Analysis of sequences involved in IE2 transactivation of a baculovirus immediate-early gene promoter and identification of a new regulatory motif

Opep-2 is a unique baculovirus early gene that has only been identified in the Orgyia pseudotsugata multiple capsid nucleopolyhedrovirus (OpMNPV). Previous analyses have shown this gene is expressed at very early times post-infection (p.i.) but is shut down by 36-48 h p.i. The promoter of opep-2 therefore, represents a class of early genes that is temporally regulated. In this study, a detailed analysis of the opep-2 promoter is performed to analyze the role individual motifs play in early gene expression. A new 13 base pair regulatory element was identified and shown to be essential in controlling high-level expression of this gene. In addition, mutational analysis revealed that GATA and CACGTG motifs, which have been shown to bind cellular factors in Sf9 and Ld652Y cells, played minor roles in influencing opep-2 expression in the absence of other viral factors. The OpMNPV transactivator IE2 causes a significant activation of the opep-2 promoter. Cotransfection of an extensive number of promoter deletions and mutations did not show any sequence specificity for IE2 transactivation. This is the first detailed analysis of the sequence requirements for IE2 transactivation, and these results suggest that IE2 does not bind directly to specific elements in the opep-2 promoter.     

Ecdysone response element in a baculovirus immediate-early gene, ie1, promoter

A computer-assisted analysis identified tentative target sequences for regulatory proteins including ecdysone-inducible factors such as BmFTZ-F1 and Broad-Complex Z4 (BR-C Z4) in the ie1 promoter of BmNPV. A transient expression experiment using BmN cells and a series of truncated ie1 promoter constructs demonstrated that the activity of the ie1 promoter responded to alpha-ecdysone and 20-hydroxyecdysone, which required a tridecameric nucleotide stretch (ie1EcRE, 5′-GTGTTATCGACCT-3′) homologous to the ecdysone response element reported for Drosophila (DmEcRE). RT-PCR demonstrated the expression of BmEcR and BmUSP, which are required as ecdysone-specific activators for EcRE-mediated activation, in BmN cells. Furthermore, the ie1 EcRE-mediated response was confirmed by using a recombinant BmNPV possessing a luciferase gene under the control of the ie1 promoter with or without ie1 EcRE. This is the first report of an ecdysone response element in a baculoviral gene promoter. These results also suggested that the regulation of the ie1 by ecdysone may militate viral replication at least under certain conditions during natural infections in vivo.     

Analysis of baculovirus aggregates using flow cytometry

Aggregation of viral particles represents a significant problem for baculoviral stock processing and storage. Aggregation may also affect the results of viral particle counting. A method using flow cytometry was previously developed in our lab to measure the concentration of baculovirus particles produced in insect cell cultures. In the present study, the use of the flow cytometry method was extended to the detection of baculovirus aggregates. Flow cytometry analysis of freshly prepared baculovirus stocks, stained with SYBR Green, generally exhibited a single unimodal distribution; while, baculovirus stocks stored at 4 degrees C for a few months exhibited a bimodal distribution of the fluorescent intensity signal. The bimodal distribution was associated with a decrease in the size of the original viral population and an emergence of a new viral population with a high fluorescence intensity. Treatment of these samples with an endonuclease (Benzonase) confirmed that the new population observed in the flow cytometry analysis is not free cellular DNA. Filtration through 0.22 and 0.45 microm membranes of the stored samples prior to flow cytometry analysis confirmed that the high fluorescence intensity population involved particles larger than a single baculovirus. Exposing freshly amplified baculovirus stocks with a unimodal distribution to a pH of 5.3, a condition known to induce aggregation, showed the emergence of a second population with a bimodal distribution. These results suggest that flow cytometry analysis could be used to detect baculovirus aggregates. The aggregates were associated with high fluorescence intensity populations and the mean green fluorescence intensity of these populations could be used as an indicator of the mean aggregate size.     

Analysis of baculovirus genomes with restriction endonucleases

The viral DNAs from nine wild-type insect baculoviruses have been isolated and the EcoR-1 restriction endonuclease fragment patterns compared. Genomic heterogeneity could be detected in the DNA restriction patterns of four of these wild-type baculoviruses. Three infectious virus forms (two that are occluded in the nucleus and an extracellular virus that has budded from the plasma membrane of infected cells) of a nuclear polyhedrosis virus with multiple nucleocapsids per envelope (MNPV) from the lepidopteran insect, Autographa californica are shown to be phenotypically distinct by comparison of viral structural polypeptides by polyacrylamide gel electrophoresis and autoradiography of L[35S]methio-nine-labeled virus proteins. The three phenotypic forms were cloned by successive plaque purification and eight distinct variants were identified from 11 plaque-purified viruses by genotypec analysis with EcoR-1 and HindIII restriction endonuclease. Isolation of variants from the three phenotypic forms has shown that each of the infectious forms is heterogeneous and that no segregation of genotypes among the three forms was evident. The characteristic restriction fragment patterns of several variants were maintained upon multiple passage in cell culture.     

Putative gene promoter sequences in the chlorella viruses

Three short (7 to 9 nucleotides) highly conserved nucleotide sequences were identified in the putative promoter regions (150 bp upstream and 50 bp downstream of the ATG translation start site) of three members of the genus Chlorovirus, family Phycodnaviridae. Most of these sequences occurred in similar locations within the defined promoter regions. The sequence and location of the motifs were often conserved among homologous ORFs within the Chlorovirus family. One of these conserved sequences (AATGACA) is predominately associated with genes expressed early in virus replication.     

转染双启动子杆状病毒表达载体在昆虫细胞共表 …

目的 通过体外表达ｒｈＩＬ－１２,以供研究其在体内外的生物学效应。方法 外周血单个核细胞（ＰＢＭＣ）、粘时细胞和人口腔表皮样癌细胞ＫＢ分别经ＳＡＣ（含Ａ蛋白的金黄色葡萄球菌ＣｏｗａｎⅠ菌株）、ＩＦＮ－γ＋ＬＰＳ和ＰＤＢｕ（１２、１３－二丁酸佛波酯）刺激,以ＧＩＴ（异硫氰酸胍）一步法提取细胞总ＲＮＡ,经ＲＴ－ＰＣＲ技术获得ＩＬ－１２ Ｐ４０和Ｐ３５ ｃＤＮＡ,将两条基因分别插入双启动子杆状病毒转染载     

Analysis of minimal promoter sequences for plus-strand synthesis by the Cucumber necrosis virus RNA-dependent RNA polymerase

Tombusviruses are small, plus-sense, single-stranded RNA viruses of plants. A partially purified RNA-dependent RNA polymerase (RdRp) preparation of Cucumber necrosis virus (CNV), which is capable of de novo initiation of complementary RNA synthesis from either plus-strand or minus-strand templates, was used to dissect minimal promoter sequences for tombusviruses and their defective interfering (DI) RNAs. In vitro RdRp assay revealed that the core plus-strand initiation promoter included only the 3'-terminal 11 nucleotides. A hypothetical promoter-like sequence, which has been termed consensus sequence by Wu and White (1998, J. Virol. 72, 9897-9905), is recognized less efficiently by the CNV RdRp than the core plus-strand initiation promoter. The CNV RdRp can efficiently recognize the core plus-strand initiation promoter for a satellite RNA associated with the distantly related Turnip crinkle virus, while artificial AU- or GC-rich 3'-terminal sequences make poor templates in the in vitro assays. Comparison of the "strength" of minimal plus-strand and minus-strand initiation promoters reveals that the latter is almost twice as efficient in promoting complementary RNA synthesis. Template competition experiments, however, suggest that the minimal plus-strand initiation promoter makes an RNA template more competitive than the minimal minus-strand initiation promoter. Taken together, these results demonstrate that promoter recognition by the tombusvirus RdRp requires only short sequences present at the 3' end of templates.     

Efficient prediction methods for selecting effective siRNA sequences

Although short interfering RNA (siRNA) has been widely used for studying gene functions in mammalian cells, its gene silencing efficacy varies markedly and there are only a few consistencies among the recently reported design rules/guidelines for selecting siRNA sequences effective for mammalian genes. Another shortcoming of the previously reported methods is that they cannot estimate the probability that a candidate sequence will silence the target gene. This paper first reviewed the recently reported siRNA design guidelines and clarified the problems concerning the guidelines. It then proposed two prediction methods—Radial Basis Function (RBF) network and decision tree learning—and their combined method for selecting effective siRNA target sequences from many possible candidate sequences. They are quite different from the previous score-based siRNA design techniques and can predict the probability that a candidate siRNA sequence will be effective. The methods imply high estimation accuracy for selecting candidate siRNA sequences.     

Probabilistic prediction of protein-protein interactions from the protein sequences

Prediction of protein-protein interactions is very important for several bioinformatics tasks though it is not a straightforward problem. In this paper, employing only protein sequence information, a framework is presented to predict protein-protein interactions using a probabilistic-based tree augmented nai ve (TAN) Bayesian network. Our framework also provides a confidence level for every predicted interaction, which is useful for further analysis by the biologists. The framework is applied to the yeast interaction datasets for predicting interactions and it is shown that our framework gives better performance than support vector machine (SVM). The framework is implemented as a webserver and is available for prediction.     

Analysis of the fowlpoxvirus gene encoding the 4b core polypeptide and demonstration that it possesses efficient promoter sequences

The gene encoding the fowlpox virus 4b core polypeptide has been identified by analogy with the vaccinia 4b gene. It has been cloned, and its nucleotide sequence determined. The gene, which is 1971 nucleotides long, can encode a protein of 75,200 Da (75.2K polypeptide), slightly longer than its vaccinia counterpart with which it shares 52% identity. Sequences upstream of the fowlpox virus 4b gene correspond to the consensus sequence determined for vaccinia late promoters, suggesting that late promoter signals may be shared by the different genera of poxviruses. Upstream sequences have been cloned into a beta-galactosidase translational fusion vector and shown to promote the efficient expression of beta-galactosidase in a transient assay system. This expression was abolished in the presence of araC, an inhibitor of DNA replication which blocks late gene expression in poxviruses. The fowlpoxvirus 4b promoter should be a useful component of genetically engineered fowlpox virus vaccines.     

An assessment of gene prediction accuracy in large DNA sequences

One of the first useful products from the human genome will be a set of predicted genes. Besides its intrinsic scientific interest, the accuracy and completeness of this data set is of considerable importance for human health and medicine. Though progress has been made on computational gene identification in terms of both methods and accuracy evaluation measures, most of the sequence sets in which the programs are tested are short genomic sequences, and there is concern that these accuracy measures may not extrapolate well to larger, more challenging data sets. Given the absence of experimentally verified large genomic data sets, we constructed a semiartificial test set comprising a number of short single-gene genomic sequences with randomly generated intergenic regions. This test set, which should still present an easier problem than real human genomic sequence, mimics the approximately 200kb long BACs being sequenced. In our experiments with these longer genomic sequences, the accuracy of GENSCAN, one of the most accurate ab initio gene prediction programs, dropped significantly, although its sensitivity remained high. Conversely, the accuracy of similarity-based programs, such as GENEWISE, PROCRUSTES, and BLASTX was not affected significantly by the presence of random intergenic sequence, but depended on the strength of the similarity to the protein homolog. As expected, the accuracy dropped if the models were built using more distant homologs, and we were able to quantitatively estimate this decline. However, the specificities of these techniques are still rather good even when the similarity is weak, which is a desirable characteristic for driving expensive follow-up experiments. Our experiments suggest that though gene prediction will improve with every new protein that is discovered and through improvements in the current set of tools, we still have a long way to go before we can decipher the precise exonic structure of every gene in the human genome using purely computational methodology.