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Biochim Biophys Acta
2002 Aug 19;15771:81-92. doi: 10.1016/s0167-4781(02)00416-5.
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The Xenopus laevis beta TrCP gene: genomic organization, alternative splicing, 5' and 3' region characterization and comparison of its structure with that of human beta TrCP genes.
Ballarino M
,
Marchioni M
,
Carnevali F
.
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beta TrCP plays a relevant role in the control of stability of several key protein factors. In Xenopus, beta TrCP acts as an inhibitor of Wnt signaling and dorsal axis formation. We determined the primary structure of the frog beta TrCP gene, which consists of 14 exons and 13 introns, spanning over 34 kb. Isoforms of x-beta TrCP have been found, which show differences in the NH(2) and COOH regions. NH(2) isoforms differ for the presence or absence of a 30 aa sequence, coded by exon III. In COOH isoforms, 19 C-terminal amino acids are replaced by three different amino acids. Occurrence of two 5' splice donor sites for splicing of intron XIII provides an explanation for these isoforms, based on alternative splicing. The DNA region of the putative beta TrCP promoter contains several TATA elements, one GCCAAT box, and putative binding sites for Ets, Tcf/Lef and NF-kappa B transcription factors. Two transcription initiation sites have been mapped downstream of TATA boxes proximal to ATG for start of translation. Comparison of the Xenopus and human beta TrCP genes indicates high conservation of exon nucleotide and amino acid sequences, size and organization; differences are limited to exons coding for N- and C-terminal regions.
Fig. 1. NH2 isoforms of βTrCP. (A) The diagram represents the first four exons of βTrCP and their sizes, indicated as base pairs. The location of the âsenseâ primer is indicated by a bent arrow above the diagram. The âantisenseâ primers are denoted by bent arrows below the diagram. (B) Electrophoretic profile of RT-PCR amplification products obtained starting from X. laevis oocyte mRNAs and using, as primers, the oligonucleotide pair specific to exons I and II (lane 2) and the oligonucleotide pair specific to exon I and IV (lane 3) primer sets are depicted at the top of each lane. M in lane 1, the size marker, is 1 kb ladder. DNA band sizes, expressed in base pair and determined by DNA sequencing, are shown on the right side of the panel. The arrowhead points to the DNA band containing the 90 nt insertion coding for additional 30 amino acids. (C) Alignment of the human βTrCP variant 1 and 2 amino acid sequences with Xenopus βTrCP/pF112 (17), and βTrCP isoform 2. The sequence in the figure is limited to the NH2 region of βTrCP. Positions with amino acid identities are shown as black boxes. Positions with conservative changes are shown in shaded boxes.
Fig. 4. The Xenopus βTrCP gene. The structure of the frog βTrCP gene and alternative splicing. Black and white boxes represent translated exon and 5â² and 3â²UTR regions, respectively. Single lines represent introns. Alternative splicing is represented by dashed lines.
Fig. 6. Transcription initiation site mapping. 5â²RACE assay was performed using X. laevis oocyte polyA+ RNA. The nucleotide sequences, depicted on the left side of each panel, were derived from the autoradiograms of the coding strand (5â² to 3â² direction from top to bottom) shown in the (A) and (B) panels. Nucleotides of the ATG translation initiation codon are enclosed in open ellipses. Arrows and +1 indicate the first nucleotide after the stretch of G residues of the âsenseâ primer used in the amplification reactions.
