[PDF] Analysis Of The 5 End Of The Drosophila Muscle Myosin Heavy Chain Gene eBook

Author : Dorothy Dianne Hodges
Publisher :
Page : 558 pages
File Size : 11,43 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 :
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Page : 520 pages
File Size : 44,75 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
Publisher :
Page : 336 pages
File Size : 10,87 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.

Author : Linda Wells
Publisher :
Page : 204 pages
File Size : 36,34 MB
Release : 1996
Category : Drosophila melanogaster
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Muscle is a dynamic and complex tissue, composed of many different types of proteins. The major protein component of the muscle thick filament is myosin heavy chain (MHC). Current research reveals that muscle proteins, including MHC, have different isoforms, potentially increasing the complexity and versatility of muscle tissue. The functional significance of these MHC isoforms in vivo is unknown. Drosophila is an ideal organism in which to address this question because of its genetic simplicity, transformation capacity, short lifespan, and single Mhc gene. To dissect the function of the muscle proteins, scientists utilize transgenic Drosophila. Different Mhc transcripts are cloned into a P-element and used for germline transformation of Drosophila. The resulting phenotypes are analyzed to elucidate the functional qualities of the isoform encoded by that transcript. In addition to exploring the functionality of in vivo isoforms, the role of specific alternative exons can be studied. By changing a single exon and performing physiological studies, the role of that exonic region can be illuminated. This approach was utilized for three experiments. When Drosophila were transformed with an all embryonic-type Mhc, muscle function was severely affected, while no change in muscle assembly was detected. Drosophila transformed with a C-terminal tailpiece reversion construct showed an increase in muscle function, but wild-type muscle phenotypes were not restored. Another construct, composed of completely embryonic exons except the Mhc hinge region, proved lethal in Drosophila. Thus, the type of MHC isoform expressed does have functional significance in vivo, as do both alternatively spliced rod exons. Once functional regions are located by the approach described above, directed mutational analysis, combined with transgenic technology, will be used to determine the contribution of specific amino acids to muscle function. This type of information is valuable in the quest to understand how muscles work. A detailed knowledge of muscle function will be invaluable when studying the causes and cures for muscle diseases.

Author : Michelle Mardahl-Dumesnil
Publisher :
Page : 428 pages
File Size : 11,73 MB
Release : 1998
Category : Drosophila melanogaster
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Manipulation of muscle genes to cause their under-, over- and mis-expression and subsequent assessment of resultant phenotypes offers a comprehensive approach to understand muscle assembly, development and function. These techniques are readily applied to the fruit fly, Drosophila melanogaster, because of the relative ease of mutant isolation and germ-line transformation. The consequences of altered muscle gene expression on muscle function and ultrastructure can be well characterized in this genetic system. This dissertation describes experiments to examine the roles of two thick filament proteins and a metabolic enzyme on Drosophila muscle structure and function. In the first chapter, I have determined the genetic lesion for the Mhc2 mutant and performed detailed ultrastructural analysis of the indirect flight muscle (IFM) of mutant and transgenic lines. This investigation reveals the negative effects of over-expression and under-expression of the Mhc gene on muscle function and structure. In Chapter Two, I characterize an enhancer detection line that exhibits strong IFM specific reporter gene activity. The P element of the enhancer detection line lies downstream of the enolase gene. Two interesting complementation groups result when the P element is used to mutagenize this locus. One complementation group is the first identification of a Drosophila enolase mutant, and the other is an unknown mutation that affects flight ability presumably by disrupting mitochondrial function in the IFM. In Chapter Three, I identify both standard (PM) and mini-paramyosin (mPM) mutants. Although thick filaments are present in embryonic body-wall muscle that is lacking PM, the sarcomere is unordered, indicating that PM is needed for its normal structure and function. Low levels of mPM significantly impair flight ability and viability. In addition, more thick filaments incorporate into IFM myofibrils of the mPM mutant than those of wild-type. Over-expression of either PM or mPM affects IFM structure and function. It also appears that equivalent stoichiometric levels of mPM and PM are important for correct sarcomeric structure in the IFM. From these studies, we determine that both PM and mPM confer specific structural qualities to the thick filament and myofibril morphology.

Author :
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Page : 166 pages
File Size : 29,58 MB
Release : 1996
Category : Drosophila melanogaster
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Muscle is a dynamic and complex tissue, composed of many different types of proteins. The major protein component of the muscle thick filament is myosin heavy chain (MHC). Current research reveals that muscle proteins, including MHC, have different isoforms, potentially increasing the complexity and versatility of muscle tissue. The functional significance of these MHC isoforms in vivo is unknown. Drosophila is an ideal organism in which to address this question because of its genetic simplicity, transformation capacity, short lifespan, and single Mhc gene. To dissect the function of the muscle proteins, scientists utilize transgenic Drosophila. Different Mhc transcripts are cloned into a P-element and used for germline transformation of Drosophila. The resulting phenotypes are analyzed to elucidate the functional qualities of the isoform encoded by that transcript. In addition to exploring the functionality of in vivo isoforms, the role of specific alternative exons can be studied. By changing a single exon and performing physiological studies, the role of that exonic region can be illuminated. This approach was utilized for three experiments. When Drosophila were transformed with an all embryonic-type Mhc, muscle function was severely affected, while no change in muscle assembly was detected. Drosophila transformed with a C-terminal tailpiece reversion construct showed an increase in muscle function, but wild-type muscle phenotypes were not restored. Another construct, composed of completely embryonic exons except the Mhc hinge region, proved lethal in Drosophila. Thus, the type of MHC isoform expressed does have functional significance in vivo, as do both alternatively spliced rod exons. Once functional regions are located by the approach described above, directed mutational analysis, combined with transgenic technology, will be used to determine the contribution of specific amino acids to muscle function. This type of information is valuable in the quest to understand how muscles work. A detailed knowledge of muscle function will be invaluable when studying the causes and cures for muscle diseases.