Multiple Sequence Alignment Analysis Research Articles

Isolation and characterization of chalcone synthase (CHS) gene from Phalaenopsis and Doritaenopsis orchids

Abstract. Sukma D, Handini AS, Sudarsono. 2020. Isolation and characterization of chalcone synthase (CHS) gene from Phalaenopsis and Doritaenopsis orchids. Biodiversitas 21: 5054-5064. Chalcone synthase (CHS) is a key enzyme in flavonoid biosynthesis. The research aims to isolate and characterize the CHS gene nucleotide sequence diversity of nine Phalaenopsis and one Doritaenopsis genotypes. Genomic DNA was isolated from young leaves and used to PCR amplify the gene using CHS specific degenerate primers. Results of PCR amplification yielded DNA fragments of 700 bp. Upon sequencing and nucleotide sequence analysis, we found that the genomic fragments were partial CHS gene from Phalaenopsis and Doritaenopsis genotypes and deposited the sequences in the NCBI Genebank Database accession numbers of KR184089-KR184098. More analysis of the sequences confirmed they shared ranged from 81-99% sequence identities to known CHS genes from Phalaenopsis deposited in the NCBI Database. They also shared 84-100% amino acid residues identity to CHS polypeptides. Multiple sequence alignment (MSA) and phylogenetic analysis further revealed the determined CHS nucleotide sequences from Phalaenopsis and Doritaenopsis genotypes were closely related to CHS genes from other orchid species.

Insulin receptors in largemouth bass ( Micropterus salmoides ): Cloning, characterization, tissue expression profile and transcriptional response to glucose tolerance test

Insulin receptors (IRs) are known to be involved in the regulation of glucose metabolism in mammals; however, their role is more complicated in fish, being dependent on the carbohydrate content of their natural diet. In the present study, two IRs (IRa and IRb) of the largemouth bass (Micropterus salmoides) were cloned and characterized, and their transcriptional expression was investigated in response to the glucose tolerance test. Results showed that the full-length cDNA sequences of IRa and IRb were 6,429 bp and 6,264 bp, which by deduction, encoded 1,372 and 1,368 amino acids respectively. Structural domain analysis, together with multiple sequence alignment and phylogenetic analysis, confirmed the conserved evolution of IRs in teleosts. IRa was highly expressed in the ovary, heart and liver, while the IRb mRNA was abundant in the heart, liver and testis. In the glucose tolerance test, the serum glucose content took 12 hr to return to normal. Meanwhile, the expression of IRa and IRb in the liver of the largemouth bass was not significantly changed in response to glucose administration, which may partly account for the limited ability of this carnivorous fish to utilize glucose.

Molecular identification and characterisation of a novel chicken leukocyte immunoglobulin-like receptor A5

ABSTRACT 1. Leukocyte immunoglobulin-like receptor A5 (LILRA5) is a key molecule that regulates the immune system. However, the LILRA5 gene has not been characterised in avian species, including chickens. The present study aimed to identify and functionally characterise LILRA5 identified from two genetically disparate chicken lines, viz., Marek’s disease (MD)-resistant (R) line 6.3 and MD-susceptible (S) line 7.2. 2. Multiple sequence alignment and phylogenetic analyses confirmed that the identity and similarity homologies of amino acids of LILRA5 in chicken lines 6.3 and 7.2 ranged between 93% and 93.7%, whereas those between chicken and mammals ranged between 20.9% and 43.7% and 21.1% to 43.9%, respectively. The newly cloned LILRA5 from chicken lines 6.3 and 7.2 revealed high conservation and a close relationship with other known mammalian LILRA5 proteins. 3. The results indicated that LILRA5 from chicken lines 6.3 and 7.2 was associated with phosphorylation of Src kinases and protein tyrosine phosphatase non-receptor type 11 (SHP2), which play a regulatory role in immune functions. Moreover, the results demonstrated that LILRA5 in these lines was associated with the activation of major histocompatibility complex (MHC) class I and β2-microglobulin and induced the expression of the transporter associated with antigen processing. In addition, LILRA5 in both chicken lines activated and induced Janus kinase (JAK)–signal transducer and the activator of transcription (STAT), nuclear factor kappa B (NF-κB), phosphoinositide-3-kinase (PI3K)/protein kinase B (AKT) and the extracellular signal-regulated kinase (ERK)1/2 signalling pathways; toll-like receptors; and Th1-, Th2-, and Th17- cytokines. 4. The data suggested that LILRA5 has innate immune receptors essential for macrophage immune response and provide novel insights into the regulation of immunity and immunopathology.

Structure-based prediction of protein–protein interactions between GhWlim5 Domain1 and GhACTIN-1 proteins: a practical evidence with improved fibre strength

Cotton fibre quality is a multigenic trait. Genetic modification of different genes to achieve high quality fibre is difficult without knowing the mechanism lying behind genes interaction. Based on background knowledge an attempt to explore the potential structural interactions between Gossypium hirsutum Wlim5 domain1 and Gossypium hirsutum ACTIN-1 proteins was done in current study. Sequence features of the LIM domain1 of GhWlim5 protein were identified through multiple sequence alignment analysis, and a phylogenetic tree was built to identify evolutionary relationships between sequences. Conservation indicated the evolutionary importance of side chain residues and the presence of several aliphatic and/or bulky residues, which stabilize the protein core and facilitate packing of zinc fingers. The structures of GhWlim5 domain1 and GhACTIN-1 proteins were modelled and validated through computational methods. Validation of GhACTIN-1 and GhWlim5 domain1 structures indicated good structural quality with 99.7% and 100% of the favoured number of residues in allowed regions and Z-score, within the ranges of − 9.87 and − 4.17, respectively. Docking analysis indicated various possible modes of interaction between these two proteins with favourable binding affinities. Based on our strong binding interaction results between GhWlim5 domain1 and GhACTIN-1 proteins, we further investigated the role of over-expression of GhWlim5 by transformation in cotton plants under fibre specific promoter and transgenic plants displayed significant increases in fibre strength.

Effects of FOXO1 on the proliferation and cell cycle-, apoptosis- and steroidogenesis-related genes expression in sheep granulosa cells

Forkhead boxO (FOXO) transcription factors regulate diverse biological processes, including cellular metabolism, cell apoptosis, and the cell cycle. Results from several studies indicate FOXO1 regulates different granulosa cell (GC) pathways involved in proliferation, survival and differentiation. Functions and mechanisms of FOXO1 regulation of sheep GCs remain unclear. This study was conducted to analyze the function of FOXO1 in regulation of sheep GCs. In this study, the 1827 bp sheep FOXO1 coding sequence was cloned from sheep GCs. Multiple sequence alignment and phylogenetic analysis indicated that the FOXO1 protein sequence is highly homologous to FOXO1 protein sequences from other species. The results obtained from using CCK-8 assays indicated sheep GC proliferation increased when there was suppression of FOXO1 gene expression. When there was induced expression of the FOXO1 gene in sheep GCs, there was a resulting increased abundance of P21 and P27 mRNA transcript, whereas suppression of the FOXO1 gene expression had the opposite effect. Furthermore, the relative abundance in vitro of apoptosis-related protein mRNA transcripts (caspase3, caspase8, caspase9, Bax/Bcl-2) was markedly increased or decreased when there was induction or suppression of FOXO1 gene expression, respectively,(P < 0.05). Induction of FOXO1 gene expression resulted in an increase in abundance of steroidogenic protein mRNA transcripts (CYP11A1, 3β-HSD), while suppression of FOXO1 gene expresion resulted in a decrease abundance of the CYP11A1, STAR mRNA transcripts. Results from the present study indicated that FOXO1 inhibited the proliferation of sheep GCs and affected mRNA transcript abundance for proteins involved in regulation of apoptosis, the cell cycle and steroidogenesis.

Identification of a dominant linear epitope on the VP2 capsid protein of porcine parvovirus and characterization of two monoclonal antibodies with neutralizing abilities

Porcine parvovirus (PPV) is a major cause of reproductive failure in swine, and has caused huge losses throughout the world. The structural viral protein VP2, which is able to self-assemble into empty capsids, known as virus-like particles (VLPs), is crucial to induce PPV-specific neutralizing antibodies and protective immunity. In this study, twelve monoclonal antibodies (mAbs) against PPV were generated. The mAbs were characterized by indirect enzyme-linked immunosorbent assay (ELISA), western blotting (WB) and virus neutralization (VN) assay. Two mAbs were defined to be able to neutralize the standard PPV 7909 strain. Subsequently, peptide scanning was applied to identify linear epitopes. The peptide, 89ESGVAGQMV97 was defined as a precise linear epitope. Results from structural analysis showed that the epitope was exposed on the virion surface. Multiple sequence alignment analysis indicated that peptide 89ESGVAGQMV97 was not completely conserved, with a higher amino acid mutation rate at 91G, 92V and 93A position. Alanine-scanning mutagenesis further revealed that residues 89E, 90S, 91G, 92V and 94G were the core sites involved in antibody recognition. These findings may facilitate further understanding the function of the VP2 protein and development of diagnostic tools.

Phylogeography and Genetic Diversity of Human Hydatidosis in Bordering the Caspian Sea, Northern Iran by Focusing on Echinococcus granulosus Sensu Stricto Complex.

Background:Human Echinococcosisis a cyclo-zoonotic infection caused by tapeworms of the Echinococcus granulosus sensu stricto complex. The detection of mitochondrial genome data of genus Echinococcus can reflect the taxonomic status, genetic diversity, and population structure genetics.Methods:Totally, 52 formalin-fixed paraffin-embedded (FFPE) tissue samples from patients with histologically confirmed CE were collected from Mazandaran province, Iran in the period of Mar 1995 to May 2018. All extracted DNAs from (FFPE) tissue samples were subjected to amplify by polymerase chain reactions method targeting cytochrome c oxidase subunit 1 (cox1) gene. All PCR amplicons were sequenced to phylogenetic analysis and genetic diversity.Results:Molecular analysis showed that 50(96.1%) and 2 (3.84%) isolates were identified as G1 andG3 E. granulosus genotypes, respectively. DNA sequence analyses indicated a high gene diversity for G1 (Haplotype diversity: 0.830) and G3 genotypes (Hd: 1.00). Based on multiple sequence alignment analyses, 7 (13.46%; G1 genotype) and 2 (3.84%; G3 genotype) new haplotypes were unequivocally identified.Conclusion:G3 genotype (Buffalo strain) was identified from two human hydatidosis isolates in the region. Present study strengthens our knowledge about taxonomic status, transmission patterns of Echinococcus parasite to human and heterogeneity aspects of this parasite in clinical CE isolates of Northern Iran.

In silico analysis of FLP 18 gene of Anguina tritici and phylogenetic analysis with other nematodes

flp 18is a neuropeptide coding gene of FMRFamide-like peptides family having important role in behavioural regulation such as locomotory, feeding and reproduction of nematodes. Genome annotation of the newly sequenced genome of seed gall nematode (Anguina tritici) has revealed the presence of flp 18 in their genome. flp 18 gene has been proven as effective target for management of Meloidogyne incognita. In view of importance of this gene here we analyse the significance of flp 18 gene in A. tritici in different nematode species through the In silico approach. Homologus nucleotide sequences in other nematode species for the gene flp 18 were obtained on performing nucleotide BLAST in WormBase Parasite database. Multiple sequence alignment, identity and phylogenetic analysis were performed on the retrived homologus sequences using CLUSTAL W programme. The BLAST result revealed the presence of flp 18 hits in different groups of plant parasitic nematodes with best hit (76) to Ditylenchus dipsaci. Based on the finding it has been suggested that the flp 18 of A. tritici is conserved with identical homologs, and might have functional similarity among different nematodes species.

Molecular Characterization and Expression Patterns of Shabby-Related Kinase (MmSK) Gene of Mulberry (Morus multicaulis)

Shaggy-related protein kinase (SK) plays important roles in the plant growth development, signal transduction, abiotic stress and biotic stress and substance metabolism regulation. In the present paper, a cDNA sequence encoding MmSK (GenBank accession No: KY348867) was cloned from the leaves of mulberry based on mulberry expressed sequence tags (EST) and homologous cloning technology using RT-PCR, which was 1705 bp in length with a full open reading frame (ORF) of 1236 bp encoding a protein of 411 amino acids. The estimated molecular weight and isoelectric point (pI) of the putative protein were 46.55 kDa and 8.61, respectively. Conservation domain structure analysis indicated that MmSK protein had typical structure of the protein kinase domain and belonged to GSK3/shaggy protein kinase family. Multiple sequence alignment and phylogenetic analysis showed that the homology between the amino acid sequences encoded by the MmSK gene and various species was more than 89%. Quantitative real-time PCR (qRT-PCR) analysis revealed that MmSK was expressed in all the tested tissues including leaf, bud, fruit, stem, phloem and xylem of the mulberry with the highest expression in the phloem. The expression level of the mRNA has changed significantly under salt, drought, cold and ABA stress treatments compared to the normal growth environment. Overall, these results showed a better understanding of the molecular basis for the signal transduction mechanism during the stress responses in mulberry trees.

Genome-wide identification and expression analysis of SABATH methyltransferases in tea plant (Camellia sinensis): insights into their roles in plant defense responses

ABSTRACT SABATH methyltransferases convent plant small-molecule metabolites into volatile methyl esters, which play important roles in many biological processes and defense reactions in plants. In this study, a total of 32 SABATH genes were identified in the Camellia sinensis var. sinensis (CSS) genome, which were renamed CsSABATH1 to CsSABATH32. Genome location annotation suggested that tandem duplication was responsible for the expansion of SABATH genes in tea plant. Multiple sequence alignment and phylogenetic analysis showed that the CsSABATHs could be classified into three groups (I, II and III), which were also supported by gene structures and conserved motifs analysis. Group II contained only two CsSABATH proteins, which were closely related to PtIAMT, AtIAMT and OsIAMT. The group III SABATH genes of tea plant exhibited expansion on the CSS genome compared with Camellia sinensis var. assamica (CSA) genome. Based on RNA-seq data, the CsSABATHs exhibited tissue-specific expression patterns, and the members with high expression in buds and young leaves were also obviously upregulated after MeJA treatment. The expression of many transcription factors was significantly correlated with that of different members of the CsSABATH gene family, suggesting a potential regulatory relationship between them. Quantitative real-time PCR (qPCR) expression analysis showed that CsSABATHs could respond to exogenous JA, SA and MeSA treatments in tea plants. RNA-seq data analysis and qPCR validation suggested that CsSABATH8, 11, 16, 25, 29 and 32 might play a special role in plant defense against insect herbivory. These results provide references for evolutionary studies of the plant SABATH family and the exploration of the potential roles of CsSABATHs in tea plant defense responses.

An in silico structural and physiochemical analysis of C-Phycocyanin of halophile Euhalothece sp.

C-Phycocyanin is a water-soluble, light-harvesting accessory pigment known to possess excellent biotechnological applications due to its intense color, fluorescent properties and health benefits. In this study, the physicochemical properties and protein structure of C-Phycocyanin from a halophile Euhalothece sp. were predicted based on the in silico analysis of the amino acid sequences. The effect of temperature and pH on the yield, as well as stability and antioxidant activity of purified C-Phycocyanin was determined. Based on the in silico analysis, the α-subunit is composed of 162 amino acids (17.76 kDa) covalently linked to the phycocyanobilin at the Cys84 position, and the β-chain contains 172 amino acids (18.48 kDa) attached to two phycocyanobilins at Cys82 and Cys153 positions. The physicochemical properties revealed that these proteins are non-polar and stable. Multiple sequence alignment analyses of the subunits displayed significant differences amongst the amino acids of hypersaline/marine and freshwater cyanobacteria. These amino acids play a vital role in the stability of the C-Phycocyanin. The secondary structure prediction of the α- and β-subunits consisted of >50% of amino acid residues in α-helices, with 9–13% of amino acid residues in the extended strand. To further verify the results, C-Phycocyanin was found thermostable up to 45 °C with an optimum pH range between 5.0 and 7.0. The yield and thermostability of C-Phycocyanin were observed to be pH-dependent, where a lower pH (<6.0) improved the stability at higher temperatures (>50 °C) and vice-versa. Based on the in vitro assays, C-Phycocyanin had a high antioxidant activity i.e. 90.0 ± 0.045% scavenging activity at 0.08 mg mL−1 and reducing power efficiency of 85.15 ± 0.012% at 150 μg mL−1. The higher antioxidant activity levels of the C-Phycocyanin could be a result of the presence of high level of non-polar and aromatic amino acids (Ala, Gly Glu, Leu, Arg, Ser, and Val).

Curated multiple sequence alignment for the Adenomatous Polyposis Coli (APC) gene and accuracy of in silico pathogenicity predictions.

Computational algorithms are often used to assess pathogenicity of Variants of Uncertain Significance (VUS) that are found in disease-associated genes. Most computational methods include analysis of protein multiple sequence alignments (PMSA), assessing interspecies variation. Careful validation of PMSA-based methods has been done for relatively few genes, partially because creation of curated PMSAs is labor-intensive. We assessed how PMSA-based computational tools predict the effects of the missense changes in the APC gene, in which pathogenic variants cause Familial Adenomatous Polyposis. Most Pathogenic or Likely Pathogenic APC variants are protein-truncating changes. However, public databases now contain thousands of variants reported as missense. We created a curated APC PMSA that contained >3 substitutions/site, which is large enough for statistically robust in silico analysis. The creation of the PMSA was not easily automated, requiring significant querying and computational analysis of protein and genome sequences. Of 1924 missense APC variants in the NCBI ClinVar database, 1800 (93.5%) are reported as VUS. All but two missense variants listed as P/LP occur at canonical splice or Exonic Splice Enhancer sites. Pathogenicity predictions by five computational tools (Align-GVGD, SIFT, PolyPhen2, MAPP, REVEL) differed widely in their predictions of Pathogenic/Likely Pathogenic (range 17.5-75.0%) and Benign/Likely Benign (range 25.0-82.5%) for APC missense variants in ClinVar. When applied to 21 missense variants reported in ClinVar and securely classified as Benign, the five methods ranged in accuracy from 76.2-100%. Computational PMSA-based methods can be an excellent classifier for variants of some hereditary cancer genes. However, there may be characteristics of the APC gene and protein that confound the results of in silico algorithms. A systematic study of these features could greatly improve the automation of alignment-based techniques and the use of predictive algorithms in hereditary cancer genes.

In silico cloning and bioinformatics analysis of HSP21 in Manihot esculenta

HSP21 gene is a key gene to respond high temperature stress in plant and plays an important role in preventing protein denaturation, protecting cell structure and maintaining normal growth and development. Therefore, cloning HSP21 gene is the basis for revealing the molecular mechanism of resistance to high temperature stress in cassava. To obtain cassava HSP21 homologous gene and analyze the properties of predicted protein, electronic cloning technology was used to assemble and derivate new gene in this study, and bioinformatics analysis method was used to analyze the primary to highest structure, hydrophilicity/hydrophobicity, signal peptide, protein homology and phylogenetic evolution of expressed protein. HSP21 gene was 969 bp, its open reading frame was 705 bp, and the predicted protein contains 234 amino acids. The predicted protein is a non-transmembrane protein that is alkaline and hydrophilic, and is mainly localized in the chloroplast. Through multiple sequence alignment and phylogenetic analysis, it was found that the cassava HSP21 protein has high homology with other plants such as Hevea brasiliensis, Ricinus communis, and Jatropha curcas. The results could provide reference for the study of cloning and transformation of this gene.

Genome-Wide Identification and Expression Analysis of MAPK and MAPKK Gene Family in Pomegranate (Punica Granatum L.)

Mitogen-activated protein kinase (MAPK) cascade is involved in the regulation of a series of biological processes in organisms, which are composed of MAPKKKs, MAPKKs, and MAPKs. Although genome-wide analyses of it has been well described in some species, little is known about MAPK and MAPKK genes in pomegranates. In this study, we identified 18 PgMAPKs, 9 PgMAPKKs through a genome-wide search. Chromosome localization showed that 27 genes are distributed on 7 chromosomes with different densities. Multiple sequence alignment and phylogenetic analysis revealed that PgMAPKs and PgMAPKKs could be divided into 4 subfamilies (groups A, B, C, and D), respectively. In addition, exon-introns structural analysis of each candidate gene has indicated high levels of conservation within and between phylogenetic groups. Cis-acting element analysis predicted that PgMAPKs and PgMAPKKs were widely involved in the growth, development, stress and hormone response of pomegranate. Expression profile analyses of PgMAPKs and PgMAPKKs were performed in different tissues (root, leaf, flower and fruit), and PgMAPK13 was significantly expressed in all tissues. To our knowledge, this is the first genome-wide analysis of the MAPK and MAPKK gene family in pomegranate. This study provides valuable information for understanding the classification and functions of pomegranate MAPK signal.

Identification and Expression Analysis of Cold Shock Protein 3 (BcCSP3) in Non-Heading Chinese Cabbage (Brassica rapa ssp. chinensis)

A cold-related protein, cold shock protein 3 (BcCSP3), was isolated from non-heading Chinese cabbage in this study. BcCSP3 can encode 205 amino acids (aa) with an open reading frame (ORF) of 618 base pairs (bp). Multiple sequence alignment and phylogenetic tree analyses showed that BcCSP3 contains an N-terminal cold shock domain and is highly similar to AtCSP2, their kinship is recent. Real-time quantitative polymerase chain reaction (RT-qPCR) showed that the expression level of BcCSP3 in stems and leaves is higher than that in roots. Compared with other stress treatments, the change in BcCSP3 expression level was most pronounced under cold stress. In addition, a BcCSP3–GFP fusion protein was localized to the nucleus and cytoplasm. These results indicated that BcCSP3 may play an important role in response to cold stress in non-heading Chinese cabbage. This work may provide a reference for the identification and expression analysis of other CSP genes in non-heading Chinese cabbage.

Characterization of two kcnk3 genes in rabbitfish (Siganus canaliculatus): Molecular cloning, distribution patterns and their potential roles in fatty acids metabolism and osmoregulation

KCNK3 is a two-pore-domain (K2P) potassium channel involved in maintaining ion homeostasis, mediating thermogenesis, controlling breath and modulating electrical membrane potential. Although the functions of this channel have been widely described in mammals, its roles in fishes are still rarely known. Here, we identified two kcnk3 genes from the euryhaline rabbitfish (Siganus canaliculatus), and their roles related to fatty acids metabolism and osmoregulation were investigated. The open reading frames of kcnk3a and kcnk3b were 1203 and 1176 bp in length, encoding 400 and 391 amino acids respectively. Multiple sequences alignment and phylogenetic analysis revealed that the two isotypes of kcnk3 were extensively presented in fishes. Quantitative real-time PCRs indicated that both genes were widely distributed in examined tissues but showed different patterns. kcnk3a primary distributed in adipose, eye, heart, and spleen tissues, while kcnk3b was mainly detectable in heart, kidney, muscle and spleen tissues. In vivo experiments showed that fish fed diets with fish oil as dietary lipid (rich in long chain polyunsaturated fatty acids, LC-PUFA) induced higher mRNA expression levels of kcnk3 genes in comparison with fish fed with plant oil diet at two different salinity environments (32 and 15‰). Meanwhile, the expression levels of kcnk3 genes were higher in seawater (32‰) than that in brackish water (15‰) when fishes were fed with both types of feeds. In vitro experiments with rabbitfish hepatocytes showed that LC-PUFA significantly improved hepatic kcnk3a expression level compared with treatment of linolenic acid. These results suggest that two kcnk3 genes are widely existed and they might be functionally related to fatty acids metabolism and osmoregulation in the rabbitfish.

Genome-wide identification of a novel elovl4 gene and its transcription in response to nutritional and osmotic regulations in rabbitfish (Siganus canaliculatus)

Elongation of very long-chain fatty acids 4 (Elovl4) proteins, belong to the fatty acyl elongase families that are involved in long-chain polyunsaturated fatty acids (LC-PUFA) biosynthesis in animals. Thus far, very little is known about the roles of Elovl4 in fish. Previously, we reported an elovl4 gene named as elovl4b in rabbitfish (Siganus canaliculatus). In this study, a novel elovl4-like gene with a 957-bp open reading frame encoding a 318 amino-acid protein, which shared 67.3% similarity with rabbitfish Elovl4b and more than 80% similarity with Elovl4a from other teleost, was identified in S. canaliculatus, and thus was termed as elovl4a. Multiple protein sequences alignment, gene synteny and phylogenetic analysis indicated that two obviously different elovl4 genes are widespread in various teleost. Different with elovl4b (mainly expressed in eye and slightly expressed in brain), rabbitfish elovl4a was primarily distributed in brain, slightly examined in eye and gonad tissues while hardly detectable in other tissues. The transcription of cerebral elovl4a was significantly higher in fishes cultured in seawater (32‰) than that of those maintained at a brackish water environment (15‰), when fishes were fed with a vegetable oil (VO) enriched diet. However, the transcriptional pattern was totally inverse when fishes were fed with a fish oil (FO) enriched diet. In the brackish water, diet types had no effect on the transcription of cerebral elovl4a. Whereas in seawater, the transcription of cerebral elovl4a was significantly higher in fishes fed with the VO diet in comparison with those fed with the FO diet. Our findings suggest that elovl4a can be regulated by both nutritional and osmotic factors, and it may play an important role in endogenous biosynthesis of LC-PUFA in the neural system of rabbitfish. This study may help us to understand the effects of nutritional and osmotic factors on the LC-PUFA biosynthesis, as well as contribute to the optimization or enhancement of LC-PUFA biosynthesis in teleost.

A method for efficient quantitative analysis of genomic subtelomere Y' element abundance in yeasts.

Subtelomere Y' elements get amplified by homologous recombination in sustaining the survival and division of the budding yeast Saccharomyces cerevisiae. However, current method for measurement of the subtelomere structures uses Southern blotting with labelled specific probes, which is laborious and time-consuming. By multiple sequence alignment analysis of all 19 subtelomere Y' elements across the 13 chromosomes of the sequenced S288C strain deposited in the yeast genome SGD database, we identified 12 consensus and relative longer fragments and 14 pairs of unique primers for real-time quantitative PCR analysis. With a PAC2 or ACT1 located near the centromere of chromosome V and VI as internal controls, these primers were applied to real-time quantitative PCR analysis, so the relative Y' element intensity normalised to that of wild type (WT) cells was calculated for subtelomere Y' element copy numbers across all different chromosomes using the formula: 2^[-((CTmutant Y' - CTmutant control ) - (CTWT Y' - CTWT control ))]. This novel quantitative subtelomere amplification assay across chromosomes by real-time PCR proves to be a much simpler and more sensitive way than the traditional Southern blotting method to analyse the Y' element recombination events in survivors derived from telomerase deficiency or recruitment failure.

An Insight into Oligopeptide Transporter 3 (OPT3) Family Proteins.

OPT3s are involved in the transport of Fe from xylem to phloem, in loading Fe into phloem, and in the transmission of shoot-to-root iron signaling. Yet, apart from Arabidopsis, little is known about these transporters'functions in other plant species. OPT3 proteins of several plant species were characterized using bioinformatical tools. Also, a probable Fe chelating protein, GSH, was used in docking analyses to shed light on the interactions of ligand binding sites of OPT3s. The multiple sequence alignment (MSA) analysis, protein secondary and tertiary structure analyses, molecular phylogeny analysis, transcription factor binding site analyses, co-expression and docking analyses were performed using up-to-date bioinformatical tools. All OPT3s in this study appear to be transmembrane proteins. They appear to have broad roles and substrate specificities in different metabolic processes. OPT3 gene structures were highly conserved. Promoter analysis showed that bZIP, WRKY, Dof and AT-Hook Transcription factors (TFs) may regulate the expression of OPT3 genes. Consequently, they seemed to be taking part in both biotic and abiotic stress responses as well as growth and developmental processes. The results showed that OPT3 proteins are involved in ROS regulation, plant stress responses, and basal pathogen resistance. They have species-specific roles in biological processes. Lastly, the transport of iron through OPT3s may occur with GSH according to the binding affinity results of the docking analyses.

The origins and developments of sulfation-prone tyrosine-rich and acidic N- and C-terminal extensions of class ll and lll small leucine-rich repeat proteins shed light on connective tissue evolution in vertebrates

BackgroundSmall leucine-rich repeat protein (SLRP) family members contain conserved leucine-rich repeat motifs flanked by highly variable N- and C-terminal regions. Most class II and III SLRPs have tyrosine-rich N-terminal regions and some of these are sulfated. However, the evolutionary origin and conservation of the tyrosine-rich and acidic terminal regions remain undetermined. In this study, we present the most comprehensive multiple sequence alignment (MSA) analyses of all eight class II and III SLRPs to date. Based on the level of conservation of tyrosine residues and adjacent sequences, we predict which tyrosine residues are most likely to be sulfated in the terminal regions of human class II and III SLRPs.ResultsUsing this novel approach, we predict a total of 22 tyrosine sulfation sites in human SLRPs, of which only 8 sites had been experimentally identified in mammals. Our analyses suggest that sulfation-prone, tyrosine-rich and acidic terminal regions of the class II and III SLRPs emerged via convergent evolution at different stages of vertebrate evolution, coinciding with significant evolutionary events including the development of endochondral bones and articular cartilage, the aquatic to terrestrial transition, and the formation of an amnion.ConclusionsOur study suggests that selective pressures due to changes in life conditions led to the formation of sulfotyrosine-rich and acidic terminal regions. We believe the independent emergence and evolution of sulfotyrosine-rich and acidic N- and C-terminal regions have provided each class II and III SLRP member with novel vital functions required to develop new specialized extracellular matrices and tissues in vertebrate species.