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Sequence and Tissue Distribution of Chicken Cellular Nucleic Acid Binding Protein cDNA

https://doi.org/10.1016/S0305-0491(97)00276-9Get rights and content

Abstract

We sequenced cDNAs coding for chicken cellular nucleic acid binding protein (CNBP). Two slightly different variations of the open reading frame were found, each of which translates into a protein with seven zinc finger domains. The longest transcript contains an in-frame insert of 3 bp. The sequence conservation between chick CNBP cDNAs with human, rat and mouse CNBP cDNAs is extreme, especially in the coding region, where the deduced amino acid sequence identity with human, rat and mouse CNBP is 99%. CNBP-like transcripts were also found in various tissues from insect, shrimp, fish and lizard. Regions with remarkable nucleotide conservation were also found in the 3′ untranslated region, indicating important functions for these regions. Quantitative reverse transcription polymerase chain reaction (RT-PCR) indicated that in the chick, CNBP is present in all tissues examined in approximately equal ratios to total RNA. RT-PCR of total RNA isolated from different phyla indicate CNBP-like proteins are widespread throughout the animal kingdom. The extraordinary level of conservation suggests an important physiological role for CNBP.

Introduction

Cellular nucleic acid binding protein (CNBP) is a zinc finger protein of approximately 19 kDa. It contains seven zinc finger repeats of 14 amino acid residues (C-X2-C-X4-H-X4-C) with a very high homology to the zinc finger domains of retroviral nucleic acid binding proteins [8]. CNBPs have been cloned from human 5, 6, 8, mouse [9]and rat [10]. Two main isoforms of CNBP transcripts have been found, namely CNBP-α and CNBP-β. CNBP-α contains an in-frame insert of 21 nucleotides as a result of alternative splicing 5, 6and has an additional seven amino acids between the first and the second zinc finger domains. Human CNBP was first isolated by screening an expression library with oligonucleotides containing the consensus sequences for a sterol regulatory element and CNBP was therefore thought to be involved in the regulation of the cholesterol biosynthetic pathway. Subsequent studies, however, have been unable to demonstrate such a role 4, 9. Nevertheless, a role in regulation of the cholesterol biosynthetic pathway has been suggested based on the upregulated levels of CNBP under sterol repressed conditions, the different nucleic acid binding properties of two CNBP isoforms (CNBP-α and CNBP-β) for the sterol regulatory element and the finding of two sterol regulatory element binding proteins with upregulatory properties [7]. CNBP has also been shown to preferentially bind to single-stranded DNA and to interact with suppressor regions of the human β-myosin heavy chain gene in vivo. CNBP-α has a repressive effect under these conditions, whereas CNBP-β had no effect [5]. In addition, both isoforms of CNBP interact with the CT element, a segment of DNA that upregulates expression of the c-myc gene. No difference in binding affinity for the CT element between CNBP-α and CNBP-β has been observed [7]. As part of a search for transcription factors expressed in the chick cochlea, we found a 419-bp DNA fragment. The nucleotide sequence of this fragment showed high sequence homology with human sterol regulatory element-binding protein (CNBP) [8].

Here we report the cloning, sequencing and tissue distribution of chick CNBP, another member of the CNBP family. The extreme conservation of the primary structure of the protein and of parts of the 3′-untranslated region (UTR) of the mRNA indicates there is a very high selection pressure and hence an important role for CNBP in the regulation of cellular processes.

Section snippets

Experimental Animals and Tissues

Total RNA was extracted from tissues listed below using Tri Reagent (Molecular Research Centre, Inc., Cincinnati, OH) according to the manufacturer's guidelines, resuspended in diethyl pyrocarbonate (DEPC)-treated water to a final concentration of 0.5 μg/μl, DNase I digested and reverse transcribed using the Perkin Elmer reverse transcription-polymerase chain reaction (RT-PCR) core kit and random hexamer primers (Perkin Elmer, Australia).

Hatchling White Steghorn chickens were decapitated and

5′- and 3′-RACE of Chick CNBP

A cDNA clone of a 419-bp fragment was first isolated from a chick cochlear cDNA library. Sequence analysis showed this fragment shared high homology with CNBP cDNAs characterized in human, rat and mouse. 3′-RACE generated a product of 950 bp, whereas with 5′-RACE, a 850-bp product was obtained. The complete cDNA sequence was obtained from the overlapping fragments and was determined to be approximately 1600 bp long (Fig. 2). The longest 5′-UTR was 117 bp and the 3′-UTR appeared to be

Discussion

A cDNA composite nucleotide sequence of 1610 nucleotides from the cochlea of the chick was obtained. It contained the entire coding region of a putative protein with a primary structure nearly identical to CNBP-β of mouse, rat and human (Fig. 7). The longest open reading frame, starting from ATG at position 117, encodes a protein of 172 amino acids.

Interestingly, the 3′-UTRs of CNBPs show a very high degree of conservation between chicken and mouse (83%), chicken and rat (82%) and chicken and

Acknowledgements

This study was supported by the Australian Research Council, the Australian National Health and Medical Research Council and the Rebecca L. Cooper Foundation. The nucleotide sequence data reported appear in the GenBank Nucleotide Sequence Database under accession number AF004942.

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