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Chicken homeo box genes: Developmental regulation and evolution
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TRITffORAX (TRX) A~D FEMALE STERILE HOMEOTIC (FSH): M O L E C O L A R CHARACTEHIZATIOW AWD IRTE-'---RACTIONS WITH OTHER HOMEOTIC LOCI. Igor B. Dawid, Susan R. Haynes, D e r - H w a Huang, Brian A. Mozer, N. Bhatia-Dey, and Alexander M. Mazo. Laboratory of Molecular G e n e t i c s , N a t i o n a l Institute of Child Health and Human Development, NIH, Bethesda, Maryland. Mutations in the maternal-effect locus fsh lead to missing organs and bithorax-like transformations in the progeny; these homeotic effects are inereased by interaction with zygotic mutations in trx. In t h e thorax m u t a t i o n s in trx cause transformations of the first and third segments toward the second segment; these effects are m o d u l a t e d by dosage of the bithorax complex (BX-C), implying that trx regulates the activity of the BX-C. We h a v e d e m o n s t r a t e d c h a n g e d p a t t e r n s of expression of Kr, eve and Ubx in progeny of fsh mutant mothers, and of Ubx expression in the nerve cord of trx mutant embryos, supporting the view that fsh and trx are regulatory loci. Both fsh and trx were cloned and s e q u e n c e d . The m a j o r t w o products of f s h a r e p r e d i c t e d to be membrane proteins; the major predicted trx product is a very large (400 kDa) p r o t e ~ w i t h a cysteine-rich region that may be folded into a series of zinc finger-like domains.
CHICKEN HOMEO BOX GENES; DEVELOPMENTAL REGUlATION AND EVOLUTION
603 POSSIBLE INVOLVEMENT OF HOMEOBOX CONTAINING PROTEINS ON THE CONTROL OF VIMENTIN EXPRESSION Philippe Duprey, Ward Odenwald, Alain Lilienbaum, and Denise Paulin Laboratoire de Biologie Mol~-ulalre de la Diff&enciation, Universit~ Paris 7, UFR de Biochimie 2, place Jussieu 75005 Paris France Laboratory of Molecular Genetics, National Institute of Health, Bethesda, Maryland 20892 USA Expression of intermediate filaments of the vimentin type was studied in early' mouse embryos. Although vimefitln is expressed in most mesodermal cells in vitro, it was not found in vivo in derivatives of the segmented mesoderm, such as somites, sclerotomes prevertebrae and embryonic rips, while lose connective tissue cells did express this marker. As segmented mesoderm cells heavily express transcripts derived from murine homeobox containing genes, we have started to look wether the lack of vimentin gene expression could be linked to the abundant expression of routine homeogenes. Structural and functional characterization of human vimentin promoter has enabled to define multiple regulatory elements, whose respective contributions to the final activity of the promoter differ according to cell types and growth conditions. One of these elements located for upstream from the cap site contains 4 clustered sequences homologous to the binding sites far the homeogene Hoxl.3 product. We have observed that a recombinant baeulovirus derived Hoxl.3 protein interacts strongly with these sequences and are currently testing '. the functional signifiance of this binding.
Fainsod, A.I, Z. Rangini2 , A. Frumkin I, E. Shapira I, A. Berr-YehudaI and ~ Gruenbaum 2. IDepartment of Cellular Biochemistry, Hebrew University-Hadassah Medical School, Jerusalem 91010 Israel, and 2Department of Genetics, Hebrew University, Jerusalem 91904 Israel. About 20 independent chicken homeo box sequences have been cloned by screening two genomic libraries with the Drosophila Antp and en homeo boxes, the murine Hox 1.5 and 3.1 homeo boxes and the chicken CHox3 homeo box under reduced stringency conditons. Six of the homeo box regions have been sequenced revealing all of them to be distant members of the Antp type of homeo boxes. Interestingly and in contrast to all other vertebrate homeo boxes, three of them are interrupted by an iron after amino acid 44 similar to the positon of the intron in the Drosophila lab homeo box. The levels of similarity to homeo boxes from other organisms are about 4075% at the DNA level and 40-70% at the protein level except for one homeo box. The CHox4 homeo box is highly similar to the Drosophila cad and the murine cdxl homeo boxes but in addition is one of the sequences interrupted by an intron. The developmental regulation of the chicken homeo box genes has been studied by northern blot analysis. Studies of the temporal pattern of expression was performed on RNA from chicken embryos incubated up to five days. CHox3 expression was studied from the time the egg is laid in which the embryo is at the blastoderm stage to five days of incubation which represents an embryo well into organogenesis. CHox3 codes for five transcripts 1.3, 1.9, 2.6, 5.6 and 7.9kb in size. The most abundant are the 1.3 and 1.9 kb transcripts and they are already present at the time the egg is laid, their abundance peaks at the time the primitive streak reaches full length and then decreases. The levels of the other transcripts also change during development. CHoxl codes for three transcripts 2.2, 2.8 and 6kb in size, all of them of low abundance and are present while the embryo develops from 22 to 40-43 somites. Transcription of CHox7 involves four transcripts 1.4, 1.9, 2.4 and 3.5kb in size. The two abundant transcripts are 1.4 and 1.9kb in size, each one peaks at a different time. The 1.4kb transcripts peaks at the time the neural folds appear while the 1.9kb transcript peaks towards day 3 of incubation. Expression of CHox4 results in two transcripts 1.6 and 2.6kb. CHox4 expression begins very close to the time the egg is laid. Both transcripts are extinguished at different rates, by day four the 1.6kb transcript is absent and by day five the 2.6kb transcript disappears. In order to clone the cDNAs for some of the chicken homeo box genes we prepared a cDNA library from embryos at 2-2.5 days of incubation. Screening of the library with gene specific probes allowed us to clone cDNAs for the abundant transcripts of CHox3, 7 and 4.
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TRITffORAX (TRX) A~D FEMALE STERILE HOMEOTIC (FSH): M O L E C O L A R CHARACTEHIZATIOW AWD IRTE-'---RACTIONS WITH OTHER HOMEOTIC LOCI. Igor B. Dawid, Susan R. Haynes, D e r - H w a Huang, Brian A. Mozer, N. Bhatia-Dey, and Alexander M. Mazo. Laboratory of Molecular G e n e t i c s , N a t i o n a l Institute of Child Health and Human Development, NIH, Bethesda, Maryland. Mutations in the maternal-effect locus fsh lead to missing organs and bithorax-like transformations in the progeny; these homeotic effects are inereased by interaction with zygotic mutations in trx. In t h e thorax m u t a t i o n s in trx cause transformations of the first and third segments toward the second segment; these effects are m o d u l a t e d by dosage of the bithorax complex (BX-C), implying that trx regulates the activity of the BX-C. We h a v e d e m o n s t r a t e d c h a n g e d p a t t e r n s of expression of Kr, eve and Ubx in progeny of fsh mutant mothers, and of Ubx expression in the nerve cord of trx mutant embryos, supporting the view that fsh and trx are regulatory loci. Both fsh and trx were cloned and s e q u e n c e d . The m a j o r t w o products of f s h a r e p r e d i c t e d to be membrane proteins; the major predicted trx product is a very large (400 kDa) p r o t e ~ w i t h a cysteine-rich region that may be folded into a series of zinc finger-like domains.
CHICKEN HOMEO BOX GENES; DEVELOPMENTAL REGUlATION AND EVOLUTION
603 POSSIBLE INVOLVEMENT OF HOMEOBOX CONTAINING PROTEINS ON THE CONTROL OF VIMENTIN EXPRESSION Philippe Duprey, Ward Odenwald, Alain Lilienbaum, and Denise Paulin Laboratoire de Biologie Mol~-ulalre de la Diff&enciation, Universit~ Paris 7, UFR de Biochimie 2, place Jussieu 75005 Paris France Laboratory of Molecular Genetics, National Institute of Health, Bethesda, Maryland 20892 USA Expression of intermediate filaments of the vimentin type was studied in early' mouse embryos. Although vimefitln is expressed in most mesodermal cells in vitro, it was not found in vivo in derivatives of the segmented mesoderm, such as somites, sclerotomes prevertebrae and embryonic rips, while lose connective tissue cells did express this marker. As segmented mesoderm cells heavily express transcripts derived from murine homeobox containing genes, we have started to look wether the lack of vimentin gene expression could be linked to the abundant expression of routine homeogenes. Structural and functional characterization of human vimentin promoter has enabled to define multiple regulatory elements, whose respective contributions to the final activity of the promoter differ according to cell types and growth conditions. One of these elements located for upstream from the cap site contains 4 clustered sequences homologous to the binding sites far the homeogene Hoxl.3 product. We have observed that a recombinant baeulovirus derived Hoxl.3 protein interacts strongly with these sequences and are currently testing '. the functional signifiance of this binding.
Fainsod, A.I, Z. Rangini2 , A. Frumkin I, E. Shapira I, A. Berr-YehudaI and ~ Gruenbaum 2. IDepartment of Cellular Biochemistry, Hebrew University-Hadassah Medical School, Jerusalem 91010 Israel, and 2Department of Genetics, Hebrew University, Jerusalem 91904 Israel. About 20 independent chicken homeo box sequences have been cloned by screening two genomic libraries with the Drosophila Antp and en homeo boxes, the murine Hox 1.5 and 3.1 homeo boxes and the chicken CHox3 homeo box under reduced stringency conditons. Six of the homeo box regions have been sequenced revealing all of them to be distant members of the Antp type of homeo boxes. Interestingly and in contrast to all other vertebrate homeo boxes, three of them are interrupted by an iron after amino acid 44 similar to the positon of the intron in the Drosophila lab homeo box. The levels of similarity to homeo boxes from other organisms are about 4075% at the DNA level and 40-70% at the protein level except for one homeo box. The CHox4 homeo box is highly similar to the Drosophila cad and the murine cdxl homeo boxes but in addition is one of the sequences interrupted by an intron. The developmental regulation of the chicken homeo box genes has been studied by northern blot analysis. Studies of the temporal pattern of expression was performed on RNA from chicken embryos incubated up to five days. CHox3 expression was studied from the time the egg is laid in which the embryo is at the blastoderm stage to five days of incubation which represents an embryo well into organogenesis. CHox3 codes for five transcripts 1.3, 1.9, 2.6, 5.6 and 7.9kb in size. The most abundant are the 1.3 and 1.9 kb transcripts and they are already present at the time the egg is laid, their abundance peaks at the time the primitive streak reaches full length and then decreases. The levels of the other transcripts also change during development. CHoxl codes for three transcripts 2.2, 2.8 and 6kb in size, all of them of low abundance and are present while the embryo develops from 22 to 40-43 somites. Transcription of CHox7 involves four transcripts 1.4, 1.9, 2.4 and 3.5kb in size. The two abundant transcripts are 1.4 and 1.9kb in size, each one peaks at a different time. The 1.4kb transcripts peaks at the time the neural folds appear while the 1.9kb transcript peaks towards day 3 of incubation. Expression of CHox4 results in two transcripts 1.6 and 2.6kb. CHox4 expression begins very close to the time the egg is laid. Both transcripts are extinguished at different rates, by day four the 1.6kb transcript is absent and by day five the 2.6kb transcript disappears. In order to clone the cDNAs for some of the chicken homeo box genes we prepared a cDNA library from embryos at 2-2.5 days of incubation. Screening of the library with gene specific probes allowed us to clone cDNAs for the abundant transcripts of CHox3, 7 and 4.
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