Cloning of Mix-related homeodomain proteins using fast retrieval of gel shift activities, (FROGS), a technique for the isolation of DNA-binding proteins

Cloning of Mix-related homeodomain proteins using fast retrieval of gel shift activities, (FROGS), a technique for the isolation of DNA-binding proteins

^*Department of Pediatrics, Division of Hematology/Oncology, Harvard Medical School and ^†Howard Hughes Medical Institute, Children’s Hospital, Enders 780, 300 Longwood Avenue, Boston, MA 02115; and ^‡Department of Cell Biology, Harvard Medical School, 25 Shattuck Street, Boston, MA 02115

Contributed by Marc W. Kirschner

Abstract

We have developed a technique, fast retrieval of gel shift activities (FROGS), that allows for the rapid isolation of proteins that interact with DNA. Using this technique, we have isolated two proteins that are structurally similar to Mix.1, a PAX class homeodomain protein with ventralizing activity in Xenopus. The Mix family of proteins are expressed during late blastula and gastrula stages of Xenopus development. During gastrulation, these genes are expressed at high levels in distinct, yet overlapping regions in mesoderm and endoderm. The members of the Mix family heterodimerize with each other and overexpression of each results in severe axial abnormalities. Mix.3 and Mix.4 can directly induce primitive ectoderm to become endoderm whereas Mix.1 cannot. Injection of Mix.3 or Mix.4 RNA in the whole embryo results in extensive ectopic endodermin mRNA expression. The expression of the Mix family homeoproteins is differentially regulated by activin, Vg1, BMP-4, and fibroblast growth factor, supporting a model in which the Mix homeoproteins are downstream effectors of growth factor signaling during endoderm and ventral mesoderm formation.

Footnotes

↵ § To whom reprint requests should be addressed. e-mail: zon{at}rascal.med.harvard.edu.
Data deposition: The sequences reported in this paper have been deposited in the GenBank database [accession nos. AF081350 (Mix.3) and AF081351 (Mix.4)].
ABBREVIATIONS:

FGF,

fibroblast growth factor;

TGF-β,

transforming growth factor β;

FROGS,

fast retrieval of gel shift activities;

RT,

reverse transcription;

HA,

homeodomain alone;

edd,

endodermin