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Expression Enhancement of a Rice Polyubiquitin Gene Promoter 总被引:11,自引:0,他引:11
An 808 bp promoter from a rice polyubiquitin gene, rubi3, has been isolated. The rubi3 gene contained an open reading frame of 1140 bp encoding a pentameric polyubiquitin arranged as five tandem, head-to-tail
repeats of 76 aa. The 1140 bp 5′ UTR intron of the gene enhanced its promoter activity in transient expression assays by 20-fold.
Translational fusion of the GUS reporter gene to the coding sequence of the ubiquitin monomer enhanced GUS enzyme activity in transient expression assays
by 4.3-fold over the construct containing the original rubi3 promoter (including the 5′ UTR intron) construct. The enhancing effect residing in the ubiquitin monomer coding sequence
has been narrowed down to the first 9 nt coding for the first three amino acid residues of the ubiquitin protein. Mutagenesis
at the third nucleotide of this 9 nt sequence still maintains the enhancing effect, but leads to translation of the native
GUS protein rather than a fusion protein. The resultant 5′ regulatory sequence, consisting of the rubi3 promoter, 5′ UTR exon and intron, and the mutated first 9 nt coding sequence, has an activity nearly 90-fold greater than
the rubi3 promoter only (without the 5′ UTR intron), and 2.2-fold greater than the maize Ubi1 gene promoter (including its 5′ UTR intron). The newly created expression vector is expected to enhance transgene expression
in monocot plants. Considering the high conservation of the polyubiquitin gene structure in higher plants, the observed enhancement
in gene expression may apply to 5′ regulatory sequences of other plant polyubiquitin genes. 相似文献
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Szabó A Pál M Deák P Kiss P Ujfaludi Z Pankotai T Lipinszki Z Udvardy A 《Molecular genetics and genomics : MGG》2007,278(1):17-29
The function and the molecular properties of the Rpt1/p48B ATPase subunit of the regulatory particle of the Drosophila melanogaster 26S proteasome have been studied by analyzing three mutant Drosophila stocks in which P-element insertions occurred in the 5′-non-translated region of the Rpt1/p48B gene. These P-element insertions
resulted in larval lethality during the second instar larval phase. Since the Rpt1/p48B gene resides within a long intron
of an annotated, but uncharacterized Drosophila gene (CG17985), the second instar larval lethality may be a consequence of a combined damage to two independent genes. To
analyze the phenotypic effect of the mutations affecting the Rpt1/p48B gene alone, imprecise P-element excision mutants were
selected. One of them, the pupal lethal P1 mutation, is a hypomorphic allele of the Rpt1/p48B gene, in which the displacement
of two essential regulatory sequences of the gene occurred due to the insertion of a 32 bp residual P-element sequence. This
mutation caused a 30-fold drop in the cellular concentration of the Rpt1/p48B mRNA. The decline in the cellular Rpt1/p48B
protein concentration induced serious damage in the assembly of the 26S proteasomes, the accumulation of multiubiquitinated
proteins, a change in the phosphorylation pattern of the subunit and depletion of this ATPase protein from the chromatin. 相似文献
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Transgenic technologies provide a promising means by which desirable traits can be introduced into cultured fish species within
a single generation thus accelerating the production of genetically superior broodstock for aquaculture. However, before such
fish are allowed to be marketed as food they must receive government regulatory approval. Two pivotal regulatory requirements
are: (1) complete characterization of the genomically integrated transgene and, (2) demonstration that the transgene remains
stable over multiple generations. We have generated a stable line of growth hormone (GH) transgenic Atlantic salmon (Salmo salar) using an “all fish” gene construct (opAFP-GHc2) containing a growth hormone cDNA from chinook salmon whose expression is
regulated by the 5′ promoter and 3′ termination regions derived from an ocean pout antifreeze protein (AFP) gene. In this
study we show that a reorganized form of the opAFP-GHc2 construct (termed EO-1α) integrated as a single functional copy into
a 35 bp repeat region of the genomic DNA. PCR based mapping revealed that the linear sequence of the EO-1α integrant was organized
as follows: base pairs 1580–2193 of the ocean pout promoter region followed by the intact chinook salmon GH cDNA, the complete
ocean pout antifreeze 3′ region, and the first 1678 bp of the ocean pout antifreeze 5′ region. Sequence analysis of the EO-1α
integrant and genomic flanking regions in F2 and F4 generation salmon revealed that they were identical. In addition, apart from the disruption at the integration sites, the
consensus sequences of the integrant in these two generations of salmon were identical to the sequence of the opAFP-GHc2 construct.
These results indicate that the EO-1α transgene codes for the chinook salmon GH, and that the transgene and the integration
site have remained stable over multiple generations. 相似文献
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Development and characterization of a chimaeric tissue-specific promoter in wheat and rice endosperm
Maria Oszvald Mark Gardonyi Cecília Tamas Imre Takacs Barnabas Jenes Laszlo Tamas 《In vitro cellular & developmental biology. Plant》2008,44(1):1-7
The recently achieved significant improvement of cereal transformation protocols provides facilities to alter the protein
composition of the endosperm, for example, to increase or decrease the quantity of one of its protein components or to express
foreign molecules. To achieve this goal, strong endosperm-specific promoters have to be available. The aim of our work was
to develop a more efficient tissue-specific promoter which is currently used. A chimaeric promoter was assembled using the
5′ UTR (1,900 bp) of the gene coding for the 1Bx17 HMW glutenin subunit protein, responsible for tissue-specific expression
and the first intron of the rice actin gene (act1). The sequence around of the translation initial codon was optimized. The effect of the intron and promoter regulatory sequences,
using different lengths of 1Bx17 HMW-GS promoter, were studied on the expression of uidA gene. The function of promoter elements, promoter length, and the first intron of the rice actin gene were tested by a transient expression assay in immature wheat endosperm and in stable transgenic rice plants. Results
showed that insertion of the rice act1 first intron increased GUS expression by four times in transient assay. The shortest 1Bx17 HMW-GS promoter fragment (173 bp) linked to the intron and GUS reporter gene provided almost the same expression level than
the intronless long 1Bx17 HMW-GS promoter. Analysis of the stable transformant plants revealed that 173 nucleotides were sufficient for endosperm-specific
expression of the uidA gene, despite 13 nucleotides missing from the HMW enhancer sequence, a relevant regulatory element in the promoter region. 相似文献
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de Boer Gert-Jan Testerink Christa Pielage Gerlof Nijkamp H. John J. Stuitje Antoine R. 《Plant molecular biology》1999,39(6):1197-1207
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Intron loss and its evolutionary significance have been noted in Drosophila. The current study provides another example of intron loss within a single-copy Dfak gene in Drosophila. By using polymerase chain reaction (PCR), we amplified about 1.3 kb fragment spanning intron 5–10, located in the position
of Tyr kinase (TyK) domain of Dfak gene from Drosophila melanogaster species group, and observed size difference among the amplified DNA fragments from different species. Further sequencing
analysis revealed that D. melanogaster and D. simulans deleted an about 60 bp of DNA fragment relative to other 7 Drosophila species, such as D. elegans, D. ficusphila, D. biarmipes, D. takahashii, D. jambulina, D. prostipennis and D. pseudoobscura, and the deleted fragment located precisely in the position of one intron. The data suggested that intron loss might have
occurred in the Dfak gene evolutionary process of D. melanogaster and D. simulans of Drosophila melanogaster species group. In addition, the constructed phylogenetic tree based on the Dfak TyK domains clearly revealed the evolutionary relationships between subgroups of Drosophila melanogaster species group, and the intron loss identified from D. melanogaster and D. simulans provides a unique diagnostic tool for taxonomic classification of the melanogaster subgroup from other group of genus Drosophila. 相似文献
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Tomatoes (Lycopersicon esculentum Mill cv. Ailsa Craig) were transformed with a gene construct having 244 bp of the 5 end of a polygalacturonase (PG) cDNA, coding for a 71 amino acid N-terminal extension to the mature protein, fused to 1320 bp of a pectinesterase (PE) cDNA encoding the full sequence of the mature PE protein. This chimaeric gene was inserted in a sense orientation between a CaMV 35S promoter and terminator for constitutive expression. In transformed tomato plants expression of the endogenous PG and PE genes in the fruit was inhibited; there was little or no observable PG and PE mRNA and a substantial reduction in the level of PG and PE enzyme activity. The transgene was expressed in the leaves of the transformed plants as demonstrated by the accumulation of mRNA, but no protein product could be identified. However, no transgene mRNA or protein were observed in the transgenic fruit.This paper represents the first report of the down-regulation of two non-homologous endogenous genes using a single gene construct. A sense gene construct was responsible for these effects. These findings are discussed in relation to possible mechanisms of action of co-suppression. 相似文献
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Ulrike Swida Eduardo Thüroff Norbert F. Käufer 《Molecular & general genetics : MGG》1986,203(2):300-304
Summary To define the extent of intervening sequences required for efficient splicing of the CYH2 gene in Saccharomyces cerevisiae, we have constructed a series of intron mutations. Artificial intron extensions of more than 300 bp of the natural intron lead to an inhibition of splicing where-as intron deletions lead to a drastic improvement of the splicing efficiency. It is shown that deletion of a 32 bp sequence element within the intron is responsible for this drastic improvement. 相似文献
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Intron-dependent transient expression of the maize GapA1 gene 总被引:2,自引:0,他引:2
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S J Du Z Gong C L Hew C H Tan G L Fletcher 《Molecular Marine Biology and Biotechnology》1992,1(4-5):290-300
To develop an all-fish gene cassette suitable for gene transfer in aquaculture, the antifreeze protein (AFP) gene promoter from the ocean pout (Macrozoarces americanus) was analyzed for its ability to direct exogenous gene expression both in vitro and in vivo. The ocean pout AFP (opAFP) gene promoter fused to the bacterial chloramphenicol acetyltransferase (CAT) was functionally analyzed in two fish cell lines and in Japanese medaka embryos. The opAFP gene promoter was active in these systems, as demonstrated by the transient expression of CAT activity. These results suggest that the opAFP gene promoter is useful for many other gene transfer experiments. To facilitate use of the opAFP gene promoter as a common and versatile vehicle for fish gene transfers, an expression vector, opAFP-V, was constructed by linking the 2.1-kb opAFP gene promoter, the 63-bp opAFP gene 5' untranslated sequence, and the 1.2-kb opAFP gene 3' sequence by two unique restriction sites, Bg/II and HpaI, respectively. Thus, genes of interest can be inserted into either the Bg/II site or the HpaI site depending on the length of their 5' untranslated sequence. The complete DNA sequence of opAFP-V was determined to facilitate future detailed analysis of integration and expression of the transgene. 相似文献