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Expression of mitochondrial genes in early mouse embryos
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325
THE EXPRESSION AND PROPERTIES OF A NEW STAGE-SPECIFIC EMBRYONIC ANTIGEN TEC-4. J.Nosek and PoDr~ber, Institute o'f'Ho'lecufar Genetics, 142 20 Prague 4, C z e c h o s l o v a k i a .
CHARACTERISATION OF A LOCALISED ANTIGEN IN EARLY X E N O P U S L A E V I S DEVELOPMENT. A.Otte, A.Durston, D.Ro~ F.Hochstenbach, M. Siemerink, K.Koster & A.Tirmnermans. Hubrecht Laboratory, Uppsalalaan 8, 3584 CT Utrecht, the Netherlands.
Hybridoma, s e c r e t i n g m o n o c l o n a l a n t i b o d y TEC-04, was c o n s t r u c t e d a f t e r the i m m u n i z a t i o n o f a r a t w i t h t h e mutant P19Xl e m b r y o n a l c a r c i n o ma (EC) c e l l l i n e d e f e c t i v e i n the expression of embryoglycan. The e p i t o p e d e f i n e d by TEC-04 ant i b o d y was found o n l y on EC c e l l l i n e s , mouse eggs and t w o - c e l l embr y o s and i n a d u l t mouse k i d n e y . T h e P19X~ c e l l l i n e , e x p r e s s i n g about 2 x l O ~ m o l e c u l e s of TEC-4 a n t i g e n per c e l l , becomes TEC-4 n e g a t i v e a f t e r differentiation i n t h e lO--M r e t i n o i c a c i d f o r 48 h o u r s . The a n t i g e n i s o l a t e d by a f f i n i t y chromatography on TEC-04 column and by g e l f i l t r a t i o n i s a g l y c o p r o t e i n o f the a p p r o x i m a t e m o l e c u l a r w e i g h t 230 000. The TEC-4 e p i t o p s i s r e s i s t a n t t o o x i d a t i o n by sodium p e r i o d a t e , but can be d e s t r o y e d by b o i l i n g and by p r o t e i n - d e n a t u r i n g agents, which suggests t h a t th e TEC-04 a n t i b o d y does not b i n d t o th e s a c c h a r i d e p o r t i o n of the m o l e c u l e .
We raised monoclonal antibodies against Xenopus gastrula cells and several recognised localised antigens. One monoclonal (3D7) is partially characterised. Immunofluorescence reveals an antigen that is primarily cytoplasmic and localised peripherally in the early embryo. It is mainly in ectoderm in the neurula, in peripheral cells in the blastula and gastrula and in peripheral cytoplasm in cleavage stages and the zygote. The antigen is non-localised in the unfertilised egg. Apart from cytop]asmic antigen, there is always extracellular antigen between cells. Certain treatments, e.g. partial cell dissociation, decrease cytoplasmic antigen and increase extracellular antigen. The former may be a precursor of the latter. The antigen is protease K, trypsin and periodate sensitive and antibody binding is competed by D-fucose and D-galactose, but not by other sugars tested. Western blots of cytoplasmic antigen show a major band at 40 kD and minor bands at about 140, 220 and 240kD. Extracellular antigen consists only of 200 and 240 kD bands.
326 MAMMALIAN PARTHENOGENESIS: POSTNATAL CONTRIBUTION OF PARTHENOGENETIC ,+ .+ EMBRYO TO AGGREGATION CHIMERAS A. P ~ l d t I L. Dezso and A. Nagy. Oep, of B e h a v i o u r Genetics, L. E~tv~s Univ. H-2I)I G6d, Hungary. + Center for Agricultural Biotechnology. H-2100 G ~ d ~ l l 6 , Hungary. Oocytes of BCFI ( p i g m e n t e d : G p i B ) females were act i v a t e d parthenogenetically by incubation i n medium containing 7 g ethanol for six minutes, and were diploidized by inhibition of second polar body extrusion with cytochalasin B treatment. Embryos at B cell stage were aggregated with fertilized Balb/C (albino:GpiA) embryos of the same stage. Only fully aggregated chimeric blsstocystes were transferred to pseUdopregnant recipients. One fifth of the transferred embryos developed to term, and 50 ~ of them showed parthenogenetic cells in retinas and fur. The incidence of chimerism in our study is much higher then that reported previously. The Gpi t y p i n g r e v e a l e d e x t e n s i v e contribution of parthenogenetic cells to a l l the organs and t i s s u e s examined. The p r e s e n t s t u d y has d e m o n s t r a t e d t h a t eggs which were a c t i v a t e d w i t h e t h a n o l and a g g r e g a t e d with fertilized embryos have the p o t e n t i a l to p a r ticipate in normal mouse embryogenesis and c o n t r i bute to d i f f e r e n t t i s s u e s of the a d u l t c h i m e r a s .
327 EXPRESSION OF MITOCHONDRIAL GENES IN EARLY MOUSE EMBRYOS. L Pik6 and IC D. Taylor. Developmental Biology Laboratory, VA Medical Center, Sepulveda, CA 91343, USA The contents of mitochondrial DNA (mtDNA) and the steady-state-amounts of 12S and 16S mitochondrial rRNAs and the mRNAs for cytochrome g oxidase subunits I and II (COI and COII) were determined in dot hybridization experiments with cloned mtDNA probes during early development from the one-cell egg to the blastocyst stage. The content of mtDNA remained constant during this period at about 2.25 pg or 125,000 molecules per embryo, suggesting an absence of mtDNA replication. The amounts of mitochondrial rRNA and COI and COII mRNAs variedgreatly depending on developmental stage. ~I~ey were quite low in large oocytes and decreased further, to about one-half, between the end of oocyte growth and the late 2-cell stage. During cleavage from two-cell to early blastocyst, there was a sharp increase of about 15X in the amount of mitochondrial rRNA and an increase of about 30X in the amount of COI and COII mRNAs. These results suggest that the mitochondrial genome is largely inactive in the egg and two-cell embryo but that a high level of mltochondrial gene expression is initiated during cleavage. The activation of the mitochondrial genome seems to be under nuclear control and coincides with a pronounced structural and functional differentiation of the mitochondria. 117S
325
THE EXPRESSION AND PROPERTIES OF A NEW STAGE-SPECIFIC EMBRYONIC ANTIGEN TEC-4. J.Nosek and PoDr~ber, Institute o'f'Ho'lecufar Genetics, 142 20 Prague 4, C z e c h o s l o v a k i a .
CHARACTERISATION OF A LOCALISED ANTIGEN IN EARLY X E N O P U S L A E V I S DEVELOPMENT. A.Otte, A.Durston, D.Ro~ F.Hochstenbach, M. Siemerink, K.Koster & A.Tirmnermans. Hubrecht Laboratory, Uppsalalaan 8, 3584 CT Utrecht, the Netherlands.
Hybridoma, s e c r e t i n g m o n o c l o n a l a n t i b o d y TEC-04, was c o n s t r u c t e d a f t e r the i m m u n i z a t i o n o f a r a t w i t h t h e mutant P19Xl e m b r y o n a l c a r c i n o ma (EC) c e l l l i n e d e f e c t i v e i n the expression of embryoglycan. The e p i t o p e d e f i n e d by TEC-04 ant i b o d y was found o n l y on EC c e l l l i n e s , mouse eggs and t w o - c e l l embr y o s and i n a d u l t mouse k i d n e y . T h e P19X~ c e l l l i n e , e x p r e s s i n g about 2 x l O ~ m o l e c u l e s of TEC-4 a n t i g e n per c e l l , becomes TEC-4 n e g a t i v e a f t e r differentiation i n t h e lO--M r e t i n o i c a c i d f o r 48 h o u r s . The a n t i g e n i s o l a t e d by a f f i n i t y chromatography on TEC-04 column and by g e l f i l t r a t i o n i s a g l y c o p r o t e i n o f the a p p r o x i m a t e m o l e c u l a r w e i g h t 230 000. The TEC-4 e p i t o p s i s r e s i s t a n t t o o x i d a t i o n by sodium p e r i o d a t e , but can be d e s t r o y e d by b o i l i n g and by p r o t e i n - d e n a t u r i n g agents, which suggests t h a t th e TEC-04 a n t i b o d y does not b i n d t o th e s a c c h a r i d e p o r t i o n of the m o l e c u l e .
We raised monoclonal antibodies against Xenopus gastrula cells and several recognised localised antigens. One monoclonal (3D7) is partially characterised. Immunofluorescence reveals an antigen that is primarily cytoplasmic and localised peripherally in the early embryo. It is mainly in ectoderm in the neurula, in peripheral cells in the blastula and gastrula and in peripheral cytoplasm in cleavage stages and the zygote. The antigen is non-localised in the unfertilised egg. Apart from cytop]asmic antigen, there is always extracellular antigen between cells. Certain treatments, e.g. partial cell dissociation, decrease cytoplasmic antigen and increase extracellular antigen. The former may be a precursor of the latter. The antigen is protease K, trypsin and periodate sensitive and antibody binding is competed by D-fucose and D-galactose, but not by other sugars tested. Western blots of cytoplasmic antigen show a major band at 40 kD and minor bands at about 140, 220 and 240kD. Extracellular antigen consists only of 200 and 240 kD bands.
326 MAMMALIAN PARTHENOGENESIS: POSTNATAL CONTRIBUTION OF PARTHENOGENETIC ,+ .+ EMBRYO TO AGGREGATION CHIMERAS A. P ~ l d t I L. Dezso and A. Nagy. Oep, of B e h a v i o u r Genetics, L. E~tv~s Univ. H-2I)I G6d, Hungary. + Center for Agricultural Biotechnology. H-2100 G ~ d ~ l l 6 , Hungary. Oocytes of BCFI ( p i g m e n t e d : G p i B ) females were act i v a t e d parthenogenetically by incubation i n medium containing 7 g ethanol for six minutes, and were diploidized by inhibition of second polar body extrusion with cytochalasin B treatment. Embryos at B cell stage were aggregated with fertilized Balb/C (albino:GpiA) embryos of the same stage. Only fully aggregated chimeric blsstocystes were transferred to pseUdopregnant recipients. One fifth of the transferred embryos developed to term, and 50 ~ of them showed parthenogenetic cells in retinas and fur. The incidence of chimerism in our study is much higher then that reported previously. The Gpi t y p i n g r e v e a l e d e x t e n s i v e contribution of parthenogenetic cells to a l l the organs and t i s s u e s examined. The p r e s e n t s t u d y has d e m o n s t r a t e d t h a t eggs which were a c t i v a t e d w i t h e t h a n o l and a g g r e g a t e d with fertilized embryos have the p o t e n t i a l to p a r ticipate in normal mouse embryogenesis and c o n t r i bute to d i f f e r e n t t i s s u e s of the a d u l t c h i m e r a s .
327 EXPRESSION OF MITOCHONDRIAL GENES IN EARLY MOUSE EMBRYOS. L Pik6 and IC D. Taylor. Developmental Biology Laboratory, VA Medical Center, Sepulveda, CA 91343, USA The contents of mitochondrial DNA (mtDNA) and the steady-state-amounts of 12S and 16S mitochondrial rRNAs and the mRNAs for cytochrome g oxidase subunits I and II (COI and COII) were determined in dot hybridization experiments with cloned mtDNA probes during early development from the one-cell egg to the blastocyst stage. The content of mtDNA remained constant during this period at about 2.25 pg or 125,000 molecules per embryo, suggesting an absence of mtDNA replication. The amounts of mitochondrial rRNA and COI and COII mRNAs variedgreatly depending on developmental stage. ~I~ey were quite low in large oocytes and decreased further, to about one-half, between the end of oocyte growth and the late 2-cell stage. During cleavage from two-cell to early blastocyst, there was a sharp increase of about 15X in the amount of mitochondrial rRNA and an increase of about 30X in the amount of COI and COII mRNAs. These results suggest that the mitochondrial genome is largely inactive in the egg and two-cell embryo but that a high level of mltochondrial gene expression is initiated during cleavage. The activation of the mitochondrial genome seems to be under nuclear control and coincides with a pronounced structural and functional differentiation of the mitochondria. 117S