Twists and turns: a scientific journey.

In this perspective I look back on the twists and turns that influenced the direction of my scientific career over the past 40 years. From my early ambition to be a chemist to my training in Philadelphia and Bethesda as a molecular biologist, I benefited enormously from generous and valuable mentoring. In my independent career in Philadelphia and Princeton, I was motivated by a keen interest in the changes in gene expression that direct the development of the mammalian embryo and inspired by the creativity and energy of my students, fellows, and research staff. After twelve years as President of Princeton University, I have happily returned to the faculty of the Department of Molecular Biology.

Genes divided according to the relative position of the longest intron show increased representation in different KEGG pathways.

Despite the fact that introns mean an energy and time burden for eukaryotic cells, they play an irreplaceable role in the diversification and regulation of protein production. As a common feature of eukaryotic genomes, it has been reported that in protein-coding genes, the longest intron is usually one of the first introns. The goal of our work was to find a possible difference in the biological function of genes that fulfill this common feature compared to genes that do not. Data on the lengths of all introns in genes were extracted from the genomes of six vertebrates (human, mouse, koala, chicken, zebrafish and fugu) and two other model organisms (nematode worm and arabidopsis). We showed that more than 40% of protein-coding genes have the relative position of the longest intron located in the second or third tertile of all introns. Genes divided according to the relative position of the longest intron were found to be significantly increased in different KEGG pathways. Genes with the longest intron in the first tertile predominate in a range of pathways for amino acid and lipid metabolism, various signaling, cell junctions or ABC transporters. Genes with the longest intron in the second or third tertile show increased representation in pathways associated with the formation and function of the spliceosome and ribosomes. In the two groups of genes defined in this way, we further demonstrated the difference in the length of the longest introns and the distribution of their absolute positions. We also pointed out other characteristics, namely the positive correlation between the length of the longest intron and the sum of the lengths of all other introns in the gene and the preservation of the exact same absolute and relative position of the longest intron between orthologous genes.

Origin, evolution, and diversification of inositol 1,4,5-trisphosphate 3-kinases in plants and animals.

BACKGROUND: In Eukaryotes, inositol polyphosphates (InsPs) represent a large family of secondary messengers and play crucial roes in various cellular processes. InsPs are synthesized through a series of pohophorylation reactions catalyzed by various InsP kinases in a sequential manner. Inositol 1,4,5-trisphosphate 3-kinase (IP3 3-kinase/IP3K), one member of InsP kinase, plays important regulation roles in InsPs metabolism by specifically phosphorylating inositol 1,4,5-trisphosphate (IP3) to inositol 1,3,4,5-tetrakisphosphate (IP4) in animal cells. IP3Ks were widespread in fungi, plants and animals. However, its evolutionary history and patterns have not been examined systematically. RESULTS: A total of 104 and 31 IP3K orthologues were identified across 57 plant genomes and 13 animal genomes, respectively. Phylogenetic analyses indicate that IP3K originated in the common ancestor before the divergence of fungi, plants and animals. In most plants and animals, IP3K maintained low-copy numbers suggesting functional conservation during plant and animal evolution. In Brassicaceae and vertebrate, IP3K underwent one and two duplication events, respectively, resulting in multiple gene copies. Whole-genome duplication (WGD) was the main mechanism for IP3K duplications, and the IP3K duplicates have experienced functional divergence. Finally, a hypothetical evolutionary model for the IP3K proteins is proposed based on phylogenetic theory. CONCLUSION: Our study reveals the evolutionary history of IP3K proteins and guides the future functions of animal, plant, and fungal IP3K proteins.

Genome-wide identification and expression analysis of the glycosyl hydrolase family 1 genes in Medicago sativa revealed their potential roles in response to multiple abiotic stresses.

Glycoside hydrolase family 1 (GH1) ß-glucosidases (BGLUs), are encoded by a large number of genes, which participate in the development and stress response of plants, particularly under biotic and abiotic stresses through the activation of phytohormones. However, there are few studies systematically analyzing stress or hormone-responsive BGLU genes in alfalfa. In this study, a total of 179 BGLU genes of the glycoside hydrolase family 1 were identified in the genome of alfalfa, and then were classified into five distinct clusters. Sequence alignments revealed several conserved and unique motifs among these MsBGLU proteins. Many cis-acting elements related to abiotic stresses and phytohormones were identified in the promoter of some MsBGLUs. Moreover, RNA-seq and RT-qPCR analyses showed that these MsBGLU genes exhibited distinct expression patterns in response to different abiotic stress and hormonal treatments. In summary, this study suggests that MsBGLU genes play crucial roles in response to various abiotic stresses and hormonal responses, and provides candidate genes for stress tolerance breeding in alfalfa.

Genome-wide identification, characterization and expression profiling of TLR family genes in Chromileptes altivelis.

Toll-like receptors (TLRs) represent a prominent category of pattern recognition receptors that have been extensively investigated for their pivotal role in combating pathogen incursions. Despite this, there has been a notable absence of comprehensive identification and exploration of the immune response associated with the TLR family genes in C. altivelis. This study successfully identified and named fourteen genes as Catlr1-1, Catlr1-2, Catlr2-1, Catlr2-2, Catlr3, Catlr5, Catlr7, Catlr8, Catlr9, Catlr13-1, Catlr13-2, Catlr18, Catlr21, and Catlr22. A series of bioinformatic analysis were performed, encompassing analysis of protein properties, examination of gene structures, evolutionary assessments, and prediction of protein tertiary structures. The expression patterns of Catlr genes were analyzed in five immune tissues: liver, spleen, kidney, gill, and intestine, in both healthy and bacterial stimulated-fish. The results showed that different tissue and different genes showed differed expression patterns after V. harveyi infection, indicating the involvement of all Catlr members in mounting immune responses following infection in various tissues. Additionally, histological evaluations of immune tissues unveiled varying levels of damage. In conclusion, this investigation into the TLR gene family offers novel information that contribute to a more profound comprehension of the immune response mechanisms in C. altivelis.

Structure and function analyses of the SRC gene in Pacific white shrimp Litopenaeus vannamei.

SRC gene encodes scavenger receptor class C, a member of the scavenger receptor family, and has only been identified and investigated in invertebrates. Our previous studies have revealed that SRC is a novel candidate gene associated with body weight in Pacific white shrimp (Litopenaeus vannamei). In order to comprehend the underlying mechanism by which LvSRC affects shrimp growth, we analyzed the structure, phylogeny, expression profiles and RNA interference (RNAi) of this gene in L. vannamei. We found that LvSRC contains two CCP domains and one MAM domain, with the highest expression level in the heart and relatively low expression level in other tissues. Notably, LvSRC exhibited significantly higher expression levels in the fast-growing group among groups with different growth rates, suggesting its potential involvement as a gene contributing to the growth of L. vannamei. RNAi of LvSRC inhibited body length and body weight gain compared to the control groups. Moreover, through RNA-seq analysis, we identified 598 differentially expressed genes (DEGs), including genes associated with growth, immunity, protein processing and modification, signal transduction, lipid synthesis and metabolism. Furthermore, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed significant changes in the signaling pathways related to growth, lipid metabolism and immune response, suggesting that LvSRC exhibits the potential to participate in diverse physiological processes and immune regulation. These findings deepen our understanding of the structure and function of the SRC in shrimp and lay the foundation for further research into the regulatory mechanism of LvSRC. Additionally, they provide potential applications in shrimp genetics and breeding.

Benzoxazinoids Biosynthetic Gene Cluster Identification and Expression Analysis in Maize under Biotic and Abiotic Stresses.

Benzoxazinoids (BXs) are unique bioactive metabolites with protective and allelopathic properties in maize in response to diverse stresses. The production of BXs involves the fine regulations of BXs biosynthetic gene cluster (BGC). However, little is known about whether and how the expression pattern of BGC members is impacted by biotic and abiotic stresses. Here, maize BGC was systemically investigated and 26 BGC gene members were identified on seven chromosomes, for which Bin 4.00-4.01/4.03-4.04/7.02 were the most enriched regions. All BX proteins were clearly divided into three classes and seven subclasses, and ten conserved motifs were further identified among these proteins. These proteins were localized in the subcellular compartments of chloroplast, endoplasmic reticulum, or cytoplasmic, where their catalytic activities were specifically executed. Three independent RNA-sequencing (RNA-Seq) analyses revealed that the expression profiles of the majority of BGC gene members were distinctly affected by multiple treatments, including light spectral quality, low-temperature, 24-epibrassinolide induction, and Asian corn borer infestation. Thirteen differentially expressed genes (DEGs) with high and specific expression levels were commonly detected among three RNA-Seq, as core conserved BGC members for regulating BXs biosynthesis under multiple abiotic/biotic stimulates. Moreover, the quantitative real-time PCR (qRT-PCR) verified that six core conserved genes in BGC were significantly differentially expressed in leaves of seedlings upon four treatments, which caused significant increases in 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) content under darkness and wound treatments, whereas a clear decrease in DIMBOA content was observed under low-temperature treatment. In conclusion, the changes in BX metabolites in maize were regulated by BGC gene members in multiple stress presences. Therefore, the identification of key genes associated with BX accumulation under biotic/abiotic stresses will provide valuable gene resources for breeding maize varieties with enhanced capability to adapt to environmental stresses.

IsoSplitter: identification and characterization of alternative splicing sites without a reference genome.

Long-read transcriptome sequencing is designed to sequence full-length RNA molecules and advantageous for identifying alternative splice isoforms; however, in the absence of a reference genome, it is difficult to accurately locate splice sites, because of the diversity of patterns of alternative splicing (AS). Based on long-read transcriptome data we developed a versatile tool, IsoSplitter, to reverse-trace and validate AS gene "split-sites" with the following features: (1) IsoSplitter initially invokes a modified SIM4 program to find transcript split-sites; (2) each split-site is then quantified, to reveal transcript diversity, and putative isoforms are grouped into gene clusters; (3) an optional step for aligning short-reads is provided, to validate split-sites by identifying unique junction reads, and revealing and quantifying tissue-specific alternative splice isoforms. We tested IsoSplitter AS prediction using datasets from multiple model and non-model plant species, and showed that IsoSplitter pipeline is efficient to handle different transcriptomes with high accuracy. Furthermore, we evaluated the IsoSplitter pipeline compared with that of the splice junction identification tools, Program to Assemble Spliced Alignments (PASA-software needs a reference genome for AS identification) and AStrap, using data from the model plant Arabidopsis thaliana. We found that, IsoSplitter determined more than twice as many AS events than AStrap analysis; and 94.13% of the IsoSplitter predicted AS events were also identified by the PASA analysis. Starting from a simple sequence file, IsoSplitter is an assembly-free tool for identification and characterization of AS. IsoSplitter is developed and implemented in Python 3.5 using the Linux platform and is freely available at https://github.com/Hengfu-Yin/IsoSplitter.

Genome-Wide Identification, Expression, and Response to Fusarium Infection of the SWEET Gene Family in Garlic (Allium sativum L.).

Proteins of the SWEET (Sugar Will Eventually be Exported Transporters) family play an important role in plant development, adaptation, and stress response by functioning as transmembrane uniporters of soluble sugars. However, the information on the SWEET family in the plants of the Allium genus, which includes many crop species, is lacking. In this study, we performed a genome-wide analysis of garlic (Allium sativum L.) and identified 27 genes putatively encoding clade I-IV SWEET proteins. The promoters of the A. sativum (As) SWEET genes contained hormone- and stress-sensitive elements associated with plant response to phytopathogens. AsSWEET genes had distinct expression patterns in garlic organs. The expression levels and dynamics of clade III AsSWEET3, AsSWEET9, and AsSWEET11 genes significantly differed between Fusarium-resistant and -susceptible garlic cultivars subjected to F. proliferatum infection, suggesting the role of these genes in the garlic defense against the pathogen. Our results provide insights into the role of SWEET sugar uniporters in A. sativum and may be useful for breeding Fusarium-resistant Allium cultivars.

Genome-Wide Identification and Characterization of the TLP Gene Family in Phyllostachys edulis and Association with Witches' Broom Disease Resistance in Bamboo.

Thaumatin-like proteins (TLPs) are pathogenesis-related proteins with pivotal roles in plant defense mechanisms. In this study, various bioinformatics and RNA-seq methods were used to analyze the biotic and abiotic stress responses of the TLP family in Phyllostachys edulis. Overall, 81 TLP genes were identified in P. edulis; 166 TLPs from four plant species were divided into three groups and ten subclasses, with genetic covariance observed between these species. Subcellular localization in silico studies indicated that TLPs were primarily distributed in the extracellular. Analysis of the upstream sequences of TLPs demonstrated the presence of cis-acting elements related to disease defense, environmental stress, and hormonal responses. Multiple sequence alignment demonstrated that most TLPs possessed five conserved REDDD amino acid sequences with only a few amino acid residue differences. RNA-seq analysis of P. edulis responses to Aciculosporium take, the pathogenic fungus that causes witches' broom disease, showed that P. edulis TLPs (PeTLPs) were expressed in different organs, with the highest expression in buds. PeTLPs responded to both abscisic acid and salicylic acid stress. These PeTLP expression patterns were consistent with their gene and protein structures. Collectively, our findings provide a basis for further comprehensive analyses of the genes related to witches' broom in P. edulis.

Genome-Wide Identification and Evolution of the GRF Gene Family and Functional Characterization of PbGRF18 in Pear.

Proteins encoded by the G-box regulating factor (GRF, also called 14-3-3) gene family are involved in protein-protein interactions and mediate signaling transduction, which play important roles in plant growth, development, and stress responses. However, there were no detailed investigations of the GRF gene family in pear at present. In this study, we identified 25 GRF family members in the pear genome. Based on a phylogenetic analysis, the 25 GRF genes were clustered into two groups; the ε group and the non-ε group. Analyses of the exon-intron structures and motifs showed that the gene structures were conserved within each of the ε and non-ε groups. Gene duplication analysis indicated that most of the PbGRF gene expansion that occurred in both groups was due to WGD/segmental duplication. Phosphorylation sites analysis showed that the main phosphorylation sites of PbGRF proteins were serine residues. For gene expression, five PbGRF genes (PbGRF7, PbGRF11, PbGRF16, PbGRF21, and PbGRF23) were highly expressed in fruits, and PbGRF18 was highly expressed in all tissues. Further analysis revealed that eight PbGRF genes were significantly differentially expressed after treatment with different sugars; the expression of PbGRF7, PbGRF8, and PbGRF11 significantly increased, implying the involvement of these genes in sugar signaling. In addition, subcellular localization studies showed that the tested GRF proteins localize to the plasma membrane, and transgenic analysis showed that PbGRF18 can increase the sugar content in tomato leaves and fruit. The results of our research establish a foundation for functional determination of PbGRF proteins, and will help to promote a further understanding of the regulatory network in pear fruit development.

Genome-Wide Identification, Characterization and Expression Analysis of the TaDUF724 Gene Family in Wheat (Triticum aestivum).

Unknown functional domain (DUF) proteins constitute a large number of functionally uncharacterized protein families in eukaryotes. DUF724s play crucial roles in plants. However, the insight understanding of wheat TaDUF724s is currently lacking. To explore the possible function of TaDUF724s in wheat growth and development and stress response, the family members were systematically identified and characterized. In total, 14 TaDUF724s were detected from a wheat reference genome; they are unevenly distributed across the 11 chromosomes, and, according to chromosome location, they were named TaDUF724-1 to TaDUF724-14. Evolution analysis revealed that TaDUF724s were under negative selection, and fragment replication was the main reason for family expansion. All TaDUF724s are unstable proteins; most TaDUF724s are acidic and hydrophilic. They were predicted to be located in the nucleus and chloroplast. The promoter regions of TaDUF724s were enriched with the cis-elements functionally associated with growth and development, as well as being hormone-responsive. Expression profiling showed that TaDUF724-9 was highly expressed in seedings, roots, leaves, stems, spikes and grains, and strongly expressed throughout the whole growth period. The 12 TaDUF724 were post-transcription regulated by 12 wheat MicroRNA (miRNA) through cleavage and translation. RT-qPCR showed that six TaDUF724s were regulated by biological and abiotic stresses. Conclusively, TaDUF724s were systematically analyzed using bioinformatics methods, which laid a theoretical foundation for clarifying the function of TaDUF724s in wheat.

CFVisual: an interactive desktop platform for drawing gene structure and protein architecture.

BACKGROUND: When researchers perform gene family analysis, they often analyze the structural characteristics of the gene, such as the distribution of introns and exons. At the same time, characteristic structural analysis of amino acid sequence is also essential, for example, motif and domain features. Researchers often integrate these analyses into one image to dig out more information, but the tools responsible for this integration are lacking. RESULTS: Here, we developed a tool (CFVisual) for drawing gene structure and protein architecture. CFVisual can draw the phylogenetic tree, gene structure, and protein architecture in one picture, and has rich interactive capabilities, which can meet the work needs of researchers. Furthermore, it also supports arbitrary stitching of the above analysis images. It has become a useful helper in gene family analysis. The CFVisual package was implemented in Python and is freely available from https://github.com/ChenHuilong1223/CFVisual/ . CONCLUSION: CFVisual has been used by some researchers and cited by some articles. In the future, CFVisual will continue to serve as a good helper for researchers in the study of gene structure and protein architecture.

Identification of ARF family in blueberry and its potential involvement of fruit development and pH stress response.

BACKGROUND: Auxin responsive factor (ARF) family is one of core components in auxin signalling pathway, which governs diverse developmental processes and stress responses. Blueberry is an economically important berry-bearing crop and prefers to acidic soil. However, the understandings of ARF family has not yet been reported in blueberry. RESULTS: In the present study, 60 ARF genes (VcARF) were identified in blueberry, and they showed diverse gene structures and motif compositions among the groups and similar within each group in the phylogenetic tree. Noticeably, 9 digenic, 5 trigenic and 6 tetragenic VcARF pairs exhibited more than 95% identity to each other. Computational analysis indicated that 23 VcARFs harbored the miRNA responsive element (MRE) of miR160 or miR167 like other plant ARF genes. Interestingly, the MRE of miR156d/h-3p was observed in the 5'UTR of 3 VcARFs, suggesting a potentially novel post-transcriptional control. Furthermore, the transcript accumulations of VcARFs were investigated during fruit development, and three categories of transcript profiles were observed, implying different functional roles. Meanwhile, the expressions of VcARFs to different pH conditions (pH4.5 and pH6.5) were surveyed in pH-sensitive and tolerant blueberry species, and a number of VcARFs showed different transcript accumulations. More importantly, distinct transcriptional response to pH stress (pH6.5) were observed for several VcARFs (such as VcARF6s and VcARF19-3/19-4) between pH-sensitive and tolerant species, suggesting their potential roles in adaption to pH stress. CONCLUSIONS: Sixty VcARF genes were identified and characterized, and their transcript profiles were surveyed during fruit development and in response to pH stress. These findings will contribute to future research for eliciting the functional roles of VcARFs and regulatory mechanisms, especially fruit development and adaption to pH stress.

Identifying genes with conserved splicing structure and orthologous isoforms in human, mouse and dog.

BACKGROUND: In eukaryote transcriptomes, a significant amount of transcript diversity comes from genes' capacity to generate different transcripts through alternative splicing. Identifying orthologous alternative transcripts across multiple species is of particular interest for genome annotators. However, there is no formal definition of transcript orthology based on the splicing structure conservation. Likewise there is no public dataset benchmark providing groups of orthologous transcripts sharing a conserved splicing structure. RESULTS: We introduced a formal definition of splicing structure orthology and we predicted transcript orthologs in human, mouse and dog. Applying a selective strategy, we analyzed 2,167 genes and their 18,109 known transcripts and identified a set of 253 gene orthologs that shared a conserved splicing structure in all three species. We predicted 6,861 transcript CDSs (coding sequence), mainly for dog, an emergent model species. Each predicted transcript was an ortholog of a known transcript: both share the same CDS splicing structure. Evidence for the existence of the predicted CDSs was found in external data. CONCLUSIONS: We generated a dataset of 253 gene triplets, structurally conserved and sharing all their CDSs in human, mouse and dog, which correspond to 879 triplets of spliced CDS orthologs. We have released the dataset both as an SQL database and as tabulated files. The data consists of the 879 CDS orthology groups with their detailed splicing structures, and the predicted CDSs, associated with their experimental evidence. The 6,861 predicted CDSs are provided in GTF files. Our data may contribute to compare highly conserved genes across three species, for comparative transcriptomics at the isoform level, or for benchmarking splice aligners and methods focusing on the identification of splicing orthologs. The data is available at https://data-access.cesgo.org/index.php/s/V97GXxOS66NqTkZ .

The impacts of allopolyploidization on Methyl-CpG-Binding Domain (MBD) gene family in Brassica napus.

BACKGROUND: Polyploidization promotes species formation and is widespread in angiosperms. Genome changes dramatically bring opportunities and challenges to plants after polyploidy. Methyl-CpG-Binding Domain (MBD) proteins can recognize and bind to methylation sites and they play an important role in the physiological process related to methylation in animals and plants. However, research on the influence of the allopolyploidization process on the MBD gene family is still lacking, so it is necessary to conduct a comprehensive analysis. RESULTS: In this study, twenty-two, ten and eleven MBD genes were identified in the genome of allotetraploid B. napus and its diploid ancestors, B. rapa and B. oleracea, respectively. Based on the clades of the MBD gene in Arabidopsis, rice and maize, we divided the new phylogenetic tree into 8 clades. Among them, the true MBD genes in Brassica existed in only 5 clades. Clade IV and Clade VI were unique in term of MBD genes in dicotyledons. Ka/Ks calculations showed that MBD genes underwent purifying selection in Brassica and may retain genes through sequence or functional differentiation early in evolution. In the process of allopolyploidization, the number of MBD gene introns increased, and the protein motifs changed. The MBD proteins had their own special motifs in each clade, and the MBD domains were only conserved in their clades. At the same time, the MBD genes were expressed in flower, leaf, silique, and stem tissues, and the expression levels of the different genes were significantly different, while the tissue specificity was not obvious. The allopolyploidization process may increase the number of cis-acting elements and activate the transposable elements. During allopolyploidization, the expression pattern of the MBD gene changes, which may be regulated by cis-acting elements and transposable elements. The number imbalance of cis-acting elements and transposable elements in An and Cn subgenomes may also lead to biased An subgenome expression of the MBD gene in B. napus. CONCLUSIONS: In this study, by evaluating the number, structure, phylogeny and expression of the MBD gene in B. napus and its diploid ancestors, we increased the understanding of MBD genes in allopolyploids and provided a reference for future analysis of allopolyploidization.

NPM2 in malignant peritoneal mesothelioma: from basic tumor biology to clinical medicine.

BACKGROUND: This review systematically summarizes gene biology features and protein structure of nucleoplasmin2 (NPM2) and the relationship between NPM2 and malignant peritoneal mesothelioma (MPM), in order to explore the molecular pathological mechanism of MPM and explore new therapeutic targets. METHODS: NCBI PubMed database was used for the literature search. NCBI Gene and Protein databases, Ensembl Genome Browser, UniProt, and RCSB PDB database were used for gene and protein review. Three online tools (Consurf, DoGSiteScorer, and ZdockServer), the GEPIA database, and the Cancer Genome Atlas were used to analyze bioinformatics characteristics for NPM2 protein. RESULTS: The main structural domains of NPM2 protein include the N-terminal core region, acidic region, and motif and disordered region. The N-terminal core region, involved in histone binding, is the most conserved domain in the nucleoplasmin (NPM) family. NPM2 with a large acidic tract in its C-terminal tail (NPM2-A2) is able to bind histones and form large complexes. Bioinformatics results indicated that NPM2 expression was correlated with the pathology of multiple tumors. Among mesothelioma patients, 5-year survival of patients with low-NPM2-expression was significantly higher than that of the high-NPM2-expression patients. NPM2 can facilitate the formation of histone deacetylation. NPM2 may promote histone deacetylation and inhibit the related-gene transcription, thus leading to abnormal proliferation, invasion, and metastasis of MPM. CONCLUSION: NPM2 may play a key role in the development and progression of MPM.

Mesoscale modeling reveals formation of an epigenetically driven HOXC gene hub.

Gene expression is orchestrated at the structural level by nucleosome positioning, histone tail acetylation, and linker histone (LH) binding. Here, we integrate available data on nucleosome positioning, nucleosome-free regions (NFRs), acetylation islands, and LH binding sites to "fold" in silico the 55-kb HOXC gene cluster and investigate the role of each feature on the gene's folding. The gene cluster spontaneously forms a dynamic connection hub, characterized by hierarchical loops which accommodate multiple contacts simultaneously and decrease the average distance between promoters by ∼100 nm. Contact probability matrices exhibit "stripes" near promoter regions, a feature associated with transcriptional regulation. Interestingly, while LH proteins alone decrease long-range contacts and acetylation alone increases transient contacts, combined LH and acetylation produce long-range contacts. Thus, our work emphasizes how chromatin architecture is coordinated strongly by epigenetic factors and opens the way for nucleosome resolution models incorporating epigenetic modifications to understand and predict gene activity.

The anaplerotic pyruvate carboxylase from white shrimp Litopenaeus vannamei: Gene structure, molecular characterization, protein modelling and expression during hypoxia.

Hypoxic zones are spreading worldwide in marine environments affecting many organisms. Shrimp and other marine crustaceans can withstand environmental hypoxia using several strategies, including the regulation of energy producing metabolic pathways. Pyruvate carboxylase (PC) catalyzes the first reaction of gluconeogenesis to produce oxaloacetate from pyruvate. In mammals, PC also participates in lipogenesis, insulin secretion and other processes, but this enzyme has been scarcely studied in marine invertebrates. In this work, we characterized the gene encoding PC in the white shrimp Litopenaeus vannamei, modelled the protein structure and evaluated its gene expression in hepatopancreas during hypoxia, as well as glucose and lactate concentrations. The PC gene codes for a mitochondrial protein and has 21 coding exons and 4 non-coding exons that generate three transcript variants with differences only in the 5'-UTR. Total PC expression is more abundant in hepatopancreas compared to gills or muscle, indicating tissue-specific expression. Under hypoxic conditions of 1.53 mg/L dissolved oxygen, PC expression is maintained in hepatopancreas, indicating its key role even in energy-limited conditions. Finally, both glucose and lactate concentrations were maintained under hypoxia for 24-48 h in hepatopancreas.

Identification and Expression Analysis of miR160 and Their Target Genes in Cucumber.

miR160 plays a crucial role in various biological processes by regulating their target gene auxin response factor (ARF) in plants. However, little is known about miR160 and ARF in cucumber fruit expansion. Here, 4 Csa-MIR160 family members and 17 CsARFs were identified through a genome-wide search. Csa-miR160 showed a closer relationship with those in melon. Phylogenetic analysis revealed that CsARFs were divided into four classes and most of CsARFs presented a closer evolutionary relationship with those from tomato. Putative cis-elements analysis predicted that Csa-MIR160 and CsARFs were involved in light, phytohormone and stress response, which proved that they might take part in light, phytohormone and stress signaling pathway by the miR160-ARF module. In addition, CsARF5, CsARF11, CsARF13 and CsARF14 were predicted as the target genes of Csa-miR160. qRT-PCR revealed that Csa-miR160 and their target gene CsARFs were differentially expressed in differential cucumber tissues and developmental stages. Csa-miR160d was only expressed in the expanded cucumber fruit. CsARF5, CsARF11 and CsARF13 exhibited the lower expression in the expanded fruit than those in the ovary, while, CsARF14 showed the reverse trend. Our results suggested that Csa-miR160d might play a crucial role in cucumber fruit expansion by negatively targeting CsARF5, CsARF11 and CsARF13. This is the first genome-wide analysis of miR160 in cucumber. These findings provide useful information and resources for further studying the role of miR160 and ARF in cucumber fruit expansion.