The homeobox containing transcription factor
plays crucial roles in embryonic development/proliferation and/or maintenance of spermatogonial stem cells (SSCs) via interacting with transcription ...factors such as
and
in mammals. However, knowledge of its exact mechanistic pathways remains unexploited. Very little is known about teleost
. Information on the
gene of farmed rohu carp (
) is lacking. We cloned and characterized the
gene of rohu carp to understand the expression pattern in early developmental stages and also deduced the genomic organization including promoter elements.
Rohu
(
) cDNA comprised an open reading frame of 1,161 nucleotides bearing a structural homeodomain; whereas, the genomic structure contained four exons and three introns suggesting that it is homologous to mammalian counterparts. Phylogenetically, it was closely related to freshwater counterparts. Protein sequence (386 AA of 42.65 kDa) comparison revealed its low similarity with other vertebrate counterparts except that of the conserved homeodomain. Tissue distribution analysis revealed the existence of
transcripts only in adult gonads. The heightened abundances in the ovary and proliferating spermatogonia suggested its participations in maternal inheritance and male germ cell development. The potentiating abundances from fertilized egg onwards peaking at blastula stage vis- à-vis decreasing levels from gastrula stage onwards demonstrated its role in embryonic stem cell development. We also provided evidence of its presence in SSCs by western blotting analysis. Further, the promoter region was characterized, predicting a basal core promoter and other consensus elements.
The molecular characterization of
and its documented expression profiling at transcript and protein levels are indicative of its functional linkage with embryonic/spermatogonial stem cell maintenance. This is the first report of
genomic organization including its promoter sequence information with predicted regulatory elements of a large-bodied carp species. This will be useful for elucidating its mechanism expression in future. Nanog could be used as a potential biomarker for proliferating carp SSCs.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Expressed genes and polymorphisms were identified in lines of rohu
Labeo rohita
selected for resistance or susceptibility to
Aeromonas hydrophila
, an important bacterial pathogen causing ...aeromoniasis. All animals were grown in a common environment and RNA from ten individuals from each line pooled for Illumina mRNA-seq. De novo transcriptome assembly produced 137,629 contigs with 40× average coverage. Forty-four percent of the assembled sequences were annotated with gene names and ontology terms. Of these, 3,419 were assigned biological process terms related to “stress response” and 1,939 “immune system”. Twenty-six contigs containing 38 single nucleotide polymorphisms (SNPs) were found to map to the
Cyprinus carpio
mitochondrial genome and over 26,000 putative SNPs and 1,700 microsatellite loci were detected. Seventeen percent of the 100 transcripts with coverage data most indicative of higher-fold expression (>5.6 fold) in the resistant line pool showed homology to major histocompatibility (MH), heat shock proteins (HSP) 30, 70 and 90, glycoproteins or serum lectin genes with putative functions affecting immune response. Forty-one percent of these 100 transcripts showed no or low homology to known genes. Of the SNPs identified, 96 showing the highest allele frequency differences between susceptible and resistant line fish included transcripts with homology to MH class I and galactoside-binding soluble lectin, also with putative functions affecting innate and acquired immune response. A comprehensive sequence resource for
L. rohita
, including annotated microsatellites and SNPs from a mixture of
A. hydrophila
-susceptible and -resistant individuals, was created for subsequent experiments aiming to identify genes associated with
A. hydrophila
resistance.
The high-throughput sequencing technology provides a platform for revealing the expressed genes within a tissue at a specific time. The giant freshwater prawn,
Macrobrachium rosenbergii,
is an ...economically important species, which is surviving in a wide-range of salinity. In this study, to understand the physiological mechanism of adaptability with respect to moulting and salinity; transcriptome sequencing of larvae and post-larvae of
M. rosenbergii
was performed using the Illumina GAIIx platform. The generated raw read-data comprised 71,391,946 and 75,276,622 paired-end reads (PE) for larvae and post-larvae respectively. Using CLC bio Genomic Workbench version 7.5 (CGWB), 71.39 million and 75.27 million of each 72 base paired-end, high quality reads were assembled into 43,383 (N50 1852) and 44,960 (N50 1874) transcripts, respectively, for larvae and post-larvae. The nucleotide level annotation of both transcriptomes showed significant similarity with unigenes of closely related species. The Gene Ontology analysis suggested enrichment of transcripts involving several biological processes linked to transcriptional regulation, signal transduction, immune response, ion-binding. Differential gene expression analysis using CGWB and DESeq identified 9680 deregulated genes of which 3454 unigenes were up-regulated and 3068 down-regulated by ≥1.5 fold (
p
< 0.05) in larval stage compared to post-larval stage. However, in larval stage 938 genes were down regulated and 1599 genes up-regulated by ≥3 fold with
p
< 0.05. GO enrichment of differentially expressed genes was shown several molecular functions for maintaining homeostasis against salinity stress. To validate the expression patterns, few transcripts were chosen for quantitative real-time PCR that showed the consistency and exactness of our analysis. In addition, we also speculated the enzymatic pathway using KEGG, which depicted that up-regulated genes are involved in several significant metabolic pathways and those are critical for maintaining osmoregulation and linked with metamorphosis. Therefore, we have generated valuable information of salinity tolerant genes in the larval and post larval stage of
M. rosenbergii
during salt- and freshwater compliances, which will be further harnessed for gene targeting. The present finding would provide the basis for further screening of salt tolerant genes associated markers for selective breeding.
Immune response mediated by toll-like receptor 22 (TLR22), only found in teleost/amphibians, is triggered by double-stranded RNA binding to its LRR (leucine-rich repeats) ecto-domain. Accumulated ...evidences suggested that missense mutations in TLR genes affect its function. However, information on mutation linked pathogen recognition for TLR22 was lacking. The present study was commenced for predicting the effect of non-synonymous single-nucleotide polymorphisms (nsSNPs) on the pathogen recognizable LRR domain of TLR22 of farmed carp,
Labeo rohita.
The sequence-based algorithms (SIFT, PROVEAN and I-Mutant2.0) indicated that three SNPs (out of 27) such as p.L159F (rs76759876) and p.L529P (rs749355507) of LRR, and p.I836M (rs750758397) of intracellular motifs could potentially disrupt protein function. The 3D structure was generated using MODELLER 9.13 and further validated by SAVEs server. The simulated molecular docking of native TLR22 and mutants with poly I:C ligand indicated that mutations positioned at p.L159F and p.L529P of the LRR region affects the binding affinity significantly. This is the first kind of study of predicting nsSNPs of teleost TLR22 with disturbed ligand binding affinity with its extra-cellular LRR domain and thereby likely hindrance in subsequent signal transduction. This study serves as a guide for in vivo evaluation of impact of mutation on immune response mediated by teleost TLR22 gene.
Advancements in the DNA sequencing technologies and computational biology have revolutionized genome/transcriptome sequencing of non-model fishes at an affordable cost. This has led to a paradigm ...shift with regard to our heightened understandings of structure-functional relationships of genes at a global level, from model animals/fishes to non-model large animals/fishes. Whole genome/transcriptome sequencing technologies were supplemented with the series of discoveries in gene editing tools, which are being used to modify genes at pre-determined positions using programmable nucleases to explore their respective in vivo functions. For a long time, targeted gene disruption experiments were mostly restricted to embryonic stem cells, advances in gene editing technologies such as zinc finger nuclease, transcriptional activator-like effector nucleases and CRISPR (clustered regulatory interspaced short palindromic repeats)/CRISPR-associated nucleases have facilitated targeted genetic modifications beyond stem cells to a wide range of somatic cell lines across species from laboratory animals to farmed animals/fishes. In this review, we discuss use of different gene editing tools and the strategic implications in fish species for basic and applied biology research.
Asf1 (anti-silencing function 1), a well conserved protein from yeast to humans, acts as a histone chaperone and is predicted to participate in a variety of chromatin-mediated cellular processes. To ...investigate the physiological role of vertebrate Asf1 in vivo, we generated a conditional Asf1-deficient mutant from chicken DT40 cells. Induction of Asf1 depletion resulted in the accumulation of cells in S phase with decreased DNA replication and increased mitotic aberrancy forming multipolar spindles, leading to cell death. In addition, nascent chromatin in Asf1-depleted cells showed increased nuclease sensitivity, indicating impaired nucleosome assembly during DNA replication. Complementation analyses revealed that the functional domain of Asf1 for cell viability was confined to the N-terminal core domain (amino acids 1-155) that is a binding platform for histones H3/H4, CAF-1p60, and HIRA, whereas Asf1 mutant proteins, abolishing binding abilities with both p60 and HIRA, exhibit no effect on viability. These results together indicate that the vertebrate Asf1 plays a crucial role in replication-coupled chromatin assembly, cell cycle progression, and cellular viability and provide a clue of a possible role in a CAF-1- and HIRA-independent chromatin-modulating process for cell proliferation.
Histone acetyltransferase 1 (HAT1) is implicated for diacetylation of Lys-5 and Lys-12 of newly synthesized histone H4, the biological significance of which remains unclear. To investigate the in ...vivo role of HAT1, we generated HAT1-deficient DT40 clone (
HAT1
−/−).
HAT1
−/− cells exhibited greatly reduced diacetylation levels of Lys-5 and Lys-12, and acetylation level of Lys-5 of cytosolic and chromatin histones H4, respectively. The in vitro nucleosome assembly assay and in vivo MNase digestion assay revealed that HAT1 and diacetylation of Lys-5 and Lys-12 of histone H4 are dispensable for replication-coupled chromatin assembly.
HAT1
−/− cells had mild growth defect, conferring sensitivities to methyl methanesulfonate and camptothecin that enforce replication blocks creating DNA double strand breaks. Such heightened sensitivities were associated with prolonged late-S/G2 phase. These results indicate that HAT1 participates in recovering replication block-mediated DNA damages, probably through chromatin modulation based on acetylation of Lys-5 and Lys-12 of histone H4.
Amounts of soluble histones in cells are tightly regulated to ensure supplying them for the newly synthesized DNA and preventing the toxic effect of excess histones. Prior to incorporation into ...chromatin, newly synthesized histones H3 and H4 are highly acetylated in pre-deposition complex, wherein H4 is di-acetylated at Lys-5 and Lys-12 residues by histone acetyltransferase-1 (Hat1), but their role in histone metabolism is still unclear. Here, using chicken DT 40 cytosolic extracts, we found that histones H3/H4 and their chaperone Asf1, including RbAp48, a regulatory subunit of Hat1 enzyme, were associated with Hat1. Interestingly, in HAT1-deficient cells, cytosolic histones H3/H4 fractions on sucrose gradient centrifugation, having a sedimentation coefficient of 5–6S in DT40 cells, were shifted to lower molecular mass fractions, with Asf1. Further, sucrose gradient fractionation of semi-purified tagged Asf1-complexes showed the presence of Hat1, RbAp48 and histones H3/H4 at 5–6S fractions in the complexes. These findings suggest the possible involvement of Hat1 in regulating cytosolic H3/H4 pool mediated by Asf1-containing cytosolic H3/H4 pre-deposition complex.
Information on the genetic structure of cultivable fish species is essential for studying molecular systematics and optimising fisheries management and fish farming. The random amplified polymorphic ...DNA (RAPD) assay was evaluated for studying genetic relationships and diversities in four species of Indian major carps (Family Cyprinidae). Thirty-four arbitrary primers were screened to identify species-specific RAPD markers among rohu (
Labeo rohita), kalbasu (
L. calbasu), catla (
Catla catla) and mrigal (
Cirrhinus mrigala). Distinct and highly reproducible RAPD profiles with a great degree of genetic variability were detected among species. On average, 45% of the scorable RAPD bands were specific to each species. Phylogenetic analysis demonstrated that kalbasu is the closest to rohu and the farthest from mrigal. Data are presented with regard to the applications of RAPD markers for hybrid identification, genetic diversity assessment and studying taxonomic relationships at a molecular level.
PDF
