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Characterization of mammalian testis specific expressed genes homologous to drosophila genes
505
504 'Characterization o f mammalian t e s t i s specific e x p r e s s e d genes h o m o l o g o u s t o Drosophila qenes.P.Burfeind,W.Enge~ S.Hoyer-Fender.
Human
Genetics,G~tt~g~
Evolutionary conserved genes, which a r e e x p r e s s e d in t e s t i c u l a r germ c e l l s f r o m o r g a n i s m s as f a r as D r o s o p h i l a and m a m m a l i a , s h o u l d p l a y an i m p o r t a n t r o l e in m a l e germ c e l l d i f f e r e n t i a t i o n . We have used t e s t i c u l a r germ c e l l s p e cific e x p r e s s e d genes o f D r o s o p h i l a as hybridization probes to isolate homo1ogous genes f r o m mammalian l i b r a r i e s . Two o f t h e D r o s o p h i l a c l o n e s t e s t e d , showed c r o s s - h y b r i d i z a t i o n t o mRNA isolated f r o m r a t and mouse t e s t e s and n o t t o mRNA o f s o m a t i c t i s s u e s . One gene, mst(3)gl-9, isolated by U. and M. S c h e f e r , D Q s s e l d o r f , was used t o i s o a l a t e t h e h o m o l o g o u s gene f r o m a rat testis cDNA-library. One c D N A - c l o n e o f 1.2 kb c o u l d be i s o l a t e d and seq u e n c e d . The e x p r e s s i o n o f t h e r a t gene in d i f f e r e n t r a t t i s s u e s and in t e s t e s o f v a r i o u s s p e c i e s was i n v e s t i g a t e d . The o n s e t o f t r a n s c r i p t i o n in testis t i s s u e was a n a l y s e d by h y b r i d i z a t i o n t o RNA o f r a t t e s t e s o f d i f f e r e n t d e v e l o p m e n t a l s t a g e s and by i n situ-hybridization to rat testis sections. In S o u t h e r n - b l o t s t o male and f e m a l e r a t and human DNA a c o n s e r v e d hybridization p a t t e r n was f o u n d .
Chorion Antisense RNA during Oogenesis in Bombyx mori. K. Iatrou and Y.A.W. Skeiky, Department of Medical Biochemistry, University of Calgary, Calgary, Alberta, Canada. Silkworm follicular cells contain significant quantities of antisense RNA transcribed from chorion transcription units. Antisense transcripts are not polyadenylated. Elements resembling Bombyx polymerase III promoter elements are present upstream from their transcription startsite and a polymerase III "box A" promoter sequence is located internally at a distance characteristic of polymerase III split promoters. Hybridizations of senseand antisensespecific probes to RNA derived from staged follicles revealed tbat sense and antisense RNA accumulate in parallel. The antisense transcripts span most of the length of the mature chorion mRNA but not the region which contains the 5'-untranslated and the beginning of the coding sequences. This, coupled with our finding that antisense transcripts are not duplexed to chorion mRNA in foll icular cytoplasm, suggests that antisense RNA may not play a role in the translational regulation of chorion mRNA. The possibility that it is involved in the post-transcriptional transport of mRNA is suggested by the discovery of an RNA unwindase activity in the cytoplasm of follicular c e l l s Supported by the Medical Research Council of Canada.
506
507
MONOCLONALANTIBODY PRODUCTION AGAINST A SUBCELLULAR FRACTION OF VEGETAL POLE CYTOPLASM CONTAINING THE "GEF~ PLASM" OF XENOPUS. Kohji Ikenishi and Sakiko Nakazato. Department of Biology, Faculty of Science, Osaka City University, Osaka 558, Japan. In the development of a few forms of animals, a localized cytoplasm plays an importest role in the differentiation of certain cell types. In anuran amphibians it is well known that "germ platen" (GP) located at the vegetal pole region of fertilized eggs is indispensable for the formation of primordial germ cells. However, any work about the nature of substances restricted to the GP has not yet been reported so far, except for cytochemical detection of RNA in the germinal granules. Toward the understandir~g of molecular nature of substances localized in the C~P, monoelonsi antibody production was tried against a subeellular fraction of vegetal pole cytoplasm containing the GP of Xenopus 2-cell eggs. One hybridonla cell line, which produced the monoclonal antibody reacting specifically with the GP of embryos at the cleavage stages, was obtained. With this antibody distribution of the corresponding antigen was investigated irfrnunohistologicaily in en~ryos extending from the fertilized to the tadpole stage. By inlnt~loblotting analysis with 2-D gel the a n t i b o d y w a s shown to react with two acidic protein spots of ca. 40 kDa.
MIGRATORY MECHANISM OF CHICK PRIMORDIAL GERM CELLS; ANALYSIS BY INTRAVASCULAR INJECTION OF LATEX BEADS AND POLLENS. Takashi Kuwana,Yuji Kajiwara*,Minoru Inouye*, and Toyoaki FuJimoto. Dept. Anat., Kumamoto Univ. Med. School, and *Pathol. Sec., Natl. Inst. for Minamata Dis. Chick primordial germ cells (PGCs) after separation from the endoderm temporarily circulate via the blood vascular system, then leave the capillaries near the germinal ridge (GR) and migrate actively into the GR, although 15-20% of the PGCs colonize in the head region (Nakamura et al., 1988) suggesting their passive migration. In order to clarify if the PGCs reach the GR passively, latex beads/pollens (the same size as that of the PGCs) were injected into the embryonic blood stream at stages 13-19 (PGCs are in the circulating phase). The majority of the particles accumulated in the head region (60%), whereas others in the GR region (23%). After 3 days, a few particles were detected close to the gonad. These results suggest that a part of the PGCs passively reach near the gonad, and also indicate that the interspecific gonadal chimera can be produced by intravascular injection of PGCs.
S154
504 'Characterization o f mammalian t e s t i s specific e x p r e s s e d genes h o m o l o g o u s t o Drosophila qenes.P.Burfeind,W.Enge~ S.Hoyer-Fender.
Human
Genetics,G~tt~g~
Evolutionary conserved genes, which a r e e x p r e s s e d in t e s t i c u l a r germ c e l l s f r o m o r g a n i s m s as f a r as D r o s o p h i l a and m a m m a l i a , s h o u l d p l a y an i m p o r t a n t r o l e in m a l e germ c e l l d i f f e r e n t i a t i o n . We have used t e s t i c u l a r germ c e l l s p e cific e x p r e s s e d genes o f D r o s o p h i l a as hybridization probes to isolate homo1ogous genes f r o m mammalian l i b r a r i e s . Two o f t h e D r o s o p h i l a c l o n e s t e s t e d , showed c r o s s - h y b r i d i z a t i o n t o mRNA isolated f r o m r a t and mouse t e s t e s and n o t t o mRNA o f s o m a t i c t i s s u e s . One gene, mst(3)gl-9, isolated by U. and M. S c h e f e r , D Q s s e l d o r f , was used t o i s o a l a t e t h e h o m o l o g o u s gene f r o m a rat testis cDNA-library. One c D N A - c l o n e o f 1.2 kb c o u l d be i s o l a t e d and seq u e n c e d . The e x p r e s s i o n o f t h e r a t gene in d i f f e r e n t r a t t i s s u e s and in t e s t e s o f v a r i o u s s p e c i e s was i n v e s t i g a t e d . The o n s e t o f t r a n s c r i p t i o n in testis t i s s u e was a n a l y s e d by h y b r i d i z a t i o n t o RNA o f r a t t e s t e s o f d i f f e r e n t d e v e l o p m e n t a l s t a g e s and by i n situ-hybridization to rat testis sections. In S o u t h e r n - b l o t s t o male and f e m a l e r a t and human DNA a c o n s e r v e d hybridization p a t t e r n was f o u n d .
Chorion Antisense RNA during Oogenesis in Bombyx mori. K. Iatrou and Y.A.W. Skeiky, Department of Medical Biochemistry, University of Calgary, Calgary, Alberta, Canada. Silkworm follicular cells contain significant quantities of antisense RNA transcribed from chorion transcription units. Antisense transcripts are not polyadenylated. Elements resembling Bombyx polymerase III promoter elements are present upstream from their transcription startsite and a polymerase III "box A" promoter sequence is located internally at a distance characteristic of polymerase III split promoters. Hybridizations of senseand antisensespecific probes to RNA derived from staged follicles revealed tbat sense and antisense RNA accumulate in parallel. The antisense transcripts span most of the length of the mature chorion mRNA but not the region which contains the 5'-untranslated and the beginning of the coding sequences. This, coupled with our finding that antisense transcripts are not duplexed to chorion mRNA in foll icular cytoplasm, suggests that antisense RNA may not play a role in the translational regulation of chorion mRNA. The possibility that it is involved in the post-transcriptional transport of mRNA is suggested by the discovery of an RNA unwindase activity in the cytoplasm of follicular c e l l s Supported by the Medical Research Council of Canada.
506
507
MONOCLONALANTIBODY PRODUCTION AGAINST A SUBCELLULAR FRACTION OF VEGETAL POLE CYTOPLASM CONTAINING THE "GEF~ PLASM" OF XENOPUS. Kohji Ikenishi and Sakiko Nakazato. Department of Biology, Faculty of Science, Osaka City University, Osaka 558, Japan. In the development of a few forms of animals, a localized cytoplasm plays an importest role in the differentiation of certain cell types. In anuran amphibians it is well known that "germ platen" (GP) located at the vegetal pole region of fertilized eggs is indispensable for the formation of primordial germ cells. However, any work about the nature of substances restricted to the GP has not yet been reported so far, except for cytochemical detection of RNA in the germinal granules. Toward the understandir~g of molecular nature of substances localized in the C~P, monoelonsi antibody production was tried against a subeellular fraction of vegetal pole cytoplasm containing the GP of Xenopus 2-cell eggs. One hybridonla cell line, which produced the monoclonal antibody reacting specifically with the GP of embryos at the cleavage stages, was obtained. With this antibody distribution of the corresponding antigen was investigated irfrnunohistologicaily in en~ryos extending from the fertilized to the tadpole stage. By inlnt~loblotting analysis with 2-D gel the a n t i b o d y w a s shown to react with two acidic protein spots of ca. 40 kDa.
MIGRATORY MECHANISM OF CHICK PRIMORDIAL GERM CELLS; ANALYSIS BY INTRAVASCULAR INJECTION OF LATEX BEADS AND POLLENS. Takashi Kuwana,Yuji Kajiwara*,Minoru Inouye*, and Toyoaki FuJimoto. Dept. Anat., Kumamoto Univ. Med. School, and *Pathol. Sec., Natl. Inst. for Minamata Dis. Chick primordial germ cells (PGCs) after separation from the endoderm temporarily circulate via the blood vascular system, then leave the capillaries near the germinal ridge (GR) and migrate actively into the GR, although 15-20% of the PGCs colonize in the head region (Nakamura et al., 1988) suggesting their passive migration. In order to clarify if the PGCs reach the GR passively, latex beads/pollens (the same size as that of the PGCs) were injected into the embryonic blood stream at stages 13-19 (PGCs are in the circulating phase). The majority of the particles accumulated in the head region (60%), whereas others in the GR region (23%). After 3 days, a few particles were detected close to the gonad. These results suggest that a part of the PGCs passively reach near the gonad, and also indicate that the interspecific gonadal chimera can be produced by intravascular injection of PGCs.
S154