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Polyadenylation site heterogeneity of mRNA encoding serum albumin in human fetal liver
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415
F u n c t i o n a l C o o p e r a t i o n b e t w e e n Lenss p e c i f i c and N o n - s p e c i f i c E l e m e n t s in the 61-Crystallln Enhancer
FLOWCYTOMETRIC ANALYSIS OF C A L C I U M RESPONSES INDUCED BY P O L Y S A C C H A R I D E ANTIGENS
Koji GOTO and Htsato KONDOH. University, Nagoya 464, Japan.
Arjan W. Griffioen, Ben J.M. Zegers
Nagoya
Ger T. Rijkers and
Dept. of Inm~unology, U n i v e r s i t y Children's Hospital, "Het W i l h e l m i n a Kinderziekenhuie", P.O. Box 18009, Utrecht, The Netherlands.
Lens-specific regulation of t h e 61-crystallin g e n e o f t h e c h i c k e n is In l a r g e p a r t a s c r i b e d t o an e n h a n c e r l kb l o n g l o c a t e d in t h e t h i r d i n t r o n of the gene. N o n e of t h e f r a g m e n t s of t h e e n h a n c e r , e a c h a b o u t lO0 bp, s h o w e d any a c t i v i t y , b u t t a n d e m m u l t i m e r i z a tion of the fragments resulted in generation of the activity with distinct tissue specifieities. Analysis of the multimers revealed t h a t t h e e n h a n c e r is c o m p o s e d of a strictly lens-specific element ( e n h a n c e r c o r e ) and e l e m e n t s w i t h a broad cell type specificity. These t w o s o r t s of c o m p o n e n t s c o o p e r a t e in cis to generate the overall lensspecific activity. This heterologous c o o p e r a t i o n m a y a c c o u n t f o r not o n l y lens specificity but for various quantitative modulation o f ~crystallin expression. We a r e c u r r e n t l y characterizing interaction of t h e e n h a n c e r s e g m e n t s with nuclear factors of the lens cells by in v i v o c o m p e t i t i o n and biochemical analysis.
416
In early childhood (until i-2 years) there is a defective antibody response to T-cell independent antigens like p o l y s a c c h a r i d e s (PS). Little is known about rhe r e q u i r e m e n t s for B cell activation by p o l y s a c c h a r i d e (PS) antigens (TI-2 antigens). In the present study we investigated w h e t h e r it is possible to induce calcium (Ca) m o b i l i z a t i o n with PS antigens. To that end p u r i f i e d p e r i p h e r a l blood B-cells of adults immunized with Pneumovax (a vaccin which consists of a mixture of 23 different types of p n e u m o c o c c a l PS) were loaded with indo-l. The Ca assays were p e r f o r m e d using a F A C S - a n a l y z e r (Griffioen et al., 1989) because m e a s u r i n g Ca responses by flowcytometry enables v i s u a l i z a t i o n of small numbers of responding cells. A d d i n g PS type 4 or type I to indo-1 loaded cells results in a number of responding cells of up to 5%. R e s p o n d i n g cells are preferentially found in those cells with blastlike v o l u m e / s c a t t e r characteristics. The exact m e c h a n i s m of how PS induce Ca m o b i l i z a t ion is not known yet. A p e r c e n t a g e of 5% is much higher than the p r e c u r s o r frequency of PS specific B cells (1:30,000). These studies may aid in e l u c i d a t i o n of the m e c h a n i s m of B cell a c t i v a t i o n by PS antigens.
P O L Y A D E N Y L A T I O N SITE H E T E R O G E N E I T Y OF m R N A ENCODING SERUM ALBUMIN IN HUMANFETALLIVER.
G r i f f i o e n A.W. et al. In press.
Kavsan, V.M. and V.V. Dmitrenko. Institute of Molecular Biology and Geneties o f t h e Academy of Sciences of the U k r a i n i a n SSR, 252627, Kiev, Zabolotnogo Str. 150, U S S ~ H u m a n fetal liver cDNA was c l o n e d in pBR322 vector. The eDNA library was screened for liverspecific clones by means of differential hybridization. H u m a n fetal liver and h u m a n k i d n e y cDNAs were used as hybridization probes. Application of this procedure revealed twenty five liver-specific clones among about one thousand r e c o m b i n a n t s analyzed. These clones represent cDNAs corresponding abundant mRNAs. About twenty clones w e r e i d e n t i f i e d as e n c o d i n g s e r u m albumin. T w o d i f f e r e n t m R N A p e l y a d e n y l a t i o n sites w e r e found in four s e q u e n c e d plasmids. Cleavage/pelyadenylatlon site in two plasmids is s i t u a t e d fifteen nucleotides downstream from AATAAA signal, in two other plasmids this site is ten nucleotides dowDstream the same signal. The relation of the existence of two pelyadenylation sites to the mRNAstructure is discussed. It is not clear now if the such processing is important for regulation of genes expression
$130
(1989) J. Imm. Meth.
415
F u n c t i o n a l C o o p e r a t i o n b e t w e e n Lenss p e c i f i c and N o n - s p e c i f i c E l e m e n t s in the 61-Crystallln Enhancer
FLOWCYTOMETRIC ANALYSIS OF C A L C I U M RESPONSES INDUCED BY P O L Y S A C C H A R I D E ANTIGENS
Koji GOTO and Htsato KONDOH. University, Nagoya 464, Japan.
Arjan W. Griffioen, Ben J.M. Zegers
Nagoya
Ger T. Rijkers and
Dept. of Inm~unology, U n i v e r s i t y Children's Hospital, "Het W i l h e l m i n a Kinderziekenhuie", P.O. Box 18009, Utrecht, The Netherlands.
Lens-specific regulation of t h e 61-crystallin g e n e o f t h e c h i c k e n is In l a r g e p a r t a s c r i b e d t o an e n h a n c e r l kb l o n g l o c a t e d in t h e t h i r d i n t r o n of the gene. N o n e of t h e f r a g m e n t s of t h e e n h a n c e r , e a c h a b o u t lO0 bp, s h o w e d any a c t i v i t y , b u t t a n d e m m u l t i m e r i z a tion of the fragments resulted in generation of the activity with distinct tissue specifieities. Analysis of the multimers revealed t h a t t h e e n h a n c e r is c o m p o s e d of a strictly lens-specific element ( e n h a n c e r c o r e ) and e l e m e n t s w i t h a broad cell type specificity. These t w o s o r t s of c o m p o n e n t s c o o p e r a t e in cis to generate the overall lensspecific activity. This heterologous c o o p e r a t i o n m a y a c c o u n t f o r not o n l y lens specificity but for various quantitative modulation o f ~crystallin expression. We a r e c u r r e n t l y characterizing interaction of t h e e n h a n c e r s e g m e n t s with nuclear factors of the lens cells by in v i v o c o m p e t i t i o n and biochemical analysis.
416
In early childhood (until i-2 years) there is a defective antibody response to T-cell independent antigens like p o l y s a c c h a r i d e s (PS). Little is known about rhe r e q u i r e m e n t s for B cell activation by p o l y s a c c h a r i d e (PS) antigens (TI-2 antigens). In the present study we investigated w h e t h e r it is possible to induce calcium (Ca) m o b i l i z a t i o n with PS antigens. To that end p u r i f i e d p e r i p h e r a l blood B-cells of adults immunized with Pneumovax (a vaccin which consists of a mixture of 23 different types of p n e u m o c o c c a l PS) were loaded with indo-l. The Ca assays were p e r f o r m e d using a F A C S - a n a l y z e r (Griffioen et al., 1989) because m e a s u r i n g Ca responses by flowcytometry enables v i s u a l i z a t i o n of small numbers of responding cells. A d d i n g PS type 4 or type I to indo-1 loaded cells results in a number of responding cells of up to 5%. R e s p o n d i n g cells are preferentially found in those cells with blastlike v o l u m e / s c a t t e r characteristics. The exact m e c h a n i s m of how PS induce Ca m o b i l i z a t ion is not known yet. A p e r c e n t a g e of 5% is much higher than the p r e c u r s o r frequency of PS specific B cells (1:30,000). These studies may aid in e l u c i d a t i o n of the m e c h a n i s m of B cell a c t i v a t i o n by PS antigens.
P O L Y A D E N Y L A T I O N SITE H E T E R O G E N E I T Y OF m R N A ENCODING SERUM ALBUMIN IN HUMANFETALLIVER.
G r i f f i o e n A.W. et al. In press.
Kavsan, V.M. and V.V. Dmitrenko. Institute of Molecular Biology and Geneties o f t h e Academy of Sciences of the U k r a i n i a n SSR, 252627, Kiev, Zabolotnogo Str. 150, U S S ~ H u m a n fetal liver cDNA was c l o n e d in pBR322 vector. The eDNA library was screened for liverspecific clones by means of differential hybridization. H u m a n fetal liver and h u m a n k i d n e y cDNAs were used as hybridization probes. Application of this procedure revealed twenty five liver-specific clones among about one thousand r e c o m b i n a n t s analyzed. These clones represent cDNAs corresponding abundant mRNAs. About twenty clones w e r e i d e n t i f i e d as e n c o d i n g s e r u m albumin. T w o d i f f e r e n t m R N A p e l y a d e n y l a t i o n sites w e r e found in four s e q u e n c e d plasmids. Cleavage/pelyadenylatlon site in two plasmids is s i t u a t e d fifteen nucleotides downstream from AATAAA signal, in two other plasmids this site is ten nucleotides dowDstream the same signal. The relation of the existence of two pelyadenylation sites to the mRNAstructure is discussed. It is not clear now if the such processing is important for regulation of genes expression
$130
(1989) J. Imm. Meth.