Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “T7 DNA replication”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

817 records · Page 46Linked to original sources
PubMed1994

Beet yellows closterovirus: complete genome structure and identification of a leader papain-like thiol protease.

The sequence of 8734 nucleotides (nt) from the 5'-end of the beet yellows closterovirus (BYV) RNA was determined to complete the 15,480-nt sequence of the virus genome. The 5'-terminal two-thirds of the sequence are occupied by two overlapping open reading frames (ORFs) 1a and 1b, encoding products with calculated M(r) of 295K and 48K, respectively. The RNA sequence surrounding the stop codon in ORF 1a shows structural elements typical of ribosomal frameshifting signals in a number of animal and plant viruses. It is predicted that the ORF 1b product is expressed via a +1 ribosomal frameshifting as the 348K ORF 1a/1b fusion protein. This putative protein contains the array of methyltransferase, RNA helicase, and RNA-dependent RNA polymerase domains that is conserved in the Sindbis-like supergroup of positive-strand RNA viruses. The 348K protein of BYV is longer than the putative replicases of the most closely related viruses (tobra- and tobamoviruses) by about 1300 amino acids distributed between two unique regions, one at the N-terminus, and the other in the central portion. The N-terminal domain showed sequence similarity to the helper component papain-like protease of potyviruses. By using in vitro translation of the T7 transcripts encoding the N-terminal 92K peptide of the BYV ORF 1a product, we found that the N-terminal fragment of 588 amino acids is released from the translation product by cleavage at the Gly-Gly dipeptide. Site-directed mutagenesis of either of the predicted catalytic residues Cys-509 and His-569 or of the Gly-588 at the cleavage site completely abolished the cleavage. The central unique region of the 348K protein contains a domain distantly resembling the aspartic protease of HIV and other lentiviruses. As shown previously, the 3'-terminal portion of the BYV genome encompasses seven more ORFs, one of which codes for a protein related to the HSP70 cell heat shock proteins, whereas two others encode the capsid protein and its diverged copy. Thus, despite the apparent evolutionary relationship with Sindbis-like viruses, BYV comprises a collection of genomic modules absorbed from different sources and has a unique expression strategy.

Amino Acid Sequence↗
PubMed2000

Recovery of a virulent strain of newcastle disease virus from cloned cDNA: expression of a foreign gene results in growth retardation and attenuation.

A recombinant mesogenic NDV strain, Beaudette C, and an engineered recombinant NDV expressing an additional gene were generated entirely from cloned cDNAs. For this purpose, a full-length cDNA clone of the virus genome, represented in eight different subgenomic fragments, was assembled in a transcription plasmid between a T7 RNA polymerase promoter and a hepatitis delta virus ribozyme sequence. Infectious NDV could be generated in the cells infected with recombinant vaccinia virus, which expressed T7 RNA polymerase, by simultaneous expression of antigenome-sense NDV RNA from the full-length plasmid and NDV NP, P, and L proteins from cotransfected plasmids. Recombinant virus was then amplified and recovered, either after inoculation of transfection supernatant into the allantoic cavity of embryonated specific-pathogen-free eggs or after further passage in cell culture. Characterization of the recombinant NDV showed similarities in growth and pathogenicity to that of the parental wild-type virus. By using this system, a recombinant NDV containing a foreign gene encoding chloramphenicol acetyltransferase (CAT) was generated. To do this, the CAT transcription cassette containing the CAT open reading frame, flanked by NDV gene start and gene end sequence motifs, was inserted into the region between the HN and L genes of the full-length cDNA. This construct was then used in the generation of a recombinant NDV expressing CAT protein. The CAT gene was maintained stably for at least eight passages without any detectable loss of the gene from the recombinant. Generation of the recombinant virus, however, was associated with reduced plaque size, slower replication kinetics, and more than 100-fold decrease in yield. In addition, the virus showed an increase in mean death time for eggs and a lower intracerebral pathogenicity index in day-old chicks, implicating attenuation of the recombinant virus. Thus, introduction of an additional gene into the NDV genome represents a method to achieve growth retardation and attenuation. These results also indicate that NDV can be engineered to express foreign protein stably and can be manipulated in the future for use as a vaccine vector.

Animals↗
PubMed2001

Recovery of infectious human parainfluenza type 2 virus from cDNA clones and properties of the defective virus without V-specific cysteine-rich domain.

A full-length cDNA clone was constructed from the genome of the human parainfluenza type 2 virus (hPIV2). First, Vero cells were infected with recombinant vaccinia virus expressing T7 RNA polymerase, and then the plasmid encoding the antigenome sequence was transfected into Vero cells together with polymerase unit plasmids, NP, P, and L, which were under control of the T7 polymerase promoter. Subsequently, the transfected cells were cocultured with fresh Vero cells. Rescue of recombinant hPIV2 (rPIV2) from cDNA clone was demonstrated by finding the introduced genetic tag. As an application of reverse genetics, we introduced one nucleotide change (UCU to ACU) to immediate downstream of the RNA-editing site of the V gene in the full-length hPIV2 cDNA and were able to obtain infectious viruses [rPIV2V(-)] from the cDNA. The rPIV2V(-) possessed a defective V protein that did not have the unique cysteine-rich domain in its carboxyl terminus (the V-protein-specific domain). The rPIV2V(-) showed no growth in CV-1 and FL cells. Replication of the rPIV2V(-) in these cells, however, was partially recovered by adding anti-interferon (IFN)-beta antibody into the culture medium, showing that the rPIV2V(-) is highly sensitive against IFN and that no growth of rPIV2V(-) in CV-1 and FL cells is mainly due to its hypersensitivity to endogenously produced IFN. These findings indicate that the V-protein-specific domain of hPIV2 is related to IFN resistance. On the other hand, the rPIV2V(-) efficiently replicated in Vero cells, which are known as a IFN-non-producers. However, the virus yields of rPIV2V(-) in Vero cells were 10- to100-fold lower than those of control rPIV2, although syntheses of the viral-specific proteins and their mRNAs in rPIV2V(-)-infected Vero cells were augmented up to 48 p.i. in comparison with those of rPIV2. Furthermore, the rPIV2V(-) virions showed anomalous in size as compared with rPIV2 virions. These results suggest that the V protein plays an important role in the hPIV2 assembly, maturation, and morphogenesis.

Animals↗
PubMed2006

[Rescue of a recombinant Newcastle disease virus expressing the green fluorescent protein].

A recombinant Newcastle disease virus (NDV) expressing the green fluorescent protein (GFP) was generated by applying reverse genetics techniques. The GFP open reading frame flanked by NDV transcription start and stop sequences was inserted between the phosphoprotein (P) and matrix protein (M) in a full-length cDNA clone of NDV Lasota vaccine strain. This plasmid transcribing antigenome RNA was cotransfected with helper plasmids expressing viral nucleoprotein, phosphoprotein and large protein into cells stably expressing T7 RNA polymerase. The rescued virus was first propagated in 10-day-old embryonated eggs and the allantoic fluid was used to infect primary chicken embryo fibroblasts (CEF) cells. The appearance of GFP in live infected cells confirmed further the recovery of a recombinant NDV (rNDV-GFP) expressing this reporter gene. Nine successive passages in embryonated chicken eggs were performed. Allantoic fluid samples were then titrated by a microtiter plate HA test. HA positive ailantoic fluid were used for further egg passages. All the allantoic fluid samples were titrated by end point dilutions and infected cells were examined for the presence of GFP expression. To analyze virus growth, 10-day-old embryonated SPF chicken eggs were inoculated with 1 x 10(4) EID50 rNDV or rNDV-GFP. At 24,48,72 and 96 h p.i. the allantoic fluid of inoculated eggs containing live embryos was harvested and clarified by centrifugation. Supernatants were used for titration of EID50 in 10-day-old embryonated SPF chicken eggs. rNDV and rNDV-GFP grew to similar titers (10(9) EID50/mL). In order to test the virulence of rNDV-GFP, infectious allantoic fluid of rNDV-GFP were inoculated into embryonated SPF chicken eggs at 1 x 10(6) EID50. No dead embryonated egg was found within 96 hours. The replication kinetics and pathogenicity in SPF embryonated eggs of rNDV-GFP did not differ significantly from that of the parent virus. LaSota is a widely used NDV live vaccine strain. The reverse genetic system established for this LaSota vaccine strain provided a useful platform for development of novel live viral vector vaccines in future.

Animals↗
PubMed1977

Roles of the early genes of bacteriophage T7 in shutoff of host macromolecular synthesis.

Through the use of phage mutants in which various combinations of the early genes are active, and in which late gene expression is blocked, we have examined the roles of each of the five early gene products of bacteriophage T7 in regulating the synthesis of host RNA and proteins. At least two independent transcriptional controls operate during bacteriophage T7 development. The product of gene 0.7, acting alone, leads to a rapid (by 5 min) shutoff of host transcription. In the absence of gene 0.7 function, and in the absence of the phage-specified RNA polymerase, a delayed shutoff of host-dependent transcription begins at approximately 15 min after infection. This secondary control element requires either a functional gene 0.3 or gene 1.1. In the absence of any early gene products, host shutoff is not observed until much later in infection (>30 min). The delayed manner in which the products of genes 0.3 and 1.1 exert their effect suggests that their mode of action is indirect. Under conditions in which the late genes are transcribed (inefficiently) by the host RNA polymerase, gene 1.1 is observed to stimulate the synthesis of lysozyme (the product of a late phage gene). In contrast, when the late genes are transcribed by the phage-specified RNA polymerase (the product of gene 1), the kinetics of synthesis of the phage RNA polymerase itself, and of lysozyme, are not affected by the deletion of genes 0.3, 0.7, 1.1, and 1.3. We conclude that under these conditions, the products of these genes are required neither for regulation of expression of the late genes nor for the shutoff of early phage gene expression.

Bacterial Proteins↗
PubMed1995

Biochemical characterization of Escherichia coli temperature-sensitive dnaB mutants dnaB8, dnaB252, dnaB70, dnaB43, and dnaB454.

By use of PCR, the dnaB genes from the classical temperature-sensitive dnaB mutants PC8 (dnaB8), RS162 (dnaB252), CR34/454 (dnaB454), HfrH165/70 (dnaB70), and CR34/43 (dnaB43) were isolated. The mutant genes were sequenced, and single amino acid changes were identified in all cases. The mutant DnaB proteins were overexpressed in BL21 (DE3) cells by using the T7 based pET-11c expression vector system. The purified proteins were compared in regard to activities in the general priming reaction of primer RNA synthesis (with primase and single-stranded DNA [ssDNA] as the template), ATPase activity, and helicase activity at permissive (30 degrees C) and nonpermissive (42 degrees C) temperatures. The DnaB252 mutation is at amino acid 299 (Gly to Asp), and in all in vitro assays the DnaB252 protein was as active as the wild-type DnaB protein at both 30 and 42 degrees C. This region of the DnaB protein is believed to be involved in interaction with the DnaC protein. The dnaB8, dnaB454, and dnaB43 mutations, although independently isolated in different laboratories, were all at the same site, changing amino acid 130 from Ala to Val. This mutation is in the hinge region of the DnaB protein domains and probably induces a temperature-sensitive conformational change. These mutants have negligible primer RNA synthesis, ATPase activity, and helicase activity at the nonpermissive temperature. DnaB70 has a mutation at amino acid 242 (Met to Ile), which is close to the proposed ATP binding site. At 30 degrees C this mutant protein has a low level of ATPase activity (approximately 25% of that of the wild type) which is not affected by high temperature. By using a gel shift method that relies upon ssDNA substrates containing the photoaffinity analog 5-(N-(p-azidobenzoyl)-3-aminoallyl)-dUMP, all mutant proteins were shown to bind to ssDNA at both 30 and 42 degrees C. Their lack of other activities at 42 degrees C, therefore, is not due to loss of binding to the ssDNA substrate.

Adenosine Triphosphatases↗
PubMed2002

DNA adducts from a tumorigenic metabolite of benzo[a]pyrene block human RNA polymerase II elongation in a sequence- and stereochemistry-dependent manner.

Many carcinogens exert their cancer-causing effects by reacting with DNA either directly or following metabolic activation, resulting in covalently linked combination molecules known as carcinogen-DNA adducts. The presence of such lesions in the genome increases the error frequency of the replication machinery, causing mutations that contribute to the initiation and progression of cancer. Cellular DNA repair pathways remove carcinogen adducts from DNA, thus averting the mutagenic potential of many DNA lesions by reducing their presence in the genome. Bulky DNA adducts, like those derived from a number of activated environmental carcinogens such as polycyclic aromatic hydrocarbons (PAHs), are primarily repaired by the nucleotide excision repair (NER) pathway. Transcription-coupled NER (TC-NER) preferentially removes lesions from the transcribed strand of actively expressed genes, and RNA polymerase II stalled at the lesion quite possibly initiates the pathway. Among the bulky DNA adducts that are subject to TC-NER are those resulting from the reaction of the metabolically activated PAH benzo[a]pyrene (BP) with DNA. The P450 mixed-function oxygenases convert BP into a number of reactive intermediates, including tumorigenic (+)- and non-tumorigenic (-)-anti-benzo[a]pyrene diol epoxide (BPDE) that react with DNA via trans epoxide opening to form (+)-trans-anti-[BP]-N(2)-dG ((+)-ta[BP]G) and (-)-trans-anti-[BP]-N(2)-dG ((-)-ta[BP]G), respectively. To test the effect of these lesions on RNA synthesis, in vitro transcription assays using human nuclear extracts were performed with DNA templates containing an RNAPII promoter and a stereochemically pure (+)- or (-)-ta[BP]G adduct on the transcribed or non-transcribed strand. Transcription past (+)- or (-)-ta[BP]G adducts was investigated in the same sequence context to examine stereochemical effects. The (+)-ta[BP]G adduct was investigated in two different local sequence contexts to determine if the surrounding bases influence the adduct's ability to block transcription. These experiments revealed that (+)- and (-)-ta[BP]G adducts on the transcribed strand of the DNA template block RNAPII in a sequence and stereochemistry-dependent manner; however, adducts on the non-transcribed strand do not block elongation significantly but may increase pausing at innate pause sites. In order to elucidate biologically influential differences between the (+)- and (-)-ta[BP]G structures, the DUPLEX program was used to carry out potential energy minimization searches at model transcription junctions. The lowest-energy minimum for the (+)-ta[BP]G adduct gives a structure in which the benzo[a]pyrenyl ring system resides in the minor groove of the heteroduplex region. In contrast, the lowest-energy minimum for a (-)-ta[BP]G adduct shows an orientation in which the benzo[a]pyrenyl group adopts a carcinogen/base-stacked conformation. These conformational preferences may contribute to the differential treatment of (+)- and (-)-ta[BP]G adducts by human RNAPII. In addition, while previous experiments showed that BPDE adducts cause T7RNAP to produce a ladder of truncated transcripts, RNAPII is blocked entirely at only one or two positions by the (+)- and (-)-ta[BP]G adducts, depending on sequence context. It is likely that these differences between the behaviors of T7RNAP and human RNAPII are a result of the structural characteristics of the enzymes' active sites, a hypothesis that is explored in light of their known crystal structures.

Autoradiography↗