Isolation and characterization of rat cytochrome P-450IIB gene family members. Use of the polymerase chain reaction to detect expression of a novel P-450 mRNA

C. M. Giachelli, J. Lin-Jones, Curtis John Omiecinski

Research output: Contribution to journalArticle

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Abstract

The rat cytochrome P-450IIB gene family consists of at least 10 members, 2 of which, P-450b and P-450e, have been well characterized and are activated transcriptionally by phenobarbital (PB) in the liver. The remaining genes in this family have not been studied extensively. In this report, data are presented that provide additional characterization of a P-450IIB gene, termed gene IV. The complete gene IV and flanking regions were isolated from a rat liver Charon 4A genomic library and subjected to a variety of analyses. Structural homology to the P-450b gene was confirmed by comparative restriction mapping and high stringency hybridization of gene IV fragments to probes comprising the entire cDNA for P-450b. The 5'-portion of gene IV, including its flanking region, was sequenced and contained an open reading frame for 58 amino acids which were 62% related to exon 1 of the P-450b/e genes. Typical TATA and CAAT promoter elements, as well as two Sp1 core sequences and a site related to a glucocorticoid responsive element were found in gene IV. Northern blot studies with an oligomer probe specific to gene IV sequence indicated a 4.3-kilobase pair polyadenylated transcript present at low levels in untreated rat liver and inducible approximately 6-fold by PB treatment. Primer extension experiments indicated that the transcription initiation site mapped to a position on gene IV that was analogous to that reported for the structurally similar P-450e gene. Due to the low levels of hepatic RNA expression, we employed the polymerase chain reaction to facilitate characterization of gene IV transcripts. The polymerase chain reaction data verified that gene IV transcripts were elevated after PB treatment. Significantly, the polymerase chain reaction results demonstrated that gene IV transcripts were associated with hepatic polysome fractions, indicating their active utilization in this tissue. Polymerase chain reaction analysis of RNA also indicated constitutive expression of gene IV transcripts in rat lung, kidney, and testis, as well as fetal rat liver. Together, these data show that gene IV is a transcribed member of the P-450IIB gene family and, like the well characterized P-450b and P-450e genes, is positively regulated by PB in rat liver.

Original languageEnglish (US)
Pages (from-to)7046-7053
Number of pages8
JournalJournal of Biological Chemistry
Volume264
Issue number12
StatePublished - 1989

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Polymerase chain reaction
Cytochromes
Rats
Genes
Polymerase Chain Reaction
Messenger RNA
Liver
Phenobarbital
Pluto
RNA
Restriction Mapping
Gene Order
Polyribosomes
Genomic Library
Transcription Initiation Site

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

@article{d4855153dccd4809858d4a71d073b91f,
title = "Isolation and characterization of rat cytochrome P-450IIB gene family members. Use of the polymerase chain reaction to detect expression of a novel P-450 mRNA",
abstract = "The rat cytochrome P-450IIB gene family consists of at least 10 members, 2 of which, P-450b and P-450e, have been well characterized and are activated transcriptionally by phenobarbital (PB) in the liver. The remaining genes in this family have not been studied extensively. In this report, data are presented that provide additional characterization of a P-450IIB gene, termed gene IV. The complete gene IV and flanking regions were isolated from a rat liver Charon 4A genomic library and subjected to a variety of analyses. Structural homology to the P-450b gene was confirmed by comparative restriction mapping and high stringency hybridization of gene IV fragments to probes comprising the entire cDNA for P-450b. The 5'-portion of gene IV, including its flanking region, was sequenced and contained an open reading frame for 58 amino acids which were 62{\%} related to exon 1 of the P-450b/e genes. Typical TATA and CAAT promoter elements, as well as two Sp1 core sequences and a site related to a glucocorticoid responsive element were found in gene IV. Northern blot studies with an oligomer probe specific to gene IV sequence indicated a 4.3-kilobase pair polyadenylated transcript present at low levels in untreated rat liver and inducible approximately 6-fold by PB treatment. Primer extension experiments indicated that the transcription initiation site mapped to a position on gene IV that was analogous to that reported for the structurally similar P-450e gene. Due to the low levels of hepatic RNA expression, we employed the polymerase chain reaction to facilitate characterization of gene IV transcripts. The polymerase chain reaction data verified that gene IV transcripts were elevated after PB treatment. Significantly, the polymerase chain reaction results demonstrated that gene IV transcripts were associated with hepatic polysome fractions, indicating their active utilization in this tissue. Polymerase chain reaction analysis of RNA also indicated constitutive expression of gene IV transcripts in rat lung, kidney, and testis, as well as fetal rat liver. Together, these data show that gene IV is a transcribed member of the P-450IIB gene family and, like the well characterized P-450b and P-450e genes, is positively regulated by PB in rat liver.",
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journal = "Journal of Biological Chemistry",
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}

Isolation and characterization of rat cytochrome P-450IIB gene family members. Use of the polymerase chain reaction to detect expression of a novel P-450 mRNA. / Giachelli, C. M.; Lin-Jones, J.; Omiecinski, Curtis John.

In: Journal of Biological Chemistry, Vol. 264, No. 12, 1989, p. 7046-7053.

Research output: Contribution to journalArticle

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T1 - Isolation and characterization of rat cytochrome P-450IIB gene family members. Use of the polymerase chain reaction to detect expression of a novel P-450 mRNA

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N2 - The rat cytochrome P-450IIB gene family consists of at least 10 members, 2 of which, P-450b and P-450e, have been well characterized and are activated transcriptionally by phenobarbital (PB) in the liver. The remaining genes in this family have not been studied extensively. In this report, data are presented that provide additional characterization of a P-450IIB gene, termed gene IV. The complete gene IV and flanking regions were isolated from a rat liver Charon 4A genomic library and subjected to a variety of analyses. Structural homology to the P-450b gene was confirmed by comparative restriction mapping and high stringency hybridization of gene IV fragments to probes comprising the entire cDNA for P-450b. The 5'-portion of gene IV, including its flanking region, was sequenced and contained an open reading frame for 58 amino acids which were 62% related to exon 1 of the P-450b/e genes. Typical TATA and CAAT promoter elements, as well as two Sp1 core sequences and a site related to a glucocorticoid responsive element were found in gene IV. Northern blot studies with an oligomer probe specific to gene IV sequence indicated a 4.3-kilobase pair polyadenylated transcript present at low levels in untreated rat liver and inducible approximately 6-fold by PB treatment. Primer extension experiments indicated that the transcription initiation site mapped to a position on gene IV that was analogous to that reported for the structurally similar P-450e gene. Due to the low levels of hepatic RNA expression, we employed the polymerase chain reaction to facilitate characterization of gene IV transcripts. The polymerase chain reaction data verified that gene IV transcripts were elevated after PB treatment. Significantly, the polymerase chain reaction results demonstrated that gene IV transcripts were associated with hepatic polysome fractions, indicating their active utilization in this tissue. Polymerase chain reaction analysis of RNA also indicated constitutive expression of gene IV transcripts in rat lung, kidney, and testis, as well as fetal rat liver. Together, these data show that gene IV is a transcribed member of the P-450IIB gene family and, like the well characterized P-450b and P-450e genes, is positively regulated by PB in rat liver.

AB - The rat cytochrome P-450IIB gene family consists of at least 10 members, 2 of which, P-450b and P-450e, have been well characterized and are activated transcriptionally by phenobarbital (PB) in the liver. The remaining genes in this family have not been studied extensively. In this report, data are presented that provide additional characterization of a P-450IIB gene, termed gene IV. The complete gene IV and flanking regions were isolated from a rat liver Charon 4A genomic library and subjected to a variety of analyses. Structural homology to the P-450b gene was confirmed by comparative restriction mapping and high stringency hybridization of gene IV fragments to probes comprising the entire cDNA for P-450b. The 5'-portion of gene IV, including its flanking region, was sequenced and contained an open reading frame for 58 amino acids which were 62% related to exon 1 of the P-450b/e genes. Typical TATA and CAAT promoter elements, as well as two Sp1 core sequences and a site related to a glucocorticoid responsive element were found in gene IV. Northern blot studies with an oligomer probe specific to gene IV sequence indicated a 4.3-kilobase pair polyadenylated transcript present at low levels in untreated rat liver and inducible approximately 6-fold by PB treatment. Primer extension experiments indicated that the transcription initiation site mapped to a position on gene IV that was analogous to that reported for the structurally similar P-450e gene. Due to the low levels of hepatic RNA expression, we employed the polymerase chain reaction to facilitate characterization of gene IV transcripts. The polymerase chain reaction data verified that gene IV transcripts were elevated after PB treatment. Significantly, the polymerase chain reaction results demonstrated that gene IV transcripts were associated with hepatic polysome fractions, indicating their active utilization in this tissue. Polymerase chain reaction analysis of RNA also indicated constitutive expression of gene IV transcripts in rat lung, kidney, and testis, as well as fetal rat liver. Together, these data show that gene IV is a transcribed member of the P-450IIB gene family and, like the well characterized P-450b and P-450e genes, is positively regulated by PB in rat liver.

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