k. 1991 Oxford University Press
Nucleic Acids Research, Vol. 19, No. 18 5085
An STS in the human cytoskeletal y-actin
Brian C.Freeman and J.Christopher States* Center for Molecular Biology, Wayne State University, Detroit, Ml 48201, USA Submitted June 21, 1991 The actin gene family is one of the most widely studied gene families in the human genome. The human cytoskeletal 'y-actin gene has been extensively characterized and compared to other actin genes, both human and non-human (1, 2). The 3' untranslated region of the -y-actin gene is evolutionarily conserved between mammals and chickens (2). Yet, despite the high level of conservation, a sequence tagged site (STS) was developed that detected the human oy-actin gene and not the hamster 'y-actin gene when amplifying from human/hamster somatic cell hybrid DNA. Using the polymerase chain reaction (PCR) described below, a fragment of the expected size (369 bp) was amplified from human genomic DNA (Figure 1). Figure 1 also demonstrates the ability of bacteriophage T4 gene 32 protein (gp32) to enhance the specificity of amplification. Without gp32 (lane 2), several nonspecific amplified products are obtained. With increasing gp32 (lanes 3-5), the non-specific products are eliminated. The DNA fragment specifically amplified at high gp32 was sequenced to confirm that it contained the expected -y-actin sequence encoded by the 3' untranslated region of exon six from positions 2463 to 2831 in the cytoskeletal 'y-actin gene (2). Amplification of the STS using a human/hamster somatic cell hybrid chromosome panel properly mapped the oy-actin gene to chromosome 17 (data not shown). Thus, the usefulness of the STS has been confirmed. The -y-actin STS is also a reliable control during reverse transcriptase PCR (RT-PCR) from fibroblast cells (data not shown).
PCR Primers: Forward (1-16) Reverse (352 -367)
PCR Components in a 10 pi Reaction: 12.5 ng DNA, 1 j1l 10 x PCR buffer D (100 mM Tris-HCl, pH 8.8, 500 mM KCl, 60 mM MgCl2, 10 mM dithiothreitol), 100 nM of each oligonucleotide, 200 isM dNTPs, 2.0 ,ig bacteriophage T4 gene 32 protein (US Biochemical, Cleveland, OH) and 0.25 U Amplitaq (Perkin-Elmer Cetus, Norwalk, CT).
PCR Profile: 94°C for 1 minute; 58°C for 1 minute; 72°C for 3 minutes; 35 cycles followed by 10 minute incubation at 72°C.
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Sequence of PCR Product: GATCTGTGCAGGGTATTAACATGTCAGGGCTGAGTGTTCTGGGATTTCTCTAGAGGCTGGCAAGAACCAGTTGTTTTTGTCTTGCGGGTCTGTCAGGGTTGGAAAGTCCAAGCCGTAGGACCCAGTTTCC TTTCTTAGCTGATGTCTTTGGCCAGAACACCGTGGGCTGTTAACTTGCCTTGAGTTGGAAGCGGT TTGCATTTACGCCTGTAAATGTATTCATTCTTAATTTATGTAAGGTTTTTTTTGTACGCAATTCT CGATTCTTTAAAGAGATGACAACAAATTTTGGTTTTCTACTGTTATGTGAGAATATTAGGCCCCA GCAACACGTCATTGTGTAAGGAAAAATAAAAGTGCTGCCGTAAC
REFERENCES 1. Erba,H.P., Gunning,P. and Kedes,L. (1986) Nucl. Acids Res. 13,
5275-5294. 2. Erba,H.P., Eddy,R., Shows,T., Kedes,L. and Gunning,P. (1988) Mol. Cell. Biol. 4, 1775-1789.
1 2 3 4 5
Figure 1. Amplification of -y-actin STS. Lane 1, 100 bp marker DNAs (GibcoBRL, Gaithersburgh, MD), sizes of some marker fragments are given in bp in the margin; Lanes 2-5, amplification products using conditions described with increasing concentration of bacteriophage T4 gene 32 protein (gp32); Lane 2: no gp32; Lane 3: 1.0 ug p32; Lane 4: 1.5 Ag gp32; Lane 5: 2.0 Ag gp32. Specificity of amplification is achieved at high gp32.