144 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 present invention provides a series of novel dystrophin minigenes that retain the essential biological functions. The expression of the dystrophin minigenes may be controlled by a regulatory element along with a small polyadenylation signal. The entire gene expression cassettes may be readily packaged into a viral vector, preferably an AAV vector. The present invention further defines the minimal functional domains of dystrophin and provides ways to optimize and create new versions of dystrophin minigenes. Finally, the present invention provides a method of treatment for Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD).

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.

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-specific 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.

Methods and constructs for the introduction of multiple genes into plants using a single transformation event are described. Constructs contain a single 5′ promoter operably linked to DNA encoding a modified intein splicing unit. The splicing unit is expressed as a polyprotein and consists of a first protein fused to an intein fused to a second protein. The splicing unit has been engineered to promote excision of all non-essential components in the polyprotein but prevent the ligation reactions normally associated with protein splicing. Additional genetic elements encoding inteins and additional proteins can be fused in frame to the 5′-terminus of the coding region for the second protein to form a construct for expression of more than two proteins. A single 3′ termination sequence, such as a polyadenylation sequence when the construct is to be expressed in eucaryotic cells, follows the last coding sequence. These methods and constructs are particularly useful for creating plants with stacked input traits, illustrated by glyphosate tolerant plants producing BT toxin, and / or value added products, illustrated by the production of polyhydroxyalkanoates in plants.

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.

The invention relates to a kit for auxiliary diagnosis of multiple tumors by taking a micro ribonucleic acid (RNA) combination as a tumor marker, and a detection method of the kit. The invention belongs to the fields of genetic engineering and oncology. The detection method comprises the steps of (1) extracting RNA from serum; (2) carrying out polyadenylation on the 3' end of micro RNA; (3) purifying and recovering a tailing product; (4) carrying out an inverse transcription reaction on the tailing product; (5) testing the relative expression of the micro RNA by means of a fluorescent quantitative PCR (polymerase chain reaction); (6) analyzing the result of the fluorescent quantitative PCR. The kit has the characteristics of high conservative property, space-time specificity, stability, tissue specificity and the like, and plays an important role in the aspects such as disease pathogenesis research, early diagnosis, individualized treatment and prognosis; furthermore, the kit is simple to operate, convenient in material obtaining, safe and noninvasive, and is conveniently used for screening of a great deal of diseases.

The present application discloses an isolated nucleic acid sequence comprising (a) at least 700 consecutive nucleotides of SEQ ID NO: 1; (b) a nucleotide sequence with at least 80% sequence identity to the nucleotide sequence of (a); (c) a nucleotide sequence hybridizing under stringent conditions to the nucleotide sequence of (a) or (b); and (d) a nucleotide sequence complementary to the nucleotide sequence of any one of (a) to (c). Further disclosed herein is a chimeric gene comprising the isolated nucleic acid described herein operably linked to a nucleic acid coding for an expression product of interest, and a transcription termination and polyadenylation sequence. Also disclosed herein are a vector, a transgenic plant cell, a transgenic plant and a seed as characterized in the claims. Methods disclosed herein relate to the production of a transgenic plant, growing cotton, producing a seed, effecting fiber-preferential expression of a product in cotton and of altering fiber properties in a cotton plant as characterized in the claims.

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.

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.

This invention relates to an expression vector wherein said expression vector comprises a polynucleotide promoter sequence, a polynucleotide encoding a signal sequence, a polynucleotide encoding an antigen protein or peptide, a polynucleotide encoding a cell binding element, and a polynucleotide polyadenylation sequence all operatively linked. More particularly, it relates to the method of eliciting an immune response directed against an antigen in a mammal comprising the steps of introducing the expression vector into a cell, expressing the vector to produce an antigen under conditions wherein the antigen is secreted from the cell, endocytosing the secreted antigen into the cell, processing the antigen, and presenting fragments to a receptor to elicit a T-cell response. In addition, this invention relates to a vaccine and a method of use. The invention also relates to the method of identifying MHC-II restricted epitopes.

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.

The present invention relates to a coccidiosis vaccine to protect poultry against Eimeria infection, comprising a recombinant avian adenovirus vector comprising in frame a (heterologous) promoter linked to a hydrophobic signal sequence for membrane anchoring, or linked to an hydrophobic secretion signal and a cleavage site to allow secretion; a multiple cloning site for in frame insertion of an Eimeria antigen ORF such as derived from the r56, 82 kDa, and / or TFP250 antigens; a polyadenylation signal; and an avian adenovirus genome.

The present invention provides a transgenic wheat cell or wheat plant, the wheat cell or wheat plant comprising a chimeric DNA molecule which encodes a dsRNA molecule which is capable of inhibiting wheat streak mosaic virus (WSMV) replication, wherein the wheat cell or plant is immune to WSMV. The present invention also provides a chimeric DNA, the chimeric DNA comprising (i) a wheat expressible promoter; (ii) a region which encodes a dsRNA which is capable of inhibiting WSMV replication; and (iii) a transcription termination and polyadenylation signal. Finally, the present invention provides a process for producing the aforementioned transgenic wheat cell or plant, comprising (i) introducing a chimeric DNA molecule comprising (a) a wheat expressible promoter; (b) a region which encodes a dsRNA which is capable of inhibiting WSMV replication; and (c) a transcription termination and polyadenylation signal into a parental wheat cell; and optionally (ii) regenerating a wheat plant from the wheat cell comprising the chimeric DNA molecule; and (iii) identifying and / or selecting a plant which is immune to WSMV.

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.

This invention relates to an expression vector wherein said expression vector comprises a polynucleotide promoter sequence, a polynucleotide encoding a signal sequence, a polynucleotide encoding an antigen protein or peptide, a polynucleotide encoding a cell binding element, and a polynucleotide polyadenylation sequence all operatively linked. More particularly, it relates to the method of eliciting an immune response directed against an antigen in a mammal comprising the steps of introducing the expression vector into a cell, expressing the vector to produce an antigen under conditions wherein the antigen is secreted from the cell, endocytosing the secreted antigen into the cell, processing the antigen, and presenting fragments to a receptor to elicit a T-cell response. In addition, this invention relates to a vaccine and a method of use. The invention also relates to the method of identifying MHC-II restricted epitopes.

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.

The current invention describes a nucleic acid comprising in a 5′ to 3′ direction a) a first nucleic acid encoding a heterologous polypeptide without an in frame stop codon, b) a second nucleic acid beginning with a 5′ splice donor site and terminated by a 3′ splice acceptor site comprising an in frame translational stop codon and a polyadenylation signal, and c) a nucleic acid encoding i) at least a fragment of a transmembrane domain, or ii) a signal peptide for a GPI-anchor.

The invention modifies the U1 ribonucleoprotein (U1 snRNP) at the 5' end of its RNA so that it binds specifically to an mRNA of the gene to be inactivated, in such a way that its expression is inhibited. Gene inactivation is greater with the binding of several ribonucleoproteins to several binding sites on the 3' terminal exon of the mRNA of the gene to be in-activated, concretely at specific sites around the site of initiation of polyadenylation of said mRNA. This method is used for (i) inhibiting the expression of genes of unknown function so that they can be studied and (ii) inhibiting the expression of genes that are harmful to the cell.