Biomedical subjects
J C Sparrow
Publications and source records attributed to J C Sparrow.
The binding of mutant actins to profilin, ATP and DNase I.
Twenty-five mutations were created in the Drosophila melanogaster Act88F actin gene by in vitro mutagenesis and the mutant actins expressed in vitro. The affinity of the mutant actins for ATP, profilin and DNase I was determined. They were also tested for conformational changes by non-denaturing gel electrophoresis. Mutations at positions 364 (highly conserved) and 366 (invariant) caused changes in conformation, reduced ATP binding and increased profilin binding. At position 362 (invariant) only the conservative change from tyrosine to phenylalanine had no effect; other changes at this position affected conformation, ATP and profilin binding. Although only glycine or serine occur naturally at position 368, changes to threonine or glutamine had no effect on the actin. The mutant in which Asp363 was replaced by His and that in which Glu364 was replaced by Lys decreased DNase I binding, yet neither amino acid occurs in the DNase I binding site. Likewise several mutations affect ATP and profilin binding but are distant from the binding sites. We conclude that, although actin has a highly conserved amino acid sequence, individual amino acids can have variable tolerance for substitutions. Also amino acid changes can exert significant effects on the binding of ligands to distant parts of the actin structure.
Mutant actin: a dead end?
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Polymorphism in a Drosophila indirect flight muscle-specific tropomyosin isozyme does not affect flight ability.
We describe polymorphism in a Drosophila indirect flight muscle-specific tropomyosin isozyme, named TnH-34. Three variants of this protein differ in their mobilities as determined by 1-D and 2-D SDS-PAGE. Meiotic mapping places the polymorphism close to, if not within, the structural gene encoding this tropomyosin isozyme. The most likely site of the mutations is within a single C-terminal exon. Flight-testing of different genotypes reveals that this variation in TnH-34 does not affect flight ability. These results suggest that some sequence variation may be tolerated in this section of the protein and correlate with the variability of this protein in different insect species.
Drosophila actin mutants and the study of myofibrillar assembly and function.
The use of Drosophila mutations in the indirect flight muscle-specific actin gene, Act88F, to study actin structure/function and its assembly into thin filaments during myofibrillogenesis is described. Mutants with different phenotypic effects are discussed and attempts made to correlate the different properties of the mutants in vivo-myofibrillar structure, actin synthesis, accumulation and stability, heat shock response induction-with properties of the same mutations expressed by in vitro transcription/translation of the cloned actin genes-co-polymerisation, thermostability and protein conformation. Few of the properties show a complete correlation between the different classes of mutants. The nature of the diversity of the mutant effects is discussed. Questions as to how this will help in elucidating the molecular effects of the mutations and the assembly of thin filaments and myofibrils are considered. In addition, the efficacy of the co-polymerisation assay is examined. The post-translational processing of this actin-by N-terminal processing, methylation and ubiquitination-are described. Data is presented that inhibition of the N-terminal processing of actin in vitro affects the ability of the actin to copolymerise, and makes unprocessed actin behave as a capping protein. The possible in vivo importance of this phenomenon is discussed.
Post-translational processing of the amino terminus affects actin function.
We have studied the importance of N-terminal processing for normal actin function using the Drosophila Act88F actin gene transcribed and translated in vitro. Despite having different charges as determined by two-dimensional (2D) gel electrophoresis, Act88F expressed in vivo and in vitro in rabbit reticulocyte lysate bind to DNase I with equal affinity and are able to copolymerise with bulk rabbit actin equally well. Using peptide mapping and thin-layer electrophoresis we have shown that bestatin [( 3-amino-2-hydroxy-4-phenyl-butanoyl]-L-leucine), an inhibitor of aminopeptidases, can inhibit actin N-terminal processing in rabbit reticulocyte lysate. Although processed and unprocessed actins translated in vitro are able to bind to DNase I equally well, unprocessed actins are less able to copolymerise with bulk actins. This effect is more pronounced when bulk rabbit actin is used but is still seen with bulk Lethocerus actin. Also, the unprocessed actins reduce the polymerisation of the processed actin translated in vitro with the bulk rabbit actin. This suggests that individual actins do interact, even in non-polymerising conditions. The reduced ability of unprocessed actin to polymerise shows that correct post-translational modification of the N terminus is required for normal actin function.
Stability of mutant actins.
Mutants of the Drosophila Act88F actin gene were transcribed and translated in vitro and their relative stabilities were examined using urea gradient gel electrophoresis. Most of the mutant actins (E334K, E364K, G366D, G368E and R372H) were as stable as the wild-type. V339I had a slight decrease in stability, and E316K was the least stable. The causes of the differences are discussed and contrasted with the behaviour of the mutants in vivo, where E316K has normal stability and V339I is the least stable.
Characterisation of missense mutations in the Act88F gene of Drosophila melanogaster.
We have created missense mutations in the indirect flight muscle (IFM)-specific Act88F actin gene of Drosophila melanogaster by random in vitro mutagenesis. Following P element-mediated transformation into wild-type flies and subsequent transfer of the inserts into Act88F null strains, the effects of the actin mutants on the structure and function of the IFMs were examined. All of the mutants were antimorphic for flight ability. E316K and G368E formed muscle with only relatively small defects in structure whilst the others produced IFMs with large amounts of disruption. E334K formed filaments but lacked Z discs. V339I formed no muscle structure in null flies and did not accumulate actin. E364K and G366D both had relatively stable actin but did not form myofibrils. Using an in vitro polymerisation assay we found no significant effects on the ability of the mutant actins to polymerise. E364K and G366D also caused a strong induction of heat shock protein (hsp) synthesis at normal temperatures and accumulated large amounts of hsp22 which, together with the mutant actin, was resistant to detergent extraction. Both E316K and E334K caused a weak induction of hsp synthesis. We discuss how the stability, structure and function of the different mutant actins affects myofibril assembly and function, and the induction of hsps.
Alteration in crossbridge kinetics caused by mutations in actin.
The generation of force during muscle contraction results from the interaction of myosin and actin. The kinetics of this force generation vary between different muscle types and within the same muscle type in different species. Most attention has focused on the role of myosin isoforms in determining these differences. The role of actin isoforms has received little attention, largely because of the lack of a suitable cell type in which the myosin isoform remains constant yet the actin isoforms vary. An alternative approach would be to examine the effect of actin mutations, however, most of these cause such gross disruption of muscle structure that mechanical measurements are impossible. We have now identified two actin mutations which, despite involving conserved amino acids, can assemble into virtually normal myofibrils. These amino-acid changes in actin significantly affect the kinetics of force generation by muscle fibres. One of the mutations is not in the putative myosin-binding site, demonstrating the importance of long-range effects of amino acids on actin function.
Physiological properties of the dorsal longitudinal flight muscle and the tergal depressor of the trochanter muscle of Drosophila melanogaster.
A prerequisite for using muscle mutants to study contraction in Drosophila melanogaster is a description of the mechanics of wild-type muscles. Here we describe the mechanics of two different wild-type muscles; the dorsal longitudinal flight muscle which is asynchronous (nerve impulses are not synchronised with each contraction), and a leg muscle, the tergal depressor of the trochanter, which is synchronous. We have compared their mechanics to those of the asynchronous flight and the synchronous leg muscle from the giant waterbug Lethocerus indicus. We found that the mechanics of the asynchronous flight muscles from the two species were similar. At rest length both muscles had a high relaxed stiffness, were partially activated by Ca2+ (low steady-state active tension) and, once activated, had a large delayed increase in tension, which was well maintained, in response to a rapid stretch. The rate constant for the delayed increase in tension was about 10 times greater for D. melanogaster than for L. indicus under the same conditions. The mechanics of the synchronous leg muscles from both species were different from those of the flight muscles and resembled those of other synchronous muscles such as vertebrate striated muscle. At rest length, both muscles had a lower relaxed stiffness than the flight muscles, were fully activated by Ca2+ (high steady-state active tension) and, once activated, had a small delayed increase in tension, which was less well maintained, in response to a rapid stretch. The rate constant for the delayed increase in tension was similar for the leg muscles of both species. The different mechanical properties of the flight and leg muscles must arise from differences in their contractile proteins. The demonstration that satisfactory mechanical responses can be obtained from the small (less than 1 mm long) muscles of D. melanogaster will enable future responses from mutant muscles to be tested.
Arthrin, a myofibrillar protein of insect flight muscle, is an actin-ubiquitin conjugate.
Flight muscles of some insects contain a myofibrillar protein termed arthrin, which is closely related to actin (mw 43,000). Here we demonstrate that arthrin (mw 55,000) is ubiquitinated actin. We show that in Act88FM342, a flightless Drosophila mutant wherein the Act88F actin gene specifies a glu93----lys replacement, isoelectric points of both actin III and arthrin are shifted, revealing that both are encoded by the same gene. Arthrin reacts with an anti-ubiquitin antibody, which demonstrates that its extra mass results from ubiquitin ligation. Approximately one-seventh of myofibrillar actin is stably ubiquitinated, suggesting that there may be one arthrin molecule per actin-tropomyosin-troponin cooperative unit. Arthrin formation lags several hours behind that of actin III, implying that ubiquitination coincides with some aspect of myofibril assembly.
The recovery and preliminary examination of a temperature sensitive suppressor of the cryptocephal mutant of Drosophila melanogaster.
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The selection for mutants in Drosophila melanogaster hypersensitive to alpha-methyl dopa, a dopa decarboxylase inhibitor.
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The genetics of some second chromosome melanotic tumour mutants of Drosophila melanogaster.
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Physiological genetics of melantoic tumours in Drosophila melanogaster. VIII. The role of choline in the expression of the tumor gene tu bw and of its suppressor, su-tu.
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A spectrophotometric assay for Drosophila dopa decarboxylase.
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Amblyopia: a long-term follow-up.
This report concerns the late results of therapy in the treatment of strabismic amblyopia. Of the patients who achieved a visual acuity of 20/40 or better (56%), 40 percent maintained this on prolonged follow-up. Those whose visual acuity deteriorated, the majority lost two lines or less in their acuity. Close follow-up and repeated patching of such patients is indicated. Eccentric fixation at the onset of amblyopia treatment is a poor prognostic sign. Two-thirds of those who were eccentric at the onset were eccentric at the final follow-up examination.