139 results about "Polyadenylation" patented technology

Chimeric gene for conferring to plants an increased tolerance to a herbicide having as its target EPSPS comprises, in the direction of transcription, a promoter region, a transit peptide region, a coding sequence for glyphosate tolerance and a polyandenylation signal region, wherein the transit peptide region comprises, in the direction of translation, at least one transit peptide of a plant gene encoding a plastid-localized enzyme and then a second transit peptide of a plant gene encoding, a plastid-localized enzyme. Production of glyphosate-tolerant plants is disclosed.

The invention relates to an artificial nucleic acid molecule comprising an open reading frame and a 3′-UTR comprising at least one poly(A) sequence or a polyadenylation signal. The invention further relates to a vector comprising the artificial nucleic acid molecule comprising an open reading frame and a 3′-UTR comprising at least one poly(A) sequence or a polyadenylation signal, to a cell comprising the artificial nucleic acid molecule or the vector, to a pharmaceutical composition comprising the artificial nucleic acid molecule or the vector and to a kit comprising the artificial nucleic acid molecule, the vector and/or the pharmaceutical composition. The invention also relates to a method for increasing protein production from an artificial nucleic acid molecule and to the use of a 3′-UTR for a method for increasing protein production from an artificial nucleic acid molecule. Moreover, the invention concerns the use of the artificial nucleic acid molecule, the vector, the kit or the pharmaceutical composition as a medicament, as a vaccine or in gene therapy.

One-step RT-PCR methods, compositions and kits for the detection and quantification of small RNAs in a sample are disclosed. The one-step RT-PCR approach involves polyadenylation of a small RNA followed by reverse transcription with a first primer containing a poly(T) sequence and at least two 3′ nucleotides complementary to the 3′ terminal end nucleotides of the small RNA, to produce a cDNA. This may be followed by PCR amplification using the same first primer as the revere primer and a second, forward primer in which a portion of its sequence is complementary to the 3′ terminal end of the cDNA. This may be then followed by detection and/or quantification of the amplified product.

Snow Mountain Virus (SMV) belongs to the Norovirus genus of the Caliciviridae family. SMV is a genogroup II (GII) reference strain of human enteric caliciviruses associated with epidemic gastroenteritis. The positive sense RNA genome sequence of SMV was determined to be 7,537 nucleotides in length excluding the 3′ polyadenylated tract. The genome is organized into three open reading frames. Pairwise sequence alignments showed SMV ORF1 is highly conserved with other GII noroviruses, and most closely related to GII strains Melksham and Hawaii viruses. Comparative sequence analyses showed the SMV is a recombinant norovirus. VP1/NP2 proteins assembled into virus-like particles (VLPs) when expressed in insect cells by a recombinant baculovirus. Characterization of one clone that expressed VP1 but failed to assemble into VLPs, identified histidine residue 91 as important for particle assembly.

A method for isolating nucleic acid molecules having a repeating nucleotide sequence or a homopolymeric nucleotide sequence, e.g. a poly A stretch, is described. In particular, the method uses oligomeric capture probes spiked with various amounts of locked nucleic acid (LNA). The invention further describes methods for the isolation of RNA molecules, for example polyadenylated mRNA molecules, which overcome the problems of rapid RNA degradation during isolation and analysis of such nucleic acid molecules. This is of major clinical and diagnostic importance, especially when dealing with RNA viruses, such as retroviruses or when analyzing rare or low-abundant mRNAs or mRNAs from biopsies or tissues enriched with RNases.

Methods and means are provided for reducing the phenotypic expression of a nucleic acid of interest in eukaryotic cells, particularly in plant cells, by providing aberrant, preferably unpolyadenylated, target-specific RNA to the nucleus of the host cell. Preferably, the unpolyadenylated target-specifc RNA is provided by transcription of a chimeric gene comprising a promoter, a DNA region encoding the target-specific RNA, a self-splicing ribozyme and a DNA region involved in 3′ end formation and polyadenylation.

The human soluble neuropilin-1 (sNRP) polyadenylation signal (sNRP-poly(A)), situated downstream of the GT splice donor site of intron 12 of the full-length neuropilin-1 gene, also functions as the termination codon for sNRP. This 17 nucleotide sequence efficiently facilitates addition of poly(A) tails to RNAs expressed in cells. The present invention shows that this optimally succinct sequence has similar activity to the SV40 polyadenylation signal that is currently used in expression vectors. By using this shorter dual termination/polyadenylation signal and avoiding the need for large and cumbersome polyadenylation signals, expression vectors may be engineered to carry considerably larger genes.

The present invention provides novel isolated nucleic acids that comprise an avian nucleic acid sequence comprising a ovomucoid gene expression control region. The ovomucoid promoter region of the present invention will allow expression of an operably linked heterologous nucleic acid insert in a transfected avian cell such as, for example, an oviduct cell. The isolated avian ovomucoid of the present invention may be operably linked with a selected nucleic acid insert, wherein the nucleic acid insert encodes a polypeptide desired to be expressed in a transfected cell. The recombinant DNA of the present invention may further comprise a polyadenylation signal sequence. The present invention further includes expression vectors comprising an isolated avian ovomucoid gene expression control region of the present invention, and transfected cells and transgenic avians comprising the expression vectors.

Disclosed are materials and methods for treating diseases of the mammalian eye, and in particular, Usher syndrome 1B (USH1B). The invention provides AAV-based, dual-vector systems that facilitate the expression of full-length proteins whose coding sequences exceed that of the polynucleotide packaging capacity of an individual AAV vector. In one embodiment, vector systems are provided that include i) a first AAV vector polynucleotide that includes an inverted terminal repeat at each end of the polynucleotide and a suitable promoter followed by a partial coding sequence that encodes an N-terminal portion of a full-length polypeptide; and ii) a second AAV vector polynucleotide that includes an inverted terminal repeat at each end of the polynucleotide and a partial coding sequence that encodes a C-terminal portion of a full-length polypeptide, optionally followed by a polyadenylation (pA) signal sequence. In another embodiment, the vector system includes i) a first AAV vector polynucleotide comprising an inverted terminal repeat at each end, a suitable promoter followed by a partial coding sequence that encodes an N-terminal portion of a full-length polypeptide followed by a splice donor site and intron and ii) a second AAV vector polynucleotide comprising an inverted terminal repeat at each end, followed by an intron and a splice-acceptor site for the intron, followed by a partial coding sequence that encodes a C-terminal portion of a full-length polypeptide, optionally followed by a polyadenylation (pA) signal sequence. The coding sequence or the intron sequence in the first and second AAV vectors preferably includes a sequence region that overlaps.

The invention provides a DNA plasmid comprising: (a) a first transcriptional unit comprising a nucleotide sequence that encodes a first polypeptide operably linked to regulatory elements including a first promoter and a first polyadenylation signal; (b) a second transcriptional unit comprising a nucleotide sequence that encodes a second polypeptide operably linked to regulatory elements including a second promoter and a second polyadenylation signal; (c) a third transcriptional unit comprising a nucleotide sequence that encodes a third polypeptide operably linked to regulatory elements including a third promoter and a third polyadenylation signal; and wherein said first, said second and said third promoters are each derived from different transcriptional units; and wherein said first, said second and said third polyadenylation signals are each derived from different transcriptional units. The invention further relates to immunogenic compositions for inducing an immune response to HIV comprising combinations of two, three, or four plasmids, where each plasmid is expressing a defined antigen, which may be a single antigen or a fusion of two or three antigens.

A lentiviral vector system is described. The system comprises a lentiviral transfer vector and a packaging construct. The transfer vector comprises (a) a 5′ LTR; (b) a 3′ LTR comprising a polyadenylation signal; (c) a minimal packaging signal, (d) (i) at least one heterologous upstream enhancer (UE) sequences, and/or (ii) at least one additional copy of endogenous UE sequences operatively associated with said polyadenylation signal; and (e) a PRE. The packaging construct comprises a nucleic acid encoding and expressing a lentiviral Gag nucleic acid (preferably Gag and Pol). Preferably the lentiviral Gag nucleic acid is a mutated Gag nucleic acid containing one or more substitution mutations, wherein said mutated Gag nucleic acid encodes the same amino acid sequence as the corresponding unmutated Gag nucleic acid, but differs from the nucleic acid sequence of said corresponding unmutated Gag nucleic acid sequence due to the degeneracy of the genetic code. Preferably the packaging construct further comprises a heterologous nucleic acid encoding and expressing an adenovirus VA RNA. The transfer vector and packaging construct can be used together in a producer cell to produce viral particles.

Methods are described to derive design sequences for the production of nucleic acid microarrays. The present methods use high throughput 3′ sequencing of transcripts in a tissue sample or diseased state to design probes for nucleic acid microarrays. Also described are nucleic acid microarrays that possess probes directed to the extreme 3′ end of transcripts in a tissue. These microarrays preferably represent alternate polyadenylation sequences that are specific to the tissue from which the transcripts are derived. Also described are methods of using the microarrays directed to the extreme 3′ end of the transcript for evaluating gene expression in a tissue where there are reduced false positive and false negative results.

A method for conditionally knocking out and altering gene function and genetic sequences that can be used in such methods, for use in gene trapping and gene targeting. Specifically, the genetic sequence is a inducible gene silencer comprising: (a) a splice acceptor sequence; (b) an internal ribosomal entry site (IRES) sequence; (c) a nucleotide sequence coding for a reporter protein; (d) a polyadenylation sequence; and (e) a pair of oppositely oriented recombination site sequences, which cause single cycle inversions in the presence of a suitable recombinase enzyme, flanking elements (a) through (d).

Proviral plasmids contain a modular gene expression cassette with one or a combination of (i) a wildtype 5′ AAV2 ITR sequence flanked by unique restriction sites that permit ready removal or replacement of said ITR; (ii) a promoter flanked by unique restriction sites that permit ready removal or replacement of the entire promoter sequence; (iii) a polylinker sequence that permits insertion of a gene coding sequence without modification thereof, wherein the gene is operatively linked to, and under the regulatory control of, the aforementioned promoter; (iv) a bovine growth hormone polyadenylation sequence flanked by unique restriction sites that permit ready removal or replacement of said polyA sequence; and (v) a wildtype 3′ AAV2 ITR sequence flanked by unique restriction sites that permit ready removal or replacement of the 3′ ITR. These plasmids enable rapid manipulation of the components of the cassette, e.g., rapid mutation and/or replacement of any component, and thereby increase the efficiency of recombinant viral vector, e.g., rAAV, production.

The present invention includes compositions and methods related to the structure and function of the cellular polyadenylation and specificity factor 30 (CPSF30) binding site on the surface of the influenza A non-structural protein 1 (NS1). Specifically, critical biochemical reagents, conditions for crystallization and NMR analysis, assays, and general processes are described for (i) discovering, designing, and optimizing small molecule inhibitors of influenza A (avian flu) viruses and (ii) creating attenuated influenza virus strains suitable for avian and human flu vaccine development.

It is intended to provide an overexpression vector which makes it possible to easily acquire a high-level producing strain with the use of mammalian cells, in particular, Chinese hamster ovary cells (CHO cells) as a host. Namely, an expression vector capable of inducing high productivity of a genetically modified protein in animal host cells which comprises a promoter strongly inducing expression, a multicloning site for gene integration and a polyadenylation signal sequence, in this order starting from the upstream, and a drug-resistance gene having no promoter functioning in animal cells in the downstream thereof.

An artificially synthetic insecticiding gene Bt used for insect-resisting transgenic plant is prepared from the high-toxic Bt CryIIe protein of lepidopterous pest through insecticiding activity deficiency, amplifying the termination-deficient gene fragment, configuring expression carrier, transforming it to obtain recombinant engineered bacteria, inducing expression analyzing insecticiding activity, determining the minimal terminal active region and active region, reinforming polyadenylation signal sequence and ATTTA sequence, optimiizng plant prefaverable codon, and artificial synthesis.

The present inventors conducted dedicated studies and successfully constructed expression vectors that enable high-level production of foreign gene-derived proteins in mammalian host cells, which comprise a translation-impaired dihydrofolate reductase gene cistron whose expression has been attenuated by altering the codons to the least frequently used codons in mammals; and a gene cassette which has a cloning site for incorporation of a foreign gene between a highly transcriptionally active promoter and a highly stable polyadenylation signal.