Genomic sequence analysis of Fugu rubripes CFTR and flanking genes in a 60 kb region conserving synteny with 800 kb of human chromosome 7 - PubMed (original) (raw)

Genomic sequence analysis of Fugu rubripes CFTR and flanking genes in a 60 kb region conserving synteny with 800 kb of human chromosome 7

H Davidson et al. Genome Res. 2000 Aug.

Abstract

To define control elements that regulate tissue-specific expression of the cystic fibrosis transmembrane regulator (CFTR), we have sequenced 60 kb of genomic DNA from the puffer fish Fugu rubripes (Fugu) that includes the CFTR gene. This region of the Fugu genome shows conservation of synteny with 800-kb sequence of the human genome encompassing the WNT2, CFTR, Z43555, and CBP90 genes. Additionally, the genomic structure of each gene is conserved. In a multiple sequence alignment of human, mouse, and Fugu, the putative WNT2 promoter sequence is shown to contain highly conserved elements that may be transcription factor or other regulatory binding sites. We have found two putative ankyrin repeat-containing genes that flank the CFTR gene. Overall sequence analysis suggests conservation of intron/exon boundaries between Fugu and human CFTR and revealed extensive homology between functional protein domains. However, the immediate 5' regions of human and Fugu CFTR are highly divergent with few conserved sequences apart from those resembling diminished cAMP response elements (CRE) and CAAT box elements. Interestingly, the polymorphic polyT tract located upstream of exon 9 is present in human and Fugu but absent in mouse. Similarly, an intron 1 and intron 9 element common to human and Fugu is absent in mouse. The euryhaline killifish CFTR coding sequence is highly homologous to the Fugu sequence, suggesting that upregulation of CFTR in that species in response to salinity may be regulated transcriptionally.

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Figures

Figure 1

Genomic conservation and organization. Percentage identity plot (PIP) analysis of Fugu CFTR genomic region compared with the equivalent human region. Protein-coding exons are indicated by black rectangles. Gray and white boxes indicate 0.75 and 0.60 CpG:GpC ratios, respectively. Horizontal lines show blocks of homology between the Fugu and human sequences. Only blocks of homology occurring in the same relative order in both sequences are indicated by horizontal lines. Genes identified within the Fugu genomic sequence are annotated and transcriptional orientation indicated above the appropriate exons. Exons of genes are numbered where space permits.

Figure 2

Sequence homology and conservation of exon/intron boundaries. Alignment of human, mouse, killifish, and Fugu CFTR. The triangles mark approximate intron/exon boundaries. Domains of CFTR are as indicated: NBD = nucleotide-binding domain; R-domain = regulatory domain; TM = transmembrane segment. Horizontal lines linking transmembrane segments represent extracellular loops. Diamonds above the phosphorylated residues predict dibasic cAMP-dependent phosphorylation sites. Short horizontal bars marked with asterisks above exon 15 indicate glycosylation sites. In the background shading at each position, phosphorylation and glycosylation sites: black indicates perfect homology, dark grey three out of four, and light grey two out of four residues matching.

Figure 3

Putative human CFTR regulatory domains. Relative positions of proposed human CFTR transcription regulatory sequences and their conservation between mammals. (A) CFTR Intron 9/exon 10 3′ splice site aligned in mouse, human, and Fugu. Bold type indicates blocks of sequence conserved between human and Fugu, but not in mouse. Italic sequence shows consensus branch-point and 3′ splice site sequences. The proposed branch-point A is marked with *. $ indicates the first nucleotide of exon 10. Capitalized letters of consensus sequences summarize positions where both Fugu and human match the consensus. (B) Alignments 1 to 4 illustrate the conservation of selected CFTR promoter elements between human (Homo sapiens), gibbon (Hylobates lar), monkey (Saimiri sciureus), cow (Bos taurus) and rabbit (Oryctolagus cuniculus) at coordinates relative to the start of translation of human CFTR. Asterisks indicated nucleotides conserved between all aligned species. Background shading marks the extent of proposed elements. (C) The relative position of sequence motifs implicated in CFTR regulation, DNase I hypersensitive sites, and exons one and two are indicated with respect to the translational start site (ATG). Annotated elements are as defined in: 1(Smith et al. 1995), 2(Smith et al. 1996), 3(Matthews et al. 1996), 4(Chou et al. 1991), 5(Yoshimura et al. 1991a), 6(Pittman et al. 1995). (D) FA and FB Fugu CFTR intron 1 elements are shown in alignment with the conserved human HA element. Asterisks indicate nucleotides conserved in all three elements. The 10-bp perfect palindromes of FB and HA are indicated by a horizontal bar, and the axis of symmetry is indicated by a vertical arrow. Horizontal arrows indicate partially conserved direct repeats in each element. The coordinates indicate distance from the 3′ end of CFTR exon 1. Background shading marks the extent of the proposed element.

Figure 3

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Genomic sequence analysis of Fugu rubripes CFTR and flanking genes in a 60 kb region conserving synteny with 800 kb of human chromosome 7 - PubMed (original) (raw)