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nuclear factor I
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TRANSCRIPTION OF DEVELDPMENTALLY REGULATED DROSOPHILA GENES IN VITRO. R. Tiian. U. Heberlein. B.P. England. M.D. Big~n. and K.K. Perkins. Dept. of Biochemistry, Univ. of Calif., Berkeley, CA 94720,
T H E D U A L F U N C T I O N OF T G G C A - P R O T E I N / N U C L E A R F A C T O R I. J . N o w o c k I and R. M i k s i c e k 2 • I He i n r i g h - P e t t e - I nst it ut, D - 2 0 0 0 H-araburg 20, ZDKFZ,~ D - 6 9 0 0 Heid e l b e r g , FR G e r m a n y .
We have developed cell-freeU'anscriptionexuacts from staged Drosophila embryos in an attempt to recapitulate the temporal program of Alcohol dehydmgenase (Adh), and the bomeotic genes Ultrabithorax (Ubx) andAntennapedia (Amp) transcription in vitro. We find that the distal promoter of Adh
TGGCA-binding proteins represent a class o f n u c l e a r p r o t e i n s u b i q u i t o u s a m o n g h i g h e r e u c a r y o t e s . T h e y are f u n c t i o n a l l y e q u i v a l e n t to n u c l e a r factor I from H e L a cells w h i c h s t i m u lates i n i t i a t i o n o f a d e n o v i r u s D N A replication. The M M T V - L T R c o n t a i n s a T G G C A - b i n d ing site. To t e s t for its f u n c t i o n a l s i g n i f i c a n c e , this s i t e was m u t a t e d and a n a l y z e d by t r a n s i e n t e x p r e s s i o n in m o u s e L and in h u m a n M C F - 7 cells. Mutants that eliminate TGGCA-protein b i n d i n g in v i t r o s t r o n g l y r e d u c e d the b a s a l and the g l u c o c o r t i c o i d - d e p e n d e n t a c t i v i t y of the M M T V p r o m o t e r . R e s t o r a t i o n of b i n d i n g by the i n s e r t i o n of o l i g o n u c l e o t i d e s containing s y n t h e t i c and n a t u r a l T G G C A - b i n d i n g s i t e s led t o a r e s t o r a t i o n of hormoneinduced expression. The requirement of the T G G C A - b i n d i n g site for hormoned e p e n d e n t e n h a n c e m e n t can be s u b s t i t u t e d by the r e c o g n i t i o n sites o f other constitutive transcription factors.
is Iranscribed by staged extracts in a manner that follows the pattexn observed in vivo. Analysis of promoter mutants together with DNase I fontprinting experiments suggest that the regulation observed/n v/tro is at least partly due to the levels of the transcription factm Adf-l. This factor, which
binds to and activates the distal promoter of Adh has been purified to near homogeueity using slx~ific DNA-affinity chromatography. Surprisingly, the purified Adf-1 also binds strongly to the Antp P1 promoter. Its role in Amp transcription is currently being investigated. We have also analyzed the signals involved in regulating transcripdon from the Ubx promoter in embryo and Kc cell extracts. Analysis of deletion and clustered point mutants indicate that sequences located both 5' and 3' to the Ubx start site affect the efficiency of initiation/n v/fro. DNase I footprinting revealed the presence of several DNA binding proteins, including several embryo specific factors, that selectively recognize these cis-regulatory sequences. We are in the process of purifying and characterizing these factors in order to study their role in regulating transcription during Drosophila embryogenesis. 6 Expression of four different mouse homeobox genes in embryos and during pluripotent stem cell differentiation. Jacqueline Deschamps, Rozalia de Laaf, Lilian Joosen Olivier Destr4e and Frits Meijlink. Hubrecht Laboratory, Netherlands Institute for Developmental Biology 5 Utrecht. We cloned and characterized a mouse gene containing an homeobox very similar to the Drosophila Antennapedia homeobox. We followed its expression and compared it to that of two other Antennapedia-like and to the engrailed-1 m~ise homeobox genes. The pattern of expression was characterized in dissected embryos and in differentiating mouse stem cells. The highest level of transcripts was found in neural tissues (brain - spinal cord) for all four genes~ the region of maximal expression being specific for each of them. Expression of these four genes was followed during differentiation of embryonal carcinoma and embryo stem cells. Alternative treatments in the presence or in the absence of retinoic acid were used to trigger cell differentiation. Induction to high levels of expression of all four genes occured only in the presence of retinoic acid. We conclude that accumulation of high amounts of homeobox transcripts is not required for cell differentiation along many pathways~ and correlates with the presence of RA.
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TRANSCRIPTION OF DEVELDPMENTALLY REGULATED DROSOPHILA GENES IN VITRO. R. Tiian. U. Heberlein. B.P. England. M.D. Big~n. and K.K. Perkins. Dept. of Biochemistry, Univ. of Calif., Berkeley, CA 94720,
T H E D U A L F U N C T I O N OF T G G C A - P R O T E I N / N U C L E A R F A C T O R I. J . N o w o c k I and R. M i k s i c e k 2 • I He i n r i g h - P e t t e - I nst it ut, D - 2 0 0 0 H-araburg 20, ZDKFZ,~ D - 6 9 0 0 Heid e l b e r g , FR G e r m a n y .
We have developed cell-freeU'anscriptionexuacts from staged Drosophila embryos in an attempt to recapitulate the temporal program of Alcohol dehydmgenase (Adh), and the bomeotic genes Ultrabithorax (Ubx) andAntennapedia (Amp) transcription in vitro. We find that the distal promoter of Adh
TGGCA-binding proteins represent a class o f n u c l e a r p r o t e i n s u b i q u i t o u s a m o n g h i g h e r e u c a r y o t e s . T h e y are f u n c t i o n a l l y e q u i v a l e n t to n u c l e a r factor I from H e L a cells w h i c h s t i m u lates i n i t i a t i o n o f a d e n o v i r u s D N A replication. The M M T V - L T R c o n t a i n s a T G G C A - b i n d ing site. To t e s t for its f u n c t i o n a l s i g n i f i c a n c e , this s i t e was m u t a t e d and a n a l y z e d by t r a n s i e n t e x p r e s s i o n in m o u s e L and in h u m a n M C F - 7 cells. Mutants that eliminate TGGCA-protein b i n d i n g in v i t r o s t r o n g l y r e d u c e d the b a s a l and the g l u c o c o r t i c o i d - d e p e n d e n t a c t i v i t y of the M M T V p r o m o t e r . R e s t o r a t i o n of b i n d i n g by the i n s e r t i o n of o l i g o n u c l e o t i d e s containing s y n t h e t i c and n a t u r a l T G G C A - b i n d i n g s i t e s led t o a r e s t o r a t i o n of hormoneinduced expression. The requirement of the T G G C A - b i n d i n g site for hormoned e p e n d e n t e n h a n c e m e n t can be s u b s t i t u t e d by the r e c o g n i t i o n sites o f other constitutive transcription factors.
is Iranscribed by staged extracts in a manner that follows the pattexn observed in vivo. Analysis of promoter mutants together with DNase I fontprinting experiments suggest that the regulation observed/n v/tro is at least partly due to the levels of the transcription factm Adf-l. This factor, which
binds to and activates the distal promoter of Adh has been purified to near homogeueity using slx~ific DNA-affinity chromatography. Surprisingly, the purified Adf-1 also binds strongly to the Antp P1 promoter. Its role in Amp transcription is currently being investigated. We have also analyzed the signals involved in regulating transcripdon from the Ubx promoter in embryo and Kc cell extracts. Analysis of deletion and clustered point mutants indicate that sequences located both 5' and 3' to the Ubx start site affect the efficiency of initiation/n v/fro. DNase I footprinting revealed the presence of several DNA binding proteins, including several embryo specific factors, that selectively recognize these cis-regulatory sequences. We are in the process of purifying and characterizing these factors in order to study their role in regulating transcription during Drosophila embryogenesis. 6 Expression of four different mouse homeobox genes in embryos and during pluripotent stem cell differentiation. Jacqueline Deschamps, Rozalia de Laaf, Lilian Joosen Olivier Destr4e and Frits Meijlink. Hubrecht Laboratory, Netherlands Institute for Developmental Biology 5 Utrecht. We cloned and characterized a mouse gene containing an homeobox very similar to the Drosophila Antennapedia homeobox. We followed its expression and compared it to that of two other Antennapedia-like and to the engrailed-1 m~ise homeobox genes. The pattern of expression was characterized in dissected embryos and in differentiating mouse stem cells. The highest level of transcripts was found in neural tissues (brain - spinal cord) for all four genes~ the region of maximal expression being specific for each of them. Expression of these four genes was followed during differentiation of embryonal carcinoma and embryo stem cells. Alternative treatments in the presence or in the absence of retinoic acid were used to trigger cell differentiation. Induction to high levels of expression of all four genes occured only in the presence of retinoic acid. We conclude that accumulation of high amounts of homeobox transcripts is not required for cell differentiation along many pathways~ and correlates with the presence of RA.
5S