26 research outputs found
Efficient method for site-directed mutagenesis in large plasmids without subcloning
Commonly used methods for site-directed DNA mutagenesis require copying the entire target plasmid. These methods allow relatively easy modification of DNA sequences in small plasmids but become less efficient and faithful for large plasmids, necessitating full sequence verification. Introduction of mutations in larger plasmids requires subcloning, a slow and labor-intensive process, especially for multiple mutations. We have developed an efficient DNA mutagenesis technique, UnRestricted Mutagenesis and Cloning (URMAC) that replaces subcloning steps with quick biochemical reactions. URMAC does not suffer from plasmid size constraints and allows simultaneous introduction of multiple mutations. URMAC involves manipulation of only the mutagenesis target site(s), not the entire plasmid being mutagenized, therefore only partial sequence verification is required. Basic URMAC requires two PCR reactions, each followed by a ligation reaction to circularize the product, with an optional third enrichment PCR step followed by a traditional cloning step that requires two restriction sites. Here, we demonstrate URMAC's speed, accuracy, and efficiency through several examples, creating insertions, deletions or substitutions in plasmids ranging from 2.6 kb to 17 kb without subcloning
Identification and characterization of a novel non-structural protein of bluetongue virus
Bluetongue virus (BTV) is the causative agent of a major disease of livestock (bluetongue). For over two decades, it has been widely accepted that the 10 segments of the dsRNA genome of BTV encode for 7 structural and 3 non-structural proteins. The non-structural proteins (NS1, NS2, NS3/NS3a) play different key roles during the viral replication cycle. In this study we show that BTV expresses a fourth non-structural protein (that we designated NS4) encoded by an open reading frame in segment 9 overlapping the open reading frame encoding VP6. NS4 is 77–79 amino acid residues in length and highly conserved among several BTV serotypes/strains. NS4 was expressed early post-infection and localized in the nucleoli of BTV infected cells. By reverse genetics, we showed that NS4 is dispensable for BTV replication in vitro, both in mammalian and insect cells, and does not affect viral virulence in murine models of bluetongue infection. Interestingly, NS4 conferred a replication advantage to BTV-8, but not to BTV-1, in cells in an interferon (IFN)-induced antiviral state. However, the BTV-1 NS4 conferred a replication advantage both to a BTV-8 reassortant containing the entire segment 9 of BTV-1 and to a BTV-8 mutant with the NS4 identical to the homologous BTV-1 protein. Collectively, this study suggests that NS4 plays an important role in virus-host interaction and is one of the mechanisms played, at least by BTV-8, to counteract the antiviral response of the host. In addition, the distinct nucleolar localization of NS4, being expressed by a virus that replicates exclusively in the cytoplasm, offers new avenues to investigate the multiple roles played by the nucleolus in the biology of the cell
Complete Genome Sequence of Avian Paramyxovirus (APMV) Serotype 5 Completes the Analysis of Nine APMV Serotypes and Reveals the Longest APMV Genome
Avian paramyxoviruses (APMV) consist of nine known serotypes. The genomes of representatives of all APMV serotypes except APMV type 5 have recently been fully sequenced. Here, we report the complete genome sequence of the APMV-5 prototype strain budgerigar/Kunitachi/74.APMV-5 Kunitachi virus is unusual in that it lacks a virion hemagglutinin and does not grow in the allantoic cavity of embryonated chicken eggs. However, the virus grew in the amniotic cavity of embryonated chicken eggs and in twelve different established cell lines and two primary cell cultures. The genome is 17,262 nucleotides (nt) long, which is the longest among members of genus Avulavirus, and encodes six non-overlapping genes in the order of 3'N-P/V/W-M-F-HN-L-5' with intergenic regions of 4-57 nt. The genome length follows the 'rule of six' and contains a 55-nt leader sequence at the 3'end and a 552 nt trailer sequence at the 5' end. The phosphoprotein (P) gene contains a conserved RNA editing site and is predicted to encode P, V, and W proteins. The cleavage site of the F protein (G-K-R-K-K-R downward arrowF) conforms to the cleavage site motif of the ubiquitous cellular protease furin. Consistent with this, exogenous protease was not required for virus replication in vitro. However, the intracerebral pathogenicity index of APMV-5 strain Kunitachi in one-day-old chicks was found to be zero, indicating that the virus is avirulent for chickens despite the presence of a polybasic F cleavage site.Phylogenetic analysis of the sequences of the APVM-5 genome and proteins versus those of the other APMV serotypes showed that APMV-5 is more closely related to APMV-6 than to the other APMVs. Furthermore, these comparisons provided evidence of extensive genome-wide divergence that supports the classification of the APMVs into nine separate serotypes. The structure of the F cleavage site does not appear to be a reliable indicator of virulence among APMV serotypes 2-9. The availability of sequence information for all known APMV serotypes will facilitate studies in epidemiology and vaccinology
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An Ultra Deep Field survey with WFIRST
Studying the formation and evolution of galaxies at the earliest cosmic
times, and their role in reionization, requires the deepest imaging possible.
Ultra-deep surveys like the HUDF and HFF have pushed to mag \mAB30,
revealing galaxies at the faint end of the LF to 911 and
constraining their role in reionization. However, a key limitation of these
fields is their size, only a few arcminutes (less than a Mpc at these
redshifts), too small to probe large-scale environments or clustering
properties of these galaxies, crucial for advancing our understanding of
reionization. Achieving HUDF-quality depth over areas 100 times larger
becomes possible with a mission like the Wide Field Infrared Survey Telescope
(WFIRST), a 2.4-m telescope with similar optical properties to HST, with a
field of view of 1000 arcmin, 100 the area of the
HST/ACS HUDF.
This whitepaper motivates an Ultra-Deep Field survey with WFIRST, covering
100300 the area of the HUDF, or up to 1 deg, to
\mAB30, potentially revealing thousands of galaxies and AGN at the
faint end of the LF, at or beyond \,\,910 in the epoch of
reionization, and tracing their LSS environments, dramatically increasing the
discovery potential at these redshifts.
(Note: This paper is a somewhat expanded version of one that was submitted as
input to the Astro2020 Decadal Survey, with this version including an Appendix
(which exceeded the Astro2020 page limits), describing how the science drivers
for a WFIRST Ultra Deep Field might map into a notional observing program,
including the filters used and exposure times needed to achieve these depths.
Transcriptional response of chicken embryo cells to Newcastle disease virus (D58 strain) infection
Nuclear localization of the p17 protein of avian reovirus is correlated with autophagy induction and an increase in viral replication
Ubiquitin-Regulated Nuclear-Cytoplasmic Trafficking of the Nipah Virus Matrix Protein Is Important for Viral Budding
Expression At The Cell-Surface Of Native Fusion Protein Of The Newcastle-Disease Virus (Ndv) Strain Italien From Cloned Cdna
A cDNA library was constructed with poly(A)+-mRNAs from NDV-Italien infected
BHK-21 cells. A clone, that hybridized to the F gene mRNA, was sequenced. A long
open reading frame encodes for a protein of 553 amino acids, with a calculated
molecular weight of 59,153, consisting of twelve cysteine residues and six
potential glycosylation sites. The protein sequence contains a hydrophobic region
at the N-terminus of F1 and a presumptive long transmembrane fragment near the
C-terminus. Comparison of the F proteins from NDV strains Italien and
Australia-Victoria shows that the sequences are very similar, with conservation
of most cysteine residues and of the potential glycosylation sites. The F coding
sequence was inserted into the genome of vaccinia virus under the control of
vaccinia P7.5 transcriptional regulatory sequences. Expression of F protein was
demonstrated by indirect immunofluorescence with five anti-F monoclonal
antibodies known to react with conformational epitopes