Modification of flower colour by suppressing ß-ring carotene hydroxylase genes in Oncidium.

Oncidium 'Gower Ramsey' (Onc. GR) is a popular cut flower, but its colour is limited to bright yellow. The ß-ring carotene hydroxylase (BCH2) gene is involved in carotenoid biogenesis for pigment formation. However, the role of BCH2 in Onc. GR is poorly understood. Here, we investigated the functions of three BCH2 genes, BCH-A2, BCH-B2 and BCH-C2 isolated from Onc. GR, to analyse their roles in flower colour. RT-PCR expression profiling suggested that BCH2 was mainly expressed in flowers. The expression of BCH-B2 remained constant while that of BCH-A2 gradually decreased during flower development. Using Agrobacterium tumefaciens to introduce BCH2 RNA interference (RNAi), we created transgenic Oncidium plants with down-regulated BCH expression. In the transgenic plants, flower colour changed from the bright yellow of the wild type to light and white-yellow. BCH-A2 and BCH-B2 expression levels were significantly reduced in the transgenic flower lips, which make up the major portion of the Oncidium flower. Sectional magnification of the flower lip showed that the amount of pigmentation in the papillate cells of the adaxial epidermis was proportional to the intensity of yellow colouration. HPLC analyses of the carotenoid composition of the transgenic flowers suggested major reductions in neoxanthin and violaxanthin. In conclusion, BCH2 expression regulated the accumulation of yellow pigments in the Oncidium flower, and the down-regulation of BCH-A2 and BCH-B2 changed the flower colour from bright yellow to light and white-yellow.

Construction of a Der p2-transgenic plant for the alleviation of airway inflammation.

In clinical therapy, the amount of antigen administered to achieve oral tolerance for allergic diseases is large, and the cost is a major consideration. In this study, we used tobacco plants to develop a large-scale protein production system for allergen-specific immunotherapy, and we investigated the mechanisms of oral tolerance induced by a transgenic plant-derived antigen. We used plants (tobacco leaves) transgenic for the Dermatophagoides pteronyssinus 2 (Der p2) antigen to produce Der p2. Mice received total protein extract from Der p2 orally once per day over 6 days (days 0-2 and days 6-8). Mice were also sensitized and challenged with yeast-derived recombinant Der p2 (rDer p2), after which the mice were examined for airway hyper-responsiveness and airway inflammation. After sensitization and challenge with rDer p2, mice that were fed with total protein extracted from transgenic plants showed decreases in serum Der p2-specific IgE and IgG1 titers, decreased IL-5 and eotaxin levels in bronchial alveolar lavage fluid, and eosinophil infiltration in the airway. In addition, hyper-responsiveness was also decreased in mice that were fed with total protein extracted from transgenic plants, and CD4(+)CD25(+)Foxp3(+) regulatory T cells were significantly increased in mediastinal and mesenteric lymph nodes. Furthermore, splenocytes isolated from transgenic plant protein-fed mice exhibited decreased proliferation and increased IL-10 secretion after stimulation with rDer p2. The data here suggest that allergen-expressing transgenic plants could be used for therapeutic purposes for allergic diseases.

Fabrication of high-aspect-ratio Fresnel zone plates by e-beam lithography and electroplating.

The fabrication of gold Fresnel zone plates, by a combination of e-beam lithography and electrodeposition, with a 30 nm outermost zone width and a 450 nm-thick structure is described. The e-beam lithography process was implemented with a careful evaluation of applied dosage, tests of different bake-out temperatures and durations for the photoresist, and the use of a developer without methylisobutylketone. Electrodeposition with a pulsed current mode and with a specially designed apparatus produced the desired high-aspect-ratio nanostructures. The fabricated zone plates were examined by electron microscopy and their performances were assessed using a transmission X-ray microscope. The results specifically demonstrated an image resolution of 40 nm.

Binding affinity of T7 RNA polymerase to its promoter in the supercoiled and linearized DNA templates.

A promoter competition assay was used to measure the stability of T7 RNA polymerase with its promoter. When T7 RNA polymerase was incubated with GTP for 5 minutes before the elongation of transcription in either the supercoiled or linearized template, the half-lives of T7 RNA polymerase-DNA complexes were reduced. The transcription product increased when T7 RNA polymerase preincubated with GTP in the supercoiled DNA but not in the linearized DNA template. On the other hand, preincubation of ATP with T7 RNA polymerase decreased the stability of T7 RNA polymerase with the supercoiled DNA, but did not affect the stability of T7 RNA polymerase with the linearized DNA. Furthermore, the production of RNA transcript was increased when T7 RNA polymerase was incubated with ATP in either supercoiled or linearized template before transcription elongation. This study is important to understand the relationship between the transcription initiation and the stability of the ternary complex, and to produce large quantities of RNA transcript in vitro.

Transcription termination by bacteriophage T3 and SP6 RNA polymerases at Rho-independent terminators.

Transcription termination of T3 and SP6 DNA-dependent RNA polymerases have been studied on the DNA templates containing the threonine (thr) attenuator and its variants. The thr attenuator is from the regulatory region of the thr operon of Escherichia coli. The DNA template, encoding the thr attenuator, contains specific features of the rho-independent terminators. It comprises a dG + dC rich dyad symmetry, encoding a stem-and-loop RNA, which is followed by a poly(U) region at the 3'-end. Thirteen attenuator variants have been analyzed for their ability to terminate transcription and the results indicated that the structure as well as the sequence in the G + C rich region of RNA hairpin affect termination of both RNA polymerases. Also, a single base change in the A residues of the hairpin failed to influence termination, whereas changes in the poly(U) region significantly reduced the termination of both T3 and SP6 RNA polymerases. The requirement of a poly(U) region for termination by T3 and SP6 RNA polymerases was studied with nested deletion mutants in this region. The minimum number of U residues required for termination of SP6 and T3 RNA polymerases was five and three, respectively. However, both RNA polymerases needed at least eight U residues to reach a termination efficiency close to that achieved by wild-type thr attenuator encoding nine U residues. In addition, the orientation of the loop sequences of the RNA hairpin did not affect the transcription termination of either of the bacteriophage RNA polymerases.

Transcription termination at the Escherichia coli thra terminator by spinach chloroplast RNA polymerase in vitro is influenced by downstream DNA sequences.

We have investigated the mechanism of transcription termination in vitro by spinach chloroplast RNA polymerase using templates encoding variants of the transcription-termination structure (attenuator) of the regulatory region of the threonine (thr) operon of Escherichia coli. Fourteen sequence variants located within its d(G+C) stem-loop and d(A+T)-rich regions were studied. We found that the helix integrity in the stem-loop structure is necessary for termination but that its stability is not directly correlated with termination efficiency. The sequence of the G+C stem-loop itself also influences termination. Moreover, the dA template stretch at the 3' end of the terminator plays a major role in termination efficiency, but base pairing between the A and U tract of the transcript does not. From the studies using deletion variants and a series of mutants that alter the sequences immediately downstream from the transcription termination site, we found that termination of transcription by spinach chloroplast RNA polymerase was also modulated by downstream DNA sequences in a sequence-specific manner. The second base immediately following the poly(T) tract is crucial for determining the termination efficiency by chloroplast RNA polymerase, but not of the T7 or E.coli enzymes.

Selection of mutations altering specificity in restriction-modification enzymes using the bacteriophage P22 challenge-phage system.

A method for selecting mutants of site-specific DNA-binding proteins has been applied to the study of the EcoRI and RsrI restriction-modification enzymes. Catalytically inactive variants of both endonucleases are shown to function as pseudo-repressors in the bacteriophage P22 challenge-phage assay, and, upon further mutagenesis of the gene encoding R.EcoRI, a variant of that enzyme has been selected which appears to bind EcoRI-methylated GAATTC sequences to the exclusion of unmethylated sites: this specificity is the opposite of that belonging to the native enzyme. Variants of the EcoRI methylase have also been found that lack either catalytic activity or both binding and catalytic activities.

Transcription termination in vitro by bacteriophage T7 RNA polymerase. The role of sequence elements within and surrounding a rho-independent transcription terminator.

rho-Independent transcription terminators in Escherichia coli contain a dG+dC-rich dyad-symmetrical structure that encodes an RNA hairpin structure and an adjacent, downstream dA+dT-rich region which encodes uridines at the 3'-end of the transcript. In the threonine (thr) attenuator, there are at least six sequence segments in the DNA that might affect termination: the sequence upstream of the attenuator, the deoxythymidine-rich stretch immediately preceding the G+C-rich region, the G+C-rich region itself and its hairpin loop-encoding region, the deoxyadenosine tract following the G+C-rich region, and the following downstream sequence. Our previous studies (Jeng, S.-T., Gardner, J.F., and Gumport, R.I. (1990) J. Biol. Chem. 265, 3823-3830) indicate that both the stability and sequence of the RNA hairpin formed by the G+C-rich region and the length of the uridine tract encoded by the deoxyadenosine stretch influence the termination of T7 RNA polymerase in vitro. In this report, we demonstrate that the template deoxythymidine run upstream of the G+C-rich region, the loop-encoding segment, and the sequences upstream and downstream of the thr attenuator also affect termination. These results indicate that: 1) a deoxythymidine tract is not absolutely required for termination, but increasing the number of deoxythymidines from one to nine base pairs causes T7 RNA polymerase to terminate more efficiently; 2) a template with the natural loop sequence reversed results in a higher termination efficiency than one encoded by the the wild-type attenuator; 3) the termination of T7 RNA polymerase is affected by sequences both proximal and distal to the thr attenuator.

Transcription termination by bacteriophage T7 RNA polymerase at rho-independent terminators.

We have investigated the mechanism of transcription termination by T7 RNA polymerase using templates encoding variants of the transcription-termination structure (attenuator) of the regulatory region of the threonine (thr) operon of Escherichia coli. The thr attenuator comprises the following two distinct structural elements: a G + C-rich inverted repeat, which encodes an RNA hairpin structure, and A + T-rich regions, one of which contains a continuous sequence of template deoxyadenosine residues within which the transcription terminates. Fourteen attenuator variants were analyzed and we find that not only the hairpin structure itself but also its sequence influences termination. Furthermore, the formation of a hairpin in the RNA encoded by the A + T-rich regions of the attenuator is not mandatory for termination. A series of seven deletion variants that successively shorten the deoxyadenosine tract in the attenuator template were also analyzed. Results from these experiments indicate that complete readthrough occurs when there are four or fewer deoxyadenosine residues. With 5 template deoxyadenosine residues there is 5% termination increasing to 32% with 8 deoxyadenosines, the value produced by the wild-type attenuator. In addition, a comparison with E. coli RNA polymerase shows that T7 RNA polymerase requires a more perfect region of dyad symmetry and a longer deoxyadenosine tract than does the bacterial enzyme to terminate with maximum efficiency.