共查询到20条相似文献,搜索用时 15 毫秒
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D. C. Fargo M. Zhang N. W. Gillham J. E. Boynton 《Molecular & general genetics : MGG》1998,257(3):271-282
Initiation of translation in Escherichia coli and related eubacteria involves well-defined interactions between a conserved Shine-Dalgarno (SD) sequence immediately upstream
of the initiation codon in the mRNA leader and an equally conserved anti-SD sequence at the 3′ end of the 16S rRNA. SD-like
sequences found in the leaders of many, but not all, mRNAs from cyanobacteria and chloroplasts are hypervariable in location,
size, and base composition compared to those in E. coli, while anti-SD sequences in the respective 16S rRNAs remain highly conserved. We have examined the function of the SD-like
sequences found in the leaders of four chloroplast genes of the green alga Chlamydomonas reinhardtii using replacement mutagenesis to eliminate complementarity with the anti-SD sequences and insertion of canonical SD sequences
(GGAGG) at positions −9 to −5 relative to the initiation codon. Promoter-leader regions of the atpB, atpE, rps4, and rps7 genes representing the diversity of chloroplast SD-like sequences were fused to aadA and uidA reporter genes encoding spectinomycin resistance and GUS activity respectively. Analysis of chloroplast transformants of
C. reinhardtii and transformants of E. coli carrying the wild-type and mutant reporter constructs revealed that mutagenic replacement of the putative SD sequences had
no effect on the expression of either the aadA or uidA reporter genes. Chloroplast transformants with the canonical SD sequence also showed no differences in reporter gene expression,
whereas expression of the reporter genes was increased by 10 to 30% in the E. coli transformants. Collectively our results suggest that even though SD-dependent initiation predominates in E. coli, this bacterium also has the capacity to initiate translation by an SD-independent mechanism. In contrast, plant chloroplasts,
and very probably their cyanobacterial ancestors, appear to have adopted the SD-independent mechanism for translational initiation
of most mRNAs.
Received: 8 July 1997 / Accepted: 9 September 1997 相似文献
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Summary The trmD operon of Escherichia coli consists of the genes for the ribosomal protein (r-protein) S16, a 21 kDa protein (21K) of unknown function, the tRNA(m1G37)methyltransferase (TrmD), and r-protein L19, in this order. Previously we have shown that the steady-state amount of the two r-proteins exceeds that of the 21K and TrmD proteins 12- and 40-fold, respectively, and that this differential expression is solely explained by translational regulation. Here we have constructed translational gene fusions of the trmD operon and lacZ. The expression of a lacZ fusion containing the first 18 codons of the 21K protein gene is 15-fold higher than the expression of fusions containing 49 or 72 codons of the gene. This suggests that sequences between the 18th and the 49th codon may act as a negative element controlling the expression of the 21K protein gene. Evidence is presented which demonstrates that this regulation is achieved by reducing the efficiency of translation. 相似文献
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Patakottu BR Singh PK Malhotra P Chauhan VS Patankar S 《Molecular biology reports》2012,39(3):2225-2232
Regulation of gene expression in the malaria parasite Plasmodium falciparum is tightly controlled and little is known about the many steps involved. One step i.e. translation initiation is also poorly
understood and in P. falciparum, choice of the translation initiation site (TIS) is a critical decision largely due to the high frequency of AUGs in the
relatively long 5′ untranslated regions of parasite mRNAs. The sequences surrounding the TIS have a major role to play in
translation initiation and this report evaluates these sequences by mutational analysis of the heat shock protein 86 gene,
transient transfection and reporter assays in the parasite. We find that purines at the −3 and +4 positions are essential
for efficient translation in P. falciparum, similar to other eukaryotes. Interestingly, a U at the −1 position results in 2.5-fold higher reporter activity compared
to wild type. Certain classes of protein biosynthetic genes show higher frequencies of U at the −1 position, suggesting that
these genes may exhibit higher levels of translation. This work defines the optimal sequences for TIS choice and has implications
for the design of efficient expression vectors in an important human pathogen. 相似文献
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B. Díez E. Mellado M. Rodríguez E. Bernasconi J. L. Barredo 《Applied microbiology and biotechnology》1999,52(2):196-207
The gdhA gene encoding the NADP-dependent glutamate dehydrogenase activity from Penicillium chrysogenum has been isolated and characterized for its use in gene expression. The nucleotide sequence of a 2816-bp genomic fragment
was determined, showing an open reading frame of 1600 bp interrupted by two introns, of 160 bp and 57 bp respectively, with
fungal consensus splice-site junctions. The predicted amino acid sequence revealed a high degree of identity to glutamate
dehydrogenase enzymes, especially to those from the fungi Aspergillus nidulans (82%) and Neurospora crassa (78%). The gdhA gene was found to be present in a single copy in the genome of several P. chrysogenum strains with different penicillin productivity. The use of the gdhA promoter for homologous and heterologous gene expression in fungi and Escherichia coli was analyzed. Heterologous gene expression was ascertained by the construction of gene fusions with the lacZ gene from E. coli and the bleomycin-resistance determinant (ble
R) from Streptoalloteichus hindustanus. Homologous gene expression was shown through the use of the penicillin-biosynthetic genes pcbC and penDE from P. chrysogenum and the cephalosporin biosynthetic genes cefEF and cefG from Acremonium chrysogenum.
Received: 2 November 1998 / Received revision: 15 January 1999 / Accepted: 5 March 1999 相似文献
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Xiaoping Chen Zhangying Wang Jianhua Wang Maoyan Wang Li Zhao Guoying Wang 《Plant Cell, Tissue and Organ Culture》2007,88(1):11-20
ADP-glucose pyrophosphorylase (AGPase) represents a key regulatory step in starch synthesis. A 0.9 kb of 5′ flanking region
preceding Brittle2 gene, encoding the small subunit of maize endosperm AGPase, was cloned from maize genome and its expression pattern was studied
via the expression of β-glucuronidase (GUS) gene in transgenic tobacco. Analysis of GUS activities showed that the 0.9 kb
fragment flanking Brittle2 gene was sufficient for driving the seed-preferred expression of the reporter gene. The activity of the 0.9 kb 5′ flanking
fragment was compared with that of the tandem promoter region from a zein gene (zE19, encoding a maize 19 kDa zein protein). The results indicated that both promoters were seed-preferred in a dicotyledonous
system as tobacco and the activity of zE19 promoter was three to fourfold higher than that of the 0.9 kb fragment flanking Brittle2 gene in transgenic tobacco seeds. At the same time, zE19-driven GUS gene expressed earlier than Brittle2 promoter during seed development. Histochemical location of GUS activity indicated that both promoters showed high expression
in embryos, which is different from similar promoters tested in maize. 相似文献
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The expression of some Saccharomyces cerevisiae genes is induced as cells enter stationary phase. Their mRNAs are translated during a period in the growth cycle when the
translational apparatus is relatively inert, thereby raising the possibility that these mRNAs compete effectively for a limiting
pool of translation factors. To test this idea, the translation of mRNAs carrying different 5′-leaders was compared during
exponential growth and after entry into stationary phase upon glucose starvation. Closely related sets of lacZ mRNAs, carrying 5′-leaders from the PYK1, PGK1, RpL3, Rp29, HSP12, HSP26 or THI4 mRNAs, were studied. These mRNAs displayed differing translational efficiencies during exponential growth, but their relative
translatabilities were not significantly affected by entry into stationary phase, indicating that they compete just as effectively
under these conditions. Polysome analysis revealed that the wild-type PYK1, ACT1 and HSP26 mRNAs are all translated efficiently during stationary phase, when the translational apparatus is relatively inert. Also,
significant levels of the translation initiation factors eIF-2α, eIF-4E and eIF-4A were maintained during the growth cycle.
These data are consistent with the idea that, while translational activity decreases dramatically during entry into stationary
phase, yeast cells maintain excess translational capacity under these conditions.
Received: 31 March 1998 / Accepted: 4 May 1998 相似文献
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Quantitative evaluation of six different viral suppressors of silencing using image analysis of transient GFP expression 总被引:1,自引:0,他引:1
The effects of six different plant viral suppressors of gene silencing were compared using an automated image collection and
analysis system developed for continual monitoring of GFP expression. Suppressors were introduced into lima bean cotyledonary
tissues either as 3′-GFP translational fusions or as co-introductions with the GFP gene on a separate plasmid. The resultant transient expression profiles for each suppressor depended on whether the suppressor
was introduced as a fusion or co-introduced on separate plasmids. As co-introductions, the silencing suppressors HCPro (from Tobacco etch virus), p19 (from Tomato bushy stunt virus), γb (from Barley stripe mosaic virus) and p21 (from Beet yellows virus) led to an almost twofold increase in initial GFP expression levels, followed by a rapid decline. In contrast, fusions of
HCPro, p19, and γb to the 3′-end of GFP resulted in slightly lower but more prolonged GFP expression. Compared with the co-introductions, all GFP::Suppressor translational fusions gave reduced GFP fluorescence levels, suggesting interference of the fusion partner with GFP fluorescence.
Regardless of the configuration, introductions of the silencing suppressors AL2 (from Tomato golden mosaic virus) and 126-kDa protein (from Tobacco mosaic virus) resulted in very low GFP fluorescence. This is the first report that directly compares the effects of a large number of
viral suppressors of silencing on transient transgene expression using both translational fusions and co-introductions.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
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M. J. Pettinari G. J. Vazquez N. Krüger P. S. Vary A. Steinbüchel B. S. Méndez 《Applied microbiology and biotechnology》1998,49(6):737-742
A Bacillus megaterium genomic fragment, which encoded an activator homologous to σ54 regulators and which was capable of activating Escherichia coli ato genes in trans, was detected in a gene library of B.␣megaterium screened for β-ketothiolase activity. The fragment presented only one complete open reading frame (ORF1), which encoded a
protein of 398 amino acids. The recombinant plasmid complemented mutations in the Escherichia coli atoC regulatory gene. The constitutive expression of the E. coli ato operon mediated by ORF1 could be useful for the synthesis of polyhydroxyalkanoates with different flexibility properties
by recombinant E. coli strains.
Received: 20 October 1997 / Received revision: 18 February 1998 / Accepted: 23 February 1998 相似文献