Polymorphism in the Dirofilaria immitis immunodominant antigen gene

1999 ◽  
Vol 73 (3) ◽  
pp. 265-272 ◽  
Author(s):  
K. Sugane ◽  
K. Nakayama ◽  
H. Kato
Keyword(s):  
Sequence Analysis ◽  
Larval Stage ◽  
Genomic Dna ◽  
Dirofilaria Immitis ◽  
Gene Encoding ◽  
Spliced Leader ◽  
Dna Library ◽  
Immunodominant Antigen ◽  
Genomic Dna Library ◽  
Rapid Amplification

Dg2, a gene encoding a 34 kDa immunodominant antigen of Dirofilaria immitis was cloned and demonstrated to be specifically expressed in the larval stage. In this study, a newly constructed genomic DNA library was screened by hybridization with Dg2. One of the resulting positive clones was similar to Dg2 in the structure of its exonic regions but different in number, position, size and sequence of introns. This was designated DgK. Full-length cDNA was isolated using the rapid amplification of cDNA ends (RACE) method to study the transcript corresponding to DgK. Sequence analysis revealed that the mRNA corresponding to DgK is trans-spliced during post-transcriptional processing because the 5′ end of the amplified cDNA contains seven nucleotides of the nematode-spliced leader (SL) sequence.

Complete structure of the gene encoding an immunodominant antigen of Dirofilaria immitis and larva-specific synthesis of primary transcript

1994 ◽  
Vol 68 (3) ◽  
pp. 259-264 ◽  
Author(s):  
S. Sun ◽  
K. Sugane
Keyword(s):  
Transcription Initiation ◽  
Genomic Dna ◽  
Dirofilaria Immitis ◽  
Signal Sequence ◽  
Open Reading Frame ◽  
Primary Transcript ◽  
Gene Encoding ◽  
Reading Frame ◽  
Immunodominant Antigen ◽  
Nuclease Mapping

AbstractThe complete gene encoding an immunodominant antigen of Dirofilaria immitis was isolated from a Charomid 9–36 genomic DNA library. This genomic DNA clone termed ‘Dg2’ was characterized by restriction mapping, DNA sequencing of the 5′ flanking region, the exon/intron boundaries and the polyadenylation addition site. The Dg2 with 4872 bp in length consisted of five exons interspersed with four introns. These exons reveal a single open reading frame followed by a long 3′ non-coding region of 1383 bp. The open reading frame of 969 bp encodes a polypeptide of 322 amino acids with a molecular weight of 34,400. The ATG translation initiation codon starts 22 nucleotides downstream from the 5′ end of the first exon. The polyadenylation signal sequence, AATAAA, is located at the 3′ end of the last exon. The transcription initiation site was determined by primer extension technique. S1 nuclease mapping analysis demonstrated that the primary transcript derived from Dg2 is synthesized in microfilariae but not in male or female adult worms. The result suggests that the stage-specific expression of Dg2 is regulated at the level of primary transcript.

Eimeria species: studies using rRNA and rDNA probes

Parasitology ◽  
1990 ◽  
Vol 101 (1) ◽  
pp. 1-6 ◽  
Author(s):  
J. Ellis ◽  
J. Bumstead
Keyword(s):  
Genomic Dna ◽  
Southern Hybridization ◽  
Eimeria Species ◽  
Small Subunit ◽  
Rrna Genes ◽  
Rdna Probe ◽  
Dna Library ◽  
Rdna Sequences ◽  
Genomic Dna Library

SUMMARYrRNA and a heterologous cloned rDNA probe have been used to detect the rRNA genes of Eimeria species which infe the chicken, and has allowed the isolation and preliminary characterization of cloned rDNA sequences from a genomic DNA library of Eimeria tenella. It is demonstrated that rRNA and rDNA probes can be used to identify individual Eimeria species by the restriction fragment patterns detected after Southern hybridization. In addition, studies have shown that the large and small subunit rRNAs are expressed throughout sporulation.

Genomic DNA Library Preparation for Resistance Gene Enrichment and Sequencing (RenSeq) in Plants

2014 ◽  
pp. 291-303 ◽  
Author(s):  
Florian Jupe ◽  
Xinwei Chen ◽  
Walter Verweij ◽  
Kamel Witek ◽  
Jonathan D. G. Jones ◽  
...  
Keyword(s):  
Resistance Gene ◽  
Genomic Dna ◽  
Library Preparation ◽  
Dna Library ◽  
Gene Enrichment ◽  
Genomic Dna Library

scholarly journals Polyphosphate:AMP Phosphotransferase as a Polyphosphate-Dependent Nucleoside Monophosphate Kinase in Acinetobacter johnsonii 210A

2005 ◽  
Vol 187 (5) ◽  
pp. 1859-1865 ◽  
Author(s):  
Toshikazu Shiba ◽  
Hiromichi Itoh ◽  
Atsushi Kameda ◽  
Keiju Kobayashi ◽  
Yumi Kawazoe ◽  
...  
Keyword(s):  
Genomic Dna ◽  
Kinase Activity ◽  
Phosphate Binding ◽  
Binding Motifs ◽  
E Coli ◽  
Acinetobacter Johnsonii ◽  
Dna Library ◽  
Genomic Dna Library ◽  
Coupled Reactions ◽  
Coupled Reaction

ABSTRACT We have cloned the gene for polyphosphate:AMP phosphotransferase (PAP), the enzyme that catalyzes phosphorylation of AMP to ADP at the expense of polyphosphate [poly(P)] in Acinetobacter johnsonii 210A. A genomic DNA library was constructed in Escherichia coli, and crude lysates of about 6,000 clones were screened for PAP activity. PAP activity was evaluated by measuring ATP produced by the coupled reactions of PAP and purified E. coli poly(P) kinases (PPKs). In this coupled reaction, PAP produces ADP from poly(P) and AMP, and the resulting ADP is converted to ATP by PPK. The isolated pap gene (1,428 bp) encodes a protein of 475 amino acids with a molecular mass of 55.8 kDa. The C-terminal region of PAP is highly homologous with PPK2 homologs isolated from Pseudomonas aeruginosa PAO1. Two putative phosphate-binding motifs (P-loops) were also identified. The purified PAP enzyme had not only strong PAP activity but also poly(P)-dependent nucleoside monophosphate kinase activity, by which it converted ribonucleoside monophosphates and deoxyribonucleoside monophosphates to ribonucleoside diphosphates and deoxyribonucleoside diphosphates, respectively. The activity for AMP was about 10 times greater than that for GMP and 770 and about 1,100 times greater than that for UMP and CMP.

scholarly journals Extra Copies of the Aspergillus fumigatus Squalene Epoxidase Gene Confer Resistance to Terbinafine: Genetic Approach to Studying Gene Dose-Dependent Resistance to Antifungals in A. fumigatus

2004 ◽  
Vol 48 (7) ◽  
pp. 2490-2496 ◽  
Author(s):  
Wei Liu ◽  
Gregory S. May ◽  
Michail S. Lionakis ◽  
Russell E. Lewis ◽  
Dimitrios P. Kontoyiannis
Keyword(s):  
Aspergillus Fumigatus ◽  
Genomic Dna ◽  
Specific Resistance ◽  
Squalene Epoxidase ◽  
Molecular Approach ◽  
Genetic Approach ◽  
Mechanisms Of Resistance ◽  
Dna Library ◽  
Genomic Dna Library ◽  
Dose Dependent

ABSTRACT With the increasing use of antifungals such as amphotericin B, itraconazole, voriconazole, caspofungin, and terbinafine (TRB) in patients at high risk for invasive aspergillosis, resistance of Aspergillus fumigatus to these agents will ultimately emerge. Due to the limited availability of molecular genetics for A. fumigatus, few studies have addressed its mechanisms of resistance to antifungals. We transformed A. fumigatus protoplasts with a pyrG-based A. fumigatus genomic DNA library (constructed in the multicopy nonintegrating vector pRG3-AMA1-NotI, which also has the pyr-4 gene for selection). We obtained one pyrG+ transformant that grew in medium containing a fungicidal concentration (0.625 μg/ml) of TRB. To determine whether TRB resistance in that transformant was plasmid dependent, we evicted the plasmid and found concomitant loss of uracil prototrophy and TRB resistance. DNA sequence analysis identified the gene responsible for TRB resistance as the A. fumigatus squalene epoxidase gene (ERG1), which encodes the target enzyme of TRB. Authentic A. fumigatus ERG1, amplified from the genome and cloned into pRG3-AMA1-NotI, also conferred TRB-specific resistance. This molecular approach has the potential to enhance our knowledge of the mechanisms of A. fumigatus resistance to modern antifungals.

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