Novel mutations of cholesteryl ester transfer protein (CETP) gene in Japanese hyperalphalipoproteinemic subjects
Rumiko OhtaniAkihiro InazuYoshihiro NojiTakanobu WAKASUGIKenji MiwaHayato TadaMasa‐aki KawashiriTohru NoguchiAtsushi NoharaJunji KobayashiJunji KoizumiMasakazu YamagishiHiroshi Mabuchi
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Minigene
Cholesterylester transfer protein
Compound heterozygosity
Splice site mutation
To define elements critical for 5' splice selection in dicot plant nuclei, wild-type and mutant transcripts containing the first intron of the pea rbcS3A gene were expressed in vivo by using an autonomously replicating plant expression vector. Mutations within the normal 5' splice site (+1) of this intron demonstrate that 5' splice sites at the normal exon-intron boundary having only limited agreement with a 5' splice site consensus sequence can be spliced quite effectively in dicot nuclei. Inactivation of the normal 5' splice site occurs only by point mutations of the G at position +1 of the intron (+1G) or +2U or by multiple mutations at other positions and results in the activation of three cryptic 5' splice sites in the adjacent exon and intron. cis competition of cryptic sites having consensus 5' splice site sequences with the normal 5' splice site demonstrates that cryptic splice sites in the exon, but not the intron, can compete to some extent with the normal site. Replacement of the sequences between the cryptic and normal 5' splice sites with heterologous exon or intron sequences demonstrates that the 5' boundary of this plant intron is defined by its position relative to the AU transition point between exon and intron. These results suggest that potential 5' splice sites upstream of the AU transition point are accessible for recognition by the plant pre-mRNA splicing machinery and that those downstream in the AU-rich intron are masked from recognition.
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To define elements critical for 5' splice selection in dicot plant nuclei, wild-type and mutant transcripts containing the first intron of the pea rbcS3A gene were expressed in vivo by using an autonomously replicating plant expression vector. Mutations within the normal 5' splice site (+1) of this intron demonstrate that 5' splice sites at the normal exon-intron boundary having only limited agreement with a 5' splice site consensus sequence can be spliced quite effectively in dicot nuclei. Inactivation of the normal 5' splice site occurs only by point mutations of the G at position +1 of the intron (+1G) or +2U or by multiple mutations at other positions and results in the activation of three cryptic 5' splice sites in the adjacent exon and intron. cis competition of cryptic sites having consensus 5' splice site sequences with the normal 5' splice site demonstrates that cryptic splice sites in the exon, but not the intron, can compete to some extent with the normal site. Replacement of the sequences between the cryptic and normal 5' splice sites with heterologous exon or intron sequences demonstrates that the 5' boundary of this plant intron is defined by its position relative to the AU transition point between exon and intron. These results suggest that potential 5' splice sites upstream of the AU transition point are accessible for recognition by the plant pre-mRNA splicing machinery and that those downstream in the AU-rich intron are masked from recognition.
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Efficient expression of many mammalian genes depends on the presence of at least one intron. We previously showed that addition of almost any of the introns from the mouse thymidylate synthase (TS) gene to an intronless TS minigene led to a large increase in expression. However, addition of intron 4 led to a reduction in minigene expression. The goal of the present study was to determine why TS intron 4 was unable to stimulate expression. Insertion of intron 4 into an intron-dependent derivative of the ribosomal protein L32 gene did not lead to a significant increase in expression, suggesting that its inability to stimulate expression was due to sequences within the intron. Deleting most of the interior of intron 4, improving the putative branch point, removing purines from the pyrimidine stretch at the 3' end of the intron, or removing possible alternative splice acceptor or donor sites within the intron each had little effect on the level of expression. However, when the splice donor sequence of intron 4 was modified so that it was perfectly complementary to U1 snRNA, the modified intron 4 stimulated expression approximately 6-fold. When the splice donor site of TS intron 1 (a stimulatory intron) was changed to that of TS intron 4, the modified intron 1 was spliced very inefficiently and lost the ability to stimulate mRNA production. Our observations support the idea that introns can stimulate gene expression by a process that depends directly on the splicing reaction.
Minigene
Group II intron
Splice site mutation
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Minigene
Cholesterylester transfer protein
Splice site mutation
Phospholipid transfer protein
Proband
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Splicing of small introns in lower eucaryotes can be distinguished from vertebrate splicing by the inability of such introns to be expanded and by the inability of splice site mutations to cause exon skipping—properties suggesting that the intron rather than the exon is the unit of recognition. Vertebrates do contain small introns. To see if they possess properties similar to small introns in lower eucaryotes, we studied the small second intron from the human α-globin gene. Mutation of the 5′ splice site of this intron resulted in in vivo intron inclusion, not exon skipping, suggesting the presence of intron bridging interactions. The intron had an unusual base composition reflective of a sequence bias present in a collection of small human introns in which multiple G triplets stud the interior of the introns. Each G triplet represented a minimal sequence element additively contributing to maximal splicing efficiency and spliceosome assembly. More importantly, G triplets proximal to a duplicated splice site caused preferential utilization of the 5′ splice site upstream of the triplets or the 3′ splice site downstream of the triplets; i.e., sequences containing G triplets were preferentially used as introns when a choice was possible. Thus, G triplets internal to a small intron have the ability to affect splice site decisions at both ends of the intron. Each G triplet additively contributed to splice site selectivity. We suggest that G triplets are a common component of human 5′ splice sites and aid in the definition of exon-intron borders as well as overall splicing efficiency. In addition, our data suggest that such intronic elements may be characteristic of small introns and represent an intronic equivalent to the exon enhancers that facilitate recognition of both ends of an exon during exon definition.
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The biosynthesis of coagulation factor VIII (FVIII) is hampered by successive controls that limit its production. To improve this production, a truncated intron I sequence of factor IX (TFIXI1) was inserted in FVIII cDNA in place of FVIII introns 1, 12 and 13 and also as a combination between introns 1 and 12, and introns 1 and 13. The intron 12 and 13 locations were targeted because this region was previously shown to contain a transcriptional silencer. The expression of FVIII in CHO and HepG2 cells revealed important variations in the properties of the minigenes depending on the TFIXI1 insertion sites. In FVIII intron 13 location the TFIXI1 seemed to diminish the transcriptional silencer activity, whereas it was poorly spliced in intron 12 position. Among the five constructs, FVIII I1+13 leaded to a significant improvement in FVIII secretion (13 times) that was associated with a dramatic intracellular accumulation in cells. Therefore, the FVIII I1+13 minigene could represent a particular interest to produce recombinant FVIII in vitro as well as in the aim of gene therapy of haemophilia A.
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Factor IX
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In vivo analyses of cis-acting sequence requirements for pre-mRNA splicing in tobacco nuclei have previously demonstrated that the 5' splice sites are selected by their position relative to AU-rich elements within plant introns and by their degree of complementarity to the U1 small nuclear RNA. To determine whether the presence of adjacent introns affects 5' splice site recognition in plant nuclei, we have analyzed the in vivo splicing patterns of two-intron constructs containing 5' splice site mutations in the second intron. These experiments indicated that the splice site selection patterns in plant nuclei are defined primarily by sequences within the intron (intron definition) and secondarily by weak interactions across exons (exon definition). The effects of these secondary interactions became evident only when mutations in the downstream 5' splice site decreased its functionality and differed depending on the availability of cryptic splice sites close to the mutant site. In beta-conglycinin chimeric transcripts containing multiple cryptic 5' splice sites, the presence of an intact upstream intron significantly increased splicing at the downstream 5' splice sites in a polar fashion without activating exon skipping. In a natural beta-conglycinin transcript, which does not contain cryptic 5'splice sites, mutation of the first nucleotide of the downstream intron activated an array of noncanonical 5' and 3' splice sites and some exon skipping.
Splice site mutation
splice
Group II intron
Exon skipping
Exon shuffling
Exonic splicing enhancer
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Minigene
splice
Group II intron
Splice site mutation
Exonic splicing enhancer
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Minigene
Splice site mutation
splice
Polypyrimidine tract
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