Molecular cloning, sequence and structure analysis of hamster apurinic/apyrimidinic endonuclease (chAPE1) gene.
We have cloned a 13 kb genomic DNA fragment from the Chinese hamster ovary cell line, CHO-KI, and determined the nucleotide sequence of a 4 kb stretch of DNA which encompasses the complete sequence (2.277 kb) of the hamster apurinic/apyrimidinic endonuclease (chAPE1) gene. The intron/exon boundaries, identified by RT-PCR, follow GT/AG rule. The structure of the chAPE1 gene is similar to other mammalian apurinic/apyrimidinic (AP) endonuclease (hAPE1, BAP1, rAPEN and mAPE1) genes in that it has five exons and four introns with the first exon unexpressed. This structure, however, differs from one of the two structures that have been proposed for mAPE1 gene. Three transcription start sites (TSS) for the chAPE1 gene were identified by primer extension analysis at +1, +14 and +18 positions. The sequence also includes 1.72 kb of the upstream region of the chAPE1 gene. In this region, a CCAAT box but no TATA box that could initiate the transcription at the initiation sites was identified. The upstream region also includes the binding sites for a variety of other transcription factors. A polyadenylation site, 13 nucleotides downstream to the polyadenylation signal, was identified by 3'-RACE analysis. The observed 1.28 kb transcript of the chAPE1 gene is smaller than the 1.5 kb transcript of the human AP endonuclease gene. The translation of chAPE1 gene starts within the second exon with ATG and terminates in the fifth exon with UGA codons, 318 and 2121 nucleotides downstream to the first TSS, respectively. The encoded peptide of 317 amino acid residues is similar in size and is highly homologous in its amino acid sequence to mouse, rat, human, and bovine AP endonucleases.
Purohit
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S
Department of Biology, University of Texas at El Paso, El Paso, TX 79902, USA. spurohit@utep.edu
Arenaz
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P
D020121:5' Untranslated Regions / Q000737:chemistry / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D019757:Carbon-Oxygen Lyases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D002417:Cattle; D003001:Cloning, Molecular; D006224:Cricetinae; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D043223:Deoxyribonuclease IV (Phage T4-Induced); D005091:Exons; D006801:Humans; D007438:Introns; D051379:Mice; D008969:Molecular Sequence Data; D051381:Rats; D017422:Sequence Analysis, DNA; D014158:Transcription, Genetic
D020121:5' Untranslated Regions; D043223:Deoxyribonuclease IV (Phage T4-Induced); D019757:Carbon-Oxygen Lyases; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase