Construction, Characterization, and Preliminary BAC-End Sequence Analysis of a Bacterial Artificial Chromosome Library of the Tea Plant (Camellia sinensis)

We describe the construction and characterization of a publicly available BAC library for the tea plant, Camellia sinensis. Using modified methods, the library was constructed with the aim of developing public molecular resources to advance tea plant genomics research. The library consists of a total of 401,280 clones with an average insert size of 135 kb, providing an approximate coverage of 13.5 haploid genome equivalents. No empty vector clones were observed in a random sampling of 576 BAC clones. Further analysis of 182 BAC-end sequences from randomly selected clones revealed a GC content of 40.35% and low chloroplast and mitochondrial contamination. Repetitive sequence analyses indicated that LTR retrotransposons were the most predominant sequence class (86.93%–87.24%), followed by DNA retrotransposons (11.16%–11.69%). Additionally, we found 25 simple sequence repeats (SSRs) that could potentially be used as genetic markers

Transforming growth factor β-activated kinase 1 transcriptionally suppresses hepatitis B virus replication

Hepatitis B Virus (HBV) replication in hepatocytes is restricted by the host innate immune system and related intracellular signaling pathways. Transforming growth factor β-activated kinase 1 (TAK1) is a key mediator of toll-like receptors and pro-inflammatory cytokine signaling pathways. Here, we report that silencing or inhibition of endogenous TAK1 in hepatoma cell lines leads to an upregulation of HBV replication, transcription, and antigen expression. In contrast, overexpression of TAK1 significantly suppresses HBV replication, while an enzymatically inactive form of TAK1 exerts no effect. By screening TAK1-associated signaling pathways with inhibitors and siRNAs, we found that the MAPK-JNK pathway was involved in TAK1-mediated HBV suppression. Moreover, TAK1 knockdown or JNK pathway inhibition induced the expression of farnesoid X receptor α, a transcription factor that upregulates HBV transcription. Finally, ectopic expression of TAK1 in a HBV hydrodynamic injection mouse model resulted in lower levels of HBV DNA and antigens in both liver and serum. In conclusion, our data suggest that TAK1 inhibits HBV primarily at viral transcription level through activation of MAPK-JNK pathway, thus TAK1 represents an intrinsic host restriction factor for HBV replication in hepatocytes

An Integrated Physical, Genetic and Cytogenetic Map of Brachypodium distachyon, a Model System for Grass Research

The pooid subfamily of grasses includes some of the most important crop, forage and turf species, such as wheat, barley and Lolium. Developing genomic resources, such as whole-genome physical maps, for analysing the large and complex genomes of these crops and for facilitating biological research in grasses is an important goal in plant biology. We describe a bacterial artificial chromosome (BAC)-based physical map of the wild pooid grass Brachypodium distachyon and integrate this with whole genome shotgun sequence (WGS) assemblies using BAC end sequences (BES). The resulting physical map contains 26 contigs spanning the 272 Mb genome. BES from the physical map were also used to integrate a genetic map. This provides an independent vaildation and confirmation of the published WGS assembly. Mapped BACs were used in Fluorescence In Situ Hybridisation (FISH) experiments to align the integrated physical map and sequence assemblies to chromosomes with high resolution. The physical, genetic and cytogenetic maps, integrated with whole genome shotgun sequence assemblies, enhance the accuracy and durability of this important genome sequence and will directly facilitate gene isolation

High prevalence and clonal dissemination of OXA-72-producing Acinetobacter baumannii in a Chinese hospital:a cross sectional study

Background: Carbapenem resistance in Acinetobacter baumannii in China was mainly mediated by OXA-23-like carbapenemases, while OXA-24/40-like carbapenemases were rarely identified. OXA-72 is one variant of OXA-24/40-like carbapenemases. This study aimed to demonstrate the epidemiology and characterizations of OXA-72-producing A. baumannii in a Chinese hospital. Methods: A total of 107 clinical A. calcoaceticus-A. baumannii (Acb) complex isolates were collected in a Chinese hospital during between 2014 and 2016. These isolates were identified using Vitek 2 system and gyrB multiplex PCR. Vitek 2 system was used for antibiotic susceptibility testing. Genes encoding for major classes of carbapenemases were investigated by PCR. Rep-PCR was used for genotyping of all the A. baumannii isolates. The risk factors for carriage of OXA-72-producing or OXA-23-producing A. baumannii were analyzed through univariate and multivariate logistic regression. Results: Of the 107 Acb isolates collected, 101 isolates (94.4%) and 6 isolates (5.6%) were identified as A. baumannii and A. pittii, respectively. 78 A. baumannii isolates (77.2%) were carbapenem resistant and mainly cultured from intensive care unit (ICU). blaOXA-72 and blaOXA-23 genes were identified in 45(57.7%) and 33(42.3%) carbapenem-resistant A. baumannii (CRAB), respectively. Multivariate risk factor analyses showed that prior carbapenem usage and nasogastric intubation were significantly associated with carriage of OXA-72-producing A. baumannii or OXA-23-producing A. baumannii. Rep-PCR analysis showed that 9 and 22 Rep-PCR types were assigned to 78 CRAB isolates and 23 carbapenem-susceptible A. baumannii (CSAB) isolates, respectively. A higher diverstiy of Rep-PCR patterns was observed among OXA-72-producing A. baumannii isolates than OXA-23-producing A. baumannii isolates, but all of them belonged to the same clone complex. MLST analysis suggested that the OXA-72 isolates from this study correspond to CC92/CC2 clone complex. Conclusions: This study demonstrates high prevalence and potential clonal spread of closely related genotypes of OXA-72-producing A. baumannii within a Chinese hospital. Continuous surveillance is necessary to monitor the dissemination of these strains in other healthcare settings to guide infection control policies in order to curb the spread of this bacterium

Detailed Analysis of a Contiguous 22-Mb Region of the Maize Genome

Most of our understanding of plant genome structure and evolution has come from the careful annotation of small (e.g., 100 kb) sequenced genomic regions or from automated annotation of complete genome sequences. Here, we sequenced and carefully annotated a contiguous 22 Mb region of maize chromosome 4 using an improved pseudomolecule for annotation. The sequence segment was comprehensively ordered, oriented, and confirmed using the maize optical map. Nearly 84% of the sequence is composed of transposable elements (TEs) that are mostly nested within each other, of which most families are low-copy. We identified 544 gene models using multiple levels of evidence, as well as five miRNA genes. Gene fragments, many captured by TEs, are prevalent within this region. Elimination of gene redundancy from a tetraploid maize ancestor that originated a few million years ago is responsible in this region for most disruptions of synteny with sorghum and rice. Consistent with other sub-genomic analyses in maize, small RNA mapping showed that many small RNAs match TEs and that most TEs match small RNAs. These results, performed on ∼1% of the maize genome, demonstrate the feasibility of refining the B73 RefGen_v1 genome assembly by incorporating optical map, high-resolution genetic map, and comparative genomic data sets. Such improvements, along with those of gene and repeat annotation, will serve to promote future functional genomic and phylogenomic research in maize and other grasses

Cloning and developmental expression analysis of a basic helix loop helix transcription factor gene for epigallocatechin-3-gallate biosynthesis in tea plant (<em>Camellia sinensis</em> L.)

594-600Epigallocatechin-3-gallate (EGCG) is the most effective active tea polyphenol, with a wide usage in food and medicine. Basic helix-loop-helix (bHLH) transcription factors play an important role in the regulation of flavonoid biosynthesis, but it remains unclear how the bHLH gene regulates the EGCG biosynthesis in the tea plant. We isolated and cloned the full-length bHLH gene from Camellia sinensis (CS_bHLH), which is 1,599 bp in length, and encodes a 379 amino acid protein of predicted molecular weight of 41.69 kDa. Bioinformatics analysis showed that the encoded protein is acidic, hydrophilic, unstable, and without the signal peptide. Its secondary structure mainly consists of random coils (70.2%) and α-helixes (22.4%). It shares 68.97% protein sequence identity with bHLH from other plants. The EGCG content of ‘1005’ exceeded that of its parent cultivars in the shoot and second leaf, and that’s up to 12.48% in shoot of ‘1005’. Comparing the expression level of CS_bHLH and the EGCG content at different developmental stages of three cultivars, it was found a similar trend for each cultivar: bud > second leaf > fourth leaf. For the samples of each cultivar, the higher the EGCG content, the higher the CS_bHLH expression level. That revealed that CS_bHLH may positively regulate the biosynthesis of EGCG

SALP, a new single-stranded DNA library preparation method especially useful for the high-throughput characterization of chromatin openness states

Abstract Background Next-generation sequencing (NGS) is fundamental to the current biological and biomedical research. Construction of sequencing library is a key step of NGS. Therefore, various library construction methods have been explored. However, the current methods are still limited by some shortcomings. Results This study developed a new NGS library construction method, Single strand Adaptor Library Preparation (SALP), by using a novel single strand adaptor (SSA). SSA is a double-stranded oligonucleotide with a 3′ overhang of 3 random nucleotides, which can be efficiently ligated to the 3′ end of single strand DNA by T4 DNA ligase. SALP can be started with any denatured DNA fragments such as those sheared by Tn5 tagmentation, enzyme digestion and sonication. When started with Tn5-tagmented chromatin, SALP can overcome a key limitation of ATAC-seq and become a high-throughput NGS library construction method, SALP-seq, which can be used to comparatively characterize the chromatin openness state of multiple cells unbiasly. In this way, this study successfully characterized the comparative chromatin openness states of four different cell lines, including GM12878, HepG2, HeLa and 293T, with SALP-seq. Similarly, this study also successfully characterized the chromatin openness states of HepG2 cells with SALP-seq by using 105 to 500 cells. Conclusions This study developed a new NGS library construction method, SALP, by using a novel kind of single strand adaptor (SSA), which should has wide applications in the future due to its unique performance