Evolution of cis elements in the differential expression of two Hoxa2 coparalogous genes in pufferfish ( Takifugu rubripes )

Tümpel et al. 10.1073/pnas.0600993103.

Supporting Information

Files in this Data Supplement:

Supporting Figure 6
Supporting Figure 7
Supporting Figure 8
Supporting Table 1

Fig. 6. Alignment of Hoxa2 r3/5 control regions directing r3/5 expression. Local alignment of the two regions of identity in the r3/5 expression module is shown. The colors within the sequence alignment indicate the degree of identity: yellow, complete conservation of sequence; blue, bases conserved in >50% of species in the alignment; and white, bases conserved in <50% of species. Dashes indicate gaps introduced to maintain maximal alignment. The locations of the key regulatory elements in the enhancer module, including the newly discovered RE5 element, are boxed in the alignment, with the names given above the boxes.

Supporting Figure 7

Fig. 7. Alignment of Hoxa2 r4 control regions directing r4 expression. Local alignment of the r4 expression module is shown. The colors within the sequence alignment indicate the degree of identity: yellow, complete conservation of sequence; blue, bases conserved in >50% of species in the alignment; and white, bases conserved in <50% of species. Dashes indicate gaps introduced to maintain maximal alignment. The locations of the critical control elements in the enhancer are boxed in the alignment, with the names given above the boxes.

Supporting Figure 8

Fig. 8. Alignment of Hoxa2 r2 control regions directing r2 expression. Local alignment of the r2 expression module is shown. The colors within the sequence alignment indicate the degree of identity: yellow, complete conservation of sequence; blue, bases conserved in >50% of species in the alignment; and white, bases conserved in <50% of species. Dashes indicate gaps introduced to maintain maximal alignment. The locations of the essential r2-enhancer elements are boxed in the alignment, with the names given above the boxes.

Table 1. Primers used to generate PCR fragments for cloning of wild-type and mutated regulatory regions

Construct no.	Forward sequence	Reverse sequence
1	TGC TGT AAT GCC AAA ACC TC	CCT GCC TCG CCT TCG TGC CG
2	TGG CTT AAT GCA AAC GCT AT	CCA TTA AGT TAA CAC TGA CAG ATA T
9	TGA TGA TTA ATA ACC TCT ATG TAA A	GAG CCT GAC TGG GAA GAT TT
10	TTC CCC AAA AGG TGG GTG AT	ATC TCA GGC GAA CCT AGA AA
14	GGC AGT TCC AGC AAA ACC AAT	GCT TGC TTC TTT TGC AAA CA
15	AGA TAC CTG CTC CTT TCA GA	TAG GCC TAT TAT TGT AT

Construct no.	Mutagenesis primer sequence (forward)
3	CCA CAG TTT GTA GGA GAG GCA GAG CTG CAC TGA AAG CCA ACA CCC ACT CAC CTC CTT GGA CAC AAA GCC TGT GCG TAA TTC
4	GCG ACA GCC TGG CTG TGA CTC CGA GCA GAA AAT GTG TCC TAT GCA CAC CTT GCT TGG TCC ACG TCC TGG CTG CAT TTG ATC CGG GGG AGA GTT AGA AGC
5	CTG AGT GGC CAG TGT TTC TCG CCG TTT CCA GGC ACT ATA TGA TCC CAG GGA GTG TTG GAT GCT TTA AAT GTG TTG CGA GGG CAC CGA GCT GTC AGA CC
6	CTG AGT GGC CAG TGT TTC TCG CCG TTT CCT GGC TGC ATT TGA TCC GGG GGA GAG TTA GAA GCC TTA AAT GTG TTC TTA GGG CAG GGA GCT GTC AGA CC
7	GCT GTC AGA CCT TTT GGC GAG TAA GAT TGA TCA CAC TCA GGG ACC GAG GTC TTT GTT TAG AGT CCG AGC AAC AAA CCT AGA GAG GCC TAC C
8	CCA GCA CTC TTT GTT TGG TAT ATA AGC AAT AAA CAG CCA TCA GAT CCC ACC TTT CCT CTG CTT CCT CTC TCC CTC ACT TCC TTA CTC TCC C
RE5 del	GGA ATA AAA GCA AGA AAA CTG GAA AAA CCC TTA CAT AAA ATA GCA TCT CTA TCT GCA AGG TAA TGC TCA GAG CTG G
11	GCT TCG GGG CAA AAT GGG TAA TGA TTT AAG CAA GTT TGG TGG TGA TGA GAA ATG ATT TAT TCC
12	CGC GTG TGA CAG TAA TGA AGA GTG ATA GAT TAC CGT TGC CGA GGA GGG CAG CTG G
13	GGA GTG CCG TTG CCG AGG AGG GCA GCT GGC ATG TTC ATT AGT ATC CCA CAC TGG CCC TTG CTG C
18	CCT TTC AGA AAA AGT CTC ACG GTT CGG CGC AGC AGC ACG GGA ACA ATG GGG AGA GCC AAA GGC TTT GCT GCT GCG CCG CTG AAC AGC
19	GCA GCA GCA CCA CAA TAG CGT GTC CAT GGC CTG AGC GCA GCC CTT TTG AAC AGC AAT GAC AAA AAT CTG AAA CAT TTT CC
20	GCA ATG TCT GAA CGG GAA CAA TGG GGA GAG CAT GGG CCT GAG CGC AGC CCT GCT GAA CAG CAA TGA GAA AAA TCT GAA ACA TTT TCC
21	CCA CAA TAG CGT GTG CCA AAG GCT TTG CTG CTG CGC CTT TGA ACA GCA ATG ACA AAA ATC TGA AAC ATT TTC CCA ACC C

The left column gives the construct number for which the relevant primers were used. All sequences are listed 5' to 3'. For mutagenesis primers, the reverse primer is the direct complement of the forward primer. Primers were designed by using the Elgar-hosted fugu database at http://fugu.biology.qmul.ac.uk (August 2005).