Complete Open Reading Frame Sequences Research Articles

Molecular characterization and expression profiling of foxl2 in zig-zag eel (Mastacembelus armatus)

Zig-zag eel (Mastacembelus armatus) is an important aquaculture fish species that is very popular as a table fish. However, the genetic regulation of gonad development and differentiation is not well understood. Foxl2 has been demonstrated to play a critical role in gonad development and differentiation in fish. In our study, foxl2 was first cloned and characterized from the gonads of zig-zag eel. The complete open reading frame sequence of foxl2 was 918 bp, encoding 305 amino acids. Multiple sequence alignments revealed that foxl2 gene was highly conserved in fish, especially in forkhead domain. Tissue distribution analysis revealed that foxl2 expressed mainly in ovary, gill, eye and brain. At five different embryonic stages, foxl2 mainly expressed at the gastrula stage. Besides, gonad expression pattern revealed the expression of foxl2 increased to peak in stage V of the ovary, while the expression level was highest in stage III of the testis. These results indicated that the foxl2 might play an important role in gonadal development of zig-zag eel.

Inferring Evolutionary Timescale of Omsk Hemorrhagic Fever Virus.

Until 2020, there were only three original complete genome (CG) nucleotide sequences of Omsk hemorrhagic fever virus (OHFV) in GenBank. For this reason, the evolutionary rate and divergence time assessments reported in the literature were based on the E gene sequences, but notably without temporal signal evaluation, such that their reliability is unclear. As of July 2022, 47 OHFV CG sequences have been published, which enables testing of temporal signal in the data and inferring unbiased and reliable substitution rate and divergence time values. Regression analysis in the TempEst software demonstrated a stronger clocklike behavior in OHFV samples for the complete open reading frame (ORF) data set (R2 = 0.42) than for the E gene data set (R2 = 0.11). Bayesian evaluation of temporal signal indicated very strong evidence, with a log Bayes factor of more than 5, in favor of temporal signal in all data sets. Our results based on the complete ORF sequences showed a more precise OHFV substitution rate (95% highest posterior density (HPD) interval, 9.1 × 10-5-1.8 × 10-4 substitutions per site per year) and tree root height (416-896 years ago) compared with previous assessments. The rate obtained is significantly higher than tick-borne encephalitis virus by at least 3.8-fold. The phylogenetic analysis and past population dynamics reconstruction revealed the declining trend of OHFV genetic diversity, but there was phylogenomic evidence that implicit virus subpopulations evolved locally and underwent an exponential growth phase.

Characterization of dmrts and their potential role in gonadal development of mandarin fish (Siniperca chuatsi)

Dmrt gene family plays a very critical role in sex determination and differentiation in animals. In this study, dmrt1, dmrt2 and dmrt3 were first cloned from the gonads of mandarin fish (Siniperca chuatsi). The complete open reading frame sequences of dmrt1, dmrt2 and dmrt3 were 891, 1581 and 1419 bp, encoding 296, 526 and 473 amino acids. Multiple sequence alignments revealed that dmrt proteins were highly conserved in teleosts, especially in DNA-binding domain. Tissue distribution analysis showed that dmrt1 expressed mainly in testis, dmrt2 expressed in ovary and dmrt3 expressed in heart, muscle and gonad. Gonad expression pattern revealed dmrt1 mainly expressed in testis after 15 day after hatching, while dmrt2 mainly expressed after 60 dah. After 17α-methyltestosterone treatment, expression levels of dmrt1 and dmrt3 increased significantly, while dmrt2 expression was repressed. Furthermore, luciferase assay showed dmrt1 could activate gsdf-promotor and inhibit cyp19a1a-promotor alone in vitro. These results suggest that the dmrt1 play a most important role in testis differentiation/development and dmrt2 is critical in ovary development in mandarin fish.

PacBio single molecule long-read sequencing provides insight into the complexity and diversity of the Pinctada fucata martensii transcriptome

BackgroundThe pearl oyster Pinctada fucata martensii is an economically valuable shellfish for seawater pearl production, and production of pearls depends on its growth. To date, the molecular mechanisms of the growth of this species remain poorly understood. The transcriptome sequencing has been considered to understanding of the complexity of mechanisms of the growth of P. f. martensii. The recently released genome sequences of P. f. martensii, as well as emerging Pacific Bioscience (PacBio) single-molecular sequencing technologies, provide an opportunity to thoroughly investigate these molecular mechanisms.ResultsHerein, the full-length transcriptome was analysed by combining PacBio single-molecule long-read sequencing (PacBio sequencing) and Illumina sequencing. A total of 20.65 Gb of clean data were generated, including 574,561 circular consensus reads, among which 443,944 full-length non-chimeric (FLNC) sequences were identified. Through transcript clustering analysis of FLNC reads, 32,755 consensus isoforms were identified, including 32,095 high-quality consensus sequences. After removing redundant reads, 16,388 transcripts were obtained, and 641 fusion transcripts were derived by performing fusion transcript prediction of consensus sequences. Alternative splicing analysis of the 16,388 transcripts was performed after accounting for redundancy, and 9097 gene loci were detected, including 1607 new gene loci and 14,946 newly discovered transcripts. The original boundary of 11,235 genes on the chromosomes was corrected, 12,025 complete open reading frame sequences and 635 long non-coding RNAs (LncRNAs) were predicted, and functional annotation of 13,482 new transcripts was achieved. Two thousand three hundred eighteen alternative splicing events were detected. A total of 228 differentially expressed transcripts (DETs) were identified between the largest (L) and smallest (S) pearl oysters. Compared with the S, the L showed 99 and 129 significantly up-and down-regulated DETs, respectively. Six of these DETs were further confirmed by quantitative real-time RT-PCR (RT-qPCR) in independent experiment.ConclusionsOur results significantly improve existing gene models and genome annotations, optimise the genome structure, and in-depth understanding of the complexity and diversity of the differential growth patterns of P. f. martensii.

Analysis of codon usage bias in potato virus Y non-recombinant strains

Potato virus Y (PVY) is a member of the genus Potyvirus, family Potyviridae, is considered one of the most devastating pest affecting economically important crops, such as potato, tobacco, tomato and pepper, representing a serious threat due to high incidence and worldwide distribution. Its economic significance as well as it biological and molecular complexities have aroused great attention, thus several studies have explore it genetic characteristics. However, little is known about PVY codon usage. To shed light on the relation of codon usage among viruses and their hosts is extremely important to understand virus survival, fitness and evolution. In this study, we performed a comprehensive analysis of codon usage and composition of PVY non-recombinant strains (PVYN−NA, PVYEu-N, PVYO, PVYO5, PVYC) based on 130 complete open reading frame sequences extracted from public databases. Furthermore, similarities between the synonymous codon usage of PVY and its main hosts were investigated. The results obtained in the current study suggest that the overall codon usage among PVY genotypes is similar and slightly biased. PVY codon usage is strongly influenced by mutational bias, but also by G + C compositional constraint and dinucleotide composition. Furthermore, similarities among codon usage preferences between PVY strains and analyzed hosts were observed.

Changes Introduced in the Open Reading Frame of Bovine Viral Diarrhea Virus During Serial Infection of Pregnant Swine

Bovine viral diarrhea virus (BVDV) is one of the most economically important viruses of cattle, but this pathogen is also able to infect pigs, camelids, and a wide range of domestic and wild ruminants. BVDV isolates circulating in animal populations are genetically and antigenically highly diverse. Acute BVDV infections in cattle cause the introduction of many substitutions in the viral genome. Serial infection of pregnant sheep with a BVDV-1b isolate of bovine origin was also associated with great numbers of substitutions. To our knowledge, genomic changes arising during BVDV infections in swine have not been investigated. The purpose of this study was to investigate the changes occurring in the open reading frame (ORF) of BVDV during serial infection of pregnant swine with a BVDV isolate of bovine origin. The BVDV-1b isolate AU526 was serially passaged in six pregnant gilts, two of which gave birth to live piglets congenitally infected with BVDV. The complete ORF sequences of 14 BVDV isolates obtained from pregnant gilts and their piglets were determined. Their analysis revealed that serial transmission of AU526 in pregnant swine resulted in many genomic changes. All isolates of porcine origin shared 32 nucleotide and 12 amino acid differences with the virus inoculum AU526. These changes were detected after a single passage in pregnant swine and were conserved during the subsequent five passages. Amino acid changes occurred primarily in genomic regions encoding the BVDV structural proteins E2 and Erns. These results suggest that BVDV infections in pregnant swine may contribute significantly to the genetic variability of BVDV and lead to the appearance of adaptive changes.

Cyclic GMP-AMP synthase is essential for cytosolic double-stranded DNA and fowl adenovirus serotype 4 triggered innate immune responses in chickens

Cyclic GMP-AMP (cGAMP) synthase (cGAS) is a predominant DNA sensor inducing the activation of the innate immune responses that produce proinflammatory cytokines and type I interferons, which has been well-investigated in mammals. However, chicken cGAS (chcGAS), which participates in avian innate immunity, has not been well-investigated. Here, we cloned the complete open reading frame sequence of chcGAS. Multiple sequence alignment and phylogenetic analysis revealed that chcGAS was homologous to mammalian cGAS. The chcGAS mRNA was highly expressed in the bone marrow and ileum. The subcellular localization of chcGAS was mainly in the cytoplasm, and partial co-localization was observed in the endoplasmic reticulum. Through overexpression and RNA interference, we demonstrated that chcGAS responded to exogenous dsDNA, HS-DNA, and poly(dA:dT), and to self dsDNA from the DNA damage response, thereby triggering the activation of STING/TBK1/IRF7-mediated innate immunity in both chicken embryonic fibroblasts and chicken liver cancer cells. Furthermore, downregulation of chcGAS enhanced the infection of fowl adenovirus serotype 4 in LMH cells. Our results demonstrated that chcGAS was an important cytosolic DNA sensor activating innate immune responses and may shed light on a strategy for preventing infectious diseases in the poultry industry.

Genome Sequence of an Unknown Subtype of Hepatitis C Virus Genotype 6: Another Piece for the Taxonomic Puzzle.

The surveillance and correct subtyping of hepatitis C virus strains require available and up-to-date publicly available reference genomes. Here, we present the complete open reading frame sequence of a hepatitis C virus genotype 6 strain of an unknown subtype that was discovered during routine subtyping of patients in the clinic.

Cloning, expression and functional characterization of a novel luteinizing hormone receptor in the orange-spotted grouper, Epinephelus coioides

Luteinizing hormone receptor (LHR) plays a critical role in reproduction by mediating LH signaling in the gonad. In this study, we cloned a novel lhr gene from the orange-spotted grouper, named glhr2. The cloned complete open reading frame sequence of glhr2 was 2082 bp in length, encoding a protein of 693 amino acids, sharing approximately 50% amino acid identity with glhr1. glhr1 and glhr2 were primarily expressed in gonad, brain and hypothalamus with low expression in other tissues such as gill, spleen, etc. The expressions of both glhr1 and glhr2 increased during vitellogenesis, while decreased during natural female to male sex change. The two gLHRs both could be activated by equine LH or human chorionic gonadotropin, but not by human follicle stimulating hormone. Both gLHR1 and gLHR2 activation stimulated the expression of cAMP response element driven reporter gene in a dose-dependent manner, while gLHR2 but not gLHR1 also activated serum response element driven reporter gene expression. This was the first study to demonstrate that two active LHRs exist in fish with possible different functional roles.

Identification of Conserved Amino Acid Substitutions During Serial Infection of Pregnant Cattle and Sheep With Bovine Viral Diarrhea Virus.

Bovine viral diarrhea virus (BVDV) is an economically important pathogen of cattle that can also infect a wide range of domestic and wild species including sheep, goats, deer, camelids, and pigs. BVDV isolates are genetically highly diverse and previous work demonstrated that many substitutions were introduced in the viral genome during acute infections in cattle. In contrast, only limited information exists regarding changes occurring during BVDV infections in species other than cattle. The purpose of this study was to determine the changes introduced in the open reading frame (ORF) of the BVDV genome during serial infection of pregnant cattle and sheep with an isolate of bovine origin. Serial experimental inoculations were performed in six pregnant heifers and six pregnant ewes using BVDV-1b isolate AU526 in the first heifer and ewe, and serum from the preceding acutely infected dam thereafter. Complete ORF sequences were determined for 23 BVDV-1b isolates including AU526, one isolate from each pregnant dam, and one isolate from each BVDV-positive offspring born to these dams. Sequence comparison revealed that greater numbers of substitutions occurred during serial infection of pregnant sheep than of pregnant cattle. Furthermore, multiple host-specific amino acid changes were gradually introduced and conserved. These changes were more abundant in ovine isolates and occurred primarily in the E2 coding region. These results suggest that BVDV infections in heterologous species may serve as a significant source of viral genetic diversity and may be associated with adaptive changes.

Molecular cloning, characterization, and expression analysis of the three cysteine and glycine-rich protein genes in the Chinese fire-bellied newt Cynops orientalis

The cysteine- and glycine-rich protein (CRP) family members, including the cysteine- and glycine-rich protein 1 (CSRP1), cysteine- and glycine-rich protein 2 (CSRP2), and the cysteine- and glycine-rich protein 3 (CSRP3), have exhibited various cellular functions during cell development and differentiation. However, the sequences of the three CSRP genes and their functions are still poorly understood in newts. In this study, we cloned the complete open reading frame (ORF) sequences of the three CSRP genes from the Chinese fire-bellied newt, Cynops orientalis (C. orientalis). The complete ORF sequences of Co-CSRP1, Co-CSRP2, and Co-CSRP3 were 582, 582, and 576bp, respectively, and encoded 193, 193, and 191 amino acids, respectively. The deduced amino acid sequences of the three CRP members showed high similarities with that of other species, particularly, with amphibians. Co-CSRP1 was highly expressed in the kidney, limb, and stomach, however, the expression was low in the spleen, heart, intestine, liver, and tail (P<0.05). The mRNA expression of Co-CSRP2 was higher in the kidney and heart than that in other organs (P<0.05). It was observed that Co-CSRP3 was only expressed in the heart, limb, and tail. The mRNA expression of Co-CSRP1 and Co-CSRP3 was lower in the digits in comparison to other limb segments. However, there was no significant difference of Co-CSRP2 mRNA expression in the four limb segments. The Co-CSRP1 and Co-CSRP2 mRNA expressions were significantly increased, whereas the expression of Co-CSRP3 was remarkably decreased during the limb regeneration. This study will provide useful information for further elucidating the role of Co-CSRP genes during newt limb regeneration.

Genetic analysis of avian paramyxovirus type I strains isolatedfrom backyard poultry in Iran

Newcastle disease (ND) is one of the most serious diseases amongst many species of birds and causes huge economic losses in the poultry sector. Backyard poultry are the main source of food in rural areas of many developing countries and play a crucial role in the epidemiology of ND. The sequence of the F protein cleavage site is the major virulence determinant of Newcastle disease virus (NDV). The aim of this study was to understand the molecular characterization of F proteins of Iranian backyard poultry in NDVs. The complete open reading frame sequences of the F gene of six isolates were amplified and sequenced. All characterized NDVs had high similarities to Chinese isolates. The phylogenetic analysis revealed that the studied strains fell into genotype VII and grouped in a specific subgenotype. All tested NDVs have a cleavage site of 112RRQKRF↓117, representing that all of them belonged to velogenic strains. Rearing methods of backyard poultry increase the chance of contact between a variety of poultry and wild birds; subsequently, new strains evolve and are transmitted between them. Therefore, continuous monitoring of NDVs in backyard poultry would help us to obtain more information about the evolution of NDVs and improve new control strategies in backyard poultry.

Prion Protein Gene Variability in Spanish Goats. Inference through Susceptibility to Classical Scrapie Strains and Pathogenic Distribution of Peripheral PrPsc

Classical scrapie is a neurological disorder of the central nervous system (CNS) characterized by the accumulation of an abnormal, partially protease resistant prion protein (PrPsc) in the CNS and in some peripheral tissues in domestic small ruminants. Whereas the pathological changes and genetic susceptibility of ovine scrapie are well known, caprine scrapie has been less well studied. We report here a pathological study of 13 scrapie-affected goats diagnosed in Spain during the last 9 years. We used immunohistochemical and biochemical techniques to discriminate between classical and atypical scrapie and bovine spongiform encephalopathy (BSE). All the animals displayed PrPsc distribution patterns and western blot characteristics compatible with classical scrapie. In addition, we determined the complete open reading frame sequence of the PRNP in these scrapie-affected animals. The polymorphisms observed were compared with those of the herd mates (n = 665) and with the frequencies of healthy herds (n = 581) of native Spanish goats (Retinta, Pirenaica and Moncaina) and other worldwide breeds reared in Spain (Saanen, Alpine and crossbreed). In total, sixteen polymorphic sites were identified, including the known amino acid substitutions at codons G37V, G127S, M137I, I142M, H143R, R151H, R154H, R211Q, Q222K, G232W, and P240S, and new polymorphisms at codons G74D, M112T, R139S, L141F and Q215R. In addition, the known 42, 138 and 179 silent mutations were detected, and one new one is reported at codon 122. The genetic differences observed in the population studied have been attributed to breed and most of the novel polymorphic codons show frequencies lower than 5%. This work provides the first basis of polymorphic distribution of PRNP in native and worldwide goat breeds reared in Spain.

Genetic characterization of Bagaza virus (BAGV) isolated in India and evidence of anti-BAGV antibodies in sera collected from encephalitis patients

During investigations into the outbreak of encephalitis in 1996 in the Kerala state in India, an arbovirus was isolated from a Culex tritaeniorhynchus mosquito pool. It was characterized as a Japanese encephalitis and West Nile virus cross-reactive arbovirus by complement fixation test. A plaque reduction-neutralization test was performed using hyperimmune sera raised against the plaque-purified arbovirus isolate. The sera did not show reactivity with Japanese encephalitis virus and were weakly reactive with West Nile virus. Complete open reading frame sequence analysis characterized the arbovirus as Bagaza virus (BAGV), with 94.80 % nucleotide identity with African BAGV strain DakAr B209. Sera collected from the encephalitic patients during the acute phase of illness showed 15 % (8/53) positivity for anti-BAGV neutralizing antibodies. This is the first report of the isolation of BAGV from India. The presence of anti-BAGV neutralizing antibodies suggests that the human population has been exposed to BAGV.

Isolation, expression patterns and SNP frequencies of the porcine PHKG2 gene

Phosphorylase kinase, gamma 2 gene ( PHKG2) encodes the catalytic subunit of phosphorylase kinase which is a regulatory enzyme in the cascade activation of glycogenolysis. In order to further understand the functions of the porcine PHKG2 gene, we obtained the complete open reading frame sequences and the determination of its expression by real-time PCR. Our spatial expression analysis showed that porcine PHKG2 gene was highly expressed in spleen, uterus and ovary. In contrast, low levels of expression of this gene were evident in heart, liver, lung, kidney, stomach, small intestine or fat, and much lower levels in skeletal muscle. Three SNPs, 785G>A, 866G>A and 875G>A were identified, their genotypes and allelic frequencies were analyzed. The frequencies of the G allele were dominant in Western pig breeds and the frequencies of the A allele were dominant in Chinese indigenous breeds in all analyzed polymorphisms. Haplotypes harboring these three polymorphisms were examined in five breeds, a total of 8 haplotypes were found in those breeds, which was equal to the theoretically expected number (2 3). The date presented here provided information about the functions of porcine PHKG2 gene.

A previously unrecognized sixth genotype of GB virus C revealed by analysis of 5′‐untranslated region sequences

GB virus C (GBV-C) is a positive-strand RNA virus that infects a large proportion of the world's human population. It has been classified tentatively as a member of the Flaviviridae family and has been shown to exist as a group of five closely related genotypes. Recently, we reported the first full-length genome sequence of a genotype 5 isolate from South Africa. As part of the analysis of that sequence, a phylogenetic tree was elucidated from the 5'-untranslated region (UTR) that showed excellent congruence to the tree produced by analysis of complete open reading frame sequences. When 5'-UTR analysis was broadened subsequently to include additional isolates from around the globe, a heretofore unrecognized GBV-C genotype was discovered in Indonesia. When first reported in 2000, these isolates were described as constituting a novel fifth genotype. However, comparison to isolates from the then-known fourth and fifth genotypes (from Myanmar/Vietnam and South Africa, respectively) was not performed. A dataset of 121 GBV-C 5'-UTR sequences was complied and included representatives of the fourth and fifth genotypes as well as the "novel" Indonesian sequences and demonstrated, with strong support via bootstrap analysis, the existence of a sixth GBV-C genotype among infected individuals in Indonesia. The discovery of this sixth genotype emphasizes the diverse nature of GBV-C isolates and may have important implications for the interpretation of studies involving GBV-C/HIV co-infected individuals.

Sodium channel mutations associated with knockdown resistance in the human head louse, Pediculus capitis (De Geer)

Pyrethroid resistance in human head louse populations is widespread in the United States and worldwide. We previously documented that the knockdown resistance of permethrin-resistant head louse populations is associated with the T929I and L932F (T917I and L920F in the numbering of the louse amino acid sequence) mutations in the voltage-sensitive sodium channel α-subunit gene. In order to identify additional sodium channel mutations potentially associated with knockdown resistance, we cloned and sequenced full-length cDNA fragments from insecticide-susceptible (Ecuador) and permethrin-resistant (Florida) head louse populations and from an insecticide-susceptible body louse population (Israel). Sequence comparisons of the complete open reading frames of the sodium channel genes identified one additional novel mutation (M815I), which was located in the IIS1-2 extracellular loop of the α-subunit, from the permethrin-resistant head louse population. Absolute conservation of the Met815 residue at the corresponding positions within sodium channels from all known susceptible populations of insect species implied that the M815I mutation likely has a functional significance in resistance. Sequence analyses of cloned cDNA fragments and genomic DNA fragments from individual louse samples, both containing the three mutation sites, confirmed that all the mutations exist en bloc as a haplotype. Northern blot analysis identified a single 7.2 kb transcript. The comparison of complete open reading frame sequences (6156 bp) of sodium channel gene between head and body lice revealed 26 polymorphic nucleotides, of which only one resulted in a conservative amino acid substitution (glutamic versus aspartic acid at 11th amino acid position). The virtual identity in nucleotide sequences indicated that both body and head lice are conspecific, and lends justification of the use of the body louse as a surrogate organism for the head louse in biochemical and molecular biology studies. Conserved point mutations resulting in knockdown resistance to the pyrethrins, the pyrethroids, and DDT are suitable for detection by various DNA-diagnostic protocols for monitoring and resistance management.

Selection of homeotic proteins for binding to a human DNA replication origin

We have previously shown that a cell cycle-dependent nucleoprotein complex assembles in vivo on a 74 bp sequence within the human DNA replication origin associated to the Lamin B2 gene. Here, we report the identification, using a one-hybrid screen in yeast, of three proteins interacting with the 74 bp sequence. All of them, namely HOXA13, HOXC10 and HOXC13, are orthologues of the Abdominal-B gene of Drosophila melanogaster and are members of the homeogene family of developmental regulators. We describe the complete open reading frame sequence of HOXC10 and HOXC13 along with the structure of the HoxC13 gene. The specificity of binding of these two proteins to the Lamin B2 origin is confirmed by both band-shift and in vitro footprinting assays. In addition, the ability of HOXC10 and HOXC13 to increase the activity of a promoter containing the 74 bp sequence, as assayed by CAT-assay experiments, demonstrates a direct interaction of these homeoproteins with the origin sequence in mammalian cells. We also show that HOXC10 expression is cell-type-dependent and positively correlates with cell proliferation.

Molecular cloning of S-protein, a link between complement, coagulation and cell-substrate adhesion.

cDNA clones coding for human S-protein have been isolated using monoclonal antibodies to screen a cDNA library in pEX. These clones are shown to be authentic S-protein clones on the basis of sequence, composition and immunological criteria. The complete open reading frame sequence for S-protein has been determined and shows it to be a single polypeptide chain of 459 amino acids preceded by a cleaved leader peptide of 19 residues. No evidence was found for polymorphism of S-protein suggesting that different molecular weight forms arise by proteolytic degradation. Of the first 44 amino-terminal residues 42 are identical with the so-called somatomedin B peptide suggesting that S-protein is the somatomedin B precursor. Striking homology is found in the rest of the sequence with the serum spreading factor, vitronectin, which has also been shown to contain somatomedin B sequences at its amino terminus. We conclude that S-protein and vitronectin are identical and discuss the relevance of this finding to the coagulation and complement pathways.