Molecular Analysis of Muscle Myosin Heavy Chain Gene Mutations in Drosophila Melanogaster PDF Download
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Author: Venetia Lynne Collier
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Category : Drosophila melanogaster
Languages : en
Pages : 190
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Author: Venetia Lynne Collier
Publisher:
ISBN:
Category : Drosophila melanogaster
Languages : en
Pages : 190
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Author: Patrick Thomas O'Donnell
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ISBN:
Category : Drosophila melanogaster
Languages : en
Pages : 278
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Author: Becky Marlene Miller
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Category : Drosophila melanogaster
Languages : en
Pages : 336
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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: Amy Maude Bejsovec
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Category :
Languages : en
Pages : 346
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Author: Hongjun Liu
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Category : Drosophila melanogaster
Languages : en
Pages : 294
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Paramyosin is a major structural component of invertebrate muscles. It forms thick filament cores with other proteins. The motor protein myosin assembles on the surface of the core to form functional thick filaments. To investigate the roles of paramyosin in thick filament assembly as well as muscle contraction, I functionally knocked out the Drosophila melanogaster paramyosin gene by mobilizing a P element localized in the promoter region. I found that homozygous paramyosin mutants die at the embryo stage. Using electron microscopy and confocal microscopy, I analyzed the phenotypic defects of a functional null allele prm1. I observed that, in the absence of paramyosin, thick filaments of embryo body wall muscles are abnormal and the striated pattern of myobibrils is disrupted. These results indicate that paramyosin is essential for thick filament assembly and myofibril formation. Surprisingly, the muscle pattern of paramyosin mutant embryos is also abnormal. Mutation of paramyosin causes random losses of muscle fibers. Using a marker for founder myoblasts and the DMEF2 antibody which recognizes all myoblasts, I proved that the muscle fiber loss is not due to defects in myoblast differentiation. Rather, it is caused by abnomal myoblast fusion. Using a paramyosin specific antibody, I revealed that paramyosin functions as a cytoplasmic protein before myofibril formation and is important for myoblast fusion. I further investigated the function of paramyosin phosphorylation in the NH2-terminal non-helical domain. I made transgenic flies in which 1, 3, or 4 phosphorylatable serine residues in this domain are substituted with alanines. I observed that mutations of paramyosin at these residues do not affect the ultrastructure of myofibrils. However, mutant flies with substitution at some specific sites are flight impaired. Mechanical studies of indirect flight muscle fibers revealed that the flight impairment is caused by reduced fiber stiffness and power output. These results indicate that paramyosin phosphorylation in the NH2-terminal domain is important for muscle contraction.
Author: Dorothy Dianne Hodges
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Category : Drosophila melanogaster
Languages : en
Pages : 558
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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: Andrew Scott Ketchum
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Category : Drosophila melanogaster
Languages : en
Pages : 498
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Author: Kenneth David Becker
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Category : Drosophila melanogaster
Languages : en
Pages : 390
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Drosophila melanogaster is a well characterized genetic system that can be easily manipulated to generate mutants affecting muscle assembly, structure and/or function. Coupling mutants with the ability to reintroduce genes into the Drosophila genome provides a powerful approach in which to study sequence specific function as it relates to muscle structural proteins. In chapter 1 we show that Drosophila melanogaster muscles contain the standard form of the thick filament protein paramyosin, as well as a novel paramyosin isoform, which we call miniparamyosin. Standard paramyosin is [approx]105 kD and cross-reacts with polyclonal antibodies made against Caenorhabditis elegans or Heliocopris dilloni paramyosin. The Heliocopris antibody also cross-reacts with a [approx]55 kD protein likely to be miniparamyosin. We have cloned and sequenced cDNA's encoding both Drosophila isoforms. The C-terminal 363 amino acids are identical in standard and miniparamyosin. However, the smaller isoform has 114 residues at the N terminus that are unique as compared to the current protein sequence data base. In-situ hybridization to Drosophila tissue sections shows that standard paramyosin is expressed in all larval and adult muscle tissues whereas miniparamyosin is restricted to a subset of the adult musculature. In chapter two we characterize the partial structure of the Drosophila paramyosin gene. This gene is single copy per haploid genome and utilizes two promoters to generate mRNA's that have either of two different 5' coding sequences joined to common 3' exons. We also show that a chromosomal deficient eliminates the function of the Drosophila paramyosin gene. This deficiency is homozygous lethal at the first larval instar stage and has a morphological phenotype which suggests that muscle function is impaired. Finally, in chapter 3 we describe the cloning of two different genomic DNA fragments that each contain the heavy chain gene (MHC) of Drosophila melanogaster. Both of these clones have been used to create transgenic lines. The expression level from these MHC transgenes has been determined to be from 0 to 60% of the wild type haploid amount. However, even multiple copies of the transgenes are unable to rescue the dominant flightless and recessive lethal phenotypes associated with Mhc hypomorphic mutants.
Author: Madhulika Achal
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Category :
Languages : en
Pages : 39
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Myosin, a motor protein, is composed of two heavy chains and four light chains. It hydrolyzes ATP to generate actin-based motility and the contractile force in muscles. A mutation in human cardiac beta myosin heavy chain that changes amino acid residue 838 from proline to leucine (P838L) results in pediatric restrictive cardiomyopathy. Restrictive cardiomyopathy (RCM) leads to rigidity of the ventricular wall and the heart is restricted from stretching and properly filling with blood. Therefore, blood flow is reduced and blood that would normally enter the heart is backed up in the circulatory system. Gradually, the patient loses the ability to pump blood efficiently, leading to heart failure. Drosophila has a rhythmically beating heart and serves as a powerful tool to study the genetic basis of heart development and disease in humans. To define the biochemical basis of myosin-based RCM and to test the hypothesis that the P838L mutation causes RCM in Drosophila, a gene encoding myosin with the P838L mutation was constructed and was expressed in place of wild-type myosin heavy chain by P element transformation. Jump tests indicated that the mutation did not have any effect on the jump muscles. However reduced flight ability was observed, indicating impairment in the indirect flight muscle expressing this mutant myosin. ATPase assays and in vitro motility assays were performed to determine the effect of the mutation at the molecular level. We found that there was a slight increase in the actin-sliding velocity and that the basal and actin-activated MgATPase activity was significantly higher for the mutants. Electron microscopy on 2 day-old mutants and the controls suggested that the myofibrils were intact but some of them were oblong in shape and had rough edges when compared to the control myofibrils. Preliminary video microscopy data suggest that the transgenic hearts display a restrictive phenotype. The P838L mutation affects an "invariant proline" located at the junction of the myosin S1 head and the S2 rod and might affect the movement of the myosin heads during the force generation step. This could lead to the observed increase in the ATPase rate. Also, the P838L mutation is located near to where the regulatory light chain (RLC) binds to the heavy chain. RLC has an important role in the ATPase cycle and the P838L mutation might alter the RLC conformation leading to the increased ATPase rate. The increased ATPase rate seen in this and another Drosophila myosin that causes a restrictive phenotype may initiate a cascade of events that result in the observed cardiac defects.
Author: Fang-Miin Sheen
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ISBN:
Category :
Languages : en
Pages : 270
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