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Distinct types of 5S rDNA repeats, differing by size and composition of its non-transcribed spacer (NTS), have been found in diverse taxa. Both concerted evolution and birth-and-death evolution have proven to play important roles in the evolution of the 5S rDNA family. In anurans, however, this subject has been underexplored as only a few anuran species had their 5S rDNA characterized and evolutionary analyzed to date. In the present study, we characterized the 5S rDNA sequences from species of two anuran families using classical molecular biology techniques and bioinformatic approaches. Based on the NTS, more than one type of 5S rDNA was identified in each analyzed species, which suggests that birth-and-death processes take part in the evolution of these sequences along the Anura tree of life. In addition, closely related species shared the same types of sequences, in accordance with the model of concerted evolution. We also found evidence of recombination between 5S rDNA and PcP190 satellite DNA, a repetitive sequence that is derived from the 5S rDNA. The interplay between 5S rDNA and PcP190 satellite DNA might favor the maintenance of the latter in the genome and respond to its presence in several species of frogs. The analysis of 5S rRNA transcripts confirmed the type I 5S rDNA of leiuperine as a functional 5S rRNA gene. Finally, the chromosome mapping of 5S rDNA sequences allowed some inferences of chromosome homology in Leiuperinae. In conclusion, our study provides additional information about the organization, differentiation and functionality of 5S rDNA in anuran species, revealing the potential participation of satellite DNA in the evolution of this family of rDNA.

In the present review, numerous experimental and theoretical data describing the properties of non-canonical DNA structures (NSs) are analyzed. NSs (G-quadruplex, i-motif, hairpin, and triplex) play an important role in epigenetic processes (including the genetic variability of viruses), are prone to energetically low-cost conformational transformations and can very effectively be used in the design of nanoscale devices. Numerous experimental data have been analyzed in connection with the so-called oligonucleotides-transformers (nucleotide sequences that able to fold not only into one, but also into several NSs). These sequences were recently predicted by our calculations using automata and graph theories ("Dafna" algorithm). Possible applications of the oligonucleotides-transformers in nanoengineering and genetic editing of organisms are considered.

Although graylag geese (A. anser) showed similar plumages of white, grey, and white with grey patches compared to those in swan geese (A. cygnoides), it was believed the substantial molecular mechanism for plumage variations were different. To date, studies on genes responsible for diverse plumages among graylag geese were limited and causal mutations remain unknown. In this study, genomes from 57 individuals belonging to six breeds showing different plumages were sequenced at ∼10X depth. Firstly, the allele frequency differences (AFD) of variants on the scaffold394 (NW_013185915.1) between grey and white goose breeds (A. anser) was calculated and a genomic region between 768,290-779,889 bp was detected to carry candidate variants associated with plumages, including one SNP (g. 775,151G T, ∼18.6 kb upstream of EDNRB2) found to be fixed in white geese. This region was overlapped with the one detected by the haplotype-based sweep analysis, in which significant signals defined a candidate region of 736,610-820,622 bp on the same scaffold. Results from the transcriptomic data showed that expression levels of EDNRB2 and many other melanogenesis-related genes were significantly decreased among white geese compared to that in grey geese, especially at late embryonic stages (E15). Modifications at transcriptional levels might result in abnormal melanocyte developments and thus the white plumages when they grow up. In addition, a frameshift mutation (C -) in exon4 of MLANA gene on scaffold176 (NW_013185876.1) was suggested as the causal mutation for sex-linked dilution phenotype in graylag geese although this requires more demonstration experiments. Together with observed white plumages caused by EDNRB2 mutations in coding regions among swan geese and chicken, our study provided new examples to study the parallel evolution.

Made available in DSpace on 2022-04-29T08:41:21Z (GMT). No. of bitstreams: 0 Previous issue date: 2022-06-05 In association with many proteins, small nuclear RNAs (snRNAs) organize the spliceosomes that play a significant role in processing precursor mRNAs during gene expression. According to snRNAs genic arrangements, two kinds of spliceosomes (major and minor) can be organized into eukaryotic cells. Although in situ localization of U1 and U2 snDNAs have been performed in fish karyotypes, studies with genomic characterization and functionality of U snRNAs integrated into chromosomal changes on Teleostei are still scarce. This study aimed to achieve a genomic characterization of the U snRNAs genes in Apareiodon sp. (2n = 54, ZZ/ZW), apply these data to recognize functional/defective copies, and map chromosomal changes involving snDNAs in Parodontidae species karyotype diversification. Nine snRNA multigene families (U1, U2, U4, U5, U6, U11, U12, U4atac and U6atac) arranged in putatively functional copies in the genome were analyzed. Proximal Sequence Elements (PSE) and TATA-box promoters occurrence, besides an entire transcribed region and conserved secondary structures, qualify them for spliceosome activity. In addition, several defective copies or pseudogenes were identified for the snRNAs that make up the major spliceosome. In situ localization of snDNAs in five species of Parodontidae demonstrated that U1, U2, and U4 snDNAs were involved in chromosomal location changes or units dispersion. The U snRNAs defective/pseudogenes units dispersion could be favored by the probable occurrence of active retrotransposition enzymes in the Apareiodon genome. The U2 and U4 snDNAs sites were involved in independent events in the differentiation of sex chromosomes among Parodontidae lineages. The study characterized U snRNA genes that compose major and minor spliceosomes in the Apareiodon sp. genome and proposes that their defective copies trigger chromosome differentiation and diversification events in Parodontidae. Programa de Pós-Graduação em Genética Universidade Federal do Paraná Centro Politécnico, Avenida Coronel Francisco H. dos Santos, 100, Paraná Departamento de Biologia Estrutural Molecular e Genética Universidade Estadual de Ponta Grossa, Av. Carlos Cavalcanti, 4748, Paraná Departamento de Genética e Evolução Universidade Federal de São Carlos, Rodovia Washington Luís, Km 235, São Paulo Departamento de Morfologia Instituto de Biociências de Botucatu Universidade Estadual Paulista, Distrito de Rubião Júnior, s/n, São Paulo Departamento de Morfologia Instituto de Biociências de Botucatu Universidade Estadual Paulista, Distrito de Rubião Júnior, s/n, São Paulo

ZNFO is a Kruppel-associated box (KRAB) containing zinc finger transcription factor, which is exclusively expressed in bovine oocytes. Previous studies have demonstrated that ZNFO possesses an intrinsic transcriptional repressive activity and is essential for early embryonic development in cattle. However, the mechanisms regulating ZNFO transcription remain elusive. In the present study, the core promoter that controls the ZNFO basal transcription was identified. A 1.7 kb 5′ regulatory region of the ZNFO gene was cloned and its promoter activity was confirmed by a luciferase reporter assay. A series of 5′ deletion in the ZNFO promoter followed by luciferase reporter assays indicated that the core promoter region has to include the sequence located within 57 bp to 31 bp upstream of the transcription start site. Sequence analysis revealed that a putative USF1/USF2 binding site (GGTCACGTGACC) containing an E-box motif (CACGTG) is located within the essential region. Depletion of USF1/USF2 by RNAi and E-box mutation analysis demonstrated that the USF1/USF2 binding site is required for the ZNFO basal transcription. Furthermore, EMSA and super-shift assays indicated that the observed effects are dependent on the specific interactions between USF proteins and the ZNFO core promoter. From these results, it is concluded that USF1 and USF2 are essential for the basal transcription of the ZNFO gene.

The true mahseer (Tor spp.) is one of the highest valued fish in the world due to its high nutritional value and great unique taste. Nevertheless, its morphological characterization and single mitochondrial gene phylogeny in the past had yet to resolve the ambiguity in its taxonomical classification. In this study, we sequenced and assembled 11 complete mahseer mitogenomes collected from Java of Indonesia, Pahang and Terengganu of Peninsular Malaysia as well as Sarawak of East Malaysia. The mitogenome evolutionary relationships among closely related Tor spp. samples were investigated based on maximum likelihood phylogenetic tree construction. Compared to the commonly used COX1 gene fragment, the complete COX1, Cytb, ND2, ND4 and ND5 genes appear to be better phylogenetic markers for genetic differentiation at the population level. In addition, a total of six population-specific mitolineage haplotypes were identified among the mahseer samples analyzed, which this offers hints towards its taxonomical landscape.

The 35S ribosomal DNA (rDNA) units that occur in tandem repeat are separated by an intergenic spacer (IGS) that plays an important role in rRNA transcription. Moreover, IGS is an important molecular marker for evolutionary research in plants. In the present study, the IGS sequence of Erianthus arundinaceus was isolated and sequenced for the first time. Structure analysis indicated the entire IGS sequence of three typical E. arundinaceus genotypes was highly conserved, with approximately 3087 bp and 67.1% mean GC content. The putative transcription termination, and initiation sites as well as a large number of methylation sites were found to be present in the IGS of E. arundinaceus compared to other plants. The phylogenic tree constructed using the E. arundinaceus IGS sequence showed that Miscanthus sinensis var. glaber was genetically close to Saccharum spp. while E. arundinaceus was close to Imperata cylindrica. Moreover, fluorescent in situ hybridization revealed that IGS and pTa71 probes had the same locus at nucleolar organizer regions. Taken together, this work enhances our current understanding of the organization of IGS in E. arundinaceus and provides a molecular evidence for an evolutionary relationship between Saccharum spp., E. arundinaceus, I. cylindrica and M. sinensis var. glaber.

Two full-length cDNAs of the cyclin-D3-1-like gene, named as BnCYCD3-1-like-1 and BnCYCD3-1-like-2 respectively were obtained from Brassica napus, both of which encoded a cell cycle protein CYCD3. Alternative splicing (AS) events of the two genes' transcripts were identified, assigned as BnCYCD3-1-like-1-1, BnCYCD3-1-like-1-2, BnCYCD3-1-like-2-1 and BnCYCD3-1-like-2-2 respectively. BnCYCD3-1-like-1-1 and BnCYCD3-1-like-2-1 were both fully-spliced transcripts which encoded a complete protein containing a LXCXE motif, two cyclin boxes and a PEST domain, while other two alternative splicing transcripts both resulted in the early termination of the protein translation. BnCYCD3-1-like-2-2 retained the third intron, lacking a PEST domain, while BnCYCD3-1-like-1-2 retained all the introns, lacking the C-terminal cyclin domain and a PEST domain. The expression pattern for tissue and development specification of the AS transcripts were investigated. The results showed that the standard splicing transcripts (BnCYCD3-1-like-1-1 and BnCYCD3-1-like-2-1) with complete structural domains were found with the most abundant expression in seeds, followed by leaves, and the least expression in stems. Both of BnCYCD3-1-like-2-1 and BnCYCD3-1-like-2-2 had the highest abundance in leaves, followed by roots. In addition, by applying various biotic and abiotic stresses on Brassica napus, the variations in the expression of each transcript under stress treatment were studied. Also, it was found that AS of the cyclin-D3-1-like gene may play an important role in helping Brassica napus respond to environmental stresses by coordinating the levels of transcripts of standard splicing and alternative splicing.

Horses (Equus caballus) have been domesticated and bred to enhance speed, strength, and agility. Members of the Equus caballus Endogenous Retrovirus (EqERV) family affect several of these abilities in horses. EqERV elements have been integrated in the horse genome during evolution and generate repeat elements such as long terminal repeats (LTRs). LTR sequences are involved in retrovirus replication and play an essential function in post-transcriptional control mechanisms, such as by providing binding sites for microRNAs (miRNAs) or generating miRNA precursors. In this study, we identified a novel miRNA derived from EqERV-E1 LTR using various bioinformatics tools. To examine the relationship between EqERV-E1 LTR and similar elements, we used BLAST2seq and phylogenetic analysis. LTR sequences were located in the untranslated region (UTR) of mRNAs and also formed the stem-loop secondary structure. The sequence was registered in the DDBJ database as LTR derived miRNA under the accession number corresponding to LC383797 (referred to eca-miR-1804). Quantitative polymerase chain reaction (qPCR) to confirm the expression of eca-miR-1804 and the similar miR-1255a, showed an almost identical expression pattern in eight different equine tissues. Therefore, these data imply that the LTR could function as an miRNA, which is expressed in the examined equine tissues. In addition, the current study provides inputs for additional functional studies concerning the LTR of other EqERV families.

To investigate whether the members of the mammalian Achaete-Scute Complex homologue (ASH) gene family have evolved functional differences, we used the patterning of bristles as a phenotypic marker. Drosophila uses a single genetic locus - the Achaete-Scute Complex - to demarcate the regions of the body where bristles can form. We found 4-5 Achaete-Scute Complex homologue genes (ASH) in the mammalian genome, which are homologous with scute in Drosophila. Although ASH2 and ASH3 have gained new functions during evolution, the function of ASH4 and its evolutionary changes are still unclear. In this study, we overexpressed mouse and human ASH1 and ASH4 in the Drosophila notum respectively. The results show that both the protein sequence and cis-regulatory elements of mammalian ASH1 have conserved an ancient proneural function during evolution. However, mouse ASH4 has lost proneural function partly due to truncation of a C-terminal amino acid domain. Interestingly, instead of a similar loss of proneural function, we found human ASH4 can actually inhibit Drosophila bristle development, implying that human ASH4 may be a potential factor relating to skin development in human being. Our results demonstrate gene duplication of the ASH family may have led to a novel function during evolution.

The mitochondrial DNA gene cytochrome oxidase subunit I (COI) has been adopted as a global bioidentification system for animals. This study represents the comprehensive molecular identification of small indigenous fish species (SIS) of Bangladesh assessed by DNA barcoding. DNA barcodes were generated from 81 SIS species belongs to the orders Clupeiformes, Cypriniforms, Siluriformes, Perciformes, Synbranchiformes, Beloniformes and Tetraodontiformes representing 55 genera and 24 families. For all the samples, %G were significantly lowered compared to other nucleotides and %GC compared to %AT. Also, a significantly lowered GC content was observed in second and third codon position compared to the first codon position in all samples. The average Kimura two-parameter (K2P) distances within genera, families, and orders were 15.83%, 19.14%, and 25.07%, respectively. The minimum and maximum K2P distance based genetic divergences were 0.19% and 57.14% respectively. Members of Cypriniformes, Siluriformes, and Perciformes were clustered separately in the neighbour-joining (NJ) tree. Nucleotide composition, GC distribution across codon positions, K2P distance, genetic divergence, and phylogenetic analyses reveal that these freshwater SIS fishes are genetically very diverse. Along with morphological data, we have confirmed the existence of seven new records of SIS fishes in Bangladesh using such barcode approach. These findings suggest that fishes can be discriminated using these barcode data without any confusion.

The tumor necrosis factor (TNF) superfamily consists of a wide variety of inflammatory cytokine, including cell-bound and secreted proteins. These TNFs function through binding and activation of the TNF receptors for modulating TNF-associated intracellular signals. A set of mammalian TNF receptor-associated factors (TRAFs) that have emerged as the major signal transducers for the TNF receptor superfamily, play an important role in both adaptive and innate immunity. However, the existence of TRAFs and their biological functions in planarian are still unknown. In this study, a new member of TRAFs, DjTRAF2, was identified in planarian Dugesia japonica. Phylogenetic analysis revealed that DjTRAF2 could be a new member of the invertebrate TRAF2 family. Sequence analysis showed that the open reading frame of DjTRAF2 had 1353 bp in length and encoded a putative protein of 450 amino acids with a predicted molecular mass of ~51.8 kDa and an isoelectric point of 7.052. Whole-mount in situ hybridization showed that DjTRAF2 was predominantly expressed in adult and regenerative pharynx, which is an important immune organ of planarian. Quantitative real-time PCR revealed that the transcriptional level of DjTRAF2 was significantly up-regulated after induced by pathogen-associated molecular patterns (polyinosinic-polycytidylic acid, lipopolysaccharide, peptidoglycan and β-glucan), suggesting that DjTRAF2 is involved in the immune response against pathogen invasion. Collectively, these results demonstrated that DjTRAF2 might play important roles in the innate immunity of planarian.

The CXCL12 gene produces a series of transcript variants through alternative splicing at the 3' end of its pre-mRNA. This study explores the biological activities of these alternative transcripts and the mechanisms involved in the regulation of CXCL12 transcription and RNA splicing. We identified a "GA" insertion mutation in the region of CXCL12α DNA encoding the conserved 3'UTR. This variant transcript was named CXCL12-3'GA+. The mutation occurred at a frequency of 13.2% in healthy Chinese individuals. However, its frequency in healthy Caucasians was 22.6%, significantly higher than what was observed in the Chinese. Genomic analysis indicated that the GA+ mutation likely encodes a G-quadruplex structure in close proximity to a cluster of important AU-rich elements (AREs) that are well-established regulators of mRNA stability at the 3'UTR. Experiments using molecular constructs encoding the 3'UTR of CXCL12 revealed that the GA+ allele can significantly increase gene expression compared to the WT allele. Further studies uncovered that the WT allele was associated with the production of a 225-bp minor transcript isoform (MTI) through alternative splicing resulting in the deletion of exon 2. ARMS-PCR using samples collected from cultured PBMCs of WT/GA+ genotype carriers indicated that the GA+ allele was preferentially transcribed compared to the WT allele. In summary, the study demonstrates that a GA insertion in the region encoding the 3'UTR of CXCL12α may affect gene expression through alternative mRNA splicing. This finding provides a basis for understanding how multiple elements in the sequence encoding the 3'UTR of the CXCL12 gene regulates its transcription and may lead to insights about diseases involving abnormal CXCL12α expression.

Chydoridae are phytophilic-benthic microcrustaceans that make up a significant proportion of species diversity and play an important role in the littoral zone of freshwater ecosystems worldwide. Here, we provide the complete mitochondrial genome of Ovalona pulchella (King, 1853), determined by next-generation sequencing. The entire mitochondrial genome is 15,362 bp in length; this is the first sequenced mitochondrial genome in the family Chydoridae. The base composition and codon usage were typical of Cladocera species. The mitochondrial gene arrangement (37 genes) was not consistent with that of other Branchiopoda. Both maximum likelihood and Bayesian analyses supported each suborder and family of Branchiopoda as monophyletic groups. The relationships among the families were as follows: [(Leptestheriidae + Limnadiidae) + (Sididae + (Bosminidae + (Chydoridae + Daphniidae)))] + Triopsidae. The newly sequenced O. pulchella was most closely related to the family Daphniidae. The complete mitochondrial genome of O. pulchella also provides valuable molecular information for further analysis of the phylogeny of the Chydoridae and the taxonomic status of the Branchiopoda.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) associated Cas protein (CRISPR-Cas) has turned out to be a very important tool for the rapid detection of viruses. This can be used for the identification of the target site in a virus by identifying a 3-6 nt length Protospacer Adjacent Motif (PAM) adjacent to the potential target site, thus motivating us to adopt CRISPR-Cas technique to identify SARS-CoV-2 as well as other members of Coronaviridae family. In this regard, we have developed a fast and effective method using k-mer technique in order to identify the PAM by scanning the whole genome of the respective virus. Subsequently, palindromic sequences adjacent to the PAM locations are identified as the potential target sites. Palindromes are considered in this work as they are known to identify viruses. Once all the palindrome-PAM combinations are identified, PAMs specific for the RNA-guided DNA Cas9/Cas12 endonuclease are identified to bind and cut the target sites. In this regard, PAMs such as 5'-TGG-3' and 5'-TTTA-3' in NSP3 and Exon for SARS-CoV-2, 5'-GGG-3' and 5'-TGG-3' in Exon and NSP2 for MERS-CoV and 5'-AGG-3' and 5'-TTTG-3' in Helicase and NSP3 respectively for SARS-CoV-1 are identified corresponding to SpCas9 and FnCas12a endonucleases. Finally, to recognise the target sites of Coronaviridae family as cleaved by SpCas9 and FnCas12a, complements of the palindromic target regions are designed as primers or guide RNA (gRNA). Therefore, such complementary gRNAs along with respective Cas proteins can be considered in assays for the identification of SARS-CoV-2, MERS-CoV and SARS-CoV-1.

Carotenoids are essential micronutrients for animals, and they can only be obtained from the diet for mollusk as well as other animals. In the body, carotenoids undergo processes including absorption, transport, deposition, and metabolic conversion; however, knowledge of the involved genes is still limited. To elucidate the molecular mechanisms of carotenoid processing and identify the related genes in Pacific oyster (Crassostrea gigas), we performed a comparative transcriptome analysis using digestive gland tissues of oysters on a beta-carotene supplemented diet or a normal diet. A total of 718 differentially expressed genes were obtained, including 505 upregulated and 213 downregulated genes in the beta-carotene supplemented group. Function Annotation and enrichment analyses revealed enrichment in genes possibly involved in carotenoid transport and storage (e.g., LOC105342035), carotenoid cleavage (e.g., LOC105341121), retinoid homeostasis (e.g., LOC105339597) and PPAR signaling pathway (e.g., LOC105323212). Notably, down-regulation of mRNA expressions of two apolipoprotein genes (LOC105342035 and LOC105342186) by RNA interference significantly decreased the carotenoid level in the digestive gland, supporting their role in carotenoid transport and storage. Based on these differentially expressed genes, we propose that there may be a negative feedback mechanism regulated by nuclear receptor transcription factors controlling carotenoid oxygenases. Our findings provide useful hints for elucidating the molecular basis of carotenoid metabolism and functions of carotenoid-related genes in the oyster.

Among ectothermic reptiles, the order Squamata has adapted most successfully to the terrestrial environment. However, the physiological background of this success remains unknown. Since the regulation of energy metabolism provides an important insight into terrestrial adaption by ectothermic animals, we focused on proglucagon-derived peptides (PGDPs). In the process of cloning proglucagon mRNA in geckos, we identified several novel proglucagon (PG) cDNA isoforms. They were tissue-specifically and strongly expressed in the pancreas and small intestine of the geckos, suggesting their biological relevance. Therefore, in order to clarify whether these novel cDNA isoforms are phylogenetically conserved, we performed the additional molecular characterization of proglucagon cDNAs from several representative species of the Squamata and Testudine clade and examined the expression of proglucagon mRNAs in the small intestine and pancreas. In the present study, a total of 7 proglucagon cDNA isoforms were identified and divided into two groups (Classes A and B) based on the 3′-UTR sequence of each isoform. The longest isoform of each group (named PG-A1 and PG-B1, respectively) had the same molecular characteristics as those previously reported from chickens and reptiles, namely, PG-A and PG-B. Other 5 isoforms were novel-type cDNAs, and were the products of exon skipping (named PG-A2, PG-A2s, PG-B2, PG-B2s, and PG-B3). Some of these isoforms coded for only one peptide hormone (GLP-1 or GLP-2). This is the first identification of single hormone-encoding proglucagon cDNAs in vertebrates. Moreover, an expression analysis of these isoforms revealed that single hormone-encoding proglucagon mRNAs were predominantly expressed with tissue and lineage specificities in the reptile clade. Collectively, the present results suggest an independent regulatory system for GLP-1 and GLP-2 secretion and indicate the plasticity of proglucagon genes in expressing different isoforms in different tissues in Squamata. These results also provide insights into the plastic energy metabolic system of Squamata in accordance with various habitats in the terrestrial environment, supporting their successful prosperity.

TMZ is the only drug that has improved survival of GBM patients although minimally. However, a number of GBM patients did not respond to TMZ-based chemotherapy. Here, we found that miR-505 inhibited tumorigenesis as a tumor suppressor in glioblastoma. In addition, TMZ could increase the levels of miR-505 and combination with pri-miR-505 and TMZ promoted the suppressive role mediated by miR-505 in GBM cells. Furthermore, miR-505 inactivated the Wnt/β-catenin signaling pathway by directly targeting and inhibiting WNT7B. In conclusion, our results demonstrated that the induction of miR-505 in combination with TMZ treatment could be a useful therapeutic strategy for suppressing GBM.

Here we present a case report of collagen VI related myopathy in a patient, 8 y.o. boy, with intermediate phenotype between severe Ullrich congenital muscular dystrophy and milder Bethlem myopathy. Whole exome sequencing revealed two novel single nucleotide variants in COL6A3 gene: paternal p.Glu2402Ter, resulting in premature translation termination codon and degradation of mRNA from this allele probably due to nonsense-mediated decay, and maternal p.Arg1660Cys leading to amino-acid substitution in N2-terminal domain. COL6A3 expression analysis of proband's fibroblasts reveals functional homozygosity of the latter variant. Paternal fibroblasts showed only WT allele expression, which could lead to a reduction in mature transcript level, while maternal fibroblasts expressed both alleles. Functional assay of immunofluorescent staining of COL6A3 protein in fibroblasts culture reveals profound changes in COL6A3 localization and reduction of protein level in studied cultures when comparing with the controls. This study not only broadens the allelic spectrum of pathogenic COL6A3 variants in myopathy but also gives an additional support to Ullrich congenital muscular dystrophy and Bethlem myopathy clinical continuum.

Krüppel-like factor 3 (KLF3), a member of the Krüppel-like factor (KLF) family, plays an important role in adipogenesis and lipid metabolism. The aim of this study was to investigate whether KLF3 could be used as a candidate gene in the breeding of cattle. The expression pattern of bovine KLF3 gene revealed that it was highly expressed in abdominal fat and perirenal fat. Using DNA sequencing, three single nucleotide polymorphisms (SNPs) within the promoter regions of KLF3 gene were identified in 448 Qinchuan cattle, which are located in the recognition sequences of 11 transcription factors and the four haplotypes representing four potential different compositions of polymorphic potential cis-acting elements. Association analysis results indicated that individuals with the Hap7/7 diplotype showed higher (P 0.05) intramuscular fat content (IFC) than those with H7/8. In addition, the H7 haplotype had much higher (P 0.05) transcriptional activity than the H8 haplotype, consistent with the association analysis. We speculated that polymorphisms in transcription factor binding sites of the KLF3 promoter region affected transcriptional activity of KLF3, which subsequently influence intramuscular fat content in Qinchuan cattle and KLF3 gene could be used as molecular markers for fat deposition traits using early marker-assisted selection (MAS) of Qinchuan cattle breeding in the future.

Autoimmune lymphoproliferative syndrome (ALPS) usually presents in childhood with fever, nonmalignant splenomegaly and lymphadenopathy along with hemocytopenia. This case report describes a 10-year-old boy presenting with signs of autoimmune disease, splenomegaly, hepatomegaly and resistant hemocytopenia. Sirolimus controlled the relapsed thrombocytopenia after splenectomy. Sequencing of the FAS gene identified two spontaneous heterozygous mutations (c.234 T > G, p.D78E) (c.236dupA, p.P80Tfs*26). The boy's homozygous missense variation (c.2588G > A, p.G863D) (rs140184929) in UNC13D gene had been identified as being related to familial hemophagocytic lymphohistiocytosis (FHL). TCRαβ + CD4/CD8 double-negative T cells (markers of ALPS) were not significantly increased from the outset. Elevated cytokines, such as interferon (IFN)-γ, interleukin (IL)-6 and tumor necrosis factor α decreased to normal levels after splenectomy whereas IL-10 remained high. Immunological analysis of the patient revealed a marked depletion of forkhead-box P3+ expressing regulatory T cells (Treg) and Th17 cells. The obtained data demonstrate that mutations to FAS and UNC13D which result in overwhelming T-cell and macrophage activation, one associated with inhibited Treg cell development and a severe ALPS-like symptom. Therefore, we propose that variations of UND13D may be a risk factor of ALPS development.

Germline-specific genes, Vasa, Dazl and Nanos1, have highly conserved functions in germline development and fertility across animal phyla. In this study, the full-length sequences of Opvasa, Opdazl and Opnanos1 were cloned and characterized from the dark sleeper (Odontobutis potamophila). Gonad-specific expression patterns of Opvasa and Opdazl were confirmed in adult tissues by quantitative real-time PCR (qRT-PCR). Different from Opvasa and Opdazl, the expression of Opnanos1 was ubiquitously detected in all examined tissues except for the liver and spleen. Time-course dynamic expressions during embryogenesis were assessed, and all three genes (Opvasa, Opdazl and Opnanos1) persisted at a high level until gastrulation. qRT-PCR and Western blotting analyses revealed that all three genes were highly expressed throughout gametogenesis. In testis, the expressions of all three genes at the mRNA and protein levels were down-regulated during spermatogenesis. In ovary, different expression patterns were found, and all three genes had a differential role in translational regulation during oogenesis. The expressions of Opvasa, Opdazl and Opnanos1 at the mRNA but not the protein level were high in stage IV. Different expression patterns were found in premeiotic gonads treated by HPG axis hormones (HCG and LHRH-A). Immunolocalization analysis demonstrated that in testis, Opvasa, Opdazl and Opnanos1 were detected in spermatogonia and spermatocytes but absent in the meiotic products, such as spermatids and spermatozoa. In ovary, Opvasa, Opdazl and Opnanos1 persisted at a high level throughout oogenesis. These findings indicated that Opvasa, Opdazl and Opnanos1 played an important role in mitotic and early meiotic phases of oogenesis and spermatogenesis, and they functioned as maternal factors in early embryogenesis. Their proteins could be used as three new markers for germ cells during gametogenesis in O. potamophila gonad. Our data laid a good foundation for improving the breeding efficiency of O. potamophila.

To elucidate the Cdk5 regulatory molecular mechanism in skin, we generated Cdk5-knockdown mice and subjected their skins to lncRNA sequencing. The results showed that there were 4533 novel lncRNAs from 142 lncRNA families. In total, 693 lncRNAs were significantly differentially expressed. Alignment analysis of the lncRNAs in miRBase identified 45 pre-mRNAs. By KEGG PATHWAY Database analysis, we found that lncRNAs (lnc-NONMMUT064276.2, lnc-NONMMUT075728.1, and lnc-NONMMUT039653.2) may regulate pigmentation by regulating target genes. To reveal potential antisense lncRNA-mRNA interactions, we searched all lncRNA-mRNA duplexes using RNAplex, and found 97 lncRNAs interacted with mRNAs. The luciferase assay confirmed that TCONS_00049140 binded to Krt80 by the co-transfection of pVAX1-TCONS_00049140 and pGL0-Krt80 expression plasmids in 293T cell, based on the bioinformatics analysis. Overexpression of TCONS_00049140 in mouse melanocytes down-regulated Krt80 and resulted in the phenotype of increased cell proliferation and increased melanin production. The results suggested that TCONS_00049140 contributed to skin thickening through Krt80. Our findings provide a direction for research of the molecular mechanism of Cdk5 function.

Mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR) gene can be involved in the development of congenital bilateral absence of the vas deferens (CBAVD). This study was aimed at investigating mutations in the promoter region of the CFTR gene and its associated effects on CFTR transcription in Chinese patients with CBAVD.To identify CFTR promoter region mutations in Chinese CBAVD patients, fragments 1.4 kb upstream of the ATG start codon of the CFTR gene were sequenced in 66 Chinese patients with CBAVD and compared to the corresponding sequences from 60 healthy subjects and sequence data present in the NCBI database. The relationship between the mutations and gene regulation was explored using Transfac analysis and a phylogenetic footprint method. Plasmids were constructed by incorporating statistically significant variant sequences. The effects of the mutations on CFTR transcription were investigated using a dual luciferase reporter gene assay.a total of six point mutations, which included c.-150G T, c.-205 T C, c.-245C T, c.-871G T, c.-966 T G and c.-1062G C, and one deletion mutation, namely c.-861delT, were identified in the promoter region of the CFTR gene in Chinese CBAVD patients. Among them, c.-966 T G had the highest frequency and presented in either a homozygous or heterozygous mutation state. The frequency of G/G genotype in the CBAVD group was 33/66 (50.00%), while the T/G and T/T genotypes had frequencies of 18/66 (27.27%) and 15/66 (22.73%), respectively. A significant difference was found between the CBAVD and control group (P 0.01). The locus for this variant was found in a conserved sequence. The Transfac tool showed that transcription factors EHF and STAT3, which are closely associated with reproduction, can bind to the sequence containing the variant locus. Two types of plasmid vectors, one carrying the G/G variant and the other the wild type T/T sequences, were constructed and respectively transfected into human cervical cancer cells (HeLa), human renal epithelial cells (HEK-293), and human colon cancer cells (SW480). It was found that the homozygous c.-966 T G mutation significantly reduced CFTR transcription efficiency by 18.75%-35.50%.Mutations in the promoter region of the CFTR gene in Chinese CBAVD patients are different from those found in comparable Caucasian patients. The homozygous c.-966 T G mutation state had the highest frequency, which reduced the CFTR transcriptional level and showed significant tissue-specificity.

The genome sequences of entomopathogenic nematode (EPN) bacteria and their functional analyses can lead to the genetic engineering of the bacteria for use as biocontrol agents. The bacterial symbiont Photorhabdus heterorhabditis strain ETL isolated from an insect pathogenic nematode, Heterorhabditis zealandica strain ETL, collected in the northernmost region of South Africa was studied to reveal information that can be useful in the design of improvement strategies for both effective and liquid production method of EPN-based pesticides. The strain ETL genome was found closely related to the type strain genome of P. australis DSM 17,609 (~60 to 99.9% CDSs similarity), but closely related to the not yet genome-sequenced type strain, P. heterorhabditis. It has a genome size of 4,866,148 bp and G + C content of 42.4% similar to other Photorhabdus. It contains 4,351 protein coding genes (CDSs) of which, at least 84% are shared with the de facto type strain P. luminescens subsp. laumondii TTO1, and has 318 unknown CDSs and the genome has a higher degree of plasticity allowing it to adapt to different environmental conditions, and to be virulent against various insects; observed through genes acquired through horizontal gene transfer mechanisms, clustered regularly interspaced short palindromic repeats, non-determined polyketide- and non-ribosomal peptide- synthase gene clusters, and many genes associated with uncharacterized proteins; which also justify the strain ETL’s genes differences (quantity and quality) compared to P. luminescens subsp. laumondii TTO1. The protein coding sequences contained genes with both bio-engineering and EPNs mass production importance, of which numerous are uncharacterized.

Enterococcal plasmids have generated renewed interest for their indispensable role in pathogenesis and dissemination of multidrug-resistance. Recently, a novel plasmid pSM409 (4303-bp, GC% = 33.6%), devoid of antibiotic-resistance and virulence genes, has been identified in Enterococcus faecium RME, isolated from raw milk by us. pSM409 contains six open reading frames encoding a replication initiator protein (RepB) and five accessory proteins: antitoxin epsilon, bacteriocin immunity protein, HsdS, and two hypothetical proteins. Comparative sequence analysis of pSM409 reveals a mosaic pattern of similarity with different loci obtained from different theta plasmids, which dictates the plasmid to be heterogeneous or mosaic, possibly due to recombination. The pSM409 comprised of a typical theta-type origin of replication with four and a half direct repeats (iterons) of 22 nucleotides. The pSM409-RepB shared 76–82% homology with the RepB of reported theta plasmids from different genera, with dissimilarities mostly in its DNA-binding and C-terminal domain. The RepB sequence-based phylogenetic tree revealed its distinct position relative to the reported ones. The RepB grouped in the same clade has identical DNA-binding domains and their cognate iterons, possibly due to their sequence-specific interaction to initiate plasmid replication. Comparative analysis of the pSM409-iteron reveals that the repeats markedly differed from their closest homologues. This clade-specific relationship provides a new concept of classifying theta plasmids. The theta-type replicon identified in pSM409 has been found to be unique to E. faecium RME, prompting us to further investigate its utility as a vector for genetic manipulation of enterococci for health and industry.

The Bifidobacterium longum 5