Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.2.1.31 (beta-glucuronidase)
7,680 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Using uidA (beta-glucuronidase; GUS) reporter gene constructs, the 5'-untranslated region (UTR) of the Chlamydomonas chloroplast rbcL gene was screened by deletion and mutational analysis for the presence of a promoter element that previous studies implied to reside within the first 63 base pairs of the UTR. Deleting a large segment of the rbcL 5'UTR in a 3'-->5' direction to position +36, changing the remaining 36 base pairs at the 5' end of the UTR, and increasing by five base pairs the distance between the rbcL 5'UTR and the basic promoter element located at position -10 did not abolish transcription from the basic rbcL promoter. It is concluded that the apparent loss of transcriptional activity found in earlier studies after deletion of sequences downstream of the transcription initiation site is due to the synthesis of very unstable transcripts that escape detection by Northern analysis and in vivo transcription assays. Chimeric rbcL:GUS transcripts containing changes in the beginning of the 5'UTR that affect RNA secondary structure are estimated to be at least 50 times less stable than rbcL:GUS transcripts containing the non-modified rbcL 5'UTR sequence.
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PMID:Changes in the 5'-untranslated region of the rbcL gene accelerate transcript degradation more than 50-fold in the chloroplast of Chlamydomonas reinhardtii. 1462 53

DNA damage occurs as a by-product of intrinsic cellular processes, like DNA replication, or as a consequence of exposure to genotoxic agents. Organisms have evolved multiple mechanisms to avoid, tolerate, or repair DNA lesions. To gain insight into these processes, we have isolated mutants hypersensitive to DNA-damaging agents in the green alga Chlamydomonas reinhardtii. One mutant, Ble-1, showed decreased survival when it was treated with methyl methanesulfonate (MMS), bleomycin, or hydrogen peroxide (H2O2) but behaved like the wild type when it was exposed to UVC irradiation. Ble-1 carries an extensive chromosomal deletion that includes the gene encoding cytosolic thioredoxin h1 (Trxh1). Transformation of Ble-1 with a wild-type copy of Trxh1 fully corrected the MMS hypersensitivity and partly restored the tolerance to bleomycin. Trxh1 also complemented a defect in the repair of MMS-induced DNA strand breaks and alkali-labile sites. In addition, a Trxh1-beta-glucuronidase fusion protein translocated to the nucleus in response to treatment with MMS. However, somewhat surprisingly, Trxh1 failed to correct the Ble-1 hypersensitivity to H2O2. Moreover, Trxh1 suppression by RNA interference in a wild-type strain resulted in enhanced sensitivity to MMS and DNA repair defects but no increased cytotoxicity to H2O2. Thioredoxins have been implicated in oxidative-stress responses in many organisms. Yet our results indicate a specific role of Chlamydomonas Trxh1 in the repair of MMS-induced DNA damage, whereas it is dispensable for the response to H2O2. These observations also suggest functional specialization among cytosolic thioredoxins since another Chlamydomonas isoform (Trxh2) does not compensate for the lack of Trxh1.
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PMID:Functional specialization of Chlamydomonas reinhardtii cytosolic thioredoxin h1 in the response to alkylation-induced DNA damage. 1570 88

The entire coding region of chlL, an essential chloroplast gene required for chlorophyll biosynthesis in the dark in Chlamydomonas reinhardtii, was precisely replaced by either the Klebsiella pneumoniae nifH (encoding the structural component of nitrogenase Fe protein) or the Escherichia coli uidA reporter gene encoding beta-glucuronidase. Homoplasmic nifH or uidA transformants were identified by Southern blots after selection on minimal medium plates for several generations. All the uidA transformants had the "yellow-in-the-dark" phenotype characteristic of chlL mutants, whereas homoplasmic nifH transformants exhibited a partial "green-in-the-dark" phenotype. NifH protein was detected in the nifH transformants but not in the wild-type strain by Western blotting. Fluorescence emission measurements also showed the existence of chlorophyll in the dark-grown nifH transformants, but not in the dark-grown uidA transformants. The nifH transplastomic form of C. reinhardtii that lacks the chlL gene can still produce chlorophyll in the dark, suggesting that the nifH product can at least partially substitute for the function of the putative "chlorophyll iron protein" encoded by chlL. Thus, introducing nitrogen fixation gene directly into a chloroplast genome is likely to be feasible and providing a possible way of engineering chloroplasts with functional nitrogenase. Notably, to introduce foreign genes without also introducing selective marker genes, a novel two-step chloroplast transformation strategy has been developed.
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PMID:The Klebsiella pneumoniae nitrogenase Fe protein gene (nifH) functionally substitutes for the chlL gene in Chlamydomonas reinhardtii. 1575 50

Chimeric genes for expression of a foreign gene in the Chlamydomonas reinhardtii chloroplast were constructed. These chimeric genes are composed of the promoter from chloroplast genes, rbcL, psbA, and atpA, 5'- and 3'-untranslated regions, and the Escherichia coli beta-glucuronidase (GUS) structural gene (uidA) as a foreign gene. Three types of chloroplast transformants (RG, PG, and AG), which contained the rbcL-uidA, psbA-uidA, and atpA-uidA chimeric genes integrated in the chloroplast genome, were generated by particle bombardment. The AG transformant grown under photoautotrophic conditions showed the highest GUS activity (130 nmol/min/mg protein) so far reported in C. reinhardtii, and the accumulated GUS protein accounted for 0.08% of the total soluble proteins. GUS activity in RG was 12% of that in AG, and no activity was detected in PG. We also measured the GUS activity from transformants grown under heterotrophic conditions, but the culture conditions made little difference in activity levels. The difference in the amount of accumulated GUS protein in the transformants was paralleled by the difference in the level of transcripts, and the pattern of gene expression was not the same as that of the endogenous genes in the chloroplast.
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PMID:Expression of a foreign gene in Chlamydomonas reinhardtii chloroplast. 1623 73

An exogenous gene, placed between the 5'-upstream regions of the Chlamydomonas reinhardtii chloroplast genes, rbcL or psbA, and the 3'-end of the rbcL gene, do not have the same expression pattern as endogenous genes in the C. reinhardtii chloroplast. Here, we chose four chloroplast genes, rbcL, psbA, psbD and atpA, and examine the effects of chloroplast gene coding regions on gene expression in C. reinhardtii. We constructed chimeric genes composed of the promoter, 5'- and 3'-untranslated regions, varying lengths of protein coding regions of the chloroplast genes, and the bacterial beta-glucuronidase (GUS) gene (uidA) as a reporter gene, and introduced into chloroplast genomes. The transformants, which contained the rbcL-uidA and psbA-uidA chimeric genes fused to the coding region of each gene, showed high expression of uidA mRNA as compared with the previously generated transformants, RG and PG, in which uidA was only fused to the promoter and 5'-UTR of each gene. The difference in the accumulation of uidA transcripts among the transformants was the result of different rates of transcription. This result indicates that the coding region is necessary for sufficient expression of rbcL and psbA. On the other hand, the psbD and atpA coding region portions did not affect chimeric gene expression.
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PMID:Effect of coding regions on chloroplast gene expression in Chlamydomonas reinhardtii. 1623 5

The 5' untranslated regions (UTR) of chloroplast mRNAs often contain regulatory sequences that control RNA stability and/or translation. The petD chloroplast mRNA in Chlamydomonas reinhardtii has three such essential regulatory elements in its 362-nt long 5' UTR. To further analyze these elements, we compared 5' UTR sequences from four Chlamydomonas species (C. reinhardtii, C. incerta, C. moewusii and C. eugametos) and five independent strains of C. reinhardtii. Overall, these petD 5' UTRs have relatively low sequence conservation across these species. In contrast, sequences of the three regulatory elements and their relative positions appear partially conserved. Functionality of the 5' UTRs was tested in C. reinhardtii chloroplasts using beta-glucuronidase reporter genes, and the nearly identical C. incerta petD functioned for mRNA stability and translation in C. reinhardtii chloroplasts while the more divergent C. eugametos petD did not. This identified what may be key features in these elements. We conclude that these petD regulatory elements, and possibly the corresponding trans-acting factors, function via mechanisms highly specific and surprisingly sensitive to minor sequence changes. This provides a new and broader perspective of these important regulatory sequences that affect photosynthesis in these algae.
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PMID:Regulatory sequences of orthologous petD chloroplast mRNAs are highly specific among Chlamydomonas species. 1651 63

We previously reported that high level of reporter gene transcript does not confer high amount of reporter protein accumulation in Chlamydomonas reinhardtii chloroplast transformants. Here, to further clarify the correlation between the level of transcript and protein accumulation, we generated the beta-glucuronidase (GUS) reporter gene (uidA) constructs with different potential for translation efficiency of the GUS protein by incorporating different 5' and 3'-untranslated regions of chloroplast genes into each construct. The relationship between mRNA stability and translation efficiency of the GUS reporter gene in each construct were then studied in C. reinhardtii stable chloroplast transformants. We found that sequences of the two nucleotides immediately upstream of the initial codon were important for translation efficiency and that transformants showing high GUS activity accumulated lower level of uidA transcripts than the transformants with low GUS activity. Moreover, accumulation and half-lives of these chimeric-uidA transcripts were increased to the same level in the presence of translation inhibitor. The accumulation and/or half-lives of several endogenous chloroplast transcripts were also increased by such inhibitor. Collectively, our results indicate that efficient translation destabilizes transcripts in chloroplasts of C. reinhardtii, and that there is an apparent negative correlation between protein accumulation and mRNA stability.
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PMID:Efficient translation destabilizes transcripts in chloroplasts of Chlamydomonas reinhardtii. 1693 48

This protocol describes the Agrobacterium tumefaciens-mediated nuclear transformation of a microalgae Chlamydomonas reinhardtii, using a gene construct carrying the genes coding for beta-glucuronidase (gus), green fluorescent protein (gfp), and hygromycin phosphotransferase (hpt). The transformation frequency with this protocol as revealed by hygromycin resistance was many fold higher (about 50-fold) than that of the commonly used glass bead method of transformation. The simplicity of Agrobacterium-mediated gene transfer and the high transformation frequency as well as the precision of T-DNA integration will enable further molecular dissection of this important model organism as well as other algal systems to understand basic plant metabolic processes as well as to exploit the systems for biotechnological applications.
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PMID:Green Alga (Chlamydomonas reinhardtii). 1703 83


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