Transformation methods for halophilic archaebacteria

1989 ◽  
Vol 35 (1) ◽  
pp. 148-152 ◽  
Author(s):  
Steven W. Cline ◽  
Wan L. Lam ◽  
Robert L. Charlebois ◽  
Leonard C. Schalkwyk ◽  
W. Ford Doolittle
Keyword(s):  
Molecular Weight ◽  
Plasmid Dna ◽  
Genomic Dna ◽  
Halobacterium Halobium ◽  
Dna Transformation ◽  
Dna Transfection ◽  
Phage Dna ◽  
Halophilic Archaebacteria ◽  
Transformation Methods ◽  
Halobacterium Volcanii

We present a practical description of polyethylene glycol mediated spheroplast transformation of Halobacterium halobium and Halobacterium volcanii. This method has been applied to phage DNA transfection, plasmid DNA transformation, and transformation with linear fragments of high molecular weight genomic DNA. Efficient spheroplast regeneration allows uncomplicated recovery of transformed progeny. Transformations can be performed equally well using fresh or frozen cell preparations. These methods should find application in molecular cloning, genetic fine mapping, and strain construction.Key words: archaebacteria, Halobacterium, transformation methods, spheroplast.

Cloning and expression of Xylella fastidiosa antigens in Escherichia coli and Erwinia stewartii

1989 ◽  
Vol 35 (4) ◽  
pp. 487-491 ◽  
Author(s):  
Paul H. Goodwin
Keyword(s):  
Escherichia Coli ◽  
Molecular Weight ◽  
Genomic Dna ◽  
Xylella Fastidiosa ◽  
Gene Bank ◽  
Cloning And Expression ◽  
Western Blots ◽  
E Coli ◽  
Cosmid Vector ◽  
Erwinia Stewartii

Xylella fastidiosa DNA, partially digested with Sau3A, was ligated into the cosmid vector, pUCD615. Approximately 4500 ampicillin-resistant Escherichia coli colonies were obtained. The frequency of complementation of leucine auxotrophy in transfected E. coli indicated that the cosmid gene bank was representative of X. fastidiosa genomic DNA. Colonies were lysed directly onto nitrocellulose membranes using a thermo-inducible λ lysogen and screened for expression of X. fastidiosa antigens. Approximately 16.5% of a random sample of clones were found to express X. fastidiosa antigens as determined by Western blots. These proteins comigrated with proteins of X. fastidiosa and ranged in molecular weight from 10 000 to 160 000. Conjugation of several of the plasmids into Erwinia stewartii resulted in expression of the similar molecular weight cloned proteins with similar levels of expression as in E. coli.Key words: Xylella fastidiosa, Pierce's disease, immunological clone screening, thermo-inducible lysogeny.

scholarly journals Structure and expression of genes for surface proteins in Paramecium.

1983 ◽  
Vol 3 (3) ◽  
pp. 466-474 ◽  
Author(s):  
J D Forney ◽  
L M Epstein ◽  
L B Preer ◽  
B M Rudman ◽  
D J Widmayer ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Restriction Enzyme ◽  
Genomic Dna ◽  
Gene Dosage ◽  
Surface Proteins ◽  
Paramecium Tetraurelia ◽  
Complementary Sequences ◽  
Expression Of Genes ◽  
Inactive Genes ◽  
Polyadenylated Rnas

Surface proteins from 11 antigenic types of Paramecium tetraurelia vary in molecular weight from 251,000 to 308,000. The size of a series of polyadenylated RNAs obtained from these types were correlated with the sizes of the proteins and judged to be the mRNAs for the proteins. The mRNAs were used to identify genomic DNA clones containing complementary sequences. The gene for antigen A was present in one copy per genome, and the data suggest that extensive introns were absent. When restriction enzyme digests of DNA from cultures of paramecia with active and inactive genes were probed with portions of the cloned genes, no evidence for rearrangements or changes in gene dosage was found.

scholarly journals A paradoxical synergism between Resveratrol and copper (II) with respect to degradation of DNA and RNA

F1000Research ◽  
2015 ◽  
Vol 4 ◽  
pp. 1145 ◽  
Author(s):  
Siddharth Subramaniam ◽  
Iqbal Vohra ◽  
Aishwarya Iyer ◽  
Naveen K Nair ◽  
Indraneel Mittra
Keyword(s):  
Synergistic Effect ◽  
Plasmid Dna ◽  
Genomic Dna ◽  
Oxygen Species ◽  
Molar Ratios ◽  
Dna Cleaving ◽  
Dna And Rna ◽  
Fixed Concentration ◽  
Anti Cancer ◽  
Low Levels

Resveratrol (R), a plant polyphenol, is known to reduce Cu (II) to Cu (I) generating reactive oxygen species that can cleave plasmid DNA. Here we report a surprising observation of a paradoxical synergistic effect between R and Cu whereby plasmid DNA cleaving / degrading activity of R-Cu increased progressively as the ratio of R to Cu was increased i.e., the concentration of Cu was successively reduced with respect to a fixed concentration R. Whereas cleavage of plasmid DNA occurred at low molar ratios of R to Cu, at higher ratios, complete degradation of DNA was achieved. By further increasing the ratio, whereby the concentration of Cu was reduced to very low levels, the DNA degrading activity of R-Cu was lost. This paradoxical synergistic effect is also seen with respect to eukaryotic genomic DNA and RNA. Since R-Cu may have anti-cancer and anti-viral activities, our findings may not only help to improve the therapeutic efficacy of R-Cu but also reduce its toxic side effects with the use of low concentration of Cu.

scholarly journals Optimization of high molecular weight DNA extraction methods in shrimp for a long-read sequencing platform

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e10340
Author(s):  
Pacharaporn Angthong ◽  
Tanaporn Uengwetwanit ◽  
Wirulda Pootakham ◽  
Kanchana Sittikankaew ◽  
Chutima Sonthirod ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Genomic Dna ◽  
Extraction Methods ◽  
Marine Organisms ◽  
High Quality ◽  
Sequencing Platform ◽  
Global Food Security ◽  
Long Read ◽  
Ctab Method ◽  
Global Food

Marine organisms are important to global food security as they are the largest source of animal proteins feeding mankind. Genomics-assisted aquaculture can increase yield while preserving the environment to ensure sufficient and sustainable production for global food security. However, only few high-quality genome sequences of marine organisms, especially shellfish, are available to the public partly because of the difficulty in the sequence assembly due to the complex nature of their genomes. A key step for a successful genome sequencing is the preparation of high-quality high molecular weight (HMW) genomic DNA. This study evaluated the effectiveness of five DNA extraction protocols (CTAB, Genomic-tip, Mollusc DNA, TIANamp Marine Animals DNA, and Sbeadex livestock kits) in obtaining shrimp HMW DNA for a long-read sequencing platform. DNA samples were assessed for quality and quantity using a Qubit fluorometer, NanoDrop spectrophotometer and pulsed-field gel electrophoresis. Among the five extraction methods examined without further optimization, the Genomic-tip kit yielded genomic DNA with the highest quality. However, further modifications of these established protocols might yield even better DNA quality and quantity. To further investigate whether the obtained genomic DNA could be used in a long-read sequencing application, DNA samples from the top three extraction methods (CTAB method, Genomic-tip and Mollusc DNA kits) were used for Pacific Biosciences (PacBio) library construction and sequencing. Genomic DNA obtained from Genomic-tip and Mollusc DNA kits allowed successful library construction, while the DNA obtained from the CTAB method did not. Genomic DNA isolated using the Genomic-tip kit yielded a higher number of long reads (N50 of 14.57 Kb) than those obtained from Mollusc DNA kits (N50 of 9.74 Kb). Thus, this study identified an effective extraction method for high-quality HMW genomic DNA of shrimp that can be applied to other marine organisms for a long-read sequencing platform.

Extracting high molecular weight genomic DNA from Saccharomyces cerevisiae

2018 ◽  
Author(s):  
Erwan DENIS ◽  
Erwan Denis ◽  
Sophie Sanchez ◽  
Barbara Mairey ◽  
Odette Beluche ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Weight ◽  
High Molecular Weight ◽  
Genomic Dna

Calcium Phosphate-Mediated Transfection of Cells with High-Molecular-Weight Genomic DNA

2019 ◽  
Vol 2019 (10) ◽  
pp. pdb.prot095448 ◽  
Author(s):  
Priti Kumar ◽  
Arvindhan Nagarajan ◽  
Pradeep D. Uchil
Keyword(s):  
Molecular Weight ◽  
Calcium Phosphate ◽  
High Molecular Weight ◽  
Genomic Dna

scholarly journals Procaryotic genomic DNA inhibits mammalian cell transformation.

1983 ◽  
Vol 3 (5) ◽  
pp. 956-959 ◽  
Author(s):  
J I Yoder ◽  
A T Ganesan
Keyword(s):  
Thymidine Kinase ◽  
Mammalian Cell ◽  
Genomic Dna ◽  
Cell Transformation ◽  
Phage Dna ◽  
Mouse Cells

Ltk- mouse cells were transformed to thymidine kinase prototrophy in the presence of carrier DNAs isolated from different organisms. Procaryotic genomic and phage DNA was consistently less effective as a carrier than was eucaryotic DNA. Mixing experiments indicate that DNA of procaryotic origin inhibits mammalian cell transformation.

scholarly journals β–Amino Ester Polymers Facilitate in Vivo DNA Transfection and Adjuvant Plasmid DNA Immunization

2005 ◽  
Vol 12 (1) ◽  
pp. 164-170 ◽  
Author(s):  
John R. Greenland ◽  
Huining Liu ◽  
David Berry ◽  
Daniel G. Anderson ◽  
Woong-Ki Kim ◽  
...  
Keyword(s):  
Plasmid Dna ◽  
Dna Immunization ◽  
Amino Ester ◽  
Dna Transfection
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