Bouwmeester T et al. (1992), Molecular cloning, expression and in vitro func...

XB-ART-23937

Mech Dev 1992 Mar 01;371-2:57-68. doi: 10.1016/0925-4773(92)90015-c.

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Molecular cloning, expression and in vitro functional characterization of Myb-related proteins in Xenopus.

Bouwmeester T , Güehmann S , el-Baradi T , Kalkbrenner F , van Wijk I , Moelling K , Pieler T .

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Two cDNAs encoding Myb-related proteins have been cloned from Xenopus laevis and they have been termed Xmyb1 and Xmyb2. The Xmyb1 cDNA clone codes for an open reading frame of 733 amino acids and exhibits a high degree of similarity over the entire predicted protein sequence with the human B-Myb protein. Xmyb2 is a partial cDNA clone encoding three copies of amino-terminal tandem repeat elements typical for the Myb DNA-binding domain. The predicted protein sequence is most closely related to the human A-Myb gene product. In vitro translation of two deletion mutants of Xmyb1, truncated in the 3'-portion of the open reading frame, results in protein products which cross-react with polyvalent as well as monoclonal antibodies directed against the human c-Myb protein. The same two XMyb1 proteins, which both contain the complete set of aminoterminal repeats, specifically bind to the c-Myb-specific DNA binding sequence as evidenced by electrophoretic mobility shift analysis in vitro. RNA expression profiles of Xmyb1 and -2 are very different from each other; Xmyb1 is present throughout oogenesis and early Xenopus embryogenesis; in adult tissue it is primarily detected in blood. In contrast, Xmyb2 is expressed at only very low levels during oogenesis, not detectable in embryonic RNA preparations, and in adult tissue it is predominantly expressed in testis, with only a very low level seen in blood.

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Species referenced: Xenopus laevis
Genes referenced: myb mybl1 mybl2
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Fig. 4. RNA profiles for Xmybl and Xmyb2 in Xenopus development. (A) Oogenesis. Four cell equivalents of total RNA from stages of oogenesis indicated (Dumont, 1972) were loaded per lane and probed for Xenopus myb related transcripts with Xmybl and Xmyb2 eDNA fragments. As a control, the same blot was rehybridized with a probe for Xenopus histone H4. (B) Embryogenesis. Four egg/embryo equivalents of total RNA were applied per lane; embryos were staged according to Nieuwkoop and Faber (1956). Hybridizations and controls were as in A, with probing for EF-la transcription as an additional control for the zygotic activation of transcription. (C) Adult tissue. 10-20 ~g of total RNA from Xenopus ovary (O), testis (T), kidney (K), lung (Lu), heart (H), intestine (I), stomach (St), brain (B), spleen (Sp), liver (Li) and blood (BI) were applied as indicated. Probing was as in B.

Fig. 5. Serological and functional analysis of Xmybl translation products. (A) Truncated versions of Xmybl and hu c-myb were transcribed in vitro and translated in a reticulocyte lysate using [35S]methionine. An aliquot (10%) of the reactions was directly applied to an SDS-polyacrylamide gel or processed for indirect immunoprecipitation using anti-Myb sheep serum $33 and a hyperimmune sheep control serum (C) or ascites fluid (2/~1) of two anti-hu c-Myb producing hybridomas, MAB 6/22 and MAB 3/7. XMyb proteins consisted of aa 1-458 (N458, abbreviated Xd) and aa 1-246 (N246, Xdd). The hu c-Myb protein (He) was full length. M, molecular weight standards. Exposure times were 15 li, except for the MABs which were exposed for 10 days. (13) DNA mobility shift analysis was performed with in vitro translation products, Xmybl N458 and N246, translated from the same RNA preparations as in (A) in the absence of labelled methionine. The translation product-containing lysates were allowed to react with a radioactively labelled 25-mer DNA oligonucleotides containing the DNA binding sequence TAACGGTT (see Materials and Methods). The reaction was performed without and with competition with increasing amounts of the identical oligonucleotide (100-, 400-, and 800-fold molar excess, respectively, blank triangle) or a mutated oligonucleotide containing one point-mutation (TATCGGTT) (dashed triangle). C indicates a control of the DNA plus non-programmed reticulocyte lysate. (C) Competition of the DNA-Myb protein interaction shown in (B) with antiserum against hu c-Myb protein was performed using the sheep serum $33 (S) and a negative hyperimmune serum (NS). The serum was added to the in vitro translation products prior to incubation with DNA.