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Fig. 1. Cloning of chicken ETR-3. (A) Chicken ETR-3 sequence. In human and mouse the ETR-3 gene has at least five different first exons encoding three different N termini (Li et al., 2001) (and unpublished observations). The open reading frame of chicken ETR-3 was amplified using primers containing the termination codon and the furthest downstream start codon. RRMs are boxed, with RNP sequences in bold. Leucine-rich regions in the divergent domain are highlighted in gray, and an arginine/lysine-rich region at the C terminus is in italics. Residue numbers shown to the right are based on variant L sequence; the four amino acids unique to variant 4 (see below) fall between residues 335 and 336 of variant L and are underlined. (B) Two full-length isoforms of ETR-3 generated by alternative splicing were amplified by RT-PCR from embryonic chicken heart RNA. Variant 4 is identical to variant L except for use of an alternative 5' splice site in an exon corresponding to human and mouse exon 10, leading to the insertion of four residues (TVNS) in the divergent domain. Exons are numbered based on comparisons to human and mouse genes. The probe used in D is shown. (C) Chicken ETR-3 variants were cloned into pcDNA3.1(+) expression vectors to express untagged proteins and transfected into quail QT35 fibroblasts. Transfected samples (4 and L) were compared to untransfected fibroblasts (U) and embryonic day 12 chicken heart (d12) by western blot with a rabbit polyclonal antibody against ETR-3. Both variants 4 and L comigrate at their predicted size (
52 kDa) with full-length ETR-3 in chicken heart. (D) RNase protection assays demonstrate that variant L is the predominant isoform expressed throughout heart development. P, undigested probe diagrammed as shown diagrammatically in B; Y, yeast RNA; d8, embryonic day 8 chicken heart RNA; A, adult chicken heart RNA. Arrowheads indicate the expected locations of the undigested probe and protected fragments for each isoform (4, variant 4 protected fragment; L, variant L protected fragment). The expected sizes for the probe and protected fragments are shown in parentheses. The two faint bands between the variant 4 and variant L protected fragment sizes are background bands, as they appear in all lanes including the yeast RNA control.
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