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. 2002 Dec 1;30(23):5110–5119. doi: 10.1093/nar/gkf664

Figure 1.

Figure 1

Schematic representation of the HD promoter region and mapping of the HD 5′ end. (A) The nucleotide sequence of the human HD promoter and 5′ UTR are shown. The initiator ATG codon of the human HD gene is shown in bold uppercase letters and the numbering scheme is relative to the adenosine of the ATG, which is set as position +1. The uORF present in the human 5′ UTR is shown in bold and the encoded amino acids are depicted below the ORF. Two transcription start sites identified by Lin et al. (31) are denoted by asterisks at positions –145 and –135. The 5′ end positions defined by 5′ RACE analysis (seven independent assays) are shown by squares above the appropriate nucleotide positions between –145 and –134. The light grey squares indicate that 5% of the clones analyzed terminated at that position. The darker grey squares indicate that 7–10% of the clones analyzed terminated at the nucleotide. The two blackened squares indicate that 16% (nucleotide position –136) and 25% (nucleotide position –134) of the clones analyzed terminated at that position. The location of the oligonucleotide primer (HD-5′ UTR) used in the 5′ RACE reactions is shown. The 5′ ends identified by S1 nuclease analysis (in B) are shown by downward arrowheads. (B) S1 nuclease mapping of the HD 5′ UTR. S1 nuclease analysis was performed as described in the Materials and Methods and the products fractionated on an 8 M urea–8% polyacrylamide gel. The gel was dried and exposed to X-Omat film (Kodak) for 3 weeks at –70°C with an intensifying screen. The input RNA used in each experiment was: lane 1, 10 µg yeast tRNA; lane 2, 50 µg yeast tRNA; lane 3, 50 µg human fetal brain poly(A–) RNA; lane 4, 50 µg human fetal brain total RNA. The arrow denotes the position of migration of undigested probe. The size of the products was determined in reference to a sequencing ladder. Radiolabeled fragments only present in total RNA from human fetal brain (lane 4) are denoted by blackened circles. The asterisks denote non-specific S1 products obtained with yeast tRNA. (C) Comparison of the polypeptide sequence encoded by the HD uORF found in humans (GenBank accession no. L27350), gorilla (GenBank accession no. Y07988), chimpanzee (GenBank accession no. Y07990), miniature pig (GenBank accession no. AB016793), rat (GenBank accession no. AJ224997) and mouse (GenBank accession no. L34008). Alignment of conserved amino acids among the HD uORFs from different mammals is shown.