Frequency Analysis of Skinned Indirect Flight Muscle From a Myosin Light Chain 2 Deficient Mutant of Drosophila Melanogaster with a Reduced Wing Beat Frequency

Author(s):  
Mineo Yamakawa ◽  
Jeffrey Warmke ◽  
Scott Falkenthal ◽  
David Maughan
1992 ◽  
Vol 119 (6) ◽  
pp. 1523-1539 ◽  
Author(s):  
J Warmke ◽  
M Yamakawa ◽  
J Molloy ◽  
S Falkenthal ◽  
D Maughan

We have used a combination of classical genetic, molecular genetic, histological, biochemical, and biophysical techniques to identify and characterize a null mutation of the myosin light chain-2 (MLC-2) locus of Drosophila melanogaster. Mlc2E38 is a null mutation of the MLC-2 gene resulting from a nonsense mutation at the tenth codon position. Mlc2E38 confers dominant flightless behavior that is associated with reduced wing beat frequency. Mlc2E38 heterozygotes exhibit a 50% reduction of MLC-2 mRNA concentration in adult thoracic musculature, which results in a commensurate reduction of MLC-2 protein in the indirect flight muscles. Indirect flight muscle myofibrils from Mlc2E38 heterozygotes are aberrant, exhibiting myofilaments in disarray at the periphery. Calcium-activated Triton X-100-treated single fiber segments exhibit slower contraction kinetics than wild type. Introduction of a transformed copy of the wild type MLC-2 gene rescues the dominant flightless behavior of Mlc2E38 heterozygotes. Wing beat frequency and single fiber contraction kinetics of a representative rescued line are not significantly different from those of wild type. Together, these results indicate that wild type MLC-2 stoichiometry is required for normal indirect flight muscle assembly and function. Furthermore, these results suggest that the reduced wing beat frequency and possibly the flightless behavior conferred by Mlc2E38 is due in part to slower contraction kinetics of sarcomeric regions devoid or partly deficient in MLC-2.


1985 ◽  
Vol 5 (11) ◽  
pp. 3058-3068
Author(s):  
V P Parker ◽  
S Falkenthal ◽  
N Davidson

Recombinant DNA clones encoding the Drosophila melanogaster homolog of the vertebrate myosin light-chain-2 (MLC-2) gene have been isolated. This single-copy gene maps to the chromosomal locus 99E. The nucleotide sequence was determined for a 3.4-kilobase genomic fragment containing the gene and for two MLC-2 cDNA clones generated from late pupal mRNA. Comparison of these sequences shows that the gene contains two introns, the positions of which are conserved in the corresponding rat sequence. Extension of a primer homologous to the mRNA reveals two start sites for transcription 12 nucleotides apart. The sequence TATA is not present ahead of the mRNA cap site. There are two major sites of poly(A) addition separated by 356 nucleotides. The protein sequence derived from translation of the cDNA sequence shows a high degree of homology with that for the DTNB myosin light chain (MLC-2) of chicken. A lower degree of sequence homology was seen in comparisons with other evolutionarily related calcium-binding proteins. RNA blots show high levels of expression of several transcripts during the developmental time stages when muscle is being produced. In vitro translation of hybrid-selected RNA produces two polypeptides which comigrate on two-dimensional gels with proteins from Drosophila actomyosin, although the cDNA sequence reveals only one 26-kilodalton primary translation product.


Genetics ◽  
2020 ◽  
Vol 216 (1) ◽  
pp. 177-190
Author(s):  
Pooneh Vaziri ◽  
Danielle Ryan ◽  
Christopher A. Johnston ◽  
Richard M. Cripps

Myosin is an essential motor protein, which in muscle is comprised of two molecules each of myosin heavy-chain (MHC), the essential or alkali myosin light-chain 1 (MLC1), and the regulatory myosin light-chain 2 (MLC2). It has been shown previously that MLC2 phosphorylation at two canonical serine residues is essential for proper flight muscle function in Drosophila; however, MLC2 is also phosphorylated at additional residues for which the mechanism and functional significance is not known. We found that a hypomorphic allele of Pkcδ causes a flightless phenotype; therefore, we hypothesized that PKCδ phosphorylates MLC2. We rescued flight disability by duplication of the wild-type Pkcδ gene. Moreover, MLC2 is hypophosphorylated in Pkcδ mutant flies, but it is phosphorylated in rescued animals. Myosin isolated from Pkcδ mutant flies shows a reduced actin-activated ATPase activity, and MLC2 in these myosin preparations can be phosphorylated directly by recombinant human PKCδ. The flightless phenotype is characterized by a shortened and disorganized sarcomere phenotype that becomes apparent following eclosion. We conclude that MLC2 is a direct target of phosphorylation by PKCδ, and that this modification is necessary for flight muscle maturation and function.


Sensors ◽  
2021 ◽  
Vol 21 (10) ◽  
pp. 3329
Author(s):  
Yuting Sun ◽  
Yueyu Lin ◽  
Guangyu Zhao ◽  
Sune Svanberg

Insects constitute a very important part of the global ecosystem and include pollinators, disease vectors, and agricultural pests, all with pivotal influence on society. Monitoring and control of such insects has high priority, and automatic systems are highly desirable. While capture and analysis by biologists constitute the gold standard in insect identification, optical and laser techniques have the potential for high-speed detection and automatic identification based on shape, spectroscopic properties such as reflectance and fluorescence, as well as wing-beat frequency analysis. The present paper discusses these approaches, and in particular presents a novel method for automatic identification of mosquitos based on image analysis, as the insects enter a trap based on a combination of chemical and suction attraction. Details of the analysis procedure are presented, and selectivity is discussed. An accuracy of 93% is achieved by our proposed method from a data set containing 122 insect images (mosquitoes and bees). As a powerful and cost-effective method, we finally propose the combination of imaging and wing-beat frequency analysis in an integrated instrument.


1956 ◽  
Vol 39 (4) ◽  
pp. 497-512 ◽  
Author(s):  
Leo Levenbook ◽  
Carroll M. Williams

1. In the present study a correlation has been sought between aging, flight muscle mitochrondria (sarcosomes), cytochrome c, and flight ability in the blowfly, Phormia regina. 2. During the 1st week of adult life, individual sarcosomes increase in mass from 2.7 x 10–7 µg. dry weight at the time of emergence, to 8.5 x 10–7 µg. by the 7th day. During this period of growth, the number of sarcosomes per fly (6.7 x 108) remains constant. When mature, the sarcosomes account for 32.6 per cent of the total muscle dry weight, or close to 40 per cent on a wet weight basis. 3. It appears probable that the high content of flight muscle cytochromes is entirely localized in the sarcosomes. The cytochromes continue to be synthesized and increase in titer within the sarcosomes for 7 days after adult emergence. 4. As determined spectroscopically, the various cytochrome components at all times maintain a constant ratio both to one another and to the sarcosomal dry weight. This suggests the possibility that the cytochrome system may be synthesized as a single entity. 5. The wing-beat frequency of Drosophila funebris and Phormia varies with the age of these flies, being lowest at the time of emergence and maximum after the 6th day. 6. The relations between wing-beat frequency, respiration during flight, and sarcosomal cytochrome c content are discussed. On the basis of some likely assumptions it is calculated that the cytrochrome c turnover number is over 5,000, and that the cytochrome c turns over once for every two wing-beat cycles.


Genetics ◽  
1989 ◽  
Vol 122 (1) ◽  
pp. 139-151 ◽  
Author(s):  
J W Warmke ◽  
A J Kreuz ◽  
S Falkenthal

Abstract Using overlapping synthetic deficiencies, we find that a haplo-insufficient locus affecting flight behavior and the myosin light chain-2 gene co-map to the Drosophila melanogaster polytene chromosome interval 99D9-E1 to 99E2-3. From screening over 9000 EMS-treated chromosomes, we obtained alleles of two complementation groups that map to this same interval. One of these complementation groups lfm(3)99Eb, exhibits dominant flightless behavior; thus, flightless behavior of the deficiency is in all likelihood due to hemizygosity of this single locus. Rescue of flightless behavior by a duplication indicates that the single allele, E38, of the Ifm(3)99Eb complementation group is a hypomorph. Based upon its map position and a reduction in concentration of myosin light chain-2 mRNA in heterozygotes, we propose that Ifm(3)Eb(E38) is a mutant allele of the myosin light chain-2 gene. Our genetic analysis also resulted in the identification of four dominant flightless alleles of an unlinked locus, l(3)nc99Eb, that exhibits dominant lethal synergism with Ifm(3)99Eb.


1985 ◽  
Vol 5 (11) ◽  
pp. 3058-3068 ◽  
Author(s):  
V P Parker ◽  
S Falkenthal ◽  
N Davidson

Recombinant DNA clones encoding the Drosophila melanogaster homolog of the vertebrate myosin light-chain-2 (MLC-2) gene have been isolated. This single-copy gene maps to the chromosomal locus 99E. The nucleotide sequence was determined for a 3.4-kilobase genomic fragment containing the gene and for two MLC-2 cDNA clones generated from late pupal mRNA. Comparison of these sequences shows that the gene contains two introns, the positions of which are conserved in the corresponding rat sequence. Extension of a primer homologous to the mRNA reveals two start sites for transcription 12 nucleotides apart. The sequence TATA is not present ahead of the mRNA cap site. There are two major sites of poly(A) addition separated by 356 nucleotides. The protein sequence derived from translation of the cDNA sequence shows a high degree of homology with that for the DTNB myosin light chain (MLC-2) of chicken. A lower degree of sequence homology was seen in comparisons with other evolutionarily related calcium-binding proteins. RNA blots show high levels of expression of several transcripts during the developmental time stages when muscle is being produced. In vitro translation of hybrid-selected RNA produces two polypeptides which comigrate on two-dimensional gels with proteins from Drosophila actomyosin, although the cDNA sequence reveals only one 26-kilodalton primary translation product.


Apidologie ◽  
2021 ◽  
Author(s):  
Antonio R. S. Parmezan ◽  
Vinicius M. A. Souza ◽  
Indrė Žliobaitė ◽  
Gustavo E. A. P. A. Batista

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