Demonstration of the indolepyruvate decarboxylase gene homologue in different auxin-producing species of the Enterobacteriaceae

1994 ◽  
Vol 40 (12) ◽  
pp. 1072-1076 ◽  
Author(s):  
Wolfgang Zimmer ◽  
Barbara Hundeshagen ◽  
Edith Niederau
Keyword(s):  
Polymerase Chain Reaction ◽  
Klebsiella Oxytoca ◽  
Pyruvate Decarboxylase ◽  
Enterobacter Agglomerans ◽  
Chain Reaction ◽  
Acetic Acid Production ◽  
Conserved Regions ◽  
Polymerase Chain ◽  
Gene Homologue ◽  
Indole 3 Acetic Acid

Different Enterobacteriaceae were assayed for their ability to produce the plant hormone indole-3-acetate with the aim to study the distribution of the indole-3-pyruvate pathway, which is known to be involved in the production of indole-3-acetate in a root-associated Enterobacter cloacae strain. Other E. cloacae strains, and also Enterobacter agglomerans strains, Pantoea agglomerans, Klebsiella aerogenes, and Klebsiella oxytoca were found to convert tryptophan into indole-3-acetate. As it was also intended to identify the conserved regions of the indole-3-pyruvate decarboxylase, which is involved in producing indole-3-acetate in the E. cloacae strain, oligonucleotide primers were synthesized for different regions of the corresponding gene. One pair of these primers allowed us to amplify a segment of the predicted size by the polymerase chain reaction with DNA of the seven different Enterobacteriaceae that produce indole-3-acetate. Segments of five strains were cloned and sequenced. All sequences showed significant homology to the indole-3-pyruvate decarboxylase gene. As in addition a positive DNA–DNA hybridization signal was detected in the seven strains using the E. cloacae or E. agglomerans segments as a probe, indole-3-acetate biosynthesis is suggested to be catalyzed via the indole-3-pyruvate pathway not only in E. cloacae but also in the other soil-living Enterobacteriaceae. Conserved regions were detected in the indole-3-decarboxylase by alignment of the now-available five different partial sequences. These regions should enable identification of the gene in other bacterial families or even in plants.Key words: indole-3-pyruvate decarboxylase, indole-3-acetic acid production, auxin, polymerase chain reaction, Enterobacteriaceae.

scholarly journals Structurally and functionally conserved regions of cytochrome P-450 reductase as targets for DNA amplification by the polymerase chain reaction. Cloning and nucleotide sequence of the Schizosaccharomyces pombe cDNA

1992 ◽  
Vol 287 (1) ◽  
pp. 195-200 ◽  
Author(s):  
J S Miles
Keyword(s):  
Polymerase Chain Reaction ◽  
Schizosaccharomyces Pombe ◽  
Binding Domain ◽  
Amino Acid Sequences ◽  
Chain Reaction ◽  
Conserved Regions ◽  
Reductase Gene ◽  
Degenerate Oligonucleotide ◽  
Polymerase Chain ◽  
Cytochrome P 450

1. Alignments of the available cytochrome P-450 reductase amino acid sequences, and comparison with the crystal structure of ferredoxin-NADP reductase, indicate that two highly conserved regions are of functional importance. 2. Degenerate oligonucleotide primers, based on these sequences, were used in the polymerase chain reaction to amplify a 309 bp fragment of the cytochrome P-450 reductase gene from Schizosaccharomyces pombe for use as an homologous probe. 3. A 2.6 kb cDNA was cloned from a lambda library, and sequencing revealed an open-reading frame of 2034 bp encoding a protein of M(r) 76774. This protein shares 38-41% identity with other eukaryotic cytochrome P-450 reductases, and 30% identity with that of Bacillus megaterium. 4. Comparison of the N-terminal FMN-binding domain with flavodoxin, and the C-terminal FAD- and NADP-binding domain with ferredoxin-NADP reductase, indicates the presence of several functionally conserved regions. 5. The Sc. pombe cytochrome P-450 reductase gene was shown to contain no introns.

scholarly journals First Canadian Outbreak of Enterobacteriaceae-ExpressingKlebsiella pneumoniaeCarbapenemase Type 3

2012 ◽  
Vol 23 (3) ◽  
pp. 117-120 ◽  
Author(s):  
Victor Leung ◽  
Vivian G Loo ◽  
Charles Frenette ◽  
Marc-Christian Domingo ◽  
Anne-Marie Bourgault ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Gel Electrophoresis ◽  
Horizontal Transmission ◽  
Klebsiella Oxytoca ◽  
Polymerase Chain Reaction Analysis ◽  
Pulsed Field Gel Electrophoresis ◽  
Chain Reaction ◽  
Pulsed Field ◽  
Vitek 2 ◽  
Polymerase Chain

BACKGROUND: Organisms expressingKlebsiella pneumoniaecarbapenemase (KPC) are found in several regions worldwide but are rarely detected in Canada. The first outbreak of KPC-expressing strains of Enterobacteriaceae clinical isolates in a university-affiliated hospital intensive care unit (ICU) in Canada is described.METHODS: Enterobacteriaceae isolates that were flagged by the Vitek 2 (bioMérieux, France) system as possible carbapenemase producers were subjected to the modified Hodge test. Modified Hodge test-positive organisms were analyzed by pulsed-field gel electrophoresis, tested for KPC and other beta-lactamase genes by polymerase chain reaction analysis and underwent subsequent nucleic acid sequencing. Antimicrobial susceptibility profiles were determined by Vitek 2 and Etest (bioMérieux, France). A chart review was conducted to establish epidemiological links.RESULTS: During the study period, 10 unique Enterobacteriaceae isolates expressing KPC were detected from nine ICU patients. Five patients had infections (three pneumonias, one surgical site infection, one urinary tract infection). Isolates includedEscherichia coli(5),Klebsiella oxytoca(2),Serratia marcescens(2) andCitrobacter freundii(1). Polymerase chain reaction analysis and sequencing confirmed the presence of KPC-3 in all isolates; four also carried TEM, two CTX-M and one CMY-2. The imipenem minimum inhibitory concentrations as determined by Etest ranged from 0.75 μg/mL to ≥32 μg/mL. Pulsed field gel electrophoresis clonal patterns and patient location in the ICU revealed presumptive horizontal transmission events.CONCLUSIONS: In the present study, Enterobacteriaceae isolates with KPC are emerging and can result in serious infections. The KPC gene can spread via plasmids to different genera of the Enterobacteriaceae family. The dissemination of KPC in Enterobacteriaceae and the consequences for treatment and infection control measures warrant a high degree of vigilance among clinicians and microbiologists.

scholarly journals Design and in silico validation of polymerase chain reaction primers to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Maria Júlia P. Davi ◽  
Selma M. B. Jeronimo ◽  
João P. M. S. Lima ◽  
Daniel C. F. Lanza
Keyword(s):  
Polymerase Chain Reaction ◽  
Severe Acute Respiratory Syndrome ◽  
In Silico ◽  
False Negative ◽  
Pcr Primers ◽  
Virus Genome ◽  
Chain Reaction ◽  
Multiple Sequence ◽  
Conserved Regions ◽  
Polymerase Chain

AbstractAccurate designing of polymerase chain reaction (PCR) primers targeting conserved segments in viral genomes is desirable for preventing false-negative results and decreasing the need for standardization across different PCR protocols. In this work, we designed and described a set of primers and probes targeting conserved regions identified from a multiple sequence alignment of 2341 Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) genomes from the Global Initiative on Sharing All Influenza Data (GISAID). We subsequently validated those primers and probes in 211,833 SARS-CoV-2 whole-genome sequences. We obtained nine systems (forward primer + reverse primer + probe) that potentially anneal to highly conserved regions of the virus genome from these analyses. In silico predictions also demonstrated that those primers do not bind to nonspecific targets for human, bacterial, fungal, apicomplexan, and other Betacoronaviruses and less pathogenic sub-strains of coronavirus. The availability of these primer and probe sequences will make it possible to validate more efficient protocols for identifying SARS-CoV-2.

scholarly journals Comparison of Mitochondrial DNA in Wolves and Coyotes in the Northern Rockies using the Polymerase Chain Reaction

1994 ◽  
Vol 18 ◽  
pp. 156-159
Author(s):  
Ernest Vyse
Keyword(s):  
Polymerase Chain Reaction ◽  
Mitochondrial Dna ◽  
National Park ◽  
Genetic Basis ◽  
Gene Activation ◽  
Evolutionary Analysis ◽  
Chain Reaction ◽  
Variable Regions ◽  
Conserved Regions ◽  
Polymerase Chain

The purpose of this study was the comparison of mitochondrial DNA (mtDNA) sequences from Northern Montana and Minnesota wolves to the extinct wolves of Yellowstone National Park (YNP). This comparison was intended to provide a genetic basis to identify potential wolf stocks for introduction into YNP. Unfortunately the only extinct YNP specimens available were dried tanned skins from the Smithsonian Museum of Natural History and YNP. The nucleic acids extracted from these skins was so badly degraded that no amplification of mtDNA was possible; so the critical comparisons could not be made. The control region of the mtDNA is 1100-1200 nucleotides long in vertebrates, contains sequences that control replication and gene activation and in terms of evolutionary analysis has the desired feature of both variable and conserved regions. The variable regions provide the genetic diversity that is the basis of taxonomic and evolutionary studies while the conserved regions provide the ability to synthesize short complementary oligonucleotides to prime the polymerase chain reaction (PCR) in the organisms studies.

scholarly journals Design and in silico validation of polymerase chain reaction primers to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)

2020 ◽  
Author(s):  
Daniel C. F. Lanza ◽  
João P. M. S. Lima ◽  
Selma M. B. Jerônimo
Keyword(s):  
Polymerase Chain Reaction ◽  
Severe Acute Respiratory Syndrome ◽  
In Silico ◽  
False Negative ◽  
Pcr Primers ◽  
Chain Reaction ◽  
Multiple Sequence ◽  
Conserved Regions ◽  
Polymerase Chain

Abstract Accurate designing of polymerase chain reaction (PCR) primers targeting conserved segments in viral genomes is desirable for preventing false negative results and decreasing the need for standardization across different PCR protocols. In this work, we designed and described a set of primers and probes targeting conserved regions identified from a multiple sequence alignment of 2341 Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) genomes from the Global Initiative on Sharing All Influenza Data (GISAID). Those primers and probes were subsequently validated in 3067 SARS-CoV-2 whole-genome sequences. From these analyses, we obtained nine systems (forward primer + reverse primer + probe) that potentially anneal to highly conserved regions of the virus genome. In silico predictions also demonstrated that those primers do not bind to nonspecific targets for human, bacterial, fungal, or apicomplexan sequences. The availability of these primer and probe sequences will make it possible to accelerate the beginning of in vitro tests in order to validate more efficient protocols for the identification of SARS-CoV-2.

High performance Nanogold™-in situ hybridzation and its use in the detection of hybridized and PCR-amplified microscopical preparations

1996 ◽  
Vol 54 ◽  
pp. 896-897
Author(s):  
G. W. Hacker ◽  
I. Zehbe ◽  
J. Hainfeld ◽  
A.-H. Graf ◽  
C. Hauser-Kronberger ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Messenger Rna ◽  
High Performance ◽  
Detection System ◽  
Direct Methods ◽  
Genetic Alterations ◽  
Chain Reaction ◽  
Protein Encoding ◽  
Polymerase Chain

In situ hybridization (ISH) with biotin-labeled probes is increasingly used in histology, histopathology and molecular biology, to detect genetic nucleic acid sequences of interest, such as viruses, genetic alterations and peptide-/protein-encoding messenger RNA (mRNA). In situ polymerase chain reaction (PCR) (PCR in situ hybridization = PISH) and the new in situ self-sustained sequence replication-based amplification (3SR) method even allow the detection of single copies of DNA or RNA in cytological and histological material. However, there is a number of considerable problems with the in situ PCR methods available today: False positives due to mis-priming of DNA breakdown products contained in several types of cells causing non-specific incorporation of label in direct methods, and re-diffusion artefacts of amplicons into previously negative cells have been observed. To avoid these problems, super-sensitive ISH procedures can be used, and it is well known that the sensitivity and outcome of these methods partially depend on the detection system used.

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