High-level bacterial cellulase accumulation in chloroplast-transformed tobacco mediated by downstream box fusions

2009 ◽  
Vol 102 (4) ◽  
pp. 1045-1054 ◽  
Author(s):  
Benjamin N. Gray ◽  
Beth A. Ahner ◽  
Maureen R. Hanson
Keyword(s):  
Downstream Box ◽  
High Level ◽  
Bacterial Cellulase ◽  
Transformed Tobacco

High-level heterologous expression and secretion inStreptomyces lividansof two major antigenic proteins fromMycobacterium tuberculosis

2002 ◽  
Vol 48 (1) ◽  
pp. 43-48 ◽  
Author(s):  
Donald Tremblay ◽  
Johanne Lemay ◽  
Michel Gilbert ◽  
Yvan Chapdelaine ◽  
Claude Dupont ◽  
...  
Keyword(s):  
Amino Acids ◽  
Mycobacterium Tuberculosis ◽  
Recombinant Proteins ◽  
Signal Sequence ◽  
Culture Conditions ◽  
Extracellular Proteins ◽  
Protein Sequence Analysis ◽  
Downstream Box ◽  
High Yields ◽  
High Level

Two major antigens from Mycobacterium tuberculosis were produced by Streptomyces lividans as secreted extracellular proteins. An expression-secretion vector had been constructed that contained the promoter of xylanase A and the signal sequence of cellulase A. The latter contained two initiation codons preceded by a Shine-Dalgarno sequence plus eight nucleotides complementary to the 16S rRNA. The genes encoding the 38-kDa (Rv0934) and 19-kDa (Rv3763) proteins, respectively, were amplified by polymerase chain reaction and cloned into that vector. The recombinant proteins were then purified from the culture supernatants of the clones. The yields after purification were 80 mg/L for the 38-kDa protein and 200 mg/L for the 19-kDa protein. Sequence analysis of the N-terminal sequences showed a deletion of seven or eight amino acids for the 38-kDa protein, while in the 19-kDa protein 22 or 23 amino acids were lost, as compared with the respective wild-type proteins. However, the 19 kDa recombinant protein had the same N-terminal sequence as the one recovered from the M. tuberculosis culture supernatant. The high yields obtained for these two proteins demonstrated the potential of S. lividans as an alternative host for the production of recombinant proteins from M. tuberculosis. The culture conditions have yet to be worked out to minimize proteolytic degradation and to recover intact products.Key words: streptomycetes, downstream box, signal peptide, protein secretion, Mycobacterium tuberculosis.

scholarly journals Correction to: A downstream box fusion allows stable accumulation of a bacterial cellulase in Chlamydomonas reinhardtii chloroplasts

2019 ◽  
Vol 12 (1) ◽  
Author(s):  
Lubna V. Richter ◽  
Huijun Yang ◽  
Mohammad Yazdani ◽  
Maureen R. Hanson ◽  
Beth A. Ahner
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Original Version ◽  
Calculation Error ◽  
Downstream Box ◽  
Order Of Magnitude ◽  
Bacterial Cellulase

In the original version of the article [1], a calculation error resulted in a 3-order of magnitude mistake for the y-axis of the data reported in Fig. 5c and d.

scholarly journals A downstream box fusion allows stable accumulation of a bacterial cellulase in Chlamydomonas reinhardtii chloroplasts

2018 ◽  
Vol 11 (1) ◽  
Author(s):  
Lubna V. Richter ◽  
Huijun Yang ◽  
Mohammad Yazdani ◽  
Maureen R. Hanson ◽  
Beth A. Ahner
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Downstream Box ◽  
Bacterial Cellulase

High Level Production of δ-Guaiene, a Bicyclic Sesquiterpene Accumulated in Agarwood, by Co-expression of δ-Guaiene Synthase and Farnesyl Diphosphate Synthase Genes in Tobacco BY-2 Cells

2018 ◽  
Vol 13 (1) ◽  
pp. 1934578X1801300
Author(s):  
Takahiro Kato ◽  
Futoshi Taura ◽  
Jung-Bum Lee ◽  
Fumiya Kurosaki
Keyword(s):  
Farnesyl Diphosphate ◽  
Farnesyl Diphosphate Synthase ◽  
Suitable Host ◽  
Tobacco Cells ◽  
Culture Volume ◽  
Appreciable Increase ◽  
Genes Encoding ◽  
High Level ◽  
Level Production ◽  
Transformed Tobacco

Two genes encoding δ-guaiene synthase ( GS) and farnesyl diphosphate synthase ( FPS) involved in δ-guaiene biosynthesis in Aquilaria microcarpa were co-expressed in tobacco ( Nicotiana tabacum) BY-2 cells by Agrobacterium -mediated transformation. GC-MS analysis revealed that the transformed tobacco cells liberated δ-guaiene, and the compound was found in the headspace of the culture but not accumulated either in the medium or in the cells. Tobacco cells transformed by solely GS produced 0.2 mg δ-guaiene /L culture, however, concentration of the compound elevated to 0.6 – 5.9 mg/L when GS and FPS were co-expressed in the cells. The stirring efficiency of the cell suspension was improved by the reduction of the culture volume in the vials, and this resulted in an appreciable increase in δ-guaiene content to the level of 102 mg/L culture. Addition of mevalonolactone as the precursor markedly activated δ-guaiene production, and content of the compound elevated to more than 400 mg/L culture. These results strongly suggested that tobacco BY-2 is a suitable host to construct the bio-production system of sesquiterpene compounds, and co-expression of FPS and appropriate sesquiterpene synthase genes in the cells should be the good strategy to establish the highly productive platform of this class of compounds.

An efficient downstream box fusion allows high-level accumulation of active bacterial beta-glucosidase in tobacco chloroplasts

2011 ◽  
Vol 76 (3-5) ◽  
pp. 345-355 ◽  
Author(s):  
Benjamin N. Gray ◽  
Huijun Yang ◽  
Beth A. Ahner ◽  
Maureen R. Hanson
Keyword(s):  
Downstream Box ◽  
Beta Glucosidase ◽  
High Level

Transcription factor/DNA interactions visualized by electron spectroscopic imaging

1992 ◽  
Vol 50 (1) ◽  
pp. 450-451
Author(s):  
David P. Bazett-Jones ◽  
Mark L. Brown
Keyword(s):  
Transcription Factor ◽  
Dna Sequences ◽  
Transcription Initiation ◽  
Molecular Mechanisms ◽  
Phosphorus Content ◽  
Spectroscopic Imaging ◽  
Electron Spectroscopic Imaging ◽  
Dna Backbone ◽  
Two Parameters ◽  
High Level

A multisubunit RNA polymerase enzyme is ultimately responsible for transcription initiation and elongation of RNA, but recognition of the proper start site by the enzyme is regulated by general, temporal and gene-specific trans-factors interacting at promoter and enhancer DNA sequences. To understand the molecular mechanisms which precisely regulate the transcription initiation event, it is crucial to elucidate the structure of the transcription factor/DNA complexes involved. Electron spectroscopic imaging (ESI) provides the opportunity to visualize individual DNA molecules. Enhancement of DNA contrast with ESI is accomplished by imaging with electrons that have interacted with inner shell electrons of phosphorus in the DNA backbone. Phosphorus detection at this intermediately high level of resolution (≈lnm) permits selective imaging of the DNA, to determine whether the protein factors compact, bend or wrap the DNA. Simultaneously, mass analysis and phosphorus content can be measured quantitatively, using adjacent DNA or tobacco mosaic virus (TMV) as mass and phosphorus standards. These two parameters provide stoichiometric information relating the ratios of protein:DNA content.

Advances in Stem Instrumentation for Biology

1990 ◽  
Vol 48 (1) ◽  
pp. 362-363
Author(s):  
J. S. Wall
Keyword(s):  
Scanning Transmission Electron Microscope ◽  
Carbon Films ◽  
Specimen Preparation ◽  
Material Deposition ◽  
Active User ◽  
Percent Improvement ◽  
Scanning Transmission ◽  
The Moment ◽  
Film Substrate ◽  
High Level

The forte of the Scanning transmission Electron Microscope (STEM) is high resolution imaging with high contrast on thin specimens, as demonstrated by visualization of single heavy atoms. of equal importance for biology is the efficient utilization of all available signals, permitting low dose imaging of unstained single molecules such as DNA.Our work at Brookhaven has concentrated on: 1) design and construction of instruments optimized for a narrow range of biological applications and 2) use of such instruments in a very active user/collaborator program. Therefore our program is highly interactive with a strong emphasis on producing results which are interpretable with a high level of confidence.The major challenge we face at the moment is specimen preparation. The resolution of the STEM is better than 2.5 A, but measurements of resolution vs. dose level off at a resolution of 20 A at a dose of 10 el/A2 on a well-behaved biological specimen such as TMV (tobacco mosaic virus). To track down this problem we are examining all aspects of specimen preparation: purification of biological material, deposition on the thin film substrate, washing, fast freezing and freeze drying. As we attempt to improve our equipment/technique, we use image analysis of TMV internal controls included in all STEM samples as a monitor sensitive enough to detect even a few percent improvement. For delicate specimens, carbon films can be very harsh-leading to disruption of the sample. Therefore we are developing conducting polymer films as alternative substrates, as described elsewhere in these Proceedings. For specimen preparation studies, we have identified (from our user/collaborator program ) a variety of “canary” specimens, each uniquely sensitive to one particular aspect of sample preparation, so we can attempt to separate the variables involved.

A Questionnaire Survey of Current Rehabilitation Practices for Adults With Normal Hearing Sensitivity Who Experience Auditory Difficulties

2020 ◽  
Vol 29 (4) ◽  
pp. 738-761
Author(s):  
Tess K. Koerner ◽  
Melissa A. Papesh ◽  
Frederick J. Gallun
Keyword(s):  
Hearing Aids ◽  
Questionnaire Survey ◽  
Patient Population ◽  
Clinical Care ◽  
Normal Hearing ◽  
Auditory Training ◽  
Training Methods ◽  
Hearing Sensitivity ◽  
Considerable Benefit ◽  
High Level

Purpose A questionnaire survey was conducted to collect information from clinical audiologists about rehabilitation options for adult patients who report significant auditory difficulties despite having normal or near-normal hearing sensitivity. This work aimed to provide more information about what audiologists are currently doing in the clinic to manage auditory difficulties in this patient population and their views on the efficacy of recommended rehabilitation methods. Method A questionnaire survey containing multiple-choice and open-ended questions was developed and disseminated online. Invitations to participate were delivered via e-mail listservs and through business cards provided at annual audiology conferences. All responses were anonymous at the time of data collection. Results Responses were collected from 209 participants. The majority of participants reported seeing at least one normal-hearing patient per month who reported significant communication difficulties. However, few respondents indicated that their location had specific protocols for the treatment of these patients. Counseling was reported as the most frequent rehabilitation method, but results revealed that audiologists across various work settings are also successfully starting to fit patients with mild-gain hearing aids. Responses indicated that patient compliance with computer-based auditory training methods was regarded as low, with patients generally preferring device-based rehabilitation options. Conclusions Results from this questionnaire survey strongly suggest that audiologists frequently see normal-hearing patients who report auditory difficulties, but that few clinicians are equipped with established protocols for diagnosis and management. While many feel that mild-gain hearing aids provide considerable benefit for these patients, very little research has been conducted to date to support the use of hearing aids or other rehabilitation options for this unique patient population. This study reveals the critical need for additional research to establish evidence-based practice guidelines that will empower clinicians to provide a high level of clinical care and effective rehabilitation strategies to these patients.

Cloning and high level expression of Cynodon dactylon (Bermuda grass) pollen profilin (Cyn d 12) in Escherichia coli: purification and characterization of the allergen

1997 ◽  
Vol 27 (11) ◽  
pp. 1307-1313 ◽  
Author(s):  
J. A. ASTURIAS ◽  
M. C. ARILLA ◽  
N. GOMEZ-BAYON ◽  
J. MARTINEZ ◽  
A. MARTINEZ ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Grass Pollen ◽  
Cynodon Dactylon ◽  
Bermuda Grass ◽  
Purification And Characterization ◽  
High Level Expression ◽  
Bermuda Grass Pollen ◽  
D 12 ◽  
High Level
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