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S Falkenthal

Publications and source records attributed to S Falkenthal.

16 recordsLinked to original sources
PubMed1992

Myosin light chain-2 mutation affects flight, wing beat frequency, and indirect flight muscle contraction kinetics in Drosophila.

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.

Allosteric Regulation↗
PubMed1989

Human ventricular/slow twitch myosin alkali light chain gene characterization, sequence, and chromosomal location.

The gene coding for the human ventricular/slow twitch myosin alkali light chain isoform was isolated and sequenced. It was found to contain a total of seven exons, the last of which is completely 3'-untranslated sequence. Comparison of this gene sequence with that of the various fast twitch skeletal isoform gene sequences revealed that the exon-intron arrangement is conserved within the myosin alkali light chain gene family. In fact the introns are in exactly the same positions within analogous codons. Comparison of the derived amino acid sequence from the human ventricular/slow twitch isoform gene with that of other isoform protein sequences indicated that the protein encoded by this gene is more homologous to the chicken cardiac isoform protein sequence than to any of the other protein sequences. These results indicate that the gene duplication which gave rise to the ventricular/slow twitch and fast twitch isoform genes must have occurred prior to the divergence of mammals and avians. We have also localized the human ventricular/slow twitch isoform gene to the short arm of human chromosome 3. Interestingly the corresponding mouse gene has been mapped to the distal region of mouse chromosome 9 which contains a conserved syntenic group of genes that map to the short arm of human chromosome 3.

Amino Acid Sequence↗
PubMed1989

Co-localization to chromosome bands 99E1-3 of the Drosophila melanogaster myosin light chain-2 gene and a haplo-insufficient locus that affects flight behavior.

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.

Alleles↗
PubMed1988

Ifm(2)2 is a myosin heavy chain allele that disrupts myofibrillar assembly only in the indirect flight muscle of Drosophila melanogaster.

Using a combination of molecular and genetic techniques we demonstrate that Ifm(2)2 is an allele of the single-copy sarcomeric myosin heavy chain gene. Flies homozygous for this allele accumulate wild-type levels of mRNA and protein in tubular muscle of adults, but fail to accumulate detectable amounts of myosin heavy chain mRNA or protein in the indirect flight muscle. We propose that the mutation interferes with either transcription of the gene or splicing of the primary transcript in the indirect flight muscle and not in other muscle tissues. Biochemical and electron microscopic analysis of flies homozygous for this mutation has revealed that thick filament assembly is abolished in the indirect flight muscle resulting in the instability of wild-type thick filament proteins. In contrast, thin filament and Z disc assembly are marginally affected. We discuss a working hypothesis for sarcomere assembly and define and experimental approach to test the predictions of this proposed pathway for sarcomere assembly.

Alleles↗
PubMed1987

The indirect flight muscle of Drosophila accumulates a unique myosin alkali light chain isoform.

Mapping of the 5' and 3' ends of the Drosophila myosin alkali light chain (MLC-ALK) mRNA by S1 nuclease and primer extension assays has shown that the primary transcripts are identical irrespective of the time in development that the RNA was prepared. As shown by S1 nuclease experiments these transcripts are alternatively spliced in a tissue-specific fashion generating mRNAs that encode tissue-specific protein isoforms. Antibodies were raised to synthetic peptides identical in sequence to the unique portion of each protein. Western blots of one-dimensional polyacrylamide gels using the type-specific antibodies confirmed and extended the results obtained from the S1 nuclease experiments. The indirect flight muscle is the only tissue in the adult that accumulates the alternatively spliced mRNA. The choice between splicing pathways involves the use of a nonconsensus 3' splice junction in larvae and in the tubular muscles of adults, whereas in the indirect flight muscle of the adult only consensus sequences are utilized. The involvement of a trans-acting factor to activate the nonconsensus splice site in the myotubes of larvae and the tubular myotubes of adults is proposed.

Animals↗
PubMed1985

Developmental variations in the splicing pattern of transcripts from the Drosophila gene encoding myosin alkali light chain result in different carboxyl-terminal amino acid sequences.

The total sequence of the Drosophila melanogaster gene encoding the myosin light chain dissociated by alkali (MLC-ALK) has been determined. By sequence comparisons with an MLC-ALK cDNA clone and by S1 nuclease analyses, the pattern of introns and exons within the gene has been deduced. There are multiple polyadenylylation signals that can account for most of the observed heterogeneity in the lengths of mRNAs. In the 3' half of the gene, there are two alternative splicing patterns which result in mRNAs that translate to give proteins with two alternative 14 amino acid carboxyl-terminal sequences. There is developmental regulation of the selection of the above splicing sites. One splicing pattern produces an mRNA that translates into a protein used for both larval and adult musculature, whereas the other splicing pattern is used for the latter stage only.

Animals↗
PubMed1985

Characterization of the myosin light-chain-2 gene of Drosophila melanogaster.

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.

Amino Acid Sequence↗
PubMed1984

Drosophila melanogaster has only one myosin alkali light-chain gene which encodes a protein with considerable amino acid sequence homology to chicken myosin alkali light chains.

A chimeric lambda DNA molecule containing the myosin alkali light-chain gene of Drosophila melanogaster was isolated. The encoded amino acid sequence was determined from the nucleic acid sequence of a cDNA homologous to the genomic clone. The identity of the encoded protein was established by two criteria: (i) sequence homology with the chicken alkali light-chain proteins and (ii) comparison of the two-dimensional gel electrophoretic pattern of the peptides synthesized by in vitro translation of hybrid-selected RNA to that of myosin alkali light-chain peptides extracted from Drosophila myofibrils. There is only one myosin alkali light-chain in D. melanogaster; its chromosomal location is region 98B . This gene is abundantly expressed during the development of larval as well as adult muscles. The Drosophila protein appears to contain one putative divalent cation-binding domain (an EF hand) as compared with the three EF hands present in chicken alkali light chains.

Amino Acid Sequence↗
PubMed1981

Measurement of ribonucleotide pool specific activities by an in vivo method: comparison with an in vitro method.

A biological assay is described for the specific activity of the nucleotides incorporated into nuclear ribonucleic acid (RNA). Under labeling conditions in which the pool specific activity reaches steady state by a process described by a continuous function, this method allows the determination of pool specific activity at all times of labeling. In Drosophila cultured cells incubated with [3H]uridine, the incorporation of radioactivity into transfer RNA (tRNA) and the absolute rate of tRNA synthesis were used to obtain an absolute value for the average specific activities of the uridine 5'-triphosphate (UTP) and cytidine 5'-triphosphate (CTP) precursor pools. The average specific activity of the UTP and CTP pool obtained by this in vivo method was 2-3 fold higher than the specific activity of the whole-cell UTP and CTP pools as assayed by an in vitro method. This suggests that the ribonucleotide pools in Drosophila cultured cells are compartmentalized. Use of the in vivo assay of ribonucleotide pool specific activities described here should avoid underestimates or overestimates which may arise when absolute rates of RNA synthesis are calculated from measurements of whole-cell ribonucleotide pool specific activities. The rate of labeling of tRNA was used to determine the rate at which the combined UTP and CTP pools equilibrate. This rate was then used to determine that the rate of processing pre-tRNA to mature tRNA is 0.21 min-1; this corresponds to a half-life of 3.3 min.

Animals↗
PubMed1980

Structure, translation, and metabolism of the cytoplasmic copia ribonucleic acid of Drosophila melanogaster.

We have characterized the copia RNA in the cytoplasm of cultured Drosophila cells. Copia RNA was detected and purified by hybridization to DNA of the plasmid cDm 1142, which contains the copia sequence. A large fraction (2.2%) of the total cytoplasmic poly(A)+ RNA was found to be copia RNA. Cytoplasmic copia RNA displays all the characteristics expected for a messenger RNA. It possesses a poly(A) tract identical in length with that of total poly(A)+ cytoplasmic RNA. It is associated with polysomes and can be released from this association by treatment with EDTA. When purified copia RNA is added to an mRNA-dependent rabbit reticulocyte lysate, three polypeptides of 51000, 33000, and 21000 daltons are seen. We have not determined if these are different polypeptides or if the two smaller polypeptides are fragments of the 51000-dalton polypeptide. The half-life of copia cytoplasmic RNA was determined in pulse--chase experiments to be 9.5 h; this is 1.6 times longer than the half-life of the intermediate decay class of total poly(A)+ cytoplasmic RNA. These properties provide strong evidence that copia RNA functions in vivo as a messenger RNA.

Animals↗