[PDF] In Vitro And In Vivo Analyses Of Alternative Splicing Of 3 End Transcripts Of The Drosophila Melanogaster Muscle Myosin Heavy Chain Gene eBook

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Page : 520 pages
File Size : 18,49 MB
Release : 1992
Category : Drosophila melanogaster
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Up to 480 isoforms of Drosophila muscle myosin heavy chain (MHC) can be generated by the process of alternative splicing. In order to better understand the regulation of MHC expression, we have analyzed the alternative splicing of MHC 3' end transcripts in vitro and in vivo. In Chapter 1 we describe the development and use of a Drosophila in-vitro splicing system to study the alternative splicing of penultimate exon 18. We demonstrate that pre-mRNA is spliced to exclude exon 18, as occurs in embryonic and larval muscle in vivo. However, when the 5' and 3' splice sites of exon 18 are modified to improve their binding to constitutive splicing factors, exon 18 is efficiently spliced to both flanking exons, as occurs in adult muscles in vivo. In Chapter 2 we express similarly modified transcripts in vivo using P element mediated germ line transformation. Mini-gene transcripts in which both splice sites of exon 18 are improved are now spliced to include exon 18 in larvae, as well as in adults. This is a complete splicing switch; all mRNAs typical of the normal larval splicing pattern have been eliminated. We also demonstrate that the correct 3' splice site of exon 18 is not utilized by the larval splicing machinery, even when the competing downstream 3' splice site is eliminated. Analysis of MHC [Delta] Int 17 mini-gene transcript splicing determined that intron 17 sequences are needed for intron 18 removal in larvae and adults. We also present results of cloning and sequencing the distantly related D. virilis MHC gene. Large stretches of non-coding sequences within exon 18 and a pyrimidine rich element in intron 17 are conserved between the D. virilis and D. melanogaster MHC genes. Mini-gene transcripts lacking most of the conserved exon 18 sequences were spliced in the correct stage-specific manner in vivo. However, analysis of splicing of mini-gene transcripts lacking the polypyrimidine sequence confirmed that it is essential for correct inclusion of exon 18 in adult mRNA, and suggests that binding of adult-specific, transacting factors to this element may mediate recognition and utilization of the weak 3' splice site of exon 18.

Author : Dorothy Dianne Hodges
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Page : 558 pages
File Size : 36,7 MB
Release : 1992
Category : Drosophila melanogaster
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Up to 480 isoforms of Drosophila muscle myosin heavy chain (MHC) can be generated by the process of alternative splicing. In order to better understand the regulation of MHC expression, we have analyzed the alternative splicing of MHC 3' end transcripts in vitro and in vivo. In Chapter 1 we describe the development and use of a Drosophila in-vitro splicing system to study the alternative splicing of penultimate exon 18. We demonstrate that pre-mRNA is spliced to exclude exon 18, as occurs in embryonic and larval muscle in vivo. However, when the 5' and 3' splice sites of exon 18 are modified to improve their binding to constitutive splicing factors, exon 18 is efficiently spliced to both flanking exons, as occurs in adult muscles in vivo. In Chapter 2 we express similarly modified transcripts in vivo using P element mediated germ line transformation. Mini-gene transcripts in which both splice sites of exon 18 are improved are now spliced to include exon 18 in larvae, as well as in adults. This is a complete splicing switch; all mRNAs typical of the normal larval splicing pattern have been eliminated. We also demonstrate that the correct 3' splice site of exon 18 is not utilized by the larval splicing machinery, even when the competing downstream 3' splice site is eliminated. Analysis of MHC [Delta] Int 17 mini-gene transcript splicing determined that intron 17 sequences are needed for intron 18 removal in larvae and adults. We also present results of cloning and sequencing the distantly related D. virilis MHC gene. Large stretches of non-coding sequences within exon 18 and a pyrimidine rich element in intron 17 are conserved between the D. virilis and D. melanogaster MHC genes. Mini-gene transcripts lacking most of the conserved exon 18 sequences were spliced in the correct stage-specific manner in vivo. However, analysis of splicing of mini-gene transcripts lacking the polypyrimidine sequence confirmed that it is essential for correct inclusion of exon 18 in adult mRNA, and suggests that binding of adult-specific, transacting factors to this element may mediate recognition and utilization of the weak 3' splice site of exon 18.

Author : Becky Marlene Miller
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Page : 336 pages
File Size : 10,79 MB
Release : 2004
Category : Drosophila melanogaster
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Drosophila melanogaster has a single myosin alkali light chain gene which encodes for two protein isoforms by developmentally regulated alternative splicing of the primary transcript. All six of the exons in the gene are present in the mRNA of larval muscles and the tubular and abdominal muscles of the adults. A novel mRNA species present exclusively in the adult and pupal Indirect Flight Muscle (IFM) lacks the fifth exon, thus encoding a MLC-ALK isoform with a variant carboxyl terminus. All introns of the transcript contain the established concensus splicing signals with the exception of intron 4. In this intron, a non-canonical polypurine stretch replaces the concensus polypyrimidine, rendering it a likely regulatory site. Because the transcripts are colinear with the gene throughout development the alternative splicing pattern in the IFM appears to be regulated at the level of splice site choice. The goal of this research is to identify the cis-regulatory sequences that control the choice between alternative larval and IFM-specific splicing pathways. I have developed a transient expression system for Drosophila Schneider 2 cultured cells utilizing the Drosophila metallothionein promoter to direct transcription of transfected MLC-ALK minigenes. This analysis demonstrated that the larval-specific splicing pathway represents the default splicing of the MLC-ALK transcripts. Analysis of mutant minigene transcripts revealed that splicing in the IFM-specific pathway is not the result of blockage or incapacitation of either splice acceptor or/and donor sequences flanking exon 5. The structures of the mutant mRNAs suggest that utilization of the IFM-specific pathway requires trans-acting factors which are absent in the cultured cells. Furthermore, analysis of mutant and hybrid minigene transcripts identified a unique cis-regulatory sequence proximal to the splice donor of intron 4, required for efficient utilization of the larval-specific splicing pathway. Mutations in intron 4 inhibit removal of the downstream intron 5 suggesting that an ordered pathway of intron removal is employed for larval-specific splicing. On the basis of these results a model of the mechanism of tissue and temporal regulation of alternative splicing of the MLC-ALK transcripts is presented.

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Page : 904 pages
File Size : 44,36 MB
Release : 1991
Category : Cells
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No. 2, pt. 2 of November issue each year from v. 19-47; 1963-70 and v. 55- 1972- contain the Abstracts of papers presented at the annual meeting of the American Society for Cell Biology, 3d-10th; 1963-70 and 12th- 1972- .