Linking pattern formation to cell-type specification: Dichaete and Ind directly repress achaete gene expression in the Drosophila CNS -- Zhao et al. 104 (10): 3847 Data Supplement - HTML Page - index.htslp -- Proceedings of the National Academy of Sciences

Zhao et al. 10.1073/pnas.0611700104.

Supporting Information

Files in this Data Supplement:

SI Data

SI Data

Oligonucleotides Used as Unlabeled Competitor DNA in the Gel-Shift Assays.
Sequences of the oligonucleotides used as unlabeled competitor DNA in the gel-shift assays (Dichaete- and Ind-binding sites are in italics):

HP1: GTGCAACCAGAATTCTGACAATGGTTTTAAGAAATA;

HP2: GAAATAATGGGGACCTCCTAAATGCTTTCAAAATGC;

HP3: TCAAAATGCTTTCGGCTGAGAGGAACAACAACTG;

HP4: GTGCAACCAGAATTCT;

HP5: ATTCTGACAATGGTTT;

HP6: TGGTTTTAAGAAATAA;

HP7: GAAATAATGGGGACCTCC;

HP8: TCCTAAATGCTTTCAAAATGC;

mHP2: GAAAGTCGACGGACCTCCTAAATGCTTTCAAAATGC;

mHP22 : GAAAACAACAGGACCTCCACAACAATTTCAAAATGC;

mHP5: ATTCTACTCTGAGTTT;

mHP7: GAAAACAACAGGACCTCC;

mHP8: TCCACAACAATTTCAAAATGC

Primers Used for Site-Directed Mutagenesis of Predicted Ind, Vnd, and Dichaete Sites in Pg7.
External Primers for the Ind- and Dichaete-binding sites:

5'-GCAAGGTCGACCGGA TATGTTTGA-3';

5'-CAGGCTATTTATAGGTATCAGGGCCTAG-3'.

Internal primer pairs (mutated site is in italics):

Primers used to mutate the H1 Ind-binding site:

5'-ACAATGGTTTTAAGAAAACAACAGGACCTCCTAAATGCTTTCAAAATGCTTTCGGCTGAGAGG-3'

5'-GCATTTAGGAGGTCCTGTTGTTTTCTTAAAACCATTGTCAGAATTCTGG-3'

Primers used to mutate the H2 Ind-binding site:

5'-ACAATGGTTTTAAGAAATAATGGGGACCTCCACAACACTTTCAAAATGCTTTCGGCTGAGAGG-3'

5'-GTGTTGTGGAGGTCCCCATTATTTCTTAAAACCATTGTCAGAATTCTGG-3'

Primers used to mutate the S2 Dichaete-binding site:

5'-TGCAACCAGAATTCTACTCTGAGTTTTAAGAAATAATGGGGACCTCC-3'

5'-CCCATTATTTCTTAAAACTCAGAGTAGAATTCTGGTTGCACGCTAGGAAAC-3'

Primers used to mutate H1 and H2:

5'-ACAATGGTTTTAAGAAAACAACAGGACCTCCGCAACACTTTCAAAATGCTTTCGGCTGAGAGG -3';

5'- GTGTTGCGGAGGTCCTGTTGTTTTCTTAAAACCATTGTCAGAATTCTGG -3';

Primers used to mutate H1 and S2

5'- TGCAACCAGAATTCTACTCTGAGTTTTAAGAAAACAACAGGACCTCCTAAATG CTTTCAAAATGCTTTCGGCTGAGAGG -3'

5'- GCATTTAGGAGGTCCTGTTGTTTTCTTAAAACTCAGAGTAGAATTCTGGTTGCACGCTAGGAAAC-3'

Primers used to mutate H2 and S2:

5'-TGCAACCAGAATTCTACTCTGAGTTTTAAGAAATAATGGGGACCTCCGCAACACTTTCAAAATGCTTTCGGCTGAGAGG-3'

5'-GTGTTGCGGAGGTCCCCATTATTTCTTAAAACTCAGAGTAGAATTCTGGTTGCACGCTAGGAAAC-3'

Primers used to mutate H1, H2, and S2:

5'-TGCAACCAGAATTCTACTCTGAGTTTTAAGAAAACAACAGGACCTCCACAACACTTTCAAAATGCTTTCGGCTGAGAGG-3'.

5'-GTGTTGCGGAGGTCCTGTTGTTTTCTTAAAACTCAGAGTAGAATTCTGGTTGCACGCTAGGAAAC-3'.

To generate mutated versions of the adjacent Vnd- and Dichaete-binding sites at the 5' end of pG7, we used the below listed pair of external primers and with three sets of internal primers to mutate each site individually and both sites together.

External primer pairs:

5'GGGTACCGGGCCCCCCCTCGAGGTCGACGGTATCGATAGATCTCAGTTCTTCCCCCCTTAAG-3'

5'-CAGTTGTTGTTCCTCTCAGCCGAAAG-3'

Internal primer pairs:

Primers used to mutate the sole predicted Vnd-binding site:

5'-AAAAGCTCAAGAAGAGCCTCGCTGGCGCTAAGCGATATTCAAAGC-3'

5'-TCGCTTAGCGCCAGCGAGGCTCTTCTTGAGCTTTTCTGCTTG-3'

Primers used to mutate the S1 Dichaete-binding site:

5'-TACATCTGACAAAAAATAATAATAAAATTGCAAATTCATAAACCTGGTTGACACTG-3'

5'-CAATTTTATTATTATTTTTTGTCAGATGTATCGCTTAGCGCCACAAGTGCTCTTCTTGAG-3'

Primers used to mutate the Vnd- and S1 Dicheate-binding sites:

5'-AGAAGAGCCTCGCTGGCGCTAAGCGATACATCTGACAAAAAATAATAATAAAATTGCAAATTCATAAACCTGGTTGACACTG-3'

5'-GTCAGATGTATCGCTTAGCGCCAGCGAGGCTCTTCTTGAGCTTTTCTGCTTG-3'