Molecular cloning, gene organization and expression of rainbow trout (Oncorhynchus mykiss) inducible nitric oxide synthase (iNOS) gene

Tiehui Wang, Mike Ward, Peter Grabowski, Christopher J Secombes

Research output: Contribution to journalArticle

56 Citations (Scopus)

Abstract

A full-length inducible nitric oxide synthase (fNOS) gene has been sequenced for the first time outside the mammals, and the gene organization compared with that already determined for human iNOS. While there are some differences from the human gene, overall the exons show remarkable conservation in sequence and organization. As in human, the trout iNOS gene has 27 exons, with 18 of the trout exons being identical in size with the equivalent human exons. The cofactor-binding domains are found in the same exons and in some cases are absolutely conserved. Differences include the start of the ORF in exon 3 instead of exon 2, resulting in a deletion at the 5' end of the trout iNOS protein. Exon 27 also shows a large difference in size and although the trout exon is larger this is due to the length of the 3'-UTR. Several non-mammalian features are notable, and include a conserved potential glycosylation site in chicken and fish, and an insertion at the boundary of exons 20 and 21 in fish. The intron sizes in trout were generally much smaller than in human iNOS, making the trout iNOS gene approximately half the size of the human gene. Analysis of RNA secondary structure revealed two regions with complementarity, which could interfere with reverse transcription. Using a trout fibroblast cell line (RTG-2 cells), it was shown by reverse transcriptase (RT)-PCR that virus infection was a good inducer of iNOS expression. However, when using a combination of Superscripts (R) II for reverse transcription and primers at the 5' end of the gene only very weak products were amplified, in contrast with the situation when primers at the 3' end of the gene were used, or ThermoScript (R) -derived cDNA was used. The impact of such results on RT-PCR analysis of iNOS expression in trout is discussed.

Original languageEnglish
Pages (from-to)747-755
Number of pages9
JournalBiochemical Journal
Volume358
Issue numberPt 3
DOIs
Publication statusPublished - 15 Sep 2001

Keywords

  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Carps
  • Cell Line
  • Chickens
  • Cloning, Molecular
  • Conserved Sequence
  • DNA Primers
  • Exons
  • Humans
  • Introns
  • Mammals
  • Molecular Sequence Data
  • Nitric Oxide Synthase
  • Nitric Oxide Synthase Type II
  • Oncorhynchus mykiss
  • Open Reading Frames
  • Phylogeny
  • Reverse Transcriptase Polymerase Chain Reaction
  • Sequence Alignment
  • Sequence Homology, Amino Acid
  • Exon Organization
  • iNOS cDNA
  • iNOS expression
  • Phylogenetic analysis
  • RNA secondary structure
  • Macrophage Activating Factor
  • Functional-Characterization
  • Virus
  • Sequence
  • Replication
  • Interferon
  • Leukocytes
  • Alignment
  • Induction
  • Catfish

Cite this

Molecular cloning, gene organization and expression of rainbow trout (Oncorhynchus mykiss) inducible nitric oxide synthase (iNOS) gene. / Wang, Tiehui; Ward, Mike; Grabowski, Peter; Secombes, Christopher J.

In: Biochemical Journal, Vol. 358, No. Pt 3, 15.09.2001, p. 747-755.

Research output: Contribution to journalArticle

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abstract = "A full-length inducible nitric oxide synthase (fNOS) gene has been sequenced for the first time outside the mammals, and the gene organization compared with that already determined for human iNOS. While there are some differences from the human gene, overall the exons show remarkable conservation in sequence and organization. As in human, the trout iNOS gene has 27 exons, with 18 of the trout exons being identical in size with the equivalent human exons. The cofactor-binding domains are found in the same exons and in some cases are absolutely conserved. Differences include the start of the ORF in exon 3 instead of exon 2, resulting in a deletion at the 5' end of the trout iNOS protein. Exon 27 also shows a large difference in size and although the trout exon is larger this is due to the length of the 3'-UTR. Several non-mammalian features are notable, and include a conserved potential glycosylation site in chicken and fish, and an insertion at the boundary of exons 20 and 21 in fish. The intron sizes in trout were generally much smaller than in human iNOS, making the trout iNOS gene approximately half the size of the human gene. Analysis of RNA secondary structure revealed two regions with complementarity, which could interfere with reverse transcription. Using a trout fibroblast cell line (RTG-2 cells), it was shown by reverse transcriptase (RT)-PCR that virus infection was a good inducer of iNOS expression. However, when using a combination of Superscripts (R) II for reverse transcription and primers at the 5' end of the gene only very weak products were amplified, in contrast with the situation when primers at the 3' end of the gene were used, or ThermoScript (R) -derived cDNA was used. The impact of such results on RT-PCR analysis of iNOS expression in trout is discussed.",
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T1 - Molecular cloning, gene organization and expression of rainbow trout (Oncorhynchus mykiss) inducible nitric oxide synthase (iNOS) gene

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AU - Ward, Mike

AU - Grabowski, Peter

AU - Secombes, Christopher J

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N2 - A full-length inducible nitric oxide synthase (fNOS) gene has been sequenced for the first time outside the mammals, and the gene organization compared with that already determined for human iNOS. While there are some differences from the human gene, overall the exons show remarkable conservation in sequence and organization. As in human, the trout iNOS gene has 27 exons, with 18 of the trout exons being identical in size with the equivalent human exons. The cofactor-binding domains are found in the same exons and in some cases are absolutely conserved. Differences include the start of the ORF in exon 3 instead of exon 2, resulting in a deletion at the 5' end of the trout iNOS protein. Exon 27 also shows a large difference in size and although the trout exon is larger this is due to the length of the 3'-UTR. Several non-mammalian features are notable, and include a conserved potential glycosylation site in chicken and fish, and an insertion at the boundary of exons 20 and 21 in fish. The intron sizes in trout were generally much smaller than in human iNOS, making the trout iNOS gene approximately half the size of the human gene. Analysis of RNA secondary structure revealed two regions with complementarity, which could interfere with reverse transcription. Using a trout fibroblast cell line (RTG-2 cells), it was shown by reverse transcriptase (RT)-PCR that virus infection was a good inducer of iNOS expression. However, when using a combination of Superscripts (R) II for reverse transcription and primers at the 5' end of the gene only very weak products were amplified, in contrast with the situation when primers at the 3' end of the gene were used, or ThermoScript (R) -derived cDNA was used. The impact of such results on RT-PCR analysis of iNOS expression in trout is discussed.

AB - A full-length inducible nitric oxide synthase (fNOS) gene has been sequenced for the first time outside the mammals, and the gene organization compared with that already determined for human iNOS. While there are some differences from the human gene, overall the exons show remarkable conservation in sequence and organization. As in human, the trout iNOS gene has 27 exons, with 18 of the trout exons being identical in size with the equivalent human exons. The cofactor-binding domains are found in the same exons and in some cases are absolutely conserved. Differences include the start of the ORF in exon 3 instead of exon 2, resulting in a deletion at the 5' end of the trout iNOS protein. Exon 27 also shows a large difference in size and although the trout exon is larger this is due to the length of the 3'-UTR. Several non-mammalian features are notable, and include a conserved potential glycosylation site in chicken and fish, and an insertion at the boundary of exons 20 and 21 in fish. The intron sizes in trout were generally much smaller than in human iNOS, making the trout iNOS gene approximately half the size of the human gene. Analysis of RNA secondary structure revealed two regions with complementarity, which could interfere with reverse transcription. Using a trout fibroblast cell line (RTG-2 cells), it was shown by reverse transcriptase (RT)-PCR that virus infection was a good inducer of iNOS expression. However, when using a combination of Superscripts (R) II for reverse transcription and primers at the 5' end of the gene only very weak products were amplified, in contrast with the situation when primers at the 3' end of the gene were used, or ThermoScript (R) -derived cDNA was used. The impact of such results on RT-PCR analysis of iNOS expression in trout is discussed.

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KW - Animals

KW - Base Sequence

KW - Carps

KW - Cell Line

KW - Chickens

KW - Cloning, Molecular

KW - Conserved Sequence

KW - DNA Primers

KW - Exons

KW - Humans

KW - Introns

KW - Mammals

KW - Molecular Sequence Data

KW - Nitric Oxide Synthase

KW - Nitric Oxide Synthase Type II

KW - Oncorhynchus mykiss

KW - Open Reading Frames

KW - Phylogeny

KW - Reverse Transcriptase Polymerase Chain Reaction

KW - Sequence Alignment

KW - Sequence Homology, Amino Acid

KW - Exon Organization

KW - iNOS cDNA

KW - iNOS expression

KW - Phylogenetic analysis

KW - RNA secondary structure

KW - Macrophage Activating Factor

KW - Functional-Characterization

KW - Virus

KW - Sequence

KW - Replication

KW - Interferon

KW - Leukocytes

KW - Alignment

KW - Induction

KW - Catfish

U2 - 10.1042/0264-6021:3580747

DO - 10.1042/0264-6021:3580747

M3 - Article

C2 - 11535135

VL - 358

SP - 747

EP - 755

JO - Biochemical Journal

JF - Biochemical Journal

SN - 0264-6021

IS - Pt 3

ER -