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Splice site choice correlates with the size of the 3′ exon in the episialin gene
Cell Biology international
w33
C-REACTIVE UNDER
w35
SBLP-GLFAS’I~
Reports, Vol. 14, Abstracts Supplement
PROTEIN GENE EXPRESSION IS CONTROL OF TRANSCRIPTION FACTOR HNFl Carlo Toniatti, Anna De Martis and Gennaro Ciliberto. Dip Biochim Biotec Med. Universita di Napoli, Via S. Pansini 5 - 80131 NAPOLI Transcription of a large number of liver-specific genes is under hormonal control during acute inflammations. C-reactive protein (CRP). is rapidly induced lOO- to l,OOO-fold during several pathologies. The CRP promoter is silent in normal conditions and is transcriptionally activated by IL-6. Two distinct segments of the CRP promoter, called acute phase responsive element (APRE) 1 and 2 are responsible for transcriptional induction. These two regions act independently of each other. APRE 2 binds at least two hepatocyte-specific nucelar factors: one of these factors is constitutively expressed in hepatic cells, while binding of the other is strongly enhanced by IL-6. Constitutive expression of several genes in hepatocytes depends on the binding to their promoters of transcription factor HNF-1. Binding sites for this protein have been mapped on several genes including albumin, al -antitrypsin, a-fetoprotein and fibrinogen. We have now evidence for the presence of two sites for HNF-1 in the CRP promoter, one in APRE 1, the other in APRE 2. Both sites are atypical because their sequence shows substantial divergence from the consensus for HNF-1. The binding site in the APRE 2 has been analyzed in greater detail by gel retardation and methylation interference both with nuclear extracts from hepatoma cells and with recombinant HNF-1 protein. This site binds specifically HNF1 at least ten-fold less efficiently than the proximal element of the albumin gene. Mutations of this site abolish CRP expression, as well as competitions in vivo with different HNF-1 binding sites. On the basis of these, data we favour the idea that the activity of the CRP promoter depends on HNF-1. Full expression during inflammation is probably the consequence of cooperation between the constitutive transcription factor HNF-1 and one or more factors induced by IL-6 which interact with adjacent sequences.
IUW IN
lRIlUKJS
F. Gremisi#, M.A. Garluccio’, P. Salvadori-, G. Cellulare e #Lab. Biologia Barsacchi#. Svi luppo, Dipt. Fisiologia e Biochimica; Dipt. Chimica, Dniversita’ di Pisa, Italy. The %gl II” FIM4s consist of cytoplasmic equal to transcripts of discrete sizes, inonaners and aultimsrs, hanologous to a dispersed, repeated DNA sequence fsmily of Dimeric “Egl II I’ transcripts undergo, Tri turus. in vitro , a site-specific self-cleavage reaction similar to that of sane viroids and We investigated the vi rusoids of plants. transcription regulation of the “Bgl II” RNA by injecting various cloned repeats, as well as their deletion derivatives, into Xenopus analysing the resulting oocytes, and by transcripts by primer elongation and IW4ase Signals for the correct promotion and nlaPPing* termination of transcription are internal to the “Egl II” DNA. A “proxitml sequence elanent” transcription of other WBE) , known to regulate mm11 RK4s. is also a regulatory element of the “Egl II” RN4 transcription. A “distal sequence elanent” @SF9 is present within the repeat units. Transcription is perfoxmed by RN4 polimsrase I I, as judged by alpha-atmni tin experiments. Brperiments self-cleavage, perfonaed on %ari~ Igiff’lq7 I@&4 or on I@I4 oligonucleotides representing the self-cleavage denmstrate that the Fegian, cleavage occurs according to a “double haamsr-head” model. lhe of the accuracy transcription regulation and the self-cleavage properties suggest for the “E&l II” RN4 a role in the cell and/or the organian.
51
1990
SPLICE SI!l'E CEOICE CO-TES WITH THS SIZE CP THE 3* SXCN IM THE EPISIALIN GENE Hans L. VOS, Annemiek M.C. Gennissen, Marjolijn J.L. Ligtenberg and John Hilkens. Dept. of Tumor Biology, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, the Netherlands. Episialin is a mucin-type glycoprotein, that is Sequenoverexpressed in mammary carcinoma cells. cing studies have revealed that the gene encodes a large transmembrane protein, that has a MM of more than 120 kD in its unprocessed form. The protein consists for more than half of its length of almost perfectly repeated tandem repeat units of twenty amino acids. The number of repeats is variable, ranging from about 40 to 100, and many different alleles exist, although numbers of about 40 and 80 are most common. Two cDMA variants were obtained, that differed from each other by 27 bp, due to an alternative splice acceptor site for exon 2. Remarkably, the selection of the splice sites appears to be allele specific, in that transcripts of long alleles, that usually encode about 80 repeats, contain the additional 27 bases, while mF?MAsof short alleles of approximately 40 repeats do not include this insertion. Nucleotide sequencing of PCR amplified genomic DNA fragments, that contained both 3' splice sites and that were derived from both types of alleles, has not yet revealed any sequence differences. This suggests a long-distance influence on splice site selection, either as a result of length variations in exon 2 (that contains all the repeats) or as a consequence of a sequence change more than 100 bp into the intron.
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STRUCTURE AND EXPRESSION OF HUMAN hnRNP PROTEIN Al GENE Silvano Riva, Massimo Buvoli, Fabio Cobianchi, Marco Bestagno, Maria T.Bassi and Giuseppe Biamonti. Istituto di Genetica Biochimica ed Evoluzionistica,C.N.R.,Via Abbiategrasso, 20727100 Pavia, Italy. Heterogeneous nuclear RNAs (hnRNAs) are associated in the nucleus with specific proteins to form complexes called hnRNP particles. We have studied hnRNP protein Al that is one of the most abundant component of the complex. At cDNA revealed a peculiar two-domain structure: an RNA-binding domain (N-terminal 195aa) and a glycine-rich C-terminal domain (125aa) probably in protein-protein interactions. A involved similar structure is observed in many RNA binding proteins. One active Al gene was selected and completely sequenced. The expression of the gene is higher in proliferating cells. Alternative splicing was revealEd in the Al gene producing a second protein (Al ; 38 kd) with a larger glycine-rich domain. Alternative splicing of the Al gene seems to be regulated. Protein Al shows a preferential affinity for intron sequences thus pointing to a role in RNA splicing.
W36
ACKNOWLEDGMENT Work supported by "Ingegneria Genetica"
the Progetto Finalizzato C.N.R., Rome, Italy.
w33
C-REACTIVE UNDER
w35
SBLP-GLFAS’I~
Reports, Vol. 14, Abstracts Supplement
PROTEIN GENE EXPRESSION IS CONTROL OF TRANSCRIPTION FACTOR HNFl Carlo Toniatti, Anna De Martis and Gennaro Ciliberto. Dip Biochim Biotec Med. Universita di Napoli, Via S. Pansini 5 - 80131 NAPOLI Transcription of a large number of liver-specific genes is under hormonal control during acute inflammations. C-reactive protein (CRP). is rapidly induced lOO- to l,OOO-fold during several pathologies. The CRP promoter is silent in normal conditions and is transcriptionally activated by IL-6. Two distinct segments of the CRP promoter, called acute phase responsive element (APRE) 1 and 2 are responsible for transcriptional induction. These two regions act independently of each other. APRE 2 binds at least two hepatocyte-specific nucelar factors: one of these factors is constitutively expressed in hepatic cells, while binding of the other is strongly enhanced by IL-6. Constitutive expression of several genes in hepatocytes depends on the binding to their promoters of transcription factor HNF-1. Binding sites for this protein have been mapped on several genes including albumin, al -antitrypsin, a-fetoprotein and fibrinogen. We have now evidence for the presence of two sites for HNF-1 in the CRP promoter, one in APRE 1, the other in APRE 2. Both sites are atypical because their sequence shows substantial divergence from the consensus for HNF-1. The binding site in the APRE 2 has been analyzed in greater detail by gel retardation and methylation interference both with nuclear extracts from hepatoma cells and with recombinant HNF-1 protein. This site binds specifically HNF1 at least ten-fold less efficiently than the proximal element of the albumin gene. Mutations of this site abolish CRP expression, as well as competitions in vivo with different HNF-1 binding sites. On the basis of these, data we favour the idea that the activity of the CRP promoter depends on HNF-1. Full expression during inflammation is probably the consequence of cooperation between the constitutive transcription factor HNF-1 and one or more factors induced by IL-6 which interact with adjacent sequences.
IUW IN
lRIlUKJS
F. Gremisi#, M.A. Garluccio’, P. Salvadori-, G. Cellulare e #Lab. Biologia Barsacchi#. Svi luppo, Dipt. Fisiologia e Biochimica; Dipt. Chimica, Dniversita’ di Pisa, Italy. The %gl II” FIM4s consist of cytoplasmic equal to transcripts of discrete sizes, inonaners and aultimsrs, hanologous to a dispersed, repeated DNA sequence fsmily of Dimeric “Egl II I’ transcripts undergo, Tri turus. in vitro , a site-specific self-cleavage reaction similar to that of sane viroids and We investigated the vi rusoids of plants. transcription regulation of the “Bgl II” RNA by injecting various cloned repeats, as well as their deletion derivatives, into Xenopus analysing the resulting oocytes, and by transcripts by primer elongation and IW4ase Signals for the correct promotion and nlaPPing* termination of transcription are internal to the “Egl II” DNA. A “proxitml sequence elanent” transcription of other WBE) , known to regulate mm11 RK4s. is also a regulatory element of the “Egl II” RN4 transcription. A “distal sequence elanent” @SF9 is present within the repeat units. Transcription is perfoxmed by RN4 polimsrase I I, as judged by alpha-atmni tin experiments. Brperiments self-cleavage, perfonaed on %ari~ Igiff’lq7 I@&4 or on I@I4 oligonucleotides representing the self-cleavage denmstrate that the Fegian, cleavage occurs according to a “double haamsr-head” model. lhe of the accuracy transcription regulation and the self-cleavage properties suggest for the “E&l II” RN4 a role in the cell and/or the organian.
51
1990
SPLICE SI!l'E CEOICE CO-TES WITH THS SIZE CP THE 3* SXCN IM THE EPISIALIN GENE Hans L. VOS, Annemiek M.C. Gennissen, Marjolijn J.L. Ligtenberg and John Hilkens. Dept. of Tumor Biology, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, the Netherlands. Episialin is a mucin-type glycoprotein, that is Sequenoverexpressed in mammary carcinoma cells. cing studies have revealed that the gene encodes a large transmembrane protein, that has a MM of more than 120 kD in its unprocessed form. The protein consists for more than half of its length of almost perfectly repeated tandem repeat units of twenty amino acids. The number of repeats is variable, ranging from about 40 to 100, and many different alleles exist, although numbers of about 40 and 80 are most common. Two cDMA variants were obtained, that differed from each other by 27 bp, due to an alternative splice acceptor site for exon 2. Remarkably, the selection of the splice sites appears to be allele specific, in that transcripts of long alleles, that usually encode about 80 repeats, contain the additional 27 bases, while mF?MAsof short alleles of approximately 40 repeats do not include this insertion. Nucleotide sequencing of PCR amplified genomic DNA fragments, that contained both 3' splice sites and that were derived from both types of alleles, has not yet revealed any sequence differences. This suggests a long-distance influence on splice site selection, either as a result of length variations in exon 2 (that contains all the repeats) or as a consequence of a sequence change more than 100 bp into the intron.
w34
STRUCTURE AND EXPRESSION OF HUMAN hnRNP PROTEIN Al GENE Silvano Riva, Massimo Buvoli, Fabio Cobianchi, Marco Bestagno, Maria T.Bassi and Giuseppe Biamonti. Istituto di Genetica Biochimica ed Evoluzionistica,C.N.R.,Via Abbiategrasso, 20727100 Pavia, Italy. Heterogeneous nuclear RNAs (hnRNAs) are associated in the nucleus with specific proteins to form complexes called hnRNP particles. We have studied hnRNP protein Al that is one of the most abundant component of the complex. At cDNA revealed a peculiar two-domain structure: an RNA-binding domain (N-terminal 195aa) and a glycine-rich C-terminal domain (125aa) probably in protein-protein interactions. A involved similar structure is observed in many RNA binding proteins. One active Al gene was selected and completely sequenced. The expression of the gene is higher in proliferating cells. Alternative splicing was revealEd in the Al gene producing a second protein (Al ; 38 kd) with a larger glycine-rich domain. Alternative splicing of the Al gene seems to be regulated. Protein Al shows a preferential affinity for intron sequences thus pointing to a role in RNA splicing.
W36
ACKNOWLEDGMENT Work supported by "Ingegneria Genetica"
the Progetto Finalizzato C.N.R., Rome, Italy.