Structure of the mouse pore-forming protein (perforin) gene: analysis of transcription initiation site, 5' flanking sequence, and alternative splicing of 5' untranslated regions.
We studied the 5' untranslated regions (UTRs) of the mouse lymphocyte pore-forming protein (PFP, perforin, and cytolysin). 5' UTRs were determined by primer extension analysis, sequencing PFP cDNA clone PFP-7, ribonuclease protection assays, and amplification of poly(A)+ RNA of cytolytic T lymphocyte using polymerase chain reaction (PCR). Two alternatively spliced 5' UTRs, designated type I and type II, of 222 and 115 bp, respectively, were found associated with PFP. Type II is identical to type I, except for being 107 bp shorter in the second exon. This deletion was generated by the use of alternative acceptor splice sites. The mouse PFP gene (Pfp) encodes three exons, is separated by two small introns, and spans a chromosomal region of approximately 7 kb. The first exon contains 79 bp of 5' UTR, the second exon contains 143 or 36 bp of 5' UTR (type I or type II UTR, respectively) plus the NH2-terminal region of the mouse PFP, and the third exon contains the rest of the COOH-terminal mouse PFP. The organization of the mouse Pfp is similar to that of the human gene. Moreover, the 5' flanking sequence of the mouse Pfp is highly homologous to that of the human Pfp. In contrast to the human sequence, the more immediate 5' flanking sequence of mouse Pfp contains two tandem "TATA" box-related elements and a GC box, but lacks a typical CAAT box-related sequence. Several other enhancer elements were found further upstream, including cAMP-, phorbol ester-, interferon-gamma-, and UV-responsive elements, and PU box-like and NFkB binding site-like elements. In addition, we found a nuclear inhibitory protein-like element, a transcriptional silencer, and a pair of purine-rich sequence motifs that were found in other T cell-specific genes, and three repeats of GGCCTG that may be a variation of a highly repetitious GCCCTG consensus sequence found in human Pfp.
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Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis 46202.
Liu
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Kim
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Young
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Kwon
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Kwon
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D000818:Animals; D001483:Base Sequence; D004742:Enhancer Elements, Genetic; D005786:Gene Expression Regulation; D005796:Genes; D008562:Membrane Glycoproteins; D008565:Membrane Proteins / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry; D054353:Perforin; D016133:Polymerase Chain Reaction; D052899:Pore Forming Cytotoxic Proteins; D011401:Promoter Regions, Genetic; D012326:RNA Splicing; D012333:RNA, Messenger / Q000235:genetics; D012045:Regulatory Sequences, Nucleic Acid; D013602:T-Lymphocytes, Cytotoxic / Q000502:physiology; D014158:Transcription, Genetic
D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009841:Oligonucleotides; D052899:Pore Forming Cytotoxic Proteins; D012333:RNA, Messenger; D054353:Perforin