RESUMEN
Transcription factors are often expressed at several times and in multiple tissues during development and regulate diverse sets of downstream target genes by varying their combinatorial interactions with other transcription factors. The Drosophila Tropomyosin I (TmI) gene is regulated by a complex of proteins within the enhancer that synergistically interacts with MEF2 to activate TmI transcription as muscle cells fuse and differentiate. One of the components of this complex is PDP1 (PAR domain protein 1), a basic leucine zipper transcription factor that is highly homologous to three vertebrate genes that are members of the PAR domain subfamily. We have isolated and describe here the structure of the Pdp1 gene. The Pdp1 gene is complex, containing at least four transcriptional start sites and producing at least six different mRNAs and PDP1 isoforms. Five of the PDP1 isoforms differ by the substitution or insertion of amino acids at or near the N-terminal of the protein. At least three of these alternately spliced transcripts are differentially expressed in different tissues of the developing embryo in which PDP1 expression is correlated with the differentiation of different cell types. A sixth isoform is produced by splicing out part of the PAR and basic DNA binding domains, and DNA binding and transient transfection experiments suggest that it functions as a dominant negative inhibitor of transcription. Furthermore, two enhancers have been identified within the gene that express in the somatic mesodermal precursors to body wall muscles and fat body and together direct expression in other tissues that closely mimics that of the endogenous gene. These results show that Pdp1 is widely expressed, including in muscle, fat, and gut precursors, and is likely involved in the transcriptional control of different developmental pathways through the use of differentially expressed PDP1 isoforms. Furthermore, the similarities between Pdp1 and the other PAR domain genes suggest that Pdp1 is the homologue of the vertebrate genes.
Asunto(s)
Proteínas de Drosophila , Drosophila/genética , Elementos de Facilitación Genéticos , Regiones Promotoras Genéticas , ARN Mensajero/genética , Factores de Transcripción/genética , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico , Clonación Molecular , Cartilla de ADN , ADN Complementario , Regulación del Desarrollo de la Expresión Génica , Inmunohistoquímica , Hibridación in Situ , Datos de Secuencia MolecularRESUMEN
In vertebrates, transcriptional control of skeletal muscle genes during differentiation is regulated by enhancers that direct the combinatorial binding and/or interaction of MEF2 and the bHLH MyoD family of myogenic factors. We have shown that Drosophila MEF2 plays a role similar to its vertebrate counterpart in the regulation of the Tropomyosin I gene in the development of Drosophila somatic muscles, however, unlike vertebrates, Drosophila MEF2 interacts with a muscle activator region that does not have binding sites for myogenic bHLH-like factors or any other known Drosophila transcription factors. We describe here the isolation and characterization of a component of the muscle activator region that we have named PDP1 (PAR domain protein 1). PDP1 is a novel transcription factor that is highly homologous to the PAR subfamily of mammalian bZIP transcription factors HLF, DBP and VBP/TEF. This is the first member of the PAR subfamily of bZIP transcription factors to be identified in Drosophila. We show that PDP1 is involved in regulating expression of the Tropomyosin I gene in somatic body-wall and pharyngeal muscles by binding to DNA sequences within the muscle activator that are required for activator function. Mutations that eliminate PDP1 binding eliminate muscle activator function and severely reduce expression of a muscle activator plus MEF2 mini-enhancer. These and previous results suggest that PDP1 may function as part of a larger protein/DNA complex that interacts with MEF2 to regulate transcription of Drosophila muscle genes. Furthermore, in addition to being expressed in the mesoderm that gives rise to the somatic muscles, PDP1 is also expressed in the mesodermal fat body, the developing midgut endoderm, the hindgut and Malpighian tubules, and the epidermis and central nervous system, suggesting that PDP1 is also involved in the terminal differentiation of these tissues.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila , Drosophila/crecimiento & desarrollo , Drosophila/genética , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Secuencia de Bases , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico , Sitios de Unión/genética , ADN Complementario/genética , Drosophila/metabolismo , Ectodermo/metabolismo , Endodermo/metabolismo , Elementos de Facilitación Genéticos , Factores de Unión a la G-Box , Regulación del Desarrollo de la Expresión Génica , Genes de Insecto , Humanos , Mesodermo/metabolismo , Datos de Secuencia Molecular , Desarrollo de Músculos , Músculo Esquelético/crecimiento & desarrollo , Músculo Esquelético/metabolismo , Homología de Secuencia de Aminoácido , Activación Transcripcional , Tropomiosina/genéticaRESUMEN
Myocyte-specific enhancer-binding factor 2 (MEF2) is a myogenic regulatory factor in vertebrates and Drosophila. Whereas the role of MEF2 in regulating vertebrate myogenesis and muscle genes has been extensively studied, little is known of the role of MEF2 in regulating Drosophila myogenesis. We have shown in a recent analysis of the regulation of the Drosophila Tropomyosin I (TmI) gene in transgenic flies that MEF2 is a positive regulator of TmI expression in the somatic body-wall muscles of embryos, larvae, and adults. To understand further the role of MEF2 in myogenesis and test the role of MEF2 in regulating TmI expression, we have used the yeast GAL4/UAS system to generate embryos in which MEF2 is ectopically expressed in tissues where it is not normally expressed or embryos in which MEF2 is overexpressed in the mesoderm and muscles. We observe that ectopic expression of MEF2 in the epidermis and the ventral midline cells in embryos activates the expression of TmI and other muscle genes in these tissues and that this activation is stage-dependent suggesting a requirement for additional factors. Furthermore, ectopic expression of MEF2 in the epidermis results in a decrease in the expression of signaling molecules in the epidermis and a failure of the embryo to properly form body-wall muscles. These results indicate that MEF2 can function out of context in the epidermis to induce the expression of muscle genes and interfere with a requirement for the epidermis in muscle development. We also find that the level of MEF2 in the mesoderm and/or muscles in embryos is critical to body-wall muscle formation; however, no effect is observed on the development of the visceral muscle or dorsal vessel.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Proteínas de Drosophila , Epidermis/metabolismo , Regulación de la Expresión Génica , Músculo Esquelético/metabolismo , Factores de Transcripción/fisiología , Animales , Diferenciación Celular , Cruzamientos Genéticos , Proteínas de Unión al ADN/biosíntesis , Drosophila , Respuesta al Choque Térmico , Proteínas de Insectos/metabolismo , Larva/genética , Larva/metabolismo , Factores de Transcripción MEF2 , Mesodermo/metabolismo , Músculo Esquelético/anomalías , Músculo Esquelético/citología , Músculo Esquelético/embriología , Factores Reguladores Miogénicos , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transducción de Señal , Factores de Transcripción/biosíntesis , Transcripción Genética , Tropomiosina/biosíntesis , Tropomiosina/genética , Proteína Wnt1RESUMEN
Developmental gene regulation in vertebrate somatic muscles involves the cooperative interaction of MEF2 (myocyte-specific enhancer-binding factor 2) and members of the b-HLH (basic helix-loop-helix) family of myogenic factors. Until recently, however, nothing was know about the factors that control the developmental regulation of muscle genes during embryogenesis in Drosophila. The Drosophila Tropomyosin I (TmI) gene contains a proximal and distal muscle enhancer within the first intron that regulates its expression in embryonic/larval and adult muscles. We have recently shown that the 355-bp proximal enhancer contains a binding site for the Drosophila homologue of vertebrate MEF2 and that MEF2 acts cooperatively with a basal level muscle activator region to direct high level muscle expression in transgenic flies. The 92-bp muscle activator region, however, does not contain any consensus E-box (CANNTG) binding site sequences for b-HLH myogenic factors, suggesting the MEF2 may interact with other factors to regulate muscle genes in Drosophila. In this study we have used mutation analysis and germ-line transformation to analyze cis-acting elements within the muscle activator region that regulate its expression in transgenic flies. We have identified a 71-bp region that is sufficient for low basal level temporal- and muscle-specific expression in the embryo, larva, and adult. Substitution mutations within the muscle activator region have identified several cis-element regions spanning 60-bp that are required for either full or partial muscle activator function. An analysis of proteins that bind to this region by gel mobility shift assay and copper nuclease footprinting has allowed us to identify the sites in this region at which multiple proteins complex and interact. We propose that these cis-elements and the proteins that they bind regulate muscle activator function and together with MEF2 are capable of regulating high level muscle expression.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Drosophila melanogaster/genética , Elementos de Facilitación Genéticos , Regulación del Desarrollo de la Expresión Génica , Genes de Insecto , Proteínas de Insectos/metabolismo , Factores de Transcripción/metabolismo , Tropomiosina/biosíntesis , Animales , Sitios de Unión , Diferenciación Celular/genética , Huella de ADN , Análisis Mutacional de ADN , Desoxirribonucleasa I , Proteínas de Drosophila , Drosophila melanogaster/embriología , Drosophila melanogaster/crecimiento & desarrollo , Embrión no Mamífero/metabolismo , Genes Reporteros , Proteínas de Insectos/genética , Larva/metabolismo , Factores de Transcripción MEF2 , Mesodermo/citología , Mesodermo/metabolismo , Desarrollo de Músculos , Músculos/embriología , Músculos/metabolismo , Factores Reguladores Miogénicos , Eliminación de Secuencia , Tropomiosina/genéticaRESUMEN
MEF2 (myocyte-specific enhancer factor 2) is a MADS box transcription factor that is thought to be a key regulator of myogenesis in vertebrates. Mutations in the Drosophila homologue of the mef2 gene indicate that it plays a key role in regulating myogenesis in Drosophila. We show here that the Drosophila tropomyosin I (TmI) gene is a target gene for mef2 regulation. The TmI gene contains a proximal and a distal muscle enhancer within the first intron of the gene. We show that both enhancers contain a MEF2 binding site and that a mutation in the MEF2 binding site of either enhancer significantly reduces reporter gene expression in embryonic, larval, and adult somatic body wall muscles of transgenic flies. We also show that a high level of proximal enhancer-directed reporter gene expression in somatic muscles requires the cooperative activity of MEF2 and a cis-acting muscle activator region located within the enhancer. Thus, mef2 null mutant embryos show a significant reduction but not an elimination of TmI expression in the body wall myoblasts and muscle fibers that are present. Surprisingly, there is little effect in these mutants on TmI expression in developing visceral muscles and dorsal vessel (heart), despite the fact that MEF2 is expressed in these muscles in wild-type embryos, indicating that TmI expression is regulated differently in these muscles. Taken together, our results show that mef2 is a positive regulator of tropomyosin gene transcription that is necessary but not sufficient for high level expression in somatic muscle of the embryo, larva, and adult.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila , Drosophila/genética , Drosophila/metabolismo , Músculos/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Tropomiosina/genética , Animales , Animales Modificados Genéticamente , Secuencia de Bases , ADN/genética , Drosophila/embriología , Elementos de Facilitación Genéticos , Eliminación de Gen , Regulación del Desarrollo de la Expresión Génica , Genes de Insecto , Intrones , Factores de Transcripción MEF2 , Datos de Secuencia Molecular , Desarrollo de Músculos , Músculos/embriología , Miocardio/metabolismo , Factores Reguladores Miogénicos , Distribución TisularRESUMEN
Myogenesis is a several step process that requires genes involved in specifying mesoderm lineage and genes involved in determining muscle identity, differentiation, and patterning. We report here on the isolation, characterization, and expression pattern of a cDNA clone encoded by the previously uncharacterized Drosophila muscle segment homeobox (msh) gene and its possible role in myogenesis. The amino acid sequence of the msh homeobox domain is highly homologous to the homeodomains of the Drosophila S59 and empty spiracles genes and the Hox 7 and Hox 8 family of vertebrate homeobox genes. In addition, the 5' end of msh has 52% sequence identity to the 5' end of the empty spiracles gene and encodes several stretches of amino acids rich in serine, alanine, proline, glutamine, and acidic amino acids, indicating potential domains of regulatory activity. The expression of msh is initially detected at about stage 6 in the dorsal lateral ectoderm of the embryo and later in the developing central (CNS) and peripheral nervous systems. During germ band retraction (stage 12), msh continues to be expressed in cells of the nervous system as well as cells of the somatic mesoderm corresponding mostly to the developing dorsal and lateral somatic body wall muscles. These mesodermal cells, which continue to express msh in daughterless mutant embryos, undergo an increase in cell number in neurogenic mutants. By late stage 14 of embryonic development, msh expression is greatly reduced or absent in most or all mesoderm and muscle but continues in CNS until hatching. Ectopic expression of msh in the mesoderm results in altered expression of the S59 and nau/Dmyd genes leading to a loss of some muscles and defects in the patterning of others, suggesting that the muscle defects are at the level of recruitment and/or patterning of muscle precursor cells. Thus the similarity of Drosophila msh expression to that of the homologous vertebrate Hox 7 and Hox 8 genes together with the effects of ectopic expression of msh in the mesoderm suggest a role for the msh-like family of genes in mesodermal and muscle differentiation and patterning.
Asunto(s)
Proteínas de Drosophila , Drosophila/embriología , Regulación del Desarrollo de la Expresión Génica , Genes Homeobox/fisiología , Proteínas de Homeodominio/genética , Músculos/embriología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Diferenciación Celular , Cricetinae , ADN Complementario/aislamiento & purificación , Femenino , Datos de Secuencia Molecular , MutaciónRESUMEN
We have characterized maternal and zygotic cytoskeletal tropomyosin mRNA expression during Drosophila embryogenesis using in situ hybridization to endogenous cytoskeletal tropomyosin mRNA and a cytoskeletal tropomyosin promoter/beta-galactosidase-encoding fusion gene mRNA in transgenic flies. A 2.0-kb maternal cytoskeletal tropomyosin mRNA is synthesized in the nurse cells and transported into the oocyte during oogenesis. During early embryogenesis, this mRNA becomes localized to the pole cell region and then to the cortex during the cellular blastoderm stage. In later embryos it is localized to the ventral and cephalic furrows and extending germ band. The major zygotic mRNA is 2.4 kb and is first detected at gastrulation. In early embryos, this mRNA is expressed in the invaginating anterior and posterior midgut, the transverse furrows, and the amnioserosa, all regions of the embryo undergoing intense cellular movement, and in later embryos, predominantly in the gut, brain, and epidermis. A transgene construct containing 1.2 kb of 5' cytoskeletal tropomyosin promoter sequences driving expression of Escherichia coli beta-galactosidase and the cytoskeletal tropomyosin 3' untranslated region in transgenic flies has the same distribution of maternal and zygotic transcripts throughout oogenesis and embryonic development as the endogenous transcripts. A transgene containing the 3' untranslated region from either the hsp70 gene or the SV40 early genes, on the other hand, does not express maternal RNA. Furthermore, none of the transgenes expressed in the follicle cells, suggesting that expression in these cells is under different transcriptional control. Our results indicate that maternal and zygotic cytoskeletal tropomyosin mRNAs are localized to specific regions of the developing embryo, particularly in regions of the embryo undergoing cell movement and invagination. Furthermore, the synthesis, transport, and accumulation of this RNA is under transcriptional and post-transcriptional control.
Asunto(s)
Proteínas de Drosophila , Drosophila melanogaster/embriología , Tropomiosina/genética , Animales , Animales Modificados Genéticamente , Compartimento Celular , Polaridad Celular , Proteínas del Citoesqueleto/genética , Drosophila melanogaster/genética , Femenino , Regulación del Desarrollo de la Expresión Génica , Proteínas HSP70 de Choque Térmico/genética , Hibridación in Situ , Morfogénesis , Proteínas Musculares/genética , Ovario/metabolismo , Regiones Promotoras Genéticas , ARN Mensajero/genética , Secuencias Reguladoras de Ácidos Nucleicos , Transcripción Genética , Cigoto/fisiologíaRESUMEN
Transcriptional control of the Drosophila tropomyosin II gene muscle promoter has been investigated by expressing TmII promoter lacZ reporter gene constructs in P-element-mediated transformed flies. A TmII/lacZ reporter gene containing 243 bp of upstream sequence, the first exon, the first intron, and 72 bp of the second exon was expressed in all muscles of embryos, larvae, and adults. Deletion of upstream sequences between -243 and -22 bp only reduced the levels of transgene expression in muscle while deletion of the intron eliminated expression. Analysis of deletions within the first intron indicated that a 454-bp muscle enhancer region, from +167 to +621, was required for high levels of transgene expression in all larval and adult muscles. When this region was deleted low levels of expression still occurred in larval and adult somatic and visceral muscles; however, there was no detectable expression in adult indirect flight and jump muscles. The 454-bp muscle enhancer region was also able to drive muscle-specific expression when placed upstream of a heterologous hsp70 promoter; however, three subfragments of the 454-bp region were unable to drive expression of the hsp70 promoter, suggesting that this region may contain multiple interacting cis-acting elements. Interestingly, the 454-bp region was inactive when placed upstream of a TmII promoter construct containing upstream DNA and most of the first exon but was active when additional exon and intron DNA was included, indicating that additional promoter elements are located in this region. Thus TmII transcription is controlled by multiple muscle type-specific cis-acting control elements and upstream and downstream promoter control elements.
Asunto(s)
Proteínas de Drosophila , Drosophila/genética , Elementos de Facilitación Genéticos , Regulación de la Expresión Génica , Músculos/metabolismo , Regiones Promotoras Genéticas , Tropomiosina/genética , Animales , Secuencia de Bases , Proteínas de Choque Térmico/genética , Intrones , Datos de Secuencia MolecularRESUMEN
Muscle development involves the coordinated regulation of transcription of muscle-type-specific genes and their encoded proteins during myogenesis. We show here that transcriptional regulation of the Drosophila tropomyosin I (TmI) gene during myogenesis is under the control of at least two muscle enhancer regions located within the first intron of the gene. Together these enhancer regions contain multiple muscle-type-specific positive and negative cis-acting elements which together contribute toward full expression of the gene. One of these enhancers is contained within a 355-bp fragment that is sufficient to direct high levels of temporally regulated expression from a heterologous promoter in all muscles of transgenic flies. Dissection of this enhancer region into smaller fragments has allowed us to identify a 91-bp enhancer fragment sufficient for directing expression in all somatic and visceral muscles of the larva and adult but not in the indirect flight muscles and tergal depressor of the trochanter or jump muscles of the adult. We also show that this somatic/visceral muscle element(s) can be repressed through an adjacent negative control region, suggesting that the regulation of expression in these muscles is under dual control during both phases of myogenesis. We propose a model in which transcriptional regulation of the Drosophila TmI gene is controlled by the cooperative interaction of multiple positive and negative cis-acting regulatory elements that control the temporal and muscle-type pattern of expression. The distribution of enhancer elements and their control of TmI gene expression are similar to those regulating transcription of the muscle promoter of the TmII gene and provide a framework for the coordinate expression of the two genes.
Asunto(s)
Proteínas de Drosophila , Drosophila/genética , Elementos de Facilitación Genéticos , Regulación de la Expresión Génica , Músculos/metabolismo , Tropomiosina/genética , Animales , Intrones , Transcripción GenéticaRESUMEN
The effects of promoter deletions on Drosophila tropomyosin I (TmI) gene expression have been determined by measuring TmI RNA levels in transformed flies. Decreases in RNA levels have been correlated with rescue of flightless and jumpless mutant phenotypes in Ifm(3)3 mutant transformed flies and changes in muscle ultrastructure. The results of this analysis have allowed us to identify a region responsible for 20% of maximal TmI expression, estimate threshold levels of TmI RNA required for indirect flight and jump muscle function, and obtain evidence suggesting that sarcomere length may be an important determinant of flight muscle function.
Asunto(s)
Músculos/fisiología , Regiones Promotoras Genéticas , Tropomiosina/genética , Animales , Drosophila/genética , Femenino , Músculos/ultraestructura , Mutación , Fenotipo , ARN/metabolismo , Transformación Genética , Tropomiosina/biosíntesisRESUMEN
The control of expression of the Drosophila melanogaster tropomyosin I (TmI) gene has been investigated by P-element transformation and rescue of the flightless and jumpless TmI mutant strain, Ifm(3)3. To localize cis-acting DNA sequences that control TmI gene expression, Ifm(3)3 flies were transformed with P-element plasmids containing various deletions and rearrangements of the TmI gene. The effects of these mutations on TmI gene expression were studied by analyzing both the extent of rescue of the Ifm(3)3 mutant phenotypes and determining TmI RNA levels in the transformed flies by primer extension analysis. The results of our analysis indicate that a region located within intron 1 of the gene is necessary and sufficient for directing muscle-specific TmI expression in the adult fly. This intron region has characteristics of a muscle regulatory enhancer element that can function in conjunction with the heterologous nonmuscle hsp70 promoter to promote rescue of the mutant phenotypes and to direct expression of an hsp70-Escherichia coli lacZ reporter gene in adult muscle. The enhancer can be subdivided further into two domains of activity based on primer extension analysis of TmI mRNA levels and on the rescue of mutant phenotypes. One of the intron domains is required for expression in the indirect flight muscle of the adult. The function of the second domain is unknown, but it could regulate the level of expression or be required for expression in other muscle.
Asunto(s)
Drosophila melanogaster/genética , Elementos de Facilitación Genéticos , Intrones , Músculos/metabolismo , Tropomiosina/genética , Animales , Elementos Transponibles de ADN , Drosophila melanogaster/fisiología , Regulación de la Expresión Génica , Mutación , Fenotipo , ARN/análisis , ARN/genética , Mapeo Restrictivo , Transformación GenéticaRESUMEN
Nuclear transcription extracts were prepared from embryos of Drosophila melanogaster to study the in vitro transcription of the tropomyosin genes. Several non-muscle gene promoters, including the non-muscle promoter of the Tropomyosin II gene, were shown to be efficiently transcribed in vitro. The Tropomyosin I gene and the muscle promoter of the Tropomyosin II gene, as well as two other contractile protein muscle genes, were not transcribed in vitro. The embryonic extract did, however, contain developmental-specific proteins that bound to the muscle enhancer regulatory region of the Tropomyosin I gene.
Asunto(s)
Proteínas de Drosophila , Drosophila melanogaster/genética , Músculos/química , Transcripción Genética , Tropomiosina/genética , Animales , Secuencia de Bases , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , ARN/análisis , Secuencias Reguladoras de Ácidos Nucleicos , TATA BoxRESUMEN
The structure of the Drosophila melanogaster tropomyosin II (TmII) gene has been determined by DNA sequencing of cDNA clones and the genomic DNA coding for the gene. Two overlapping transcriptional units produce at least four different tropomyosin isoforms. A combination of developmentally regulated promoters and alternative splicing produces both muscle and cytoskeletal tropomyosin isoforms. One promoter is a muscle-specific promoter and produces three different tropomyosin isoforms by alternative splicing of the last three 3' exons. The second promoter has the characteristics of a housekeeping promoter and produces a cytoskeletal tropomyosin isoform. Several internal exons along with a final 3' exon are alternatively spliced in the cytoskeletal transcript. The intron-exon boundaries of the TmII gene are identical to the intron-exon boundaries of all vertebrate tropomyosin genes reported, but are very different from the intron-exon boundaries of the D. melanogaster tropomyosin I gene. The TmII gene is the only reported tropomyosin gene that has two promoters and a quadruple alternative splice choice for the final exon. Models for the mechanism of D. melanogaster tropomyosin gene evolution are discussed.
Asunto(s)
Proteínas de Drosophila , Drosophila melanogaster/genética , Genes , Regiones Promotoras Genéticas , Empalme del ARN , ARN Mensajero/genética , Transcripción Genética , Tropomiosina/genética , Animales , Secuencia de Bases , Clonación Molecular , ADN/genética , Enzimas de Restricción del ADN , Exones , Datos de Secuencia Molecular , Homología de Secuencia de Ácido NucleicoRESUMEN
We report here the isolation and DNA sequence of a cDNA clone encoding a 252-amino acid non-muscle or cytoskeletal tropomyosin (cTm) isoform from Drosophila. The Drosophila cTm shows considerable homology with vertebrate cTm throughout the middle portion of the molecule. The amino-terminal end of the molecule, however, shows less homology and contains five more amino acids than the equine platelet and human tropomyosins. There is also a proline at position 6 in the Drosophila protein. The carboxyl-terminal 27 amino acids also show little homology with vertebrate non-muscle tropomyosins. This is a region of the molecule that shows considerably diversity among other Drosophila tropomyosins and vertebrate tropomyosins. A comparison of the DNA sequence of the cTm cDNA and a previously reported muscle tropomyosin II cDNA sequence shows regions of identical DNA sequence alternating with regions of nonidentical sequence, suggesting that both mRNAs are produced by alternate splicing of the same gene.
Asunto(s)
ADN/análisis , Proteínas de Drosophila , Drosophila/genética , Tropomiosina/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Datos de Secuencia Molecular , ARN Mensajero/análisisRESUMEN
In the Drosophila flightless mutant Ifm(3)3, a transposable element inserted into the alternatively spliced fourth exon of the tropomyosin I (TmI) gene prevents proper expression of Ifm-TmI, the tropomyosin isoform found in indirect flight muscle. We have rescued the flightless phenotype of Ifm(3)3 flies using P-element-mediated transformation with a segment of the Drosophila genome containing the wild-type TmI gene plus 2.5 kb of 5' flanking and 2 kb of 3' flanking DNA. The inserted TmI gene is expressed with the proper developmental and tissue specificity, although its level of expression varies among the five transformed lines examined. These conclusions are based on analyses of flight, myofibrillar morphology, and TmI RNA and protein levels. A minimum of two copies of the inserted TmI gene per cell is necessary to restore flight to most of the flies in each line. We also show that the Ifm-TmI isoform is expressed in the leg muscle of wild-type flies and is decreased in Ifm(3)3 leg muscle. Homozygous Ifm(3)3 mutants do not jump. The ability to jump can be restored with a single copy of the wild-type TmI gene per cell.
Asunto(s)
Drosophila/genética , Mutación , Transformación Genética , Tropomiosina/genética , Animales , Elementos Transponibles de ADN , Femenino , Vuelo Animal , Genes , Homocigoto , Masculino , Músculos/metabolismo , Transcripción GenéticaRESUMEN
The muscle tropomyosin I gene of Drosophila melanogaster undergoes alternative splicing in different muscles of the fly to generate two isoforms of the same protein. We report here the structural analysis and DNA sequence of the tropomyosin I gene. The gene spans 5 kilobase of DNA and is comprised of five exons and four introns. Exon 4 is alternatively spliced in RNA of different muscle, resulting in two isoforms of the same protein. The gene lacks a "TATA" box homology at the map position; it is usually found in the vast majority of eukaryotic genes characterized thus far. Instead, a series of three alternating TG stretches are located upstream from the site of initiation of transcription. The gene encodes a 5' untranslated leader of 103 base pairs, and the 3' untranslated region comprises between 30 and 50% of the transcripts. The DNA sequence is extremely G + C rich in the protein coding regions of the gene, and A + T rich in the non-coding, flanking, and intron regions. The DNA sequence upstream of the acceptor sites in the two introns which are subject to alternative splicing displays a stretch of homology which is noted. The 3' untranslated region of the fifth exon contains multiple polyadenylation sites. The 284 amino acid protein encoded by the gene is split by introns between residues 198/199 and 257/258. These sites correlate closely with two important functional domains in the tropomyosin molecule. A comparison of the first 257 amino acids and the carboxyl-terminal 27 amino acids of the Drosophila and vertebrate tropomyosins together, shows two distinct and mutually exclusive classes for these domains. The functional significance of the Drosophila tropomyosin isoforms is discussed.
Asunto(s)
ADN/análisis , Tropomiosina/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Drosophila melanogaster/genética , Conejos , Tropomiosina/análisisRESUMEN
A cDNA clone was sequenced that contains the entire coding region for the muscle tropomyosin II isoform from Drosophila. The cDNA clone is 1253 nucleotides (nt) long and contains an 88-nt 5'-leader sequence and a 310-nt 3'-untranslated sequence. The muscle tropomyosin II isoform consists of 285 amino acids and is 60% homologous with the previously reported muscle tropomyosin I isoform Drosophila and 55% homologous with rabbit muscle tropomyosin.
Asunto(s)
Clonación Molecular , ADN/aislamiento & purificación , Genes , Variación Genética , Tropomiosina/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Drosophila/genética , Músculos/metabolismoRESUMEN
We constructed a gene transfer vector containing the herpes simplex virus type 1 thymidine kinase (TK) gene flanked by Drosophila P element terminal repeats (W. R. Engels, Annu. Rev. Genet. 17:315-344). This vector was introduced into mouse LTK- cells and enhanced the frequency of stable transformation to the TK+ phenotype by approximately 50-fold relative to a similar plasmid lacking the P element terminal repeats.
Asunto(s)
Elementos Transponibles de ADN , Drosophila melanogaster/genética , Vectores Genéticos , Animales , Línea Celular , Simplexvirus/genética , Timidina Quinasa/genética , TransfecciónRESUMEN
The gene encoding muscle tropomyosin I in Drosophila is alternatively spliced in embryonic and thoracic muscle to generate two sizes classes of RNAs. By Northern blot analysis, the embryonic RNA class shows a broad RNA band of hybridization of 1.3 kb and a more sharply defined, less abundant RNA band at 1.6 kb. The thoracic class of RNAs, on the other hand, consists of a broad hybridization band at 1.7 kb and a more sharply defined band at 1.9 kb. Each size class of RNA encodes a different tropomyosin isoform. The two classes of alternatively spliced RNAs utilize the same 3' terminal exon of the gene. The DNA sequence of this exon reveals a cluster of several polyadenylation signals (AAUAAA) or polyadenylation-like signals. We show here by S1 nuclease protection analysis that at least five and possibly seven of these polyadenylation or polyadenylation-like sequences are associated with in vivo embryonic and thoracic mRNA cleavage processing sites. Six of these S1 sites are clustered within 119 bp and a seventh is located 255 bp downstream. At least one of the polyadenylation-like signal sequences appears to be an unusual AACAAA sequence. In addition we also show that these mRNAs function in vitro to synthesize muscle tropomyosins.
Asunto(s)
Poli A/análisis , ARN Mensajero/metabolismo , Tropomiosina/genética , Animales , Secuencia de Bases , Drosophila , Fluorometría , Hibridación de Ácido Nucleico , Biosíntesis de Proteínas , Empalme del ARNRESUMEN
The muscle tropomyosin I (mTm I) gene from Drosophila melanogaster has been analyzed and shown to express a complex transcription unit consisting of two sets of tissue-specific mRNAs. A 1.3- and 1.6-kilobase set of mRNAs is expressed during myogenesis in embryos, and in myogenic cell cultures. The mRNAs encode a 34,000-dalton muscle tropomyosin isoform. The same mTm I gene expresses a different set of 1.7- and 1.9-kilobase mRNAs in thoracic flight muscle tissue of the adult. The thorax RNAs encode a new tropomyosin isoform resolved on two-dimensional gels. The structure of the gene has been determined, and we show that the embryonic and thoracic mRNAs are generated by alternative splicing. The alternate exon splicing patterns determine a different 27 amino acids at the carboxy-terminal end of the two tropomyosin isoforms. These results show that the carboxy-terminal domain of tropomyosin is highly regulated in determining tropomyosin function. The results also show that contractile protein isoforms can be generated by single as well as multiple genes.