Abstract
The TREX1 and TREX2 genes encode mammalian 3'-->5' exonucleases. Expression of the TREX genes in human cells was investigated using a reverse transcription-polymerase chain reaction strategy. Our results show that TREX1 and TREX2 are expressed in all tissues tested, providing direct evidence for the expression of these genes in human cells. Potential transcription start sites are identified for the TREX genes using rapid amplification of cDNA ends to recover the 5'-flanking regions of the TREX transcripts. The 5'-flanking sequences indicate transcription initiation from consensus putative promoters identified -140 and -650 base pairs upstream of the TREX1 open reading frame (ORF) and -623 and -753 base pairs upstream of the TREX2 ORF. Novel TREX1 and TREX2 cDNAs are identified that contain protein-coding sequences generated from exons positioned in genomic DNA up to 18 kilobases 5' to the TREX1 ORF and up to 25 kilobases 5' to the TREX2 ORF. These novel cDNAs and sequences in the GenBank data base indicate that transcripts containing the TREX1 and TREX2 ORFs are produced using a variety of mechanisms that include alternate promoter usage, alternative splicing, and varied sites for 3' cleavage and polyadenylation. These initial studies have revealed previously unrecognized complexities in the structure and expression of the TREX1 and TREX2 genes.
Highlights
The multistep processes of DNA replication, repair, and genetic recombination often require the excision of 3Ј nucleotides to generate DNA 3Ј termini suitable for subsequent metabolic steps
Our results show that TREX1 and TREX2 are expressed in all tissues tested, providing direct evidence for the expression of these genes in human cells
These novel cDNAs and sequences in the GenBank data base indicate that transcripts containing the TREX1 and TREX2 open reading frame (ORF) are produced using a variety of mechanisms that include alternate promoter usage, alternative splicing, and varied sites for 3 cleavage and polyadenylation
Summary
The first-round PCR products were fractionated on agarose gels and recov-. The AML RNA or tissue specific RNA (CLONTECH Laboratories, Inc.) (5 g) was hybridized to 0.5 g of oligo(dT) primer (Promega Corp.) for 5 min at 95 °C and 10 min at 70 °C. The PCR conditions using AML cDNA and the tissue-specific cDNA were as described above for genomic DNA. A single-round PCR was performed for TREX1 and TREX2 expression analysis using the 13 human tissue-specific cDNAs and the primers indicated in the text. The PCR conditions for amplification of novel TREX1 and TREX2 cDNAs were as described above for genomic DNA. The products from the second round were resolved by agarose gel electrophoresis, recovered from the gel, and sequenced
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