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Query: UNIPROT:P06889 (Mol)
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In Chlamydomonas reinhardtii, expression of the cabII-1 gene increases dramatically in response to light (cabII-1 encodes one of the light-harvesting chlorophyll a/b-binding proteins of photosystem II). We have used a region upstream of the cabII-1 gene in translational fusions to the bacterial uidA gene (encodes beta-glucuronidase) and transcriptional fusions to the Chlamydomonas nitrate reductase gene (nit1). Chlamydomonas transformants carrying intact copies of the chimeric uidA gene do not express beta-glucuronidase at the level of enzyme activity or mRNA accumulation. Methylation in the cabII-1 promoter region of the introduced gene is extensive in these strains, suggesting that newly introduced foreign genes may be recognized and silenced by a cellular mechanism that is correlated with increased methylation. Transformants that express the chimeric cabII-1/nit1 gene have been recovered. In contrast to the endogenous nit1 gene, the chimeric cabII-1/nit1 gene is expressed in ammonium-containing medium. Moreover, nit1 mRNA accumulation is dramatically stimulated by light, with a time course that is indistinguishable from that of the endogenous cabII-1 gene. The cabII-1/nit1 gene has been used to select transformants in a nit1- nit2- Chlamydomonas strain (CC400G) and should be useful for transformation of the large number of mutants in the Ebersold-Levine lineage, which carry the same mutations.
Mol Cell Biol 1992 Nov
PMID:Expression of chimeric genes by the light-regulated cabII-1 promoter in Chlamydomonas reinhardtii: a cabII-1/nit1 gene functions as a dominant selectable marker in a nit1- nit2- strain. 140 96

A plasmid vector, pGV910, and a derived cosmid, pRG930, have been constructed. Both contain the ColE1 and pVS1 origins of replication and are stably maintained in Escherichia coli, Agrobacterium tumefaciens, and Azorhizobium caulinodans ORS571. They are compatible with commonly used IncP cloning vectors, although pVS1 was classified as an IncP plasmid, unable to replicate in E. coli (Y. Itoh, J.M. Watson, D. Haas, and T. Leisinger, Plasmid 11:206-220, 1984). Promoter selection vectors were derived from both of these plasmids by using a promoterless beta-glucuronidase and/or beta-galactosidase gene. These vectors facilitate the study of gene expression in bacteria under particular environmental conditions. This is illustrated by the expression of the gusA gene under the control of a nod promoter in A. caulinodans nodulating stem-located infection sites on Sesbania rostrata.
Mol Plant Microbe Interact
PMID:Broad host range and promoter selection vectors for bacteria that interact with plants. 142 10

From a potato genomic library a phage lambda clone was isolated that carried nucleotide sequences of two patatin genes, thus demonstrating a close physical linkage between these two members of the patatin gene family. Sequence and restriction analysis showed the genes to be oriented in tandem. The more upstream gene was a pseudogene truncated at the 3' end, whereas the downstream gene was a class II patatin gene. In addition to a 208 bp fragment also present in patatin class I promoters, the region in between both genes contained various direct repeats also found in other patatin genes. To study the promoter activity of this intergenic region, a 2.78 kb fragment was transcriptionally fused to the beta-glucuronidase gene and reintroduced into potato cultivar Bintje. Histochemical analysis revealed expression in the outermost layer of cells of the cortex, in the tuber phellogen, in or around the root vascular system, and also in the abaxial phloem layer of the vascular bundle in leaves.
Plant Mol Biol 1992 Nov
PMID:Analysis of the region in between two closely linked patatin genes: class II promoter activity in tuber, root and leaf. 145 Mar 83

A hammerhead ribozyme designed against the mRNA coding for the Escherichia coli beta-glucuronidase (GUS) reporter enzyme was constructed. The synthetic ribozyme appeared able to correctly cleave in vitro the target RNA. This catalytic molecule was then assayed for in vivo activity in plant protoplasts. Plasmids coding either for the ribozyme or for the GUS target gene were cotransfected into the cells by the PEG-calcium procedure and GUS gene expression monitored following transient expression by measuring the intracellular GUS enzymatic activity. Expression of the ribozyme to high molar excess over the GUS transcript did not lead to any significant decrease of GUS activity in the transfected protoplasts. Insertion of the ribozyme sequence in the 3'-untranslated region of the GUS mRNA also had no detectable effect on GUS reporter gene expression whereas the corresponding RNA appeared able to self-cleave in vitro. These results indicate that the ability of ribozymes to perform catalytic cleavage of their substrate mRNA in vitro is essential but clearly not sufficient to ensure that efficient inhibition of the corresponding target gene will occur upon endogenous expression of this catalytic RNA in the plant cell.
Plant Mol Biol 1992 Nov
PMID:Assaying synthetic ribozymes in plants: high-level expression of a functional hammerhead structure fails to inhibit target gene activity in transiently transformed protoplasts. 145 Mar 86

A wheat gene (A121) encoding a protein with sequence similarity to mammalian cathepsin B is regulated by gibberellic acid (GA) in aleurone layers of germinating grains. To analyse the mechanism of A121 regulation, its promoter was fused to the beta-glucuronidase reporter gene (GUS) and introduced by micro-projectile bombardment into aleurone layers of oat. With 2.3 kb of promoter sequence, the GUS expression was enhanced by GA treatment. This effect was reversed by abscisic acid (ABA). This result showed for A121, like the alpha-amylase genes, that the regulation by GA and ABA was at the level of transcription. The GA responsiveness of the promoter was retained with as little as 276 bp of promoter sequence. Sequence comparison with a GA responsive promoter of an alpha-amylase gene identified the conserved element GCAACGGCAACGATGG which is required intact for full expression of both promoters. However, there was no identifiable similarity in the cathepsin-like promoter with the GA-responsive element of alpha-amylase promoters with the consensus sequence TAACAAA, suggesting that GA affects more than one mechanism of transcriptional control.
Plant Mol Biol 1992 Dec
PMID:Analysis of the gibberellin-responsive promoter of a cathepsin B-like gene from wheat. 146 24

beta-Conglycinin, the 7S storage protein of soybean, is expressed only in seeds and is regulated predominantly by gene transcription [5]. We applied an antisense strategy to modify expression of a beta-glucuronidase (uidA or gusA) gene in seeds using a promoter from a beta-conglycinin gene. Transgenic tobacco plants harboring the gusA gene under the control of the CaMV 35S promoter were retransformed with a gene construct comprising the beta-conglycinin promoter fused to the gusA gene in the antisense orientation. Double transformants were regenerated and transformation was confirmed by Southern blot hybridization. Seed-specific repression of GUS activity was observed in lines containing high copy numbers of the antisense gusA transgene. Suppression of GUS activity was correlated with the amounts of (-) sense gusA transcript detected and concomitantly with a decrease in gusA transcript levels. Furthermore, the amount of suppression of GUS activity was greatest during mid to late stages of seed development, when expression of the alpha' promoter is high. These results indicate that suppression of GUS activity is due to expression of the antisense gene.
Plant Mol Biol 1992 Dec
PMID:Seed-specific repression of GUS activity in tobacco plants by antisense RNA. 146 41

In an effort to improve the T-DNA-mediated transformation frequency of economically important crops, we investigated the possible enhancement effect of multiple copies of virG genes contained in Agrobacterium tumefaciens strains upon the transient transformation of celery, carrot and rice tissues. Four days after A. tumefaciens infection, we performed histochemical beta-glucuronidase (GUS) assays to determine the frequency of transient transformation of calli from celery and carrot, and explants from rice and celery. Additional copies of octopine- and agropine-type virG genes in A. tumefaciens strains containing an agropine-type Ti-plasmid enhanced the frequency of transient transformation of celery and rice. This enhancement ranged from 25% to five-fold, depending upon the source of the virG gene and the plant tissues inoculated. For both rice and celery, we observed a greater enhancement of transformation using A. tumefaciens strains containing additional copies of an octopine-type virG gene than with strains harboring additional copies of an agropine-type virG gene. Multiple copies of virG genes contained in A. tumefaciens strains harboring a nopaline-type Ti-plasmid had a smaller enhancing effect upon the transformation of celery tissues, and no enhancing effect upon the transformation of rice. In contrast, we obtained a three-fold increase in the transient transformation frequency of carrot calli using an A. tumefaciens strain harboring a nopaline-type Ti-plasmid and additional copies of an octopine-type virG gene. Our results show that multiple copies of virG in A. tumefaciens can greatly enhance the transient transformation frequency of celery, carrot and rice tissues, and that this enhancement is influenced by both the type of Ti-plasmid harbored by A. tumefaciens and by the infected plant species.
Plant Mol Biol 1992 Dec
PMID:Multiple copies of virG enhance the transient transformation of celery, carrot and rice tissues by Agrobacterium tumefaciens. 146 42

Five new binary vectors have been constructed which have the following features: (1) different plant selectable markers including neomycin phosphotransferase (nptII), hygromycin phosphotransferase (hpt), dihydrofolate reductase (dhfr), phosphinothricin acetyl transferase (bar), and bleomycin resistance (ble); (2) selectable markers are located near the T-DNA left border and; (3) selectable marker and beta-glucuronidase (uidA) reporter genes are divergently organized for efficient expression, and can easily be removed or replaced as needed.
Plant Mol Biol 1992 Dec
PMID:New plant binary vectors with selectable markers located proximal to the left T-DNA border. 146 55

T-DNA vectors were constructed which carry a beta-glucuronidase (gusA) gene fused to the promoter of the nopaline synthase (nos) gene and the 3' end of the octopine synthase (ocs) gene. This reporter gene was cloned at different locations and orientations towards the right T-DNA border. For each construct, between 30 and 60 stably transformed calli were analysed for beta-glucuronidase activity. Depending on the T-DNA configuration, distinct populations of gusA-expressing calli were obtained. Placing the reporter gene in the middle of the T-DNA results in relatively low expression levels and a limited inter-transformant variability. Placing the gene with its promoter next to the right border led to an increase in both the mean activity and the variability level. With this construct, some of the calli expressed the gusA gene at levels four to five times higher than the mean. In all these series, at least 30% of the calli contained reporter gene activities that were less than half of the mean expression level. Separating the gusA gene from the right T-DNA border by an additional 3'-untranslated region, derived from the nos gene, resulted in an increase in the mean expression to a level almost four times higher than that of constructions carrying the reporter gene in the middle of the T-DNA. Moreover, the number of transformants with extremely low activities decreased by at least 50% and this resulted in significantly lower inter-transformant variability independently of the orientation of the reporter gene on the T-DNA.
Mol Gen Genet 1992 Nov
PMID:Effect of T-DNA configuration on transgene expression. 146 11

Promoter and terminator sequences from a range of species were tested for activity in the oomycetes, a group of lower fungi that bear an uncertain taxonomic affinity to other organisms and in which little is known of the sequences required for transcription. Transient assays, using the reporter gene beta-glucuronidase (GUS), were used to examine the function of these promoters and terminators in the plant pathogens Phytophthora infestans and P. megasperma f. sp. glycinea, and in the saprophytic water mold, Achlya ambisexualis. Oomycete promoters, isolated from the ham34 and hsp70 genes of Bremia lactucae and the actin gene of P. megasperma f. sp. glycinea, resulted in high levels of GUS accumulation in each of the three oomycetes. In contrast, little or no activity was detected when promoters from higher fungi (four ascomycetes and one basidiomycete), plants, and animals were tested. The terminator from the ham34 gene resulted in much higher levels of GUS accumulation than did others, although an oomycete terminator was not absolutely required for expression. Transcript mapping of RNA from stable transformants confirmed accurate initiation from the B. lactucae hsp70 promoter and termination within 3' ham34 sequences in P. infestans. Our results indicate that the transcriptional machinery of the oomycetes differs significantly from that of the higher fungi, but that enough conservation exists within the class to allow vectors developed from one oomycete species to be used for others.
Mol Gen Genet 1992 Jul
PMID:Regulatory sequences for expressing genes in oomycete fungi. 149 76


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