 and 3 764 bp respectively, which contained complete coding regions. Gene SiPHYB and SiPHYC showed closer evolutionary relationship. Photoperiod regulated all of the three genes, but showed more profound effects on diurnal expression pattern of SiPHYB, SiPHYC than that of SiPHYA. Under short-day, when near heading, the expression levels of SiPHYA and SiPHYB were significantly lower than that under long-day, indicating their roles in suppressing heading of foxtail millet under long-day. SiPHYB and SiPHYC were responsive to PEG-simulated drought, natural drought, ABA and high temperature stresses together. SiPHYA and SiPHYB responded differently to salt stress, whereas SiPHYC did not respond to salt stress. Re-sequencing of 160 foxtail millet materials revealed that SiPHYB was highly conservative. Two missense mutations of SiPHYA, such as single nucleotide polymorphism (SNP) 7 034 522C→T and SNP7 036 657G→C, led to delaying heading and increasing plant height. One missense mutation of SiPHYC, such as SNP5 414 823G→T, led to shortening heading under short-day and delaying heading under long-day, as well as increasing plant height and panicle length regardless of photo-thermal conditions. Photoperiod showed different regulatory effects on SiPHYA, SiPHYB and SiPHYC. SiPHYB and SiPHYC jointly responded to various abiotic stresses except for the salt stress. Compared with the reference genotype, mutation genotypes of SiPHYA and SiPHYC delayed heading and increased plant height and panicle length.

Cloning and Characterization of Eukaryotic Translation Initiation Factor 4E (eIF4E) Gene Family in &amp;lt;i&amp;gt;Ipomoea batatas&amp;lt;/i&amp;gt; L. (Lam) for Understanding Hexaploid Sweetpotato-Virus Interactions

Characterization of genes related to sweetpotato to viral disease resistance is critical for understanding plant-pathogen interactions, especially with feathery mottle virus infection. For example, genes encoding eukaryotic translation initiation factor (eIF)4E, its isoforms, eIF(iso)4E, and the cap-binding protein (CBP) in plants, have been implicated in viral infections aside from their importance in protein synthesis. Full-length cDNA encoding these putative eIF targets from susceptible/resistant and unknown hexaploid sweetpotato (Ipomoea batatas L. Lam) were amplified based on primers designed from the diploid wild-type relative Ipomoea trifida consensus sequences, and designated IbeIF4E, IbeIF(iso)4E and IbCBP. Comparative analyses following direct-sequencing of PCR-amplified cDNAs versus the cloned cDNA sequences identified multiple homeoalleles: one to four IbeIF4E, two to three IbeIF(iso)4E, and two IbCBP within all cultivars tested. Open reading frames were in the length of 696 bp IbeIF4E, 606 bp IbeIF(iso)4E, and 675 bp IbCBP. The encoded single polypeptide lengths were 232, 202, and 225 amino acids for IbeIF4E, IbeIF(iso)4E, and IbCBP, with a calculated protein molecular mass of 26 kDa, 22.8 kDa, and 25.8 kDa, while their theoretical isoelectric points were 5.1, 5.57, and 6.6, respectively. Although the homeoalleles had similar sequence lengths, single nucleotide polymorphisms and multi-allelic variations were detected within the coding sequences. The multi-sequence alignment performed revealed a 66.9% - 96.7% sequence similarity between the predicted amino acid sequences obtained from the homeoalleles and closely related species. Furthermore, phylogenetic analysis revealed ancestral relationships between the eIF4E homeoalleles and other species. The outcome herein on the eIF4E superfamily and its correlation in sequence variations suggest opportunities to decipher the role of eIF4E in hexaploid sweetpotato feathery mottle virus infection.

A Comprehensive Analysis of Human Endogenous Retroviruses HERV-K (HML.2) from Teratocarcinoma Cell Lines and Detection of Viral Cargo in Microvesicles.

About 8% of our genome is composed of sequences from Human Endogenous Retroviruses (HERVs). The HERV-K (HML.2) family, here abbreviated HML.2, is able to produce virus particles that were detected in cell lines, malignant tumors and in autoimmune diseases. Parameters and properties of HML.2 released from teratocarcinoma cell lines GH and Tera-1 were investigated in detail. In most experiments, analyzed viruses were purified by density gradient centrifugation. HML.2 structural proteins, reverse transcriptase (RT) activity, viral RNA (vRNA) and particle morphology were analyzed. The HML.2 markers were predominantly detected in fractions with a buoyant density of 1.16 g/cm3. Deglycosylation of TM revealed truncated forms of transmembrane (TM) protein. Free virions and extracellular vesicles (presumably microvesicles—MVs) with HML.2 elements, including budding intermediates, were detected by electron microscopy. Viral elements and assembled virions captured and exported by MVs can boost specific immune responses and trigger immunomodulation in recipient cells. Sequencing of cDNA clones demonstrated exclusive presence of HERV-K108 env in HML.2 from Tera-1 cells. Not counting two recombinant variants, four known env sequences were found in HML.2 from GH cells. Obtained results shed light on parameters and morphology of HML.2. A possible mechanism of HML.2-induced diseases is discussed.

Cloning, tissue distribution and functional characterization of the donkey (Equus asinus) oligopeptide transporter 2.

Oligopeptide transporter 2 (PepT2) is an important transporter of oligopeptides. In the present study, we describe the molecular cloning, tissue distribution and functional characterization of a donkey (Equus asinus) PepT2. The cloned cDNA sequence was 2202bp at full length, encoding a 733 amino acid peptide with a molecular weight of 81.9kDa and a theoretical pI of 8.92. Bioinformatics analysis showed that the deduced peptide sequence possessed all the characteristic features of PepT2. The expression of PepT2 in the kidney and lung was significantly higher than that observed in the ileum, duodenum, jejunum, spleen, liver, heart and stomach. Functional characterization by heterologous expression in Chinese hamster ovary cells showed that the uptake of β-Ala-Lys-N-7-amino-4-methylcoumarin-3-acetic acid (β-Ala-Lys-AMCA) by donkey PepT2-Chinese hamster ovary cells was dependent on time, pH and substrate concentration, with a low Km value of 91.51±14.14μM and a maximum velocity of 41.37±2.193pmol/min/mg protein. In the present study, for the first time, the expression and functional characteristics of donkey PepT2 were evaluated, the results of which provide new insights and a better understanding of its crucial role in oligopeptide transport in donkeys.

A Preliminary Toxicology Study on Eco-friendly Control Target of Spodoptera frugiperda.

Pyrethroid and indoxacarb are commonly used pesticides to control the fall armyworm (Spodoptera frugiperda) in the crops. There are a series of consequences caused by the use of pyrethroid and indoxacarb pesticides under emergency control, such as pest resistance development, contamination of soil, water, and farm products. This study analyzed the structure and physiological function of the sodium channel in S. frugiperda, which is an important step to elaborate the resistance mechanism of S. frugiperda to indoxacarb and pyrethroid pesticides. According to genetic analysis, the cloned cDNA sequences of sodium channel in S. frugiperda (SfNav) showed the shortest genetic distance with that of the sodium channel in Helicoverpa armigera. Under the induction of three pesticides, the expression of SfNav decreased in the first 12h and then increased after 24h. It was concluded that SfNav had a typical structure of the sodium channel of insects and its down-regulated expression can decrease the combination of S. frugiperda with pyrethroid and indoxacarb pesticides. The up-regulated expression of SfNav was conducive to the enhancement of the pesticide resistance.

Identification of the cDNA Encoding the Growth Hormone Receptor (GHR) and the Regulation of GHR and IGF-I Gene Expression by Nutritional Status in Reeves' Turtle (Chinemys reevesii).

Chinemys reevesii (Reeves’ turtle) is a slow-growing reptile that is distributed widely across China. Prior to this study, the cDNA sequence of the growth hormone receptor (GHR) in the Reeve’s turtle, or how periods of starvation might influence the gene expression of GHR and insulin-like growth factor I (IGF-I) in this species, were unknown. Here, we identified the full-length sequence of the cDNA encoding GHR in Reeves’ turtle by using RT-PCR and RACE. The full-length GHR cDNA was identified to be 3936 base-pairs in length, with a 1848 base-pair open reading frame (ORF) that encodes a 615 amino acid protein. Analysis showed that GHR mRNA was detectable in a wide range of tissues; the highest and lowest levels of expression were detected in the liver and the gonad, respectively. IGF-I was also expressed in a range of tissues, but not in the gonad; the highest levels of IGF-I expression were detected in the liver. After 4 weeks of fasting, the expression levels of GHR and IGF-I in the liver had decreased significantly; however, these gradually returned to normal after refeeding. We report the first cloned cDNA sequence for the GHR gene in the Reeve’s turtle. Our findings provide a foundation from which to investigate the specific function of the GHR in Reeve’s turtle, and serve as a reference for studying the effects of different nutrient levels on GHR expression in this species.

Construction and characterization of an infectious cDNA clone of potato virus S developed from selected populations that survived genetic bottlenecks

BackgroundInfectious cDNA clones are a powerful tool for studies on RNA viruses using reverse genetics. Potato virus S (PVS) is a carlavirus with a worldwide distribution. Although the complete genome sequences of many PVS isolates have been reported, the construction of an infectious cDNA clone of PVS is yet to be reported. The aim of this study is the development and molecular characterization of an infectious cDNA clone of PVS.MethodsA full-length cDNA clone pPVS-H-FL-AB was constructed by connecting eight cDNA clones of PVS isolate H95. Capped RNA transcripts from pPVS-H-FL-AB and a modified clone pPVS-H-FL-H, containing the consensus genome sequence of PVS-H95, proved to be non-infectious. Therefore, a full-length cDNA clone pPVS-H-FL-β was reconstructed from PVS-H00, isolated from PVS-H95 populations by repeating a single local lesion isolation in Chenopodium quinoa three times; PVS-H00 appeared to be a selected variant that survived genetic bottlenecks. The sequence of cDNA clone pPVS-H-FL-β was determined as the genome sequence of PVS-H00 and compared with the consensus sequence of PVS-H95 genome.ResultsAll Nicotiana occidentalis plants inoculated with ≥0.2 μg capped RNA transcripts from pPVS-H-FL-β developed symptoms on upper leaves, as observed with PVS-H00 inoculation. Similar levels of viral genomic and subgenomic RNAs and coat protein were detected in systemically infected leaves. Sequence comparison of PVS-H95 and PVS-H00 revealed 370 nucleotide polymorphisms (4.4% of the entire genome sequence), causing 91 amino acid substitutions in six open reading frames (ORFs). The infectivity of chimeric RNAs derived from recombinants between the two cDNA clones revealed that the lack of infectivity of pPVS-H-FL-H transcripts was due to ORF1, which encodes replicase and harbors 80 amino acid substitutions compared with pPVS-H-FL-β. Approximately 71.3% amino acid substitutions in replicase were located within the variable region of unknown function between the putative methyltransferase and ovarian tumor-like protease domains.ConclusionsThis is the first report of the development of an infectious cDNA clone of PVS. Our analyses suggest that PVS population within a plant exists as quasispecies and the replicase sequence diversity of PVS obstruct the construction of a full-length infectious cDNA clone.

Nucleotide heterogeneity at the terminal ends of the genomes of two California Citrus tristeza virus strains and their complete genome sequence analysis

BackgroundThe non-translated regions at the genome ends of RNA viruses serve diverse functions and can exhibit various levels of nucleotide (nt) heterogeneity. However, the extent of nt heterogeneity at the extreme termini of Citrus tristeza virus (CTV) genomes has not been comprehensively documented. This study aimed to characterize two widely prevalent CTV genotypes, T36-CA and T30-CA, from California that have not been sequenced or analyzed substantially. The information obtained will be used in our ongoing effort to construct the infectious complementary (c) DNA clones of these viruses.MethodsThe terminal nts of the viral genomes were identified by sequencing cDNA clones of the plus- and/or minus-strand of the viral double-stranded (ds) RNAs generated using 5′ and 3′ rapid amplification of cDNA ends. Cloned cDNAs corresponding to the complete genome sequences of both viruses were generated using reverse transcription-polymerase chain reactions, sequenced, and subjected to phylogenetic analysis.ResultsAmong the predominant terminal nts identified, some were identical to the consensus sequences in GenBank, while others were different or unique. Remarkably, one of the predominant 5′ nt variants of T36-CA contained the consensus nts “AATTTCAAA” in which a highly conserved cytidylate, seen in all other full-length T36 sequences, was absent. As expected, but never systematically verified before, unique variants with additional nt (s) incorporated upstream of the 5′ terminal consensus nts of T36-CA and T30-CA were also identified. In contrast to the extreme 5′ terminal nts, those at the extreme 3′ termini of T36-CA and T30-CA were more conserved compared to the reference sequences, although nt variants were also found. Notably, an additional thymidylate at the extreme 3′ end was identified in many T36-CA sequences. Finally, based on pairwise comparisons and phylogenetic analysis with multiple reference sequences, the complete sequences of both viruses were found to be highly conserved with those of the respective genotypes.ConclusionsThe extreme terminal nts in the T36-CA and T30-CA genomes were identified, revealing new insights on the heterogeneity of these CTV genomic regions. T36-CA and T30-CA were the first and the second genotypes, respectively, of CTV originating from California to be completely sequenced and analyzed.

CDNA cloning of gs, gogat, and gdh from Haematococcus pluvialis and transcription and enzyme level analysis in different nitrogen concentration

Haematococcus pluvialis has attracted attention due to its ability to produce astaxanthin, which has many functions such as antioxidation and anticancer. In order to further understand its nitrogen metabolism, the transcription levels and the enzyme levels of glutamine synthetase (GS), glutamate synthase (GOGAT), and glutamate dehydrogenase (GDH) in the process of nitrogen assimilation were studied under different nitrogen concentrations after cloning their cDNA sequences. The cloned cDNA sequence of gs is 1438 bp, including a 178-bp 5′ untranslatable region (UTR), a 114-bp 3′ UTR, and a 1146-bp open reading frame (ORF), which encodes a protein of 381 amino acids. The gogat had a total cDNA of 4931 bp, including a 17-bp 5′ UTR and a 4914-bp ORF, which encodes a protein of 1637 amino acids. The gdh had a total cDNA of 1529 bp, including 131-bp 3′ UTR and a 1398-bp ORF, which encodes a protein of 465 amino acids. During cultivation the transcription levels of the three genes fluctuated slightly during the first 4 days and then declined significantly. At the fourth day, the highest level of the transcription of the three genes was at the nitrogen concentration of 1000 mg L−1. The enzyme level also fluctuated with culture time, which is positively correlated with the transcription level. This study may lay foundation for optimization of nitrogen concentration for cultivation of H. pluvialis.

Cloning cDNA sequence coding the KSA-1 lectin from red alga Kappaphycus striatum cultivated in Vietnam

The red alga Kappaphycus striatum is economically important food species and extensively cultivated in Vietnam. This cultivated alga may thus now contribute as a source of not only carrageenan, but also other bioactive compounds for biochemical and medicinal uses. KSA-1 lectin from this alga has been evaluated for biochemical properties, including high-mannose binding specificity, N-terminal amino acid sequence and molecular mass. In this study, we have cloned the cDNA clone encoding the lectin KSA-1. The full-length sequence of cDNA clone of KSA-1 lectin encoded a polypeptide of 268 amino acids including initiating methionine, with four tandemly repeated domains of about 67 amino acids, and sharing 43% sequence identity. The primary structure and the amino acid residues interact with mannopentaose core structure per a repeat domain of the KSA-1 lectin highly resemble and match those of anti-virus the lectin family in lower organinsms, including bacteria [BOA from Burkholderia oklahomensis EO147, MBHA from Myxococcus xanthus and PFA from Pseudomonas fluorescens Pf0-1], cyanobacteria [OAA from Oscillatoria agardhii] and red algae [ESA-2 from Eucheuma serra, EDA-2 from Eucheuma denticulatum, KAA-1 and KAA-2 from Kappaphycus alvarezii and KSA-2 from K. striatum]. Analysis of predicted secondary structure of KSA-1 showed twenty β-strands. Each repeated domain comprises the five β-strands and matches with number of β-strands of BOA lectin. Therefore, the red alga K. striatum could be a good source for application in biomedicine and biochemistry as materials for production of the functional lectin(s).

Cloning, tissue distribution, functional characterization and nutritional regulation of Δ6 fatty acyl desaturase in chu's croaker Nibea coibor

In the present study, we successfully cloned the full-length cDNA of a Δ6 fatty acyl desaturase (Δ6 Fad) of chu's croaker Nibea coibor. The cloned cDNA sequence was 1590bp at full length, including a 5′-UTR of 137bp, a 3′-UTR of 115bp and a mean ORF of 1338bp that specified a peptide of 445 aa. Sequence analysis of the deduced polypeptide showed that it contained all the characteristics of ∆6 Fad identified such as one cytochrome b5 domain, three trans-membrane regions and three histidine-rich boxes. Tissue distribution revealed that it strongly expressed in brain, significantly high than the level in eye, spleen, intestine, heart, gill, liver, stomach, muscle, liver and kidney. Functional characterization by heterologous expression in yeast showed that the chu's croaker desaturase was a Fad2 with ∆6 Fad activity, with 9.36% conversion of 18:2n-6 to 18:3n-6 and 4.93% of 18:3n-3 to 18:4n-3, respectively. Nutritional study showed that the expression of ∆6 Fad in liver were not significantly affected among treatments.

The major constituents of the venom gland of a braconid endoparasitoid, Meteorus pulchricornis (Hymenoptera: Braconidae)

The endoparasitoid wasp, Meteorus pulchricornis (Wesmael), introduces virus-like particles [M. pulchricornis VLP (MpVLP)] and venom into its lepidopteran host larvae upon oviposition. These wasp-associated factors are considered to help impair some host functions including immune defense. To obtain the major protein repertoire of the MpVLP and venom that might support successful parasitism, we constructed a conventional cDNA library of the venom gland filament that produces both of them, and sequenced cDNA clones arbitrarily in the 5′ regions. The 5′ expressed sequence tags obtained from 473 independent cDNA clones were grouped into 228 clusters. One hundred and five of these clusters were annotated with one or more gene ontology (GO) terms by Blast2GO analyses. While the major repertoire of the M. pulchricornis venom gland shared some constituents with those of the venom glands of other parasitoid wasp species, it was distinct from those encoded by polydnaviruses. Twenty clusters selected for further analyses were fully sequenced and characterized again. Among them, 17 factors were subjected to quantitative reverse transcription polymerase chain reaction analyses, and 12 factors were suggested to be highly adult specific. Among these 17 factors, ten were tested as RNA interference targets, and effective gene silencing was observed for five factors. These five factors included two components of MpVLP. The MpVLP deficient in the two factors prepared from the knockdown wasps was less effective than the wild-type MpVLP in terms of the inhibitory activity against host hemocyte spreading estimated in vitro.

Novel Catalytically-Inactive PII Metalloproteinases from a Viperid Snake Venom with Substitutions in the Canonical Zinc-Binding Motif.

Snake venom metalloproteinases (SVMPs) play key biological roles in prey immobilization and digestion. The majority of these activities depend on the hydrolysis of relevant protein substrates in the tissues. Hereby, we describe several isoforms and a cDNA clone sequence, corresponding to PII SVMP homologues from the venom of the Central American pit viper Bothriechis lateralis, which have modifications in the residues of the canonical sequence of the zinc-binding motif HEXXHXXGXXH. As a consequence, the proteolytic activity of the isolated proteins was undetectable when tested on azocasein and gelatin. These PII isoforms comprise metalloproteinase and disintegrin domains in the mature protein, thus belonging to the subclass PIIb of SVMPs. PII SVMP homologues were devoid of hemorrhagic and in vitro coagulant activities, effects attributed to the enzymatic activity of SVMPs, but induced a mild edema. One of the isoforms presents the characteristic RGD sequence in the disintegrin domain and inhibits ADP- and collagen-induced platelet aggregation. Catalytically-inactive SVMP homologues may have been hitherto missed in the characterization of snake venoms. The presence of such enzymatically-inactive homologues in snake venoms and their possible toxic and adaptive roles deserve further investigation.

Effects and mechanisms of waterborne copper exposure influencing ovary development and related hormones secretion in yellow catfish Pelteobagrus fulvidraco

The present study was conducted to determine the effects and mechanism of waterborne copper (Cu) exposure influencing ovary development and related hormones secretion in yellow catfish Pelteobagrus fulvidraco. To this end, two experiments were conducted. In Exp. 1, the partial cDNA sequences of three steroidogenesis-related genes (androgen receptor (ar), steroidogenic factor 1 (sf-1) and steroidogenic acute regulatory protein (star)) were firstly characterized from P. fulvidraco. The predicted amino acid sequences for the P. fulvidraco ar, sf-1 and star contained the main structural features characteristic in other species. In Exp. 2, P. fulvidraco were exposed to three waterborne Cu concentrations (control, 30μg/l and 60μg/l, respectively) for 56days. Sampling occurred on day 28 and day 56, respectively. On day 28, the levels of serum sex-steroid hormones (FSH and LH) and the mRNA levels of steroidogenesis-related genes (3β-hsd, cyp11a1, cyp17, cyp19a, sf-1 and star) were significantly increased in ovary of P. fulvidraco exposed to 30μg Cu/l. The immunohistochemical analysis showed the positive reaction of ER, VTG and aromatase in low dose exposure group. These indicated that in low dose and relative short-term exposure, Cu was beneficial. In contrast, 60μg Cu/l exposure significantly reduced the levels of serum FSH, LH, E2 and P, and the mRNA levels of ovarian 20β-hsd, cyp19a and erα in P. fulvidraco. On day 56, waterborne Cu concentration exposure reduced the levels of serum gonadotropins and sex hormones, and down-regulated the mRNA levels of steroidogenesis-related genes, indicating long-term Cu exposure had toxic effect on the secretion of sex-steroid hormone in P. fulvidraco. For the first time, our study cloned cDNA sequences of ar, sf-1 and star in P. fulvidraco, and demonstrated the effects and mechanism of waterborne Cu exposure influencing hormones secretion and synthesis in dose- and time-dependent manner in P. fulvidraco, which will help to understand the Cu-induced reproductive toxicity at both protein and transcriptional levels in fish.

A variant of Runx2 that differs from the bone isoform in its splicing is expressed in spermatogenic cells.

Background. Members of the Runx gene family encode transcription factors that bind to DNA in a sequence-specific manner. Among the three Runx proteins, Runx2 comprises 607 amino acid (aa) residues, is expressed in bone, and plays crucial roles in osteoblast differentiation and bone development. We examined whether the Runx2 gene is also expressed in testes.Methods. Murine testes from 1-, 2-, 3-, 4-, and 10-week-old male mice of the C57BL/6J strain and W∕Wv strain were used throughout the study. Northern Blot Analyses were performed using extracts form the murine testes. Sequencing of cDNA clones and 5′-rapid amplification of cDNA ends were performed to determine the full length of the transcripts, which revealed that the testicular Runx2 comprises 106 aa residues coding novel protein. Generating an antiserum using the amino-terminal 15 aa of Runx2 (Met1 to Gly15) as an antigen, immunoblot analyses were performed to detect the predicted polypeptide of 106 aa residues with the initiating Met1. With the affinity-purified anti-Runx2 antibody, immunohistochemical analyses were performed to elucidate the localization of the protein. Furthermore, bioinformatic analyses were performed to predict the function of the protein.Results. A Runx2 transcript was detected in testes and was specifically expressed in germ cells. Determination of the transcript structure indicated that the testicular Runx2 is a splice isoform. The predicted testicular Runx2 polypeptide is composed of only 106 aa residues, lacks a Runt domain, and appears to be a basic protein with a predominantly alpha-helical conformation. Immunoblot analyses with an anti-Runx2 antibody revealed that Met1 in the deduced open reading frame of Runx2 is used as the initiation codon to express an 11 kDa protein. Furthermore, immunohistochemical analyses revealed that the Runx2 polypeptide was located in the nuclei, and was detected in spermatocytes at the stages of late pachytene, diplotene and second meiotic cells as well as in round spermatids. Bioinformatic analyses suggested that the testicular Runx2 is a histone-like protein.Discussion. A variant of Runx2 that differs from the bone isoform in its splicing is expressed in pachytene spermatocytes and round spermatids in testes, and encodes a histone-like, nuclear protein of 106 aa residues. Considering its nuclear localization and differentiation stage-dependent expression, Runx2 may function as a chromatin-remodeling factor during spermatogenesis. We thus conclude that a single Runx2 gene can encode two different types of nuclear proteins, a previously defined transcription factor in bone and cartilage and a short testicular variant that lacks a Runt domain.

Creation of Functional Viruses from Non-Functional cDNA Clones Obtained from an RNA Virus Population by the Use of Ancestral Reconstruction.

RNA viruses have the highest known mutation rates. Consequently it is likely that a high proportion of individual RNA virus genomes, isolated from an infected host, will contain lethal mutations and be non-functional. This is problematic if the aim is to clone and investigate high-fitness, functional cDNAs and may also pose problems for sequence-based analysis of viral evolution. To address these challenges we have performed a study of the evolution of classical swine fever virus (CSFV) using deep sequencing and analysis of 84 full-length cDNA clones, each representing individual genomes from a moderately virulent isolate. In addition to here being used as a model for RNA viruses generally, CSFV has high socioeconomic importance and remains a threat to animal welfare and pig production. We find that the majority of the investigated genomes are non-functional and only 12% produced infectious RNA transcripts. Full length sequencing of cDNA clones and deep sequencing of the parental population identified substitutions important for the observed phenotypes. The investigated cDNA clones were furthermore used as the basis for inferring the sequence of functional viruses. Since each unique clone must necessarily be the descendant of a functional ancestor, we hypothesized that it should be possible to produce functional clones by reconstructing ancestral sequences. To test this we used phylogenetic methods to infer two ancestral sequences, which were then reconstructed as cDNA clones. Viruses rescued from the reconstructed cDNAs were tested in cell culture and pigs. Both reconstructed ancestral genomes proved functional, and displayed distinct phenotypes in vitro and in vivo. We suggest that reconstruction of ancestral viruses is a useful tool for experimental and computational investigations of virulence and viral evolution. Importantly, ancestral reconstruction can be done even on the basis of a set of sequences that all correspond to non-functional variants.

Experimental confirmation of a whole set of tRNA molecules in two archaeal species.

Based on the genomic sequences for most archaeal species, only one tRNA gene (isodecoder) is predicted for each triplet codon. This observation promotes analysis of a whole set of tRNA molecules and actual splicing patterns of interrupted tRNA in one organism. The entire genomic sequences of two Creanarchaeota, Aeropyrum pernix and Sulfolobus tokodaii, were determined approximately 15 years ago. In these genome datasets, 47 and 46 tRNA genes were detected, respectively. Among them, 14 and 24 genes, respectively, were predicted to be interrupted tRNA genes. To confirm the actual transcription of these predicted tRNA genes and identify the actual splicing patterns of the predicted interrupted tRNA molecules, RNA samples were prepared from each archaeal species and used to synthesize cDNA molecules with tRNA sequence-specific primers. Comparison of the nucleotide sequences of cDNA clones representing unspliced and spliced forms of interrupted tRNA molecules indicated that some introns were located at positions other than one base 3' from anticodon region and that bulge-helix-bulge structures were detected around the actual splicing sites in each interrupted tRNA molecule. Whole-set analyses of tRNA molecules revealed that the archaeal tRNA splicing mechanism may be essential for efficient splicing of all tRNAs produced from interrupted tRNA genes in these archaea.

Cloning, molecular characterization, and expression analysis of a nucleoporin gene (rgNUP98-96) from Rehmannia glutinosa.

Nucleoporin 98 (NUP98) and nucleoporin 96 (NUP96) are essential components of the nuclear pore complex (NPC) in eukaryote cells. However, there is a lack of available information about complete Rehmannia glutinosa NUP98-96 (rgNUP98-96) sequences. Here, the full-length cDNA sequence of rgNUP96-98 was isolated from R. glutinosa using rapid amplification of cDNA ends (RACE) technology, based on a cloned cDNA sequence (GenBank accession No. JZ483329). The identified rgNUP98-96 was 3476 bp, and it encoded a 1041-amino acid peptide. The BLAST search analysis of rgNUP98-96 showed an intermediate degree of similarity (60-79%) to the NUP98-96 protein sequences of 34 other plants, including the dicotyledons Erythranthe guttata, Genlisea aurea, Coffea canephora, Nicotiana benthamiana, Solanum lycopersicum, and Solanum tuberosum. The phylogenetic analysis of NUP96-98 sequences indicated that R. glutinosa and E. guttata sequences shared the closest homology. The calculated molecular mass and predicted isolectric point of the complex protein were 117.6 kDa and 4.99, respectively. The secondary and three-dimensional structure studies illustrated that the rgNUP96-98 protein folded into a channel motif comprised of 34 alpha-helices, nine beta-strands, and several long loops. Using quantitative real-time PCR, the spatio-temporal expression patterns of rgNUP98-96 were analyzed in R. glutinosa, and the results indicated that rgNUP98-96 was highly expressed at the early stage of R. glutinosa tuberous root expansion, which is associated with a higher expression pattern in roots. The study provides a valuable foundation for further investigation of rgNUP96-98 molecular functions in R. glutinosa.

CDNA library construction from meristematic tissue of finger millet panicle

A protocol to obtain full-length cDNA using a SuperScript® Full-Length cDNA Library Construction Kit II (Invitrogen, United States) was developed, and a high quality cDNA library of meristem tissue of finger millet panicle (Eleusine coracana (L.) Gaertn) was constructed. The titer of the constructed cDNA library was 3.01 × 105 CFU/mL, the average length of the insert was approximately 1070 base pairs, and the average efficiency of insertion of cDNA fragments was 99.5%. The sequencing of randomly selected clones created cDNA library was carried out. The cDNA sequences of clones were identified by BLAST search. The cDNA library analysis and selective sequencing indicate good functionality and full size of cDNA inserts of the clones. The constructed cDNA library from meristematic tissue of finger millet panicle is a good and reliable source for isolation and identification of key genes of metabolism and development of meristem as well for creation of new genetic markers for genetic research and molecular selection.