Isolation and characterisation of microsatellite loci in the bush stone-curlew (Burhinus grallarius), a declining Australian bird

2013 ◽  
Vol 61 (6) ◽  
pp. 421
Author(s):  
Robert A. B. Mason ◽  
Catherine Price ◽  
Walter E. Boles ◽  
Karen-Anne Gray ◽  
Edwina Rickard ◽  
...  
Keyword(s):  
Microsatellite Loci ◽  
Allelic Diversity ◽  
Pcr Amplification ◽  
Habitat Modification ◽  
Microsatellite Repeat ◽  
Gene Encoding ◽  
Repeat Sequences ◽  
Introduced Predators ◽  
Ground Nesting

The bush stone-curlew (Burhinus grallarius Latham), a ground-nesting nocturnal bird, is endangered in southern Australia due to habitat modification and introduced predators. To provide tools for conservation, ecological and behavioural studies, we isolated variable microsatellite repeat sequences and designed primers for PCR amplification in this species. Primer pairs were developed and levels of diversity were assessed for eight microsatellite loci, including one locus linked to the gene encoding Microtubule-Associated Protein 2, a protein important for behavioural imprinting in birds, and one sex-linked locus. Isolated loci contained allelic diversity of between 5 and 17 alleles.

scholarly journals Plasmid-Encoded Metallo-β-Lactamase (IMP-6) Conferring Resistance to Carbapenems, Especially Meropenem

2001 ◽  
Vol 45 (5) ◽  
pp. 1343-1348 ◽  
Author(s):  
Hisakazu Yano ◽  
Akio Kuga ◽  
Ryoichi Okamoto ◽  
Hidero Kitasato ◽  
Toshimitsu Kobayashi ◽  
...  
Keyword(s):  
Point Mutation ◽  
Antimicrobial Agents ◽  
Pcr Amplification ◽  
Penicillin G ◽  
Gene Encoding ◽  
Mature Enzyme ◽  
Dna Fragment ◽  
Lactamase Gene ◽  
Reduced Activity ◽  
Northern Japan

ABSTRACT In 1996, Serratia marcescens KU3838 was isolated from the urine of a patient with a urinary tract infection at a hospital in northern Japan and was found to contain the plasmid pKU501. Previously, we determined that pKU501 carries bla IMP and the genes for TEM-1-type β-lactamases as well as producing both types of β-lactamases (H. Yano, A. Kuga, K. Irinoda, R. Okamoto, T. Kobayashi, and M. Inoue, J. Antibiot. 52:1135–1139, 1999). pKU502 is a recombinant plasmid that contains a 1.5-kb DNA fragment, including the metallo-β-lactamase gene, and is obtained by PCR amplification of pKU501. The sequence of the metallo-β-lactamase gene in pKU502 was determined and revealed that this metallo-β-lactamase gene differed from the gene encoding IMP-1 by one point mutation, leading to one amino acid substitution: 640-A in the base sequence of the IMP-1 gene was replaced by G, and Ser-196 was replaced by Gly in the mature enzyme. This enzyme was designated IMP-6. The strains that produced IMP-6 were resistant to carbapenems. The MICs of panipenem and especially meropenem were higher than the MIC of imipenem for these strains. The k cat/Km value of IMP-6 was about sevenfold higher against meropenem than against imipenem, although the MIC of meropenem for KU1917, which produced IMP-1, was lower than that of imipenem, and the MIC of panipenem was equal to that of imipenem. These results support the hypothesis that IMP-6 has extended substrate profiles against carbapenems. However, the activity of IMP-6 was very low against penicillin G and piperacillin. These results suggest that IMP-6 acquired high activity against carbapenems, especially meropenem, via the point mutation but in the process lost activity against penicillins. Although IMP-6 has reduced activity against penicillins due to this point mutation, pKU501 confers resistance to a variety of antimicrobial agents because it also produces TEM-1-type enzyme.

scholarly journals Autonomous assembly of synthetic oligonucleotides built from an expanded DNA alphabet. Total synthesis of a gene encoding kanamycin resistance

2014 ◽  
Vol 10 ◽  
pp. 2348-2360 ◽  
Author(s):  
Kristen K Merritt ◽  
Kevin M Bradley ◽  
Daniel Hutter ◽  
Mariko F Matsuura ◽  
Diane J Rowold ◽  
...  
Keyword(s):  
Solid Phase ◽  
Pcr Amplification ◽  
Kanamycin Resistance ◽  
Dna Fragments ◽  
Gene Encoding ◽  
Synthetic Dna ◽  
Information Density ◽  
Letter Alphabet ◽  
New Strategy ◽  
Autonomous Assembly

Background: Many synthetic biologists seek to increase the degree of autonomy in the assembly of long DNA (L-DNA) constructs from short synthetic DNA fragments, which are today quite inexpensive because of automated solid-phase synthesis. However, the low information density of DNA built from just four nucleotide “letters”, the presence of strong (G:C) and weak (A:T) nucleobase pairs, the non-canonical folded structures that compete with Watson–Crick pairing, and other features intrinsic to natural DNA, generally prevent the autonomous assembly of short single-stranded oligonucleotides greater than a dozen or so. Results: We describe a new strategy to autonomously assemble L-DNA constructs from fragments of synthetic single-stranded DNA. This strategy uses an artificially expanded genetic information system (AEGIS) that adds nucleotides to the four (G, A, C, and T) found in standard DNA by shuffling hydrogen-bonding units on the nucleobases, all while retaining the overall Watson–Crick base-pairing geometry. The added information density allows larger numbers of synthetic fragments to self-assemble without off-target hybridization, hairpin formation, and non-canonical folding interactions. The AEGIS pairs are then converted into standard pairs to produce a fully natural L-DNA product. Here, we report the autonomous assembly of a gene encoding kanamycin resistance using this strategy. Synthetic fragments were built from a six-letter alphabet having two AEGIS components, 5-methyl-2’-deoxyisocytidine and 2’-deoxyisoguanosine (respectively S and B), at their overlapping ends. Gaps in the overlapped assembly were then filled in using DNA polymerases, and the nicks were sealed by ligase. The S:B pairs in the ligated construct were then converted to T:A pairs during PCR amplification. When cloned into a plasmid, the product was shown to make Escherichia coli resistant to kanamycin. A parallel study that attempted to assemble similarly sized genes with optimally designed standard nucleotides lacking AEGIS components gave successful assemblies of up to 16 fragments, but generally failed when larger autonomous assemblies were attempted. Conclusion: AEGIS nucleotides, by increasing the information density of DNA, allow larger numbers of DNA fragments to autonomously self-assemble into large DNA constructs. This technology can therefore increase the size of DNA constructs that might be used in synthetic biology.

scholarly journals Characterization of Duck Microsatellite Repeat Sequences

2001 ◽  
Vol 35 (4) ◽  
pp. 217-219 ◽  
Author(s):  
Hideaki TAKAHASHI ◽  
Masahiro SATOH ◽  
Mitsuru MINEZAWA ◽  
Tresnawati PURWADARIA ◽  
Hardi PRASETYO
Keyword(s):  
Microsatellite Repeat ◽  
Repeat Sequences

scholarly journals ISOLASI FRAGMEN GEN PENYANDI PUTRESIN N-METILTRANSFERASE DAN QUINOLINAT FOSFORIBOSILTRANSFERASE ASAL TEMBAKAU LOKAL TEMANGGUNG (Nicotiana tabacum)

2020 ◽  
Vol 17 (3) ◽  
pp. 109 ◽  
Author(s):  
SESANTI BASUKI ◽  
NURHAJATI AA MATTJIK ◽  
SUWARSO SUWARSO ◽  
DESTA WIRNAS ◽  
SUDARSONO SUDARSONO
Keyword(s):  
Dna Sequences ◽  
Pcr Amplification ◽  
Data Bank ◽  
Degenerate Primer ◽  
Gene Encoding ◽  
Methyl Transferase ◽  
Dna Database ◽  
Genes Encoding ◽  
Tobacco Cultivar ◽  
Dna Template

<p>ABSTRAK</p><p>Upaya untuk menurunkan kandungan nikotin merupakan salah satuprioritas utama penelitian tembakau. Nikotin adalah senyawa alkaloidutama berpotensi dikonversi menjadi senyawa nor-nikotin yang bersifatkarsinogen. Gen PMT sebagai penyandi enzim putresin n-metiltransferase(PMT) dan gen QPT - penyandi enzim quinolinat fosforibosiltransferase(QPT) merupakan dua gen kunci yang berperan penting pada proses bio-sintesis nikotin. Penelitian ini bertujuan untuk mengisolasi potongan genPMT dan QPT asal tembakau lokal Indonesia, mengkarakterisasi danmenganalisis runutan DNA-nya. Tahapan penelitian dimulai dengan me-rancang primer degenerate berdasarkan informasi yang ada di pangkalandata Bank Gen NCBI (National Centre for Biotechnology Information),mengamplifikasi PCR menggunakan templat DNA genomik tembakaulokal cv. Sindoro1, mengklon potongan DNA hasil PCR dan menentukanrunutan DNA-nya. Hasil penelitian menunjukkan dari dua belas pasangprimer degenerate yang dirancang, hanya dua pasang primer yang meng-hasilkan potongan DNA hasil amplifikasi PCR, yaitu pasangan primerPMt-7 (F &amp; R) untuk gen PMT dan primer QPt-3 (F &amp; R) untuk gen QPT.Setelah dilakukan penentuan runutan DNA-nya, amplikon yang didapatdari hasil PCR dengan pasangan primer PMt-7 sebesar 1418 bp, sedangkanuntuk primer QPt-3 sebesar 205 bp. Runutan DNA gen PMT dan gen QPTasal tembakau lokal cv. Sindoro1 mempunyai tingkat kesamaan yang ting-gi dengan gen PMT dan gen QPT asal tembakau lainnya yang ada dipangkalan data Bank Gen NCBI.</p><p>Kata kunci : Gen PMT, gen QPT, lintasan biosintesis nikotin, perunutanDNA, amplifikasi PCR, primer degenerate</p><p>ABSTRACT</p><p>Isolation of Genes encoding Putrescine N-Methyl-transferase and Quinolinat Phosphoribosyl transferasederived from Temanggung Tobacco Cultivar (Nicotianatabacum)</p><p>Reduction of nicotine content is one of the major objective intobacco research. Nicotine is the main alcaloid compound that potentiallycould be converted into a carcinogenic compound (nor-nicotine). The PMTgene encoding putrescine N-methyl transferase (PMT) and the QPT gene -encoding quinolinate phosphoribosyl transferase (QPT) are the two keyenzymes involved in nicotine biosynthesis. The objectives of this researchwere to isolate PMT and QPT gene fragments originated from Indonesianlocal tobacco, to characterize, and to analyze their DNA sequences. Theresearch activities included: degenerate primer design based oninformation available in the GenBank DNA Database NCBI (NationalCentre for Biotechnology Information), PCR amplification usingdegenerate primer and genomic DNA template of a local tobacco cv.Sindoro1, clone the PCR amplified products, and determine their DNAnucleotide sequences. Results of the experiment indicated that from 12degenerate primer pairs synthesized, only two were able to yield positivePCR amplified products. These primer pairs were PMt-7 (F &amp; R primers)for PMT and QPt-3 (F &amp; R primers) for QPT. After DNA sequencing, theamplified DNA product amplified using PMt-7 degenerate primer pairswere 1418 bp, while that using QPt-3 primer pairs were only 205 bp.Nucleotide sequences of PMT or QPT gene fragments originated fromlocal tobacco cv. Sindoro1 showed a high nucleotide sequences identity ascompared to that of the respective genes from other tobacco species thatwere available in the GenBank DNA Database NCBI.</p><p>Key words: PMT gene, QPT gene, nicotine biosynthetic pathways, DNAsequencing, PCR amplification, degenerate primer</p>

scholarly journals Protocols for 16S rDNA Array Analyses of Microbial Communities by Sequence-Specific Labeling of DNA Probes

2003 ◽  
Vol 3 ◽  
pp. 578-584 ◽  
Author(s):  
Knut Rudi ◽  
Janneke Treimo ◽  
Hilde Nissen ◽  
Gerd Vegarud
Keyword(s):  
Microbial Communities ◽  
Pcr Amplification ◽  
Dna Probes ◽  
Dna Array ◽  
Gene Encoding ◽  
Variable Regions ◽  
Conserved Regions ◽  
Rdna Array ◽  
Array Hybridization ◽  
Array Analyses

Analyses of complex microbial communities are becoming increasingly important. Bottlenecks in these analyses, however, are the tools to actually describe the biodiversity. Novel protocols for DNA array-based analyses of microbial communities are presented. In these protocols, the specificity obtained by sequence-specific labeling of DNA probes is combined with the possibility of detecting several different probes simultaneously by DNA array hybridization. The gene encoding 16S ribosomal RNA was chosen as the target in these analyses. This gene contains both universally conserved regions and regions with relatively high variability. The universally conserved regions are used for PCR amplification primers, while the variable regions are used for the specific probes. Protocols are presented for DNA purification, probe construction, probe labeling, and DNA array hybridizations.

scholarly journals Characterization of Flagellotropic, Chi-Like Salmonella Phages Isolated from Thai Poultry Farms

Viruses ◽  
2019 ◽  
Vol 11 (6) ◽  
pp. 520 ◽  
Author(s):  
Preeda Phothaworn ◽  
Matthew Dunne ◽  
Rattaya Supokaivanich ◽  
Catherine Ong ◽  
Jiali Lim ◽  
...  
Keyword(s):  
Salmonella Enterica Serovar Typhimurium ◽  
Sequence Similarity ◽  
Phage Type ◽  
Pcr Amplification ◽  
Phylogenomic Analysis ◽  
Monophyletic Clade ◽  
Gene Encoding ◽  
Poultry Farms ◽  
Serovar Typhimurium

Despite a wealth of knowledge on Salmonella phages worldwide, little is known about poultry-associated Salmonella phages from Thailand. Here, we isolated 108 phages from Thai poultry farms that infect Salmonella enterica serovar Typhimurium. Phages STm101 and STm118 were identified as temperate Siphoviridae phages. Genome sequencing and analyses revealed these phages share approximately 96% nucleotide sequence similarity to phage SPN19, a member of the Chi-like virus genus. PCR amplification of the gene encoding capsid protein E of the Chi-like phage was positive for 50% of phage isolates, suggesting a predominance of this phage type among the sampled poultry farms. In addition to the flagella, two phages required the lipopolysaccharide to infect and lyse Salmonella. Furthermore, phylogenomic analysis demonstrated that phages STm101 and STm118 formed a monophyletic clade with phages isolated from Western countries, but not from closer isolated phages from Korea. However, further investigation and more phage isolates are required to investigate possible causes for this geographic distribution.

scholarly journals Expanding Allelic Diversity of Helicobacter pylori vacA

1998 ◽  
Vol 36 (9) ◽  
pp. 2597-2603 ◽  
Author(s):  
Leen-Jan van Doorn ◽  
Céu Figueiredo ◽  
Ricardo Sanna ◽  
Salvador Pena ◽  
Peter Midolo ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Direct Sequencing ◽  
Allelic Diversity ◽  
Pcr Primers ◽  
Epidemiological Studies ◽  
Sequence Alignments ◽  
Gene Encoding ◽  
The World ◽  
Probe Assay ◽  
H Pylori

The diversity of the gene encoding the vacuolating cytotoxin (vacA) of Helicobacter pylori was analyzed in 98 isolates obtained from different geographic locations. The studies focused on variation in the previously defined s and m regions ofvacA, as determined by PCR and direct sequencing. Phylogenetic analysis revealed the existence of four distinct types of s-region alleles: aside from the previously described s1a, s1b, and s2 allelic types, a novel subtype, designated s1c, was found. Subtype s1c was observed exclusively in isolates from East Asia and appears to be the major s1 allele in that part of the world. Three different allelic forms (m1, m2a, and m2b) were detected in the m region. On the basis of sequence alignments, universal PCR primers that allow effective amplification of the s and m regions from H. pyloriisolates from all over the world were defined. Amplimers were subsequently analyzed by reverse hybridization onto a line probe assay (LiPA) that allows the simultaneous and highly specific hybridization of the different vacA s- and m-region alleles and tests for the presence of the cytotoxin-associated gene (cagA). This PCR-LiPA method permits rapid analysis of the vacA andcagA status of H. pylori strains for clinical and epidemiological studies and will facilitate identification of any further variations.

scholarly journals Defining Blood Group Gene Reference Alleles by Long-Read Sequencing: Proof of Concept in the ACKR1 Gene Encoding the Duffy Antigens

2019 ◽  
Vol 47 (1) ◽  
pp. 23-32 ◽  
Author(s):  
Yann Fichou ◽  
Isabelle Berlivet ◽  
Gaëlle Richard ◽  
Christophe Tournamille ◽  
Lilian Castilho ◽  
...  
Keyword(s):  
Blood Group ◽  
Single Molecule ◽  
Pcr Amplification ◽  
Null Alleles ◽  
Sequencing Technology ◽  
Gene Encoding ◽  
Next Generation Sequencing Technology ◽  
Sequencing Technologies ◽  
Long Read ◽  
Long Range Pcr

Background: In the novel era of blood group genomics, (re-)defining reference gene/allele sequences of blood group genes has become an important goal to achieve, both for diagnostic and research purposes. As novel potent sequencing technologies are available, we thought to investigate the variability encountered in the three most common alleles of ACKR1, the gene encoding the clinically relevant Duffy antigens, at the haplotype level by a long-read sequencing approach. Materials and Methods: After long-range PCR amplification spanning the whole ACKR1 gene locus (∼2.5 kilobases), amplicons generated from 81 samples with known genotypes were sequenced in a single read by using the Pacific Biosciences (PacBio) single molecule, real-time (SMRT) sequencing technology. Results: High-quality sequencing reads were obtained for the 162 alleles (accuracy >0.999). Twenty-two nucleotide variations reported in databases were identified, defining 19 haplotypes: four, eight, and seven haplotypes in 46 ACKR1*01, 63 ACKR1*02, and 53 ACKR1*02N.01 alleles, respectively. Discussion: Overall, we have defined a subset of reference alleles by third-generation (long-read) sequencing. This technology, which provides a “longitudinal” overview of the loci of interest (several thousand base pairs) and is complementary to the second-generation (short-read) next-generation sequencing technology, is of critical interest for resolving novel, rare, and null alleles.

scholarly journals Rapid Depletion of Free Vancomycin in Medium in the Presence of β-Lactam Antibiotics and Growth Restoration in Staphylococcus aureus Strains with β-Lactam-Induced Vancomycin Resistance

2008 ◽  
Vol 53 (1) ◽  
pp. 63-68 ◽  
Author(s):  
Chie Yanagisawa ◽  
Hideaki Hanaki ◽  
Hidehito Matsui ◽  
Shinsuke Ikeda ◽  
Taiji Nakae ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Growth ◽  
Culture Medium ◽  
Pcr Amplification ◽  
Vancomycin Resistance ◽  
Gene Encoding ◽  
Vancomycin Concentration ◽  
Methicillin Resistant ◽  
Lactam Antibiotic ◽  
Absorption Level

ABSTRACT A class of methicillin-resistant Staphylococcus aureus strains shows vancomycin resistance in the presence of β-lactam antibiotics (β-lactam-induced VAN-resistant methicillin-resistant S. aureus [BIVR]). Two possible explanations may be offered: (i) vancomycin in culture medium is depleted, and (ii) the d-Ala-d-Ala terminal of the peptidoglycan network is replaced with d-Ala-d-lactate. We tested these hypotheses by quantifying free vancomycin in the medium through the course of cell growth and by PCR amplification of the van genes. Growth of the BIVR cells to an absorption level of ∼0.3 at 578 nm required about 24 h in the presence of vancomycin alone at the MIC (4.0 μg/ml). However, growth was achieved in only about 10 h when 1/1,000 to 1/2,000 the MIC of β-lactam antibiotic was added 2 h prior to the addition of vancomycin, suggesting that the β-lactams shortened the time to recovery from vancomycin-mediated growth inhibition. Free vancomycin in the culture medium decreased to 2.3 μg/ml in the first 8 h in the culture containing vancomycin alone, yet cell growth was undetectable. When the vancomycin concentration dropped below ∼1.5 μg/ml at 24 h, the cells began to grow. In the culture supplemented with the β-lactam 2 h prior to the addition of vancomycin, the drug concentration continuously dropped from 4 to 0.5 μg/ml in the first 8 h, and the cells began to grow at a vancomycin concentration of ∼1.7 μg/ml or at 4 h of incubation. The gene encoding the enzyme involved in d-Ala-d-lactate synthesis was undetectable. Based on these results, we concluded that BIVR is attributable mainly to a rapid depletion of vancomycin in the medium triggered or promoted by β-lactam antibiotics.

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