Molecular Cloning, Escherichia coli Expression and Genomic Organization of Squalene Synthase Gene from Artemisia annua

A 1 539 by squalene synthase (AaSQS) cDNA was cloned from a high-yield Artemisia annua L. strain 001 by reverse transcription-polymerise chain reaction (RT-PCR). The amino acid sequence of AaSQS is 70%, 77%, 44% and 39%a identical to that of squalene synthases from Arabidopsis thaliana, tobacco, human and yeast, respectively. The AaSQS genomic DNA has a complex organization containing 14 exons and 13 introns. Full-length or C-terminal truncated cDNA was subcloned into prokaryotic expression vector pET30a and the constructed plasmid was introduced to Escherichia coli strain BL21 (DE3) for induced overexpression. No squalene synthase protein with expected molecular mass was observed in E. cola containing the putative full-length squalene synthase cDNA, however, overexpression in E. coli was achieved by truncating 30 amino acids of hydrophobic region at the carboxy terminus.

3D genome organization and its study in livestock breeding

Eukaryotic genomes are hierarchically packaged into cell nucleus,affecting gene regulation.The genome is organized into multiscale structural units,including chromosome territories,compartments,topologically associating domains(TADs),and DNA loops.The identification of these hierarchical structures has benefited from the development of experimental approaches,such as 3C-based methods(Hi-C,ChIA-PET,etc.),imaging tools(2D-FISH,3D-FISH,Cryo-FISH,etc.)and ligation-free methods(GAM,SPRITE,etc.).In recent two decades,numerous studies have shown that the 3D organization of genome plays essential roles in multiple cellular processes via various mechanisms,such as regulating enhancer activity and promoter-enhancer interactions.However,there are relatively few studies about the 3D genome in livestock species.Therefore,studies for exploring the function of 3D genomes in livestock are urgently needed to provide a more comprehensive understanding of potential relationships between the genome and production traits.In this review,we summarize the recent advances of 3D genomics and its biological functions in human and mouse studies,drawing inspiration to explore the 3D genomics of livestock species.We then mainly focus on the biological functions of 3D genome organization in muscle development and its implications in animal breeding.

Distal-less homeobox genes of insects and spiders： genomic organization, function, regulation and evolution

The Distal-less （DlI） genes are homeodomain transcription factors that are present in most Metazoa and in representatives of all investigated arthropod groups. In Drosophila, the best studied insect, Dll plays an essential role in forming the proximodistal axis of the legs, antennae and analia, and in specifying antennal identity. The initiation of Dll expression in clusters of cells in mid-lateral regions of the Drosophila embryo represents the earliest genetic marker of limbs. Dll genes are involved in the development of the peripheral nervous system and sensitive organs, and they also function as master regulators of black pigmentation in some insect lineages. Here we analyze the complete genomes of six insects, the nematode Caenorhabditis elegans and Homo sapiens, as well as multiple Dll sequences available in databases in order to examine the structure and protein features of these genes. We also review the function, expression, regulation and evolution of arthropod Dll genes with emphasis on insects and spiders.

Understanding the commonalities and differences in genomic organizations across closely related bacteria from an energy perspective

The availability of a large number of sequenced bacterial genomes facilitates in-depth studies about why genes(operons)in a bacterial genome are globally organized the way they are.We have previously discovered that(the relative)transcription-activation frequencies among different biological pathways encoded in a genome have a dominating role in the global arrangement of operons.One complicating factor in such a study is that some operons may be involved in multiple pathways with different activation frequencies.A quantitative model has been developed that captures this information,which tends to be minimized by the current global arrangement of operons in a bacterial(and archaeal)genome compared to possible alternative arrangements.A study is carried out here using this model on a collection of 52 closely related Escherichia coli genomes,which revealed interesting new insights about how bacterial genomes evolve to optimally adapt to their environments through adjusting the(relative)genomic locations of the encoding operons of biological pathways once their utilization and hence transcription activation frequencies change,to maintain the above energy-efficiency property.More specifically we observed that it is the frequencies of the transcription activation of pathways relative to those of the other encoded pathways in an organism as well as the variation in the activation frequencies of a specific pathway across the related genomes that play a key role in the observed commonalities and differences in the genomic organizations of genes(and operons)encoding specific pathways across different genomes.

The Distribution of Repetitive DNAs Along Chromosomes in Plants Revealed by Self-genomic in situ Hybridization

The distribution of repetitive DNAs along chromosomes is one of the crucial elements for understanding the organization and the evolution of plant genomes. Using a modified genomic in situ hybridization （GISH） procedure, fluorescence in situ hybridization （FISH） with genomic DNA to their own chromosomes （called self-genomic in situ hybridization, self-GISH） was carried out in six selected plant species with different genome size and amount of repetitive DNA. Nonuniform distribution of the fluorescent labeled probe DNA was observed on the chromosomes of all the species that were tested. The signal patterns varied among species and were related to the genome size. The chromosomes of the small Arabidopsis genome were labeled almost only in the pericentromeric regions and the nucleolus organizer regions （NORs）. The signals in the relatively small genomes, rice, sorghum, and Brassica oleracea var. capitata L., were dispersed along the chromosome lengths, with a predominant distribution in the pericentromeric or proximal regions and some heterochromatic arms. All chromosomes of the large genomes, maize and barley, were densely labeled with strongly labeled regions and weakly labeled or unlabeled regions being arranged alternatively throughout the lengths. In addition, enhanced signal bands were shown in all pericentromeres and the NORs in B. oleracea var. capitata, and in all pericentromeric regions and certain intercalary sites in barley. The enhanced signal band pattern in barley was found consistent with the N-banding pattern of this species. The GISH with self-genomic DNA was compared with FISH with Cot-1 DNA in rice, and their signal patterns are found to be basically consistent. Our results showed that the self-GISH signals actually reflected the hybridization of genomic repetitive DNAs to the chromosomes, thus the self-GISH technique would be useful for revealing the distribution of the regions where repetitive DNAs concentrate along chromosomes and some chromatin differentiation associated with repetitive DNAs in plants.

Reorganization of 3D genome architecture across wild boar and Bama pig adipose tissues

Background:A growing body of evidence has revealed that the mammalian genome is organized into hierarchical layers that are closely correlated with and may even be causally linked with variations in gene expression.Recent studies have characterized chromatin organization in various porcine tissues and cell types and compared them among species and during the early development of pigs.However,how chromatin organization differs among pig breeds is poorly understood.Results:In this study,we investigated the 3D genome organization and performed transcriptome characterization of two adipose depots(upper layer of backfat[ULB]and greater omentum[GOM])in wild boars and Bama pigs;the latter is a typical indigenous pig in China.We found that over 95%of the A/B compartments and topologically associating domains(TADs)are stable between wild boars and Bama pigs.In contrast,more than 70%of promoterenhancer interactions(PEIs)are dynamic and widespread,involving over a thousand genes.Alterations in chromatin structure are associated with changes in the expression of genes that are involved in widespread biological functions such as basic cellular functions,endocrine function,energy metabolism and the immune response.Approximately 95%and 97%of the genes associated with reorganized A/B compartments and PEIs in the two pig breeds differed between GOM and ULB,respectively.Conclusions:We reported 3D genome organization in adipose depots from different pig breeds.In a comparison of Bama pigs and wild boar,large-scale compartments and TADs were mostly conserved,while fine-scale PEIs were extensively reorganized.The chromatin architecture in these two pig breeds was reorganized in an adipose depotspecific manner.These results contribute to determining the regulatory mechanism of phenotypic differences between Bama pigs and wild boar.

Characterization and Genomic Analysis of a Plaque Purified Strain of Cyanophage PP

Cyanophages are ubiquitous and essential components of the aquatic environment and play an important role in the termination of algal blooms.As such,they have attracted widespread interest.PP was the first isolated cyanophage in China,which infects Plectonema boryanum and Phormidium foveolarum.In this study,this cyanophage was purified three times by a double-agar overlay plaque assay and characterized.Its genome was extracted,totally sequenced and analyzed.Electron microscopy revealed a particle with an icosahedral head connected to a short stubby tail.Bioassays showed that PP was quite virulent.The genome of PP is a 42,480 base pair(bp),linear,double-stranded DNA molecule with 222 bp terminal repeats.It has high similarity with the known Pf-WMP3 sequence.It contains 41 open reading frames(ORFs),17 of which were annotated.Intriguingly,the genome can be divided into two completely different parts,which differ both in orientation and function.

A mRNA molecule encoding truncated excitatory amino acid carrier 1 (EAAC1) protein (EAAC2) is transcribed from an independent promoter but not an alternative splicing event

Glutamate transporter EAACl removes excitatory neurotransmitter in central nervous system, and also absorbs glutamate in epithelia of intestine, kidney, liver and heart for normal cell growth. When a mouse cDNA was screened using EAACl cDNA fragment as probe in our lab, a transcript (GenBank U75214) encoding an EAACl protein with 148 residues truncated at N-terminal was cloned and named as EAAC2. Sequence analysis shows that EAAC2 has it's own start code and unique 5'UTR that is different from that of EAACl. A mouse genomic library was screened and a positive clone including EAACl CDS was sequenced (GenBank AF 322393) and indicates that normal EAACl transcript (GenBank U73521) is transcribed from 10 exons in terms of exon Ⅰ, Ⅱ, Ⅲ, Ⅳ, Ⅴ, Ⅵ, Ⅶ, Ⅷ,Ⅸ, Ⅹ, and EAAC2 transcript is consisted by exons from IV to IX as same as that of EAACl and with its unique exon β upstream to exon Ⅳ and exon δ downstream to IX. EAAC2 transcript has a cluster of transcriptional start sites not overlapping with the transcriptional start sites of EAACl. These results indicate that EAAC2 is transcribed from an independent promoter but not an alternative splicing event.

Interplay between genome organization and epigenomic alterations of pericentromeric DNA in cancer

In eukaryotic genome biology,the genomic organization inside the three-dimensional(3D)nucleus is highly complex,and whether this organization governs gene expression is poorly understood.Nuclear lamina(NL)is a filamentous meshwork of proteins present at the lining of inner nuclear membrane that serves as an anchoring platform for genome organization.Large chromatin domains termed as lamina-associated domains(LADs),play a major role in silencing genes at the nuclear periphery.The interaction of the NL and genome is dynamic and stochastic.Furthermore,many genes change their positions during developmental processes or under disease conditions such as cancer,to activate certain sorts of genes and/or silence others.Pericentromeric heterochromatin(PCH)is mostly in the silenced region within the genome,which localizes at the nuclear periphery.Studies show that several genes located at the PCH are aberrantly expressed in cancer.The interesting question is that despite being localized in the pericentromeric region,how these genes still manage to overcome pericentromeric repression.Although epigenetic mechanisms control the expression of the pericentromeric region,recent studies about genome organization and genome-nuclear lamina interaction have shed light on a new aspect of pericentromeric gene regulation through a complex and coordinated interplay between epigenomic remodeling and genomic organization in Cancer.

The complete nucleotide sequence and its organization of the genome of Barley yellow dwarf virus-GAV

The complete nucleotide sequence of genomic RNA of BYDV-GAV was determined. It comprised 5685 nucleotides and contained six open reading frames and four un-translated regions. The size and organization of BYDV-GAV genome were similar to those of BYDV PAV-aus. The nucleotide and deduced amino acid sequences of the six ORFs were aligned and compared with those of other luteoviruses. The results showed that there was a high degree of identity between BYDV-GAV and MAV-PS1 in all ORFs except ORF5 and ORF6, which had only 87.4% and 70.2% identities respectively. The reported genomic nucleotide sequence of MAV was shorter than that of BYDV-GAV, but the comparison of the genomic nucleotide sequences for MAV-PS1 and GAV showed 90.4% sequence identity for the same region of the genome. Ac-cording to the level of sequence similarities, BYDV-GAV should be closely related to BYDV-MAV.

The 3D organization of genome in the nucleus: from the nucleosome to the 4D nucleome

The genetic information,stored in the linear sequences of DNA,encodes all of the genes that a living organism uses to produce proteins and RNAs essential for various cellular functions.In eukaryotes,however,genomic DNA is hierarchically and efficiently packaged into multiple-level chromatin structures within the nucleus,including the nucleosome,30 nm fibers,chromatin loops,topology associated domains(TADs).

Recent genome duplications facilitate the phenotypic diversity of Hb repertoire in the Cyprinidae

Whole-genome duplications(WGDs)are an important contributor to phenotypic innovations in evolutionary history.The diversity of blood oxygen transport traits is the perfect reflection of physiological versatility for evolutionary success among vertebrates.In this study,the evolutionary changes of hemoglobin(Hb)repertoire driven by the recent genome duplications were detected in representative Cyprinidae fish,including eight diploid and four tetraploid species.Comparative genomic analysis revealed a substantial variation in both membership composition and intragenomic organization of Hb genes in these species.Phylogenetic reconstruction analyses were conducted to characterize the evolutionary history of these genes.Data were integrated with the expression profiles of the genes during ontogeny.Our results indicated that genome duplications facilitated the phenotypic diversity of the Hb gene family;each was associated with species-specific changes in gene content via gene loss and fusion after genome duplications.This led to repeated evolutionary transitions in the ontogenic regulation of Hb gene expression.Our results revealed that genome duplications helped to generate phenotypic changes in Cyprinidae Hb systems.

Genome analysis of Heliothis virescens ascovirus 3h isolated from China

No ascovirus isolated from China has been sequenced so far. Therefore, in this study, we aimed to sequence the genome of Heliothis virescens ascovirus 3h （HvAV-3h） using the 454 pyrosequencing technology. The genome was found to be 190,519-bp long with a G＋C content of 45.5%. We also found that it encodes 185 hypothetical open reading frames （ORFs） along with at least 50 amino acids, including 181 ORFs found in other ascoviruses and 4 unique ORFs. Gene-parity plots and phylogenetic analysis revealed a close relationship between HvAV-3h and three other HvAV-3a strains and a distant relationship with Spodoptera frugiperda ascovirus la （SfAV-la）, Trichoplusia ni ascovirus 6a （TnAV-6a）, and Diadromus pulchellus ascovirus 4a （DpAV-4a）. Among the 185 potential genes encoded by the genome, 44 core genes were found in all the sequenced ascoviruses. In addition, 25 genes were found to be conserved in all ascoviruses except DpAV-4a. In the HvAV-3h genome, 24 baculovirus repeat ORFs （bros） were present, and the typical homologous repeat regions （hrs） were absent. This study supplies information important for understanding the conservation and functions of ascovirus genes as well as the variety of ascoviral genomes.

A Structural Bisulfite Assay to Identify DN Cruciforms

In the half century since the discovery of the double-helix structure of DNA, it has become increasingly clear that DNA functionality is based on much more than its sequence in a double-helical structure. Further advances have highlighted the importance of additional aspects of DNA structure： its packaging in the higher order chromatin structure, positioning of nucleosomes along the DNA, and the occurrence of non-helical DNA structures. Of these, the latter has been problematic to prove empirically. Here, we describe a method that uses non-denaturing bisulfite sequencing on isolated Arabidopsis thaliana nuclei to determine the location of cytosines positioned outside the double helix as a result of non-B-form DNA structures. We couple this with computational methods and S1 nuclease digest to reliably identify stable, non-B-form, cruciform structures. This enables us to identify a palindrome in the promoter of FLOWERING LOCUS T that forms a stable non-B-form structure. The stronger conservation of the ability to form a nonhelical secondary structure than of the sequence suggests that this structure is biologically relevant.

Web Resources for Model Organism Studies

An ever-growing number of resources on model organisms have emerged with the continued development of sequencing technologies. In this paper, we review 13 databases of model organisms, most of which are reported by the National Institutes of Health of the United States（NIH; http：//www.nih.gov/science/models/）. We provide a brief description for each database, as well as detail its data source and types, functions, tools, and availability of access. In addition,we also provide a quality assessment about these databases. Significantly, the organism databases instituted in the early 1990s––such as the Mouse Genome Database（MGD）, Saccharomyces Genome Database（SGD）, and Fly Base––have developed into what are now comprehensive, core authority resources. Furthermore, all of the databases mentioned here update continually according to user feedback and with advancing technologies.