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E Fyrberg

Publications and source records attributed to E Fyrberg.

At least 19 recordsLinked to original sources
PubMed1998

A family of Drosophila genes encoding quaking-related maxi-KH domains.

We recently identified a Drosophila gene, wings held out (who), that specifies a STAR (signal transduction and RNA activation) protein expressed within mesoderm and muscles. Genetic evidence suggests that WHO regulates muscle development and function in response to steroid hormone titer. who is related to the mouse quacking gene, essential for embryogenesis and neural myelination, and gld-1, a nematode tumor suppressor gene necessary for oocyte differentiation, both of which contain RNA binding "maxi-KH" domains presumed to link RNA metabolism to cell signaling. To initiate a broader study of Drosophila WHO-related proteins we used degenerate primers encoding peptides unique to maxi-KH domains to amplify the corresponding genes. We recovered nine genes, all specifying single maxi-KH domain proteins having tripartite regions of similarity that extend over 200 amino acids. One is located within the 54D chromosome subdivision, and one within 58C, while the remaining seven are within the 58E subdivision. At least four of these STAR proteins are expressed in a general manner, suggesting that maxi-KH domains are employed widely in Drosophila.

Amino Acid Sequence↗
PubMed1998

Characterization of lethal Drosophila melanogaster alpha-actinin mutants.

We have partially characterized four Drosophila melanogaster alpha-actinin gene mutants, I(1)2Cb1, I(1)2Cb2, I(1)2Cb4, and I(1)2Cb5. We demonstrate that in each case the mutation is caused by a chromosomal rearrangement that precludes normal protein synthesis. In the absence of alpha-actinin, flies complete embryogenesis and develop into flaccid larvae that die within approximately 24 hr. These larvae have noticeable muscle dysfunction at hatching, although they, nevertheless, are capable of escaping from the egg membranes and of subsequent crawling movements. During larval development muscles degenerate, progressively limiting mobility and ultimately causing death. Electron microscopy of mutant muscle fibers reveals that myofibrils are grossly disrupted in one day old larvae and that electron-dense structures reminiscent of those seen in human nemaline myopathies are present throughout larval life. Our work rigorously demonstrates that alpha-actinin deficiencies are the cause of I(1)2Cb muscle defects. We anticipate that the alpha-actinin mutants described herein will facilitate in vivo tests of spectrin superfamily protein domain functions using a combination of directed mutagenesis and germline transformation.

Actinin↗
PubMed1997

A Drosophila muscle-specific gene related to the mouse quaking locus.

We have characterized a novel muscle-specific gene of Drosophila melanogaster, defined by enhancer trap strain 24B of Brand and Perrimon (1993). We show that transcripts of the gene accumulate within presumptive mesoderm and persist within developing muscles, strongly suggesting that the encoded protein is involved in muscle cell determination and differentiation. cDNA sequences reveal that the Drosophila protein is similar to quaking (64% identity over 210 amino acids), a protein essential for mouse embryogenesis, and gld-1 (53% identity over 162 amino acids) a germ-line-specific tumor suppressing protein of the nematode, Caenorhabditis elegans. We demonstrate that the Drosophila gene resides within the 93F chromosome subdivision, and describe its physical map. Finally, we have used the gene, which we have named quaking-related 93F (qkr93F), to identify a family of closely related KH domains.

Amino Acid Sequence↗
PubMed1997

Green fluorescent protein/beta-galactosidase double reporters for visualizing Drosophila gene expression patterns.

We characterized 120 novel yeast Ga14-targeted enhancer trap lines in Drosophila using upstream activating sequence (UAS) reporter plasmids incorporating newly constructed fusions of Aequorea victoria green fluorescent protein (GFP) and Escherichia coli beta-galactosidase genes. Direct comparisons of GFP epifluorescence and beta-galactosidase staining revealed that both proteins function comparably to their unconjugated counterparts within a wide variety of Drosophila tissues. Generally, both reporters accumulated in similar patterns within individual lines, but in some tissues, e.g., brain, GFP staining was more reliable than that of beta-galactosidase, whereas in other tissues, most notably tests and ovaries, the converse was true. In cases of weak enhancers, we occasionally could detect beta-galactosidase staining in the absence of discernible GFP fluorescence. This shortcoming of GFP can, in most cases, be alleviated by using the more efficient S65T GFP derivative. The GFP/beta-gal reporter fusion protein facilitated monitoring several aspects of protein accumulation. In particular, the ability to visualize GFP fluorescence enhances recognition of global static and dynamic patterns in live animals, whereas beta-galactosidase histochemistry affords sensitive high resolution protein localization. We present a catalog of Ga 14-expressing strains that will be useful for investigating several aspects of Drosophila melanogaster cell and developmental biology.

Animals↗
PubMed1995

Monitoring development and pathology of Drosophila indirect flight muscles using green fluorescent protein.

We describe the use of a green fluorescent protein (GFP) reporter construct to monitor indirect flight muscle development in normal and mutant Drosophila melanogaster strains. We used polymerase chain reaction to amplify a portion of the Act88F actin gene that includes 1420 nucleotides of flanking DNA, the transcription start, first intron, and initiator codon, incorporating the fragment into the Drosophila germ line transformation vector pCaSpeR. We fused the fragment to the gene encoding green fluorescent protein of the bioluminescent jellyfish Aequorea victoria. We could detect GFP protein in transgenic strains and found that its accumulation, conveniently visualized in living flies using epifluorescence microscopy, was limited to the indirect flight muscles. GFP fluorescence can be used to visualize all stages of flight muscle development subsequent to myoblast fusion nad facilitates the detection of morphological changes in fibers caused by particular mutations.

Actins↗
PubMed1994

Genes encoding actin-related proteins of Drosophila melanogaster.

Recently, several laboratories have described proteins of yeasts, mammals and Drosophila melanogaster that are 35 to 55% identical to conventional actins, but, as yet, little is known about their functions. We have initiated a systematic study by using degenerate oligonucleotides specifying two highly conserved nucleotide-binding peptides of actin, in conjunction with polymerase chain reaction techniques, to isolate Drosophila genes that encode actin-related proteins. Here we summarize the isolation of four such genes and compare the sequences of the proteins that they encode. Computer searches of databases revealed that three of the encoded proteins are homologs of yeast or mammalian actin-related proteins, implying that the corresponding proteins participate in functions common to many cell types. The fourth gene encodes a novel protein that, apparently is expressed within testes. The four genes are located within the 14D, 53D, 66B and 87C subdivisions of polytene chromosomes.

Actins↗
PubMed1994

Drosophila melanogaster genes encoding three troponin-C isoforms and a calmodulin-related protein.

Using low-stringency hybridization and polymerase chain reaction (PCR)-based DNA amplification, we have isolated three Drosophila melanogaster genes that encode troponin-C isoforms and one specifying a protein that is closely related to calmodulin. Two of the troponin-C genes, located within the 47D and 73F subdivisions of chromosomes 2 and 3, respectively, encode very closely related isoforms. That specified by the 47D gene accumulates almost exclusively in larval muscles, while that encoded by the 73F gene is present in both larvae and adults. The third gene, located within the 41C subdivision of chromosome 2, encodes a more distantly related troponin-C isoform that accumulates only within adults. The gene that encodes the calmodulin-related protein is located within the 97A subdivision of chromosome three. The protein encoded by this gene has a different primary sequence from that of conventional calmodulin, which is specified by a gene located within the 49A subdivision of chromosome 2. Our report is the first to describe insect troponin-C isoforms and further avails genetic methods for investigating the in vivo functions of the troponin-C/myosin light-chain/calmodulin protein superfamily.

Amino Acid Sequence↗
PubMed1993

A Drosophila homologue of the Schizosaccharomyces pombe act2 gene.

Diverse proteins that are 35% to 55% identical to actins have been discovered recently in yeasts, nematodes, and vertebrates. In order to study these proteins systematically and relate their functions to those of conventional actins, we are isolating the corresponding genes from the genetically tractable eukaryote, Drosophila melanogaster. Here we report the isolation and partial characterization of a Drosophila homologue of the Schizosaccharomyces pombe act2 gene. Degenerate oligonucleotide primers specifying peptides that are highly conserved within the actin protein superfamily were used in conjunction with polymerase chain reaction (PCR) to amplify a portion of the Drosophila gene that we have named actr66B. The corresponding full-length cDNA sequence encodes a protein of 418 residues that is 65% identical to the product of the S. pombe act2 gene, 80% identical to the bovine act2 homologue, but only 48% identical to the principal Drosophila cytoplasmic actin encoded by the Act5C actin gene. Alignment of the yeast, bovine, and Drosophila actin-related proteins shows that they have four peptide insertions, relative to conventional actins, three of which are well placed to modify actin polymerization and one that is likely to perturb the binding of myosin. Locations of two of the five actr66B introns are conserved between Drosophila and yeast genes, further attesting that they evolved from a common ancestor and are likely to encode proteins having similar functions. We demonstrate that the Drosophila gene is located on the left arm of chromosome 3, within subdivision 66B. Finally, we show by RNA blot-hybridization that the gene is expressed at low levels, relative to conventional nonmuscle actin, in all developmental stages. From these and other observations we infer that the actr66B protein is a minor component of all cells, perhaps serving to modify the polymerization, structure, and dynamic behavior of actin filaments.

Actins↗
PubMed1992

Drosophila projectin: relatedness to titin and twitchin and correlation with lethal(4) 102 CDa and bent-dominant mutants.

We have investigated projectin, a large protein of insect muscles, in Drosophila melanogaster. The 5.3 kilobases of coding sequence reported here contains Class I and Class II motifs characteristic of titin and twitchin, arranged in a three domain ... [II-I-I] [II-I-I] ... pattern. Two mutants mapped to the location of the projectin gene in the 102C subdivision of chromosome 4, lethal(4) 102 CDa and bent-Dominant, have DNA rearrangements within their projectin gene. The lethal(4) 102 CDa mutant has a 141 nucleotide insertion containing stop codons in all three reading frames within an exon sequence, showing that it cannot synthesize normal projectin. Both bent-Dominant and lethal(4) 102 CDa homozygotes die at the completion of embryogenesis because they are unable to escape the egg vitelline membrane. We propose that this hatching failure is due to muscle weakness caused by projectin defects.

Alleles↗
PubMed1992

Isolation of a Drosophila gene encoding glutathione S-transferase.

We have isolated a Drosophila gene, DmGST-2, that encodes glutathione S-transferase, a homo- or heterodimeric enzyme thought to be involved in detoxification of xenobiotics, including known carcinogens. The encoded protein has a primary sequence that is more similar to mammalian placental and nematode GSTs than that of a previously described Drosophila GST gene, herein referred to as DmGST-1. We provide a physical map of the gene and show that it specifies at least two mRNAs, measuring 1.9 and 1.6 kb, which differ only in the lengths of their 3' untranslated regions. Both of the mRNAs are present during all developmental stages. In situ hybridization of the DmGST-2 gene to larval polytene chromosomes places it within the 53F subdivision of chromosome 2, and Southern blotting to chromosomal DNA indicates that the gene has no close relatives within the Drosophila genome. Our results make possible molecular genetic approaches for further elaborating the function of glutathione S-transferases in insect development and physiology, in the metabolism of plant toxins, and in conferring insecticide resistance.

Amino Acid Sequence↗
PubMed1992

Perturbations of Drosophila alpha-actinin cause muscle paralysis, weakness, and atrophy but do not confer obvious nonmuscle phenotypes.

We have investigated accumulation of alpha-actinin, the principal cross-linker of actin filaments, in four Drosophila fliA mutants. A single gene is variably spliced to generate one nonmuscle and two muscle isoforms whose primary sequence differences are confined to a peptide spanning the actin binding domain and first central repeat. In fliA3 the synthesis of an adult muscle-specific isoform is blocked in flight and leg muscles, while in fliA4 the synthesis of nonmuscle and both muscle-specific isoforms is severely reduced. Affected muscles are weak or paralyzed, and, in the case of fliA3, atrophic. Their myofibrils, while structurally irregular, are remarkably normal considering that they are nearly devoid of a major contractile protein. Also surprising is that no obvious nonmuscle cell abnormalities can be discerned despite the fact that both the fliA1- and fliA4-associated mutations perturb the nonmuscle isoform. Our observations suggest that alpha-actinin stabilizes and anchors thin filament arrays, rather than orchestrating their assembly, and further imply that alpha-actinin function is redundant in both muscle and nonmuscle cells.

Actin Cytoskeleton↗
PubMed1991

Muscle abnormalities in Drosophila melanogaster heldup mutants are caused by missing or aberrant troponin-I isoforms.

We have investigated the molecular bases of muscle abnormalities in four Drosophila melanogaster heldup mutants. We find that the heldup gene encodes troponin-I, one of the principal regulatory proteins associated with skeletal muscle thin filaments. heldup3, heldup4, and heldup5 mutants, all of which have grossly abnormal flight muscle myofibrils, lack mRNAs encoding one or more troponin-I isoforms. In contrast, heldup2, an especially interesting mutant wherein flight muscles are atrophic, synthesizes the complete mRNA complement. By sequencing mutant troponin-I cDNAs we demonstrate that the molecular basis for muscle degeneration in heldup2 is conversion of an invariant alanine residue to valine. We finally show that degeneration of heldup2 thin filament/Z-disc networks can be prevented by eliminating thick filaments from flight muscles using a null allele of the sarcomeric myosin heavy chain gene. This latter observation suggests that actomyosin interactions exacerbate the structural or functional defect resulting from the troponin-I mutation.

Amino Acid Sequence↗
PubMed1991

From genes to tensile forces: genetic dissection of contractile protein assembly and function in Drosophila melanogaster.

Myofibrils, the contractile organelles of skeletal muscle, are highly ordered and precisely regulated actomyosin networks. Investigations of myofibril assembly are revealing the cellular mechanisms by which contractile components are arranged and regulated. In order to facilitate this research we have developed formal molecular genetics for myofibrillar proteins of Drosophila flight muscle. Presently, mutations can be used systematically to perturb or eliminate any of the classical myofibrillar proteins within these fibers, and the in vivo consequences can be conveniently evaluated using protein electrophoresis, electron microscopy, or by assaying flight performance. Here we review some recent progress.

Animals↗
PubMed1990

Drosophila melanogaster troponin-T mutations engender three distinct syndromes of myofibrillar abnormalities.

In vertebrates troponin complexes interact co-operatively with tropomyosin dimers to modulate skeletal muscle contraction. In order further to investigate troponin assembly and function in vivo, we are developing molecular genetic approaches. Here we report characterization of the gene that encodes Drosophila tropinin-T and analyses of muscle defects engendered by several mutant alleles. We found that the Drosophila troponin-T locus specifies at least three proteins having sequences similar to vertebrate troponin-T. All are significantly larger than any avian or mammalian isoforms, however, due to a highly acidic carboxy-terminal extension. Comparisons of the chromosomal arrangements of vertebrate and Drosophila troponin-T genes revealed that the location of one intron-exon boundary is conserved. This observation and the similarity of vertebrate and Drosophila troponin-T primary sequences suggest that the respective proteins are homologous, and that troponin-T pre-dates the divergence of vertebrate and invertebrate organisms. In situ hybridization of the Drosophila troponin-T gene to polytene chromosomes demonstrated that it resides within subdivision 12A of the X chromosome, precisely where upheld and indented thorax flight muscle mutations have been mapped previously. We determined the nucleotide sequences of troponin-T genes in five extant mutants. All have deleterious alterations, directly establishing that upheld and indented thorax muscle abnormalities are due to defective troponin-T. Two of the alleles, upheld2 and upheld3, apparently disrupt RNA splicing and eliminate most or all troponin-T from flight and jump muscles, while the remaining three alleles change the identities of single amino acids of troponin-T. Electron microscopy of mutant muscles revealed that the two null alleles eliminate thin filaments, except where they are bound by electron-dense material presumed to be Z-disc proteins. Two of the point mutations, upheld101 and indented thorax3, do not perturb assembly of myofibrils, but cause their degeneration within days after muscles begin to be utilized. The final mutation, upheldwhu, reduces the diameter of the myofibril lattice by approximately one-half. We propose hypotheses to explain how each troponin-T mutation engenders the observed myofibrillar defects.

Alleles↗
PubMed1990

Genetic approaches to myofibril form and function in Drosophila.

Myofibrils, the contractile organelles of muscle, are apt subjects for studies on the formation and function of actomyosin networks. Molecular genetic approaches are advancing our understanding of myofibril structure and assembly, and may offer a novel and useful approach for investigating the crossbridge cycle. We review recent progress in Drosophila.

Animals↗