Distinct Roles for TBP and TBP-Like Factor in Early Embryonic Gene Transcription in Xenopus Gert Jan C. Veenstra, Daniel L. Weeks, and Alan P. Wolffe

Constructs and RT-PCR

Five individual point mutations (Fig. 1A) were introduced in the pSP64A-xTBP (1, 2) using the QuikChange Site-Directed Mutagenesis Kit (Stratagene). Mutations were confirmed by sequencing. For microinjection experiments, capped xTBP RNA was synthesized from linearized pSP64A-xTBP, using an in vitro RNA synthesis kit (Ambion). The Xenopus homolog of TLF was isolated by RT-PCR on basis of the available sequence information (accession number AJ238441). RNA was isolated from staged embryos and reverse transcribed using Superscript RT (Life), and the resultant DNA was amplified, cloned in pBlueScript, and sequenced. The sequence of the primers used for RT-PCR of TLF was as follows: TLF-1L: 5´-GCTGTAGAATTCATCCCGGGTAAGAAGCAACGGGTAAGCC-3´ TLF-1R: 5´-GTTGCAGAATTCCCGGGTTCCATTCAGCATCCACAAA-3´ RT-PCR primers for detection of MyoD and GS17 RNA were as described (3). Linearity of amplification in the quantitative RT-PCR assays was assessed by titrating the amount of input cDNA or by analyzing the amount of amplified DNA at different PCR cycles between cycles 12 and 22. RT-PCR products were detected by Southern blotting, using TLF, MyoDb, or GS17 coding fragments as probes.

Modified oligonucleotides

The sequence of the oligonucleotides used was as follows: TBP-AS3: 5´-T⁺G⁺C⁺A⁺T⁺T-T-C-G-C-G-C-T⁺C⁺T⁺A⁺A⁺G-3´ TLF-AS93: 5´-A⁺T⁺A⁺T⁺C⁺C-A-G-T-G-C-C⁺A⁺C⁺A⁺T⁺C-3´ TLF-AS57: 5´-G⁺A⁺T⁺G⁺G⁺C-T-T-A-C-C-C⁺G⁺T⁺T⁺G⁺C⁺T-3´

DEED-modified linkages are indicated with "+", and normal phosphodiester bonds are indicated with "-". N,N-diethyl-ethylenediamine (DEED)-modified oligonucleotides were synthesized using hydrogen phosphonate chemistry and oxidative amidation of hydrogen phosphonate diesters with DEED, as described by Dagle et al. (4) and references therein. Briefly, the oligonucleotide was synthesized in blocks to generate both phosphodiester and phosphoramidate linkages. The six 3´ residues were first coupled and then oxidatively amidated. The next two blocks of six residues were individually condensed and oxidized or oxidatively amidated, respectively. Between 1 and 2 ng of DEED-modified oligonucleotide was injected per embryo.

Northern blotting

Northern blot analysis was performed using Hybond N-plus (Amersham) membranes and Hybrisol I (Oncor) for blocking and hybridization solution. Three to five embryo equivalents of total RNA were loaded per lane for each sample. 28S and 18S ribosomal RNA was visualized by staining with 0.02% methylene blue in 0.3 M sodium acetate and destaining in 20% ethanol. For marker gene analysis, gel-purified DNA fragments (25 ng) were labeled using a RediPrime random labeling kit (Amersham). Blots were washed in 0.2× SSC/0.1 % SDS at 65°C.

Western blotting

Embryos were homogenized in four volumes of low-salt whole-cell extract buffer (WCE-LS: 25 mM tris-HCl (pH 7.5), 70 mM KCl, 1 mM EDTA, 20% glycerol, 5 mM DTT, 1 mg/ml of leupeptin, pepstatin A, and aprotinin). Homogenates were centrifuged at 15,000g for 5 to 10 min at 4°C. Subsequently the supernatant was frozen on dry ice and stored at -80°C for later use. Two embryo equivalents of extract were loaded per lane. Western blot analysis was performed using Hybond-ECL (Amersham) membranes, anti-TBP (58C9, Santa Cruz Biotech., use at 1:2000), and an enhanced chemiluminescence (ECL) detection kit (Pierce).

References

1. S. Hashimoto, H. Fujita, S. Hasegawa, R. G. Roeder, M. Horikoshi, Nucleic Acids Res. 20, 3788 (1992).

2. G. J. C. Veenstra, O. H. J. Destrée, A. P. Wolffe, Mol. Cell. Biol. 19, 7972 (1999).

3. R. A. W. Rupp, H. Weintraub, Cell 65, 927 (1991). 4. J. M. Dagle, J. L. Littig, L. B. Sutherland, D. L. Weeks, Nucleic Acids Res. 28, 2153 (2000).