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Identification of a functionally conserved surface region of rat cytochromes P450IA.

ABSTRACT: A region of rat cytochrome P450IA1 at residues 294-301 (Gln-Asp-Arg-Arg-Leu-Asp-Glu-Asn), equivalent to a proinhibitory region of cytochrome P450IA2, was identified by sequence alignment. Anti-peptide antibodies were successfully raised when the peptide was coupled through either its N- or its C-terminus to carrier protein, but no antibodies were produced against the so-called multiple peptide antigen, which consisted of eight copies of the peptide attached through its C-terminus to a synthetic base. Both of the anti-peptide antibodies bound specifically to cytochrome P450IA1 in the rat, as shown by e.l.i.s.a. and immunoblotting. They inhibited microsomal aryl hydrocarbon hydroxylase activity and the mutagenic activation of 2-acetylaminofluorene (these reactions are catalysed by cytochrome P450IA1), but not high-affinity phenacetin O-de-ethylation activity, which is catalysed by cytochrome P450IA2. However, there was differences in the properties of the two antisera in their binding to cytochromes P450IA1 in species other than the rat, their relative binding to the multiple peptide antigen, the yield of antibody following affinity purification using peptide coupled through its N-terminus to CNBr-activated Sepharose, and the binding of the purified preparations to N- and C-terminal-coupled peptide conjugates. These observations indicated that the antibodies were directed to the region of the peptide opposite to the end which was coupled to the carrier protein. Nevertheless, both of the antibody preparations bound equally well to the target cytochrome P450, thus indicating that, in the native protein, the whole of the peptide region is exposed on the surface of cytochrome P450IA1 and is available for binding by the antibodies. The role of this region appears to be the same in both cytochromes P450IA1 and P450IA2, despite the difference in its primary structure in the two cytochromes P450.

SUBMITTER: Edwards RJ

PROVIDER: S-EPMC1151410 | biostudies-other | 1991 Sep

REPOSITORIES: biostudies-other

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Project description:BACKGROUND:Long noncoding RNAs (lncRNAs) are involved in diverse biological processes and play an essential role in various human diseases. The number of lncRNAs identified has increased rapidly in recent years owing to RNA sequencing (RNA-Seq) technology. However, presently, most lncRNAs are not well characterized, and their regulatory mechanisms remain elusive. Many lncRNAs show poor evolutionary conservation. Thus, the lncRNAs that are conserved across species can provide insight into their critical functional roles. RESULTS:Here, we performed an orthologous analysis of lncRNAs in human and rat brain tissues. Over two billion RNA-Seq reads generated from 80 human and 66 rat brain tissue samples were analyzed. Our analysis revealed a total of 351 conserved human lncRNAs corresponding to 646 rat lncRNAs. Among these human lncRNAs, 140 were newly identified by our study, and 246 were present in known lncRNA databases; however, the majority of the lncRNAs that have been identified are not yet functionally annotated. We constructed co-expression networks based on the expression profiles of conserved human lncRNAs and protein-coding genes, and produced 79 co-expression modules. Gene ontology (GO) analysis of the co-expression modules suggested that the conserved lncRNAs were involved in various functions such as brain development (P-value?=?1.12E-2), nervous system development (P-value?=?1.26E-3), and cerebral cortex development (P-value?=?1.31E-2). We further predicted the interactions between lncRNAs and protein-coding genes to better understand the regulatory mechanisms of lncRNAs. Moreover, we investigated the expression patterns of the conserved lncRNAs at different time points during rat brain growth. We found that the expression levels of three out of four such lncRNA genes continuously increased from week 2 to week 104, which is consistent with our functional annotation. CONCLUSION:Our orthologous analysis of lncRNAs in human and rat brain tissues revealed a set of conserved lncRNAs. Further expression analysis provided the functional annotation of these lncRNAs in humans and rats. Our results offer new targets for developing better experimental designs to investigate regulatory molecular mechanisms of lncRNAs and the roles lncRNAs play in brain development. Additionally, our method could be generalized to study and characterize lncRNAs conserved in other species and tissue types.

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Identification of a functionally conserved surface region of rat cytochromes P450IA.

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