Transcriptional Regulatory Sequences Research Articles

Herpes Simplex Virus Type 1/Adeno-Associated Virus Hybrid Vectors

Herpes simplex virus type 1 (HSV-1) amplicons can accommodate foreign DNA of any size up to 150 kbp and, therefore, allow extensive combinations of genetic elements. Genomic sequences as well as cDNA, large transcriptional regulatory sequences for cell type-specific expression, multiple transgenes, and genetic elements from other viruses to create hybrid vectors may be inserted in a modular fashion. Hybrid amplicons use genetic elements from HSV-1 that allow replication and packaging of the vector DNA into HSV-1 virions, and genetic elements from other viruses that either direct integration of transgene sequences into the host genome or allow episomal maintenance of the vector. Thus, the advantages of the HSV-1 amplicon system, including large transgene capacity, broad host range, strong nuclear localization, and availability of helper virus-free packaging systems are retained and combined with those of heterologous viral elements that confer genetic stability to the vector DNA. Adeno-associated virus (AAV) has the unique capability of integrating its genome into a specific site, designated AAVS1, on human chromosome 19. The AAV rep gene and the inverted terminal repeats (ITRs) that flank the AAV genome are sufficient for this process. HSV-1 amplicons have thus been designed that contain the rep gene and a transgene cassette flanked by AAV ITRs. These HSV/AAV hybrid vectors direct site-specific integration of transgene sequences into AAVS1 and support long-term transgene expression.

Group-specific structural features of the 5′-proximal sequences of coronavirus genomic RNAs

Global predictions of the secondary structure of coronavirus (CoV) 5′ untranslated regions and adjacent coding sequences revealed the presence of conserved structural elements. Stem loops (SL) 1, 2, 4, and 5 were predicted in all CoVs, while the core leader transcription-regulating sequence (L-TRS) forms SL3 in only some CoVs. SL5 in group I and II CoVs, with the exception of group IIa CoVs, is characterized by the presence of a large sequence insertion capable of forming hairpins with the conserved 5′-UUYCGU-3′ loop sequence. Structure probing confirmed the existence of these hairpins in the group I Human coronavirus-229E and the group II Severe acute respiratory syndrome coronavirus (SARS-CoV). In general, the pattern of the 5′ cis-acting elements is highly related to the lineage of CoVs, including features of the conserved hairpins in SL5. The function of these conserved hairpins as a putative packaging signal is discussed.

Cryopreservation for Broader Production of Transgenic Mice by DNA Injection into Zygotes

Mutant mice are indispensable to biological and medical research, and transgenic mouse production by DNA injection into zygotes has been an important method for producing these mice which are used to examine the over-expression of genes and to analyze gene transcriptional regulatory sequences. Recently, cryopreservation of zygotes by a simple vitrification method has become popular, saving the labor of isolating zygotes following each injection. However, the DNA injection technique requires training in the use of special equipment, and the injection cannot be accomplished routinely in every laboratory. The exchange of live mice also risks the propagation of common murine pathogens and possible escape of the animals; therefore the transfer of mice in frozen zygotes or embryos is recommended. Here we propose injecting DNA into frozen and thawed zygotes, refreezing them before transportation to any destination, where they can be thawed and developed into pups. The rate of transgenic mouse production using this method does not decrease significantly, even if zygotes are frozen and thawed before and after their DNA injection, and will make transgenic studies more popular on a worldwide scale.

Recombinant PRRSV expressing porcine circovirus sequence reveals novel aspect of transcriptional control of porcine arterivirus

Porcine reproductive and respiratory syndrome virus (PRRSV) expresses its genes via a set of nested subgenomic (sg) mRNAs. Such discontinuous transcription is unique yet poorly understood for arterivirus. The utilization of transcription-regulating sequence (TRS) remains a puzzle, as many TRS-like sequences exist in viral genome, yet only six or seven sg mRNAs were transcribed in arterivirus infected cells. To investigate the transcriptional control of the porcine arterivirus, a recombinant PRRSV infectious cDNA clone pCPV expressing the capsid gene of porcine circovirus 2 (PCV2) between PRRSV ORF1b and ORF2a was developed. The rescued recombinant viruses contained a range of disparate deletions of the inserted PCV2 sequence, yet two stable recombinant viruses containing 41 and 275 nt of foreign sequences were generated upon plaque purification and serial passages. Further analysis of the sg RNA2 profile revealed that an array of novel sg RNA species was generated in cells infected with the recombinant virus. One group was formed by utilizing the inserted PCV2 sequence as TRS; another group was generated from cryptic TRS-like PRRSV sequences located 19, 37 and 97 nt immediately downstream of the PRRSV ORF2 AUG. These results demonstrated that (1) the recombinant virus from direct insertion of foreign sequences was genetically unstable, while two recombinant PRRSVs containing foreign sequence of 41 or 275 nt in length, respectively, became stable upon plaque purification and further serial passages; (2) PRRSV can utilize foreign TRS-like sequence as transcriptional promoter; (3) the insertion of foreign sequence provoked the generation of novel subgenomic RNAs utilizing cryptic TRS-like sequences that remain non-functional in native PRRSV.

Lentiviral Vector-Mediated Transfer of the Gamma-Globin Gene Into Normal and Beta-Thalassemic Human CD34+ Cells Results in Potentially Therapeutic Levels of Fetal Hemoglobin in Erythroid Progeny.

Abstract 3579Poster Board III-516Beta-thalassemia results from severely reduced or absent expression of the beta-globin chain of hemoglobin, leading to severe anemia which is dependent upon transfusion for survival. The pathophysiology centers on the alpha- to beta-globin chain imbalance, which results in precipitation of excess alpha chains. These precipitates caused oxidative stress, membrane damage, and apoptosis of erythroid precursors in the bone marrow (BM). Allogeneic stem cell transplant can be curative but is only available to individuals with a matched donor. It is well known that increased levels of fetal hemoglobin (α2γ2;HbF), such as in the case of hereditary persistence of fetal hemoglobin, ameliorate the clinical severity of beta-thalassemia. We therefore developed a self-inactivating (SIN), erythroid-specific, gamma-globin lentiviral vector with the goal of reducing the chain imbalance through the production of HbF in red blood cells following transduction and transplantation of autologous HSCs. The vector, termed V5m3, contains 3.1-kb of transcriptional regulatory sequences from the β-globin locus control region, a 130-bp beta-globin promoter regulating transcription of the gamma-globin gene, and 3′ untranslated sequences from the native beta-globin gene. We previously showed that this vector was effective in correcting mouse models of beta-thalassemia and sickle cell disease (SCD). We further modified V5m3 to include a 400-bp chicken HS4 insulator element. Mobilized peripheral blood (PB)- or steady state bone marrow (BM)-derived CD34+ cells from normal or thalassemic human donors were used to evaluate vector performance. A two-phase in vitro model of human erythropoiesis was used in which a seed population of CD34+ cells is first expanded and then differentiated into late stage erythroblasts over a two week period. Gene transfer is performed 48 hours after initiation of culture. In this model, we routinely observe a 1000-fold expansion in total cell numbers where the vast majority of maturing erythroid cells are late stage erythroblasts reflected by nearly complete enrichment for expression of transferrin receptor (CD71; ≥98%) and glycophorin A (CD235; ≥80%) and loss or reduced expression of CD34 and CD45. Erythroid cells generated from normal adult PB and BM CD34+ cells demonstrate an adult pattern of hemoglobin production (HbA>96%;HbF<2%), as measured by acetate gel electrophoresis and HPLC, making this model ideal to evaluate enhancement of HbF levels by gene transfer. Normal PB CD34+ cells from four independent donors were transduced with the gamma-globin vector (MOI=20) or a GFP control vector (MOI=5). Vector transduction had no effect on cell growth or differentiation as monitored by consistent increases in total cell numbers and the appearance of CD71 (transferrin receptor) and glycophorin A on most cells (≥98% and '80%, respectively). The GFP vector achieved an average transduction rate of 87+/−6% and erythroblasts expressed low levels of HbF (1.7+/−0.6). Gamma globin gene transfer with the V5m3 (N=2) and V5m3-400 (N=4) vectors resulted in HbF levels ranging from 6 to 25%, with an average vector copy number of 0.8 to 1.1. We next tested the V5m3-400 vector using BM CD34+ cells from two patients with beta-thalassemia major. High levels of gene transfer were obtained with both the GFP and the globin vector, as evidenced by bulk marking (74+/−6% GFP+) or PCR analysis of CFU for presence of the V5m3-400 vector (12/12 positive Exp 1); (18/20 Exp 2); (18/24 Exp 3). Again, gene transfer did not perturb erythroid differentiation as monitored by the appearance glycophorin A (88+/−9%; GFP control and 86+/−6%; V5m3-400) as well as cell morphology. Erythroblasts derived from GFP transduced cells had a mean HbF level of 26+/−5% whereas those derived from cells transduced with V5m3-400 demonstrated a 100% increase of HbF to 58+/−2% with an average vector copy number of 0.6-0.8. Importantly, cultures of cells transduced with the globin vector demonstrated an average of 30% reduction in apoptotic cells (10% tunnel+) cells compared to GFP transduced control cells (15% tunnel+), suggesting rescue of erythroblasts through correction of the globin chain imbalance. Our data show that potentially therapeutic levels of HbF in thalassemic erythroblasts can be obtained following gene transfer using a gamma-globin lentiviral vector. Disclosures:No relevant conflicts of interest to declare.

Screening of human gene promoter activities using transfected-cell arrays

Promoters are the best characterized transcriptional regulatory sequences in complex genomes because of their predictable location immediately upstream of transcription start sites. Despite a substantial body of literature describing transcriptional promoters, the identification of true start sites for all human transcripts is far from complete. The same is true of the key structural and functional elements responsible for promoter action in different cell types. In order to identify elements responsible for promoter activity, we applied transfected-cell array technology to functionally evaluate promoters for genes involved in inflammatory bowel disease. Seventy-four promoters were examined by reverse transfection of a promoter-fluorescent reporter constructs into a human embryonic kidney cell line (HEK293T). Sixteen (21.6%) promoters were found to be active in HEK293 T cells. Correlations between promoter activity and endogenous transcript level were calculated, and 75% of active promoters were found to be associated with transcriptional activity of their gene counterparts. These results provide experimental evidence of promoter activity, which may aid in understanding the regulation of gene expression. Moreover, this is the first large-scale functional study of regulatory sequences to use a high-throughput transfected-cell array technique.

Coronavirus N Protein N-Terminal Domain (NTD) Specifically Binds the Transcriptional Regulatory Sequence (TRS) and Melts TRS-cTRS RNA Duplexes

All coronaviruses (CoVs), including the causative agent of severe acute respiratory syndrome (SARS), encode a nucleocapsid (N) protein that harbors two independent RNA binding domains of known structure, but poorly characterized RNA binding properties. We show here that the N-terminal domain (NTD) of N protein from mouse hepatitis virus (MHV), a virus most closely related to SARS-CoV, employs aromatic amino acid-nucleobase stacking interactions with a triple adenosine motif to mediate high-affinity binding to single-stranded RNAs containing the transcriptional regulatory sequence (TRS) or its complement (cTRS). Stoichiometric NTD fully unwinds a TRS-cTRS duplex that mimics a transiently formed transcription intermediate in viral subgenomic RNA synthesis. Mutation of the solvent-exposed Y127, positioned on the β-platform surface of our 1.75 Å structure, binds the TRS far less tightly and is severely crippled in its RNA unwinding activity. In contrast, the C-terminal domain (CTD) exhibits no RNA unwinding activity. Viruses harboring Y127A N mutation are strongly selected against and Y127A N does not support an accessory function in MHV replication. We propose that the helix melting activity of the coronavirus N protein NTD plays a critical accessory role in subgenomic RNA synthesis and other processes requiring RNA remodeling.

Cloning the structure genes and expression the N gene of porcine epidemic diarrhea virus DX

The structure genes spike (S), nucleocapsid (N), membrane (M), small membrane (sM) of a porcine epidemic diarrhea virus (PEDV) strain DX isolated in Gansu province, North-west of China, were cloned, sequenced and compared with published sequences of PEDV strains. The nucleotide sequences encoding the entire S, sM, M and N genes open reading frame (ORF) of DX were 4 152, 231, 681 and 1 326 bases long respectively. There were transcription regulatory sequences (TRSs) upstream of the initiator ATG of the S, N and M genes. The amino acids sequences of S, M and N contained 30, 3 and 7 potential asparagine (N)-linked glycosylation sites. Homologous analysis and phylogenetic trees showed that DX had the closest relationship with strains LJB/06, JS-2004-2Z and CH/HLJH/06 that were also isolated from China and indicated the prevalence of some PEDV isolates in China were widespread since the JS-2004-2Z strain originated from the south of the China, and LJB/06 and CH/HLJH/06 were isolated from northeast China. The N gene was cloned using two primers which contained Nco I and BamH I restriction enzyme sites and subcloned into expression vector pET30a. The recombinant plasmid was then transformed into E.coli Rossta. SDS-PAGE showed there was a protein of about 55kDa as expected and Western blot indicated the N protein had biological activity.

Porcine reproductive and respiratory syndrome virus as a vector: Immunogenicity of green fluorescent protein and porcine circovirus type 2 capsid expressed from dedicated subgenomic RNAs

Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of PRRS, which is characterized by late-term abortions in sows and respiratory disease in young pigs. Using an infectious cDNA clone of North American PRRSV strain P129, the viral genome was engineered to transcribe an additional subgenomic RNA initiating between non-structural and structural genes. Two unique restriction sites and a copy of the transcription regulatory sequence for ORF6 (TRS6) were inserted between ORFs 1b and 2a, yielding a general purpose expression vector. The enhanced green fluorescent protein (GFP) gene was cloned between the unique sites such that the inserted gene was transcribed from TRS2 which was located upstream within ORF1b, while the copy of TRS6 drives ORF2a/b transcription. Upon transfection of cells with this plasmid, PRRSV infection was initiated and progeny virus "P129-GFP" was obtained. Cells infected with P129-GFP showed fluorescence and the inserted gene was phenotypically stable for at least 37 serial in vitro passages. Subsequently, a capsid (C) protein gene was cloned from porcine circovirus type 2 (PCV2) recovered from an outbreak of porcine multisystemic wasting syndrome (PMWS) and inserted into the PRRSV infectious clone vector, generating virus "P129-PCV". To determine the immunogenicity of the recombinant viruses, pigs were immunized intramuscularly with P129-WT (wild-type), P129-GFP, or P129-PCV2. By 5 weeks post-infection, specific antibody responses to GFP and PCV2 capsid were elicited. This is the first report of foreign gene expression using PRRSV from dedicated subgenomic RNAs and demonstrates the potential use of PRRSV as a vaccine vector for swine pathogens.

A combinatorial approach to determine the context-dependent role in transcriptional and posttranscriptional regulation in Arabidopsis thaliana

BackgroundWhile progresses have been made in mapping transcriptional regulatory networks, posttranscriptional regulatory roles just begin to be uncovered, which has arrested much attention due to the discovery of miRNAs. Here we demonstrated a combinatorial approach to incorporate transcriptional and posttranscriptional regulatory sequences with gene expression profiles to determine their probabilistic dependencies.ResultsWe applied the proposed method to microarray time course gene expression profiles and could correctly predict expression patterns for more than 50% of 1,132 genes, based on the sequence motifs adopted in the network models, which was statistically significant. Our study suggested that the contribution of miRNA regulation towards gene expression in plants may be more restricted than that of transcription factors; however, miRNAs might confer additional layers of robustness on gene regulation networks. The programs written in C++ and PERL implementing methods in this work are available for download from our supplemental data web page.ConclusionIn this study we demonstrated a combinatorial approach to incorporate miRNA target motifs (miRNA-mediated posttranscriptional regulatory sites) and TFBSs (transcription factor binding sites) with gene expression profiles to reconstruct the regulatory networks. The proposed approach may facilitate the incorporation of diverse sources with limited prior knowledge.

Correction of Murine Sickle Cell Disease Using γ-Globin Lentiviral Vectors to Mediate High-level Expression of Fetal Hemoglobin

Increased levels of red cell fetal hemogloblin, whether due to hereditary persistence of expression or from induction with hydroxyurea therapy, effectively ameliorate sickle cell disease (SCD). Therefore, we developed erythroid-specific, gamma-globin lentiviral vectors for hematopoietic stem cell (HSC)-targeted gene therapy with the goal of permanently increasing fetal hemoglobin (HbF) production in sickle red cells. We evaluated two different gamma-globin lentiviral vectors for therapeutic efficacy in the BERK sickle cell mouse model. The first vector, V5, contained the gamma-globin gene driven by 3.1 kb of beta-globin regulatory sequences and a 130-bp beta-globin promoter. The second vector, V5m3, was identical except that the gamma-globin 3'-untranslated region (3'-UTR) was replaced with the beta-globin 3'-UTR. Adult erythroid cells have beta-globin mRNA 3'-UTR-binding proteins that enhance beta-globin mRNA stability and we postulated this design might enhance gamma-globin expression. Stem cell gene transfer was efficient and nearly all red cells in transplanted mice expressed human gamma-globin. Both vectors demonstrated efficacy in disease correction, with the V5m3 vector producing a higher level of gamma-globin mRNA which was associated with high-level correction of anemia and secondary organ pathology. These data support the rationale for a gene therapy approach to SCD by permanently enhancing HbF using a gamma-globin lentiviral vector.

Transcriptional Regulation of the Salivary Histatin Gene: Finding of a Strong Positive Regulatory Element and Its Binding Protein

Histatins are salivary proteins found and expressed in human salivary glands. They play a role in the non-immune system of antimicrobial defense, for instance, against Candida albicans. The transcriptional regulatory sequences of the histatin gene, HIS1, have remained obscure for a long time. Here, we cloned the putative promoter from human genomic DNA and tested it in a luciferase reporter system. This promoter is much more active in salivary gland cells than in other cell types. Analysis of deletion mutants revealed that the region encompassing -2254 to -1748 is a strong positive transcriptional element, and its functional core sequence (termed HTN27 box) works in correct and reverse orientations in synergy with downstream sequences, the region spanning -680 to +28 and a proximal promoter. The plus single-stranded HTN27 box is specifically bound by a 100 kDa protein that is present in HSG cells, but not in HeLa cells. These findings indicate that the regulation of the histatin gene expression may be intricate, and it seems to have a cell-type preference in the salivary gland cells.

Evaluation of a γ-Globin Lentiviral Vector in Sickle Cell Mice and Pigtail Macaques

Increased levels of red cell fetal hemogloblin (α2 γ2; HbF), whether due to hereditary persistence of HbF or from induction with hydroxyurea therapy, effectively ameliorate sickle cell disease (SCD). Therefore, we developed an erythroid-specific, γ-globin lentiviral vector for hematopoietic stem cell (HSC)-targeted gene therapy with the goal of permanent, high level expression of HbF in sickle red cells. The vector contained the γ-globin gene driven by 3.1 kb of transcriptional regulatory sequences from the β-globin LCR and a 130 bp β-globin promoter. Since adult erythroid cells have β-globin mRNA 3′UTR binding proteins that enhance β-globin mRNA stability, we replaced the native γ-globin 3′UTR with its β-globin counterpart. We tested the therapeutic efficacy of this vector using the BERK sickle cell mouse model. Five months following transplant, mice that received transduced lineage-depleted sickle steady-state bone marrow (BM) cells (n=10) expressed the g-globin transgene in 95% ± 2% of RBCs. We observed levels of HbF that equaled that of the endogenous HbS (HbF 48% ± 3% of total Hb). This was achieved with an average BM vector copy number of 1.7 ± 0.2 and led to correction of both the severe anemia and end-organ damage characterizing this SCD strain. Globin vector mice had a Hb level of 12.2 ± 0.2 g/dL, compared to 7.1 ± 0.3 g/dL of mice (n=16) transplanted with cells transduced with a control GFP vector. Urine concentrating ability was normal in globin vector mice, while severely impaired in control mice. At necropsy, minimal evidence of sickle-related organ damage was found in the globin vector recipient group. In contrast, severe renal, hepatic, splenic and pulmonary pathology was observed in control, mock-transduced animals. We then transplanted the BM from 6 primary recipients of globin vector-transduced cells into 23 secondary recipients. Five months after transplant, these animals maintained HbF levels similar to those of their primary donors, along with persistent resolution of anemia. This suggested that HSCs were transduced and that vector silencing was minimal. We then evaluated this vector using non-human primate CD34+ cells. Steady-state BM CD34+ cells from several different pigtail macaques were transduced with the globin lentiviral vector or with a GFP control vector. The GFP vector achieved an average transduction rate of 57% ± 6% (n=6) into CD34+ cells and 76% ± 9% into CFU, as judged by GFP expression. Similar high levels of gene transfer were obtained with the globin vector. Bulk CD34+ cells transduced with the globin vector and then cultured for 5 days demonstrated an average vector copy number of 0.6–1.0 as judged by Southern blot analysis and qPCR. High level transduction of CFU was also obtained as 12/16 and 16/16 colonies in two separate experiments were positive for the globin vector by PCR analysis of colony DNA. We are in the process of comparing globin gene transfer and expression with that of our standard GFP vector in the pigtail macaque autologous transplant model by transplanting a graft consisting of 50% globin lentiviral vector-transduced CD34+ cells and 50% GFP lentiviral vector-transduced cells.

Promoter Analysis of Palindromic Transcription Units in the Ribosomal DNA Circle of Entamoeba histolytica

rRNA genes of Entamoeba histolytica are organized as palindromic ribosomal DNA (rDNA) units (I and II) in a 24.5-kb circle. Although the two rDNAs are identical in sequence, their upstream spacers are completely different. Since the intergenic sequences (IGS) of all rDNA copies in other organisms are conserved and contain transcription regulatory sequences, the lack of sequence conservation in the IGS prompted the question of whether both rDNAs are indeed transcriptionally active. We mapped the transcriptional start points (tsp's) and promoters of the two rDNAs. A 51-bp sequence immediately upstream of the tsp's was highly conserved in both units. In addition, both units had an A+T-rich stretch upstream of the 51-bp core. Analysis of reporter gene transcription showed promoter activity to reside in the regions from positions -86 to +123 (rDNA I) and positions -101 to +140 (rDNA II). The promoter-containing fragments from both units could bind and compete with each other for protein(s) from nuclear extracts. Protein binding was especially dependent on the A+T-rich region upstream of the 51-bp core (positions -53 to -68). The requirement of >80 bp downstream of the tsp was striking. Although this sequence was not conserved in the two units, it could potentially fold into very long stem-loops. Both rDNAs transcribed with comparable efficiency, as measured by nuclear runon. Thus, both rDNAs share very similar organization of promoter sequences, and in exponential culture both rDNAs are transcribed. It remains to be seen whether the different IGS affect the regulation of the two units under adverse conditions.

The PAZAR database of gene regulatory information coupled to the ORCA toolkit for the study of regulatory sequences

The PAZAR database unites independently created and maintained data collections of transcription factor and regulatory sequence annotation. The flexible PAZAR schema permits the representation of diverse information derived from experiments ranging from biochemical protein–DNA binding to cellular reporter gene assays. Data collections can be made available to the public, or restricted to specific system users. The data ‘boutiques’ within the shopping-mall-inspired system facilitate the analysis of genomics data and the creation of predictive models of gene regulation. Since its initial release, PAZAR has grown in terms of data, features and through the addition of an associated package of software tools called the ORCA toolkit (ORCAtk). ORCAtk allows users to rapidly develop analyses based on the information stored in the PAZAR system. PAZAR is available at http://www.pazar.info. ORCAtk can be accessed through convenient buttons located in the PAZAR pages or via our website at http://www.cisreg.ca/ORCAtk.

Comparative Analysis of Complete Genome Sequences of Three Avian Coronaviruses Reveals a Novel Group 3c Coronavirus

In this territory-wide molecular epidemiology study of coronaviruses (CoVs) in Hong Kong involving 1,541 dead wild birds, three novel CoVs were identified in three different bird families (bulbul CoV HKU11 [BuCoV HKU11], thrush CoV HKU12 [ThCoV HKU12], and munia CoV HKU13 [MuCoV HKU13]). Four complete genomes of the three novel CoVs were sequenced. Their genomes (26,396 to 26,552 bases) represent the smallest known CoV genomes. In phylogenetic trees constructed using chymotrypsin-like protease (3CL(pro)), RNA-dependent RNA polymerase (Pol), helicase, spike, and nucleocapsid proteins, BuCoV HKU11, ThCoV HKU12, and MuCoV HKU13 formed a cluster distantly related to infectious bronchitis virus and turkey CoV (group 3a CoVs). For helicase, spike, and nucleocapsid, they were also clustered with a CoV recently discovered in Asian leopard cats, for which the complete genome sequence was not available. The 3CL(pro), Pol, helicase, and nucleocapsid of the three CoVs possessed higher amino acid identities to those of group 3a CoVs than to those of group 1 and group 2 CoVs. Unique genomic features distinguishing them from other group 3 CoVs include a distinct transcription regulatory sequence and coding potential for small open reading frames. Based on these results, we propose a novel CoV subgroup, group 3c, to describe this distinct subgroup of CoVs under the group 3 CoVs. Avian CoVs are genetically more diverse than previously thought and may be closely related to some newly identified mammalian CoVs. Further studies would be important to delineate whether the Asian leopard cat CoV was a result of interspecies jumping from birds, a situation analogous to that of bat and civet severe acute respiratory syndrome CoVs.

Evolution of Arabidopsis thaliana microRNAs from random sequences

One mechanism for the origin of new plant microRNAs (miRNAs) is from inverted duplications of transcribed genes. However, even though many young MIRNA genes have recently been identified in Arabidopsis thaliana, only a subset shows evidence for having evolved by this route. We propose that the hundreds of thousands of partially self-complementary foldback sequences found in a typical plant genome provide an alternative path for miRNA evolution. Our genome-wide analyses of young MIRNA genes suggest that some arose from DNA that either has self-complementarity by chance or that represents a highly eroded inverted duplication. These observations are compatible with the idea that, following capture of transcriptional regulatory sequences, random foldbacks can occasionally spawn new miRNAs. Subsequent stabilization through coevolution with initially fortuitous targets may lead to fixation of a small subset of these proto-miRNA genes.

Genome-Wide Analysis of Protein-Protein Interactions and Involvement of Viral Proteins in SARS-CoV Replication

Analyses of viral protein-protein interactions are an important step to understand viral protein functions and their underlying molecular mechanisms. In this study, we adopted a mammalian two-hybrid system to screen the genome-wide intraviral protein-protein interactions of SARS coronavirus (SARS-CoV) and therefrom revealed a number of novel interactions which could be partly confirmed by in vitro biochemical assays. Three pairs of the interactions identified were detected in both directions: non-structural protein (nsp) 10 and nsp14, nsp10 and nsp16, and nsp7 and nsp8. The interactions between the multifunctional nsp10 and nsp14 or nsp16, which are the unique proteins found in the members of Nidovirales with large RNA genomes including coronaviruses and toroviruses, may have important implication for the mechanisms of replication/transcription complex assembly and functions of these viruses. Using a SARS-CoV replicon expressing a luciferase reporter under the control of a transcription regulating sequence, it has been shown that several viral proteins (N, X and SUD domains of nsp3, and nsp12) provided in trans stimulated the replicon reporter activity, indicating that these proteins may regulate coronavirus replication and transcription. Collectively, our findings provide a basis and platform for further characterization of the functions and mechanisms of coronavirus proteins.

Screening reveals conserved and nonconserved transcriptional regulatory elements including an E3/E4 allele-dependent APOE coding region enhancer

We performed an unbiased experimental search for enhancers and silencers in a 153-kb region containing the human apolipoprotein (APO) E/C1/C4/C2 gene cluster using shotgun cloning into a luciferase vector. A continuum of transcriptional effect sizes was observed, possibly explaining the limited success of bioinformatics in identifying regulatory regions. We identified nine statistically significant enhancers and five silencers functional in either liver or astrocyte cells, including two previously known enhancers. Only two of the fourteen elements contained conserved noncoding sequences. Within the coding sequence of the APOE gene we identified an enhancer for the E4 allele associated with Alzheimer's disease, but not E3. The single nucleotide polymorphism (SNP) causing the E4/E3 amino acid substitution was responsible for these variations, potentially explaining the higher expression levels of E4. Our results suggest a wider variety of mammalian transcriptional regulatory sequences than is currently recognized and that these may include coding region SNPs.

Genetic diversity in the yellow head nidovirus complex

Penaeus monodon shrimp collected from across the Indo-Pacific region during 1997–2004 were screened for the presence of yellow head-related viruses. Phylogenetic analyses of amplified ORF1b gene segments identified at least six distinct genetic lineages (genotypes). Genotype 1 (YHV) was detected only in shrimp with yellow head disease. Genotype 2 (GAV) was detected in diseased shrimp with the less severe condition described as mid-crop mortality syndrome and in healthy shrimp from Australia, Thailand and Vietnam. Other genotypes occurred commonly in healthy shrimp. Sequence comparisons of structural protein genes (ORF2 and ORF3), intergenic regions (IGRs) and the long 3′-UTR supported the delineation of genotypes and identified both conserved and variant structural features. In putative transcription regulating sequences (TRSs) encompassing the sub-genomic mRNA 5′-termini, a core motif (5′-GUCAAUU ACAAC-3′) is absolutely conserved. A small (83 nt) open reading frame (ORF4) in the 3′-UTR of GAV is variously truncated in all other genotypes and a TRS-like element preceding ORF4 is invariably corrupted by a A > G/U substitution in the central core motif (5′-UU( G/U) CAAC-3′). The data support previous evidence that ORF4 is a non-functional gene under construction or deconstruction. The 3′-UTRs also contain predicted 3′-terminal hairpin-loop structures that are preserved in all genotypes by compensatory nucleotide substitutions, suggesting a role in polymerase recognition for minus-strand RNA synthesis.