Characterization of the synthetic compatible solute homoectoine as a potent PCR enhancer

Schnoor, Michael; Voss, Peter; Cullen, Paul; Böking, Thomas; Galla, Hans‐Joachim; Galinski, Erwin A.; Lorkowski, Stefan

doi:10.1016/j.bbrc.2004.07.200

Cited by 86 publications

(57 citation statements)

References 23 publications

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“…L-Proline, 3-hydroxy-DL-proline, and 4-hydroxy-L-proline were obtained from Sigma-Aldrich. Two synthetic ectoine derivatives, homoectoine ((S)-4,5,6,7-tetrahydro-2-methyl-1H-(1, 3)-diazepine-4-carboxylic acid) (28) and DHMICA ((RS)-4,5-dihydro-2-methyl-imidazole-4-carboxylic acid) were kindly provided by E. A. Galinski (University of Bonn, Bonn, Germany). Anhydrotetracycline hydrochloride and d-desthiobiotin were purchased from IBA (Göttingen, Germany).…”

Section: Methodsmentioning

confidence: 99%

“…No EctD-mediated hydroxylation of L-proline was detected using a sensitive HPLC assay with either 3-or 4-hydroxy proline as reference standards for the HPLC analysis. Recently, two synthetic ectoine derivatives, homoectoine ((S)-4,5,6,7-tetrahydro-2-methyl-1H-(1,3)-diazepine-4-carboxylic acid) and DHMICA ((RS)-4,5-dihydro-2-methyl-imidazole-4-carboxylic acid), have been described that function as effective PCR enhancers (28). We also tested whether these compounds would serve as substrates for an EctD-mediated hydroxylation reaction, but this was not the case.…”

Section: Journal Of Biological Chemistry 31151mentioning

confidence: 99%

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Osmotically Induced Synthesis of the Compatible Solute Hydroxyectoine Is Mediated by an Evolutionarily Conserved Ectoine Hydroxylase

Bursy

Pierik

Pica

et al. 2007

Journal of Biological Chemistry

137

258

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By using natural abundance 13 C NMR spectroscopy, we investigated the types of compatible solutes synthesized in a variety of Bacilli under high salinity growth conditions. Glutamate, proline, and ectoine were the dominant compatible solutes synthesized by the various Bacillus species. The majority of the inspected Bacilli produced the tetrahydropyrimidine ectoine in response to high salinity stress, and a subset of these also synthesized a hydroxylation derivative of ectoine, 5-hydroxyectoine. In Salibacillus salexigens, a representative of the ectoine-and 5-hydroxyectoine-producing species, ectoine production was linearly correlated with the salinity of the growth medium and dependent on an ectABC biosynthetic operon. The formation of 5-hydroxyectoine was primarily a stationary growth phase phenomenon. The enzyme responsible for ectoine hydroxylation (EctD) was purified from S. salexigens to apparent homogeneity. The EctD protein was shown in vitro to directly hydroxylate ectoine in a reaction dependent on iron(II), molecular oxygen, and 2-oxoglutarate. We identified the structural gene (ectD) for the ectoine hydroxylase in S. salexigens. Northern blot analysis showed that the transcript levels of the ectABC and ectD genes increased as a function of salinity. Many EctDrelated proteins can be found in data base searches in various Bacteria. Each of these bacterial species also contains an ect-ABC ectoine biosynthetic gene cluster, suggesting that 5-hydroxyectoine biosynthesis strictly depends on the prior synthesis of ectoine. Our data base searches and the biochemical characterization of the EctD protein from S. salexigens suggest that the EctD-related ectoine hydroxylases are members of a new subfamily within the non-heme-containing, iron(II)-and 2-oxoglutarate-dependent dioxygenase superfamily (EC 1.14.11).

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Section: Methodsmentioning

confidence: 99%

Section: Journal Of Biological Chemistry 31151mentioning

confidence: 99%

Osmotically Induced Synthesis of the Compatible Solute Hydroxyectoine Is Mediated by an Evolutionarily Conserved Ectoine Hydroxylase

Bursy

Pierik

Pica

et al. 2007

Journal of Biological Chemistry

137

258

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“…Moreover (3), at least one organism (Halomonas Elongata) even produces different osmolytes at different temperatures. Moreover, it is well accepted that the ability of a molecule to act as biological osmolyte is mostly determined by the properties of the osmolyte/water binary system.…”

Section: Accepted M Manuscriptmentioning

confidence: 99%

Compatible solutes: Thermodynamic properties and biological impact of ectoines and prolines

Held

Neuhaus

Sadowski

2010

Biophysical Chemistry

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“…We attempted to address this problem by using recently commercialized thermostable reverse transcriptases for first strand cDNA synthesis reactions at 50°C (SuperScriptIII™) or 65°C (Thermo-X™), but with no success (data not shown). Thus, we turned to homoectoine, which has an exceptional ability to decrease the melting temperature of DNA and has been used as a potent PCR enhancer [17]. It seemed likely that homoectoine might be able to decrease the melting temperature of our extremely GC-rich mRNA template and, if it could do so without inactivating the reverse transcriptase, it should facilitate the reverse transcription step in our 5′-RACE method.…”

Section: Resultsmentioning

confidence: 99%

A new rapid amplification of cDNA ends method for extremely guanine plus cytosine-rich genes

Shi

Jarvis

2006

Analytical Biochemistry

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Rapid amplification of cDNA ends (RACE) is widely utilized to determine the 5′-and 3′-terminal nucleotide sequences of genes. Many different RACE methods have been developed to meet various requirements, but none address the difficult problems that arise when trying to isolate the ends of extremely GC-rich genes. In this study, we found that we were unable to isolate the correct 5′ or 3′ ends of an insect gene, which appeared to include extremely GC-rich sequences, using current RACE methods. Thus, we developed a new RACE method that can be used for this purpose. This new method entails first strand cDNA synthesis at 70°C with Thermo-X™ reverse transcriptase in the presence of homoectoine, followed by a polymerase chain reaction with 98°C denaturation steps and Phusion™ DNA polymerase in the presence of 1M betaine and 5% DMSO. The use of these conditions yielded 5′-and 3′-RACE products that were about 80% GC over 213 and 162 bp, respectively, and included shorter, internal regions of 82-89% GC.

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Characterization of the synthetic compatible solute homoectoine as a potent PCR enhancer

Cited by 86 publications

References 23 publications

Osmotically Induced Synthesis of the Compatible Solute Hydroxyectoine Is Mediated by an Evolutionarily Conserved Ectoine Hydroxylase

Osmotically Induced Synthesis of the Compatible Solute Hydroxyectoine Is Mediated by an Evolutionarily Conserved Ectoine Hydroxylase

Compatible solutes: Thermodynamic properties and biological impact of ectoines and prolines

A new rapid amplification of cDNA ends method for extremely guanine plus cytosine-rich genes

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