Access to a messenger RNA sequence or its protein product is not limited by tissue or species specificity.
RNA amplification with transcript sequencing (RAWTS) is a rapid and sensitive method of direct sequencing that involves complementary DNA synthesis, polymerase chain reaction (PCR) with a primer or primers containing a phage promoter, transcription from the phage promoter, and reverse transcriptase-mediated sequencing. By means of RAWTS, it was possible to sequence each of four tissue-specific human messenger RNAs (blue pigment, factor IX, phenylalanine hydroxylase, and tyrosine hydroxylase) in four cell types examined (white blood cells, liver, K562 erythroleukemia cells, and chorionic villus cells). These results indicate that there is a basal rate of transcription, splicing, and polyadenylation of tissue-specific mRNAs in adult and embryonic tissues. In addition to revealing sequence information, it is possible to generate a desired in vitro translation product by incorporating a translation initiation signal into the appropriate PCR primer. RAWTS can be used to obtain novel mRNA sequence information from other species as illustrated with a segment of the catalytic domain of factor IX. In general, the ability to obtain mRNA sequences rapidly across species boundaries should aid both the study of protein evolution and the identification of sequences crucial for protein structure and function.
Sarkar
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Department of Cell Biology and Molecular Biology, Mayo Clinic/Foundation, Rochester, MN 55905.
Sommer
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D000595:Amino Acid Sequence; D001483:Base Sequence; D002824:Chorionic Villi / Q000032:analysis; D004247:DNA / Q000096:biosynthesis; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D005164:Factor IX / Q000235:genetics*; D005784:Gene Amplification; D006801:Humans; D004915:Leukemia, Erythroblastic, Acute / Q000378:metabolism; D007962:Leukocytes / Q000032:analysis; D008099:Liver / Q000032:analysis; D008969:Molecular Sequence Data; D010651:Phenylalanine Hydroxylase / Q000235:genetics*; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics*; D012168:Retinal Pigments / Q000235:genetics*; D013045:Species Specificity; D014018:Tissue Distribution; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured; D014446:Tyrosine 3-Monooxygenase / Q000235:genetics*
D012333:RNA, Messenger; D012168:Retinal Pigments; D005164:Factor IX; D004247:DNA; D010651:Phenylalanine Hydroxylase; D014446:Tyrosine 3-Monooxygenase; D004259:DNA-Directed DNA Polymerase