Natural Zeitgebers Under Temperate Conditions Cannot Compensate for the Loss of a Functional Circadian Clock in Timing of a Vital Behavior in Drosophila

The adaptive significance of adjusting behavioral activities to the right time of the day seems obvious. Laboratory studies implicated an important role of circadian clocks in behavioral timing and rhythmicity. Yet, recent studies on clock-mutant animals questioned this importance under more naturalistic settings, as various clock mutants showed nearly normal diel activity rhythms under seminatural zeitgeber conditions. We here report evidence that proper timing of eclosion, a vital behavior of the fruit fly Drosophila melanogaster, requires a functional molecular clock under quasi-natural conditions. In contrast to wild-type flies, period01 mutants with a defective molecular clock showed impaired rhythmicity and gating in a temperate environment even in the presence of a full complement of abiotic zeitgebers. Although period01 mutants still eclosed during a certain time window during the day, this time window was much broader and loosely defined, and rhythmicity was lower or lost as classified by various statistical measures. Moreover, peak eclosion time became more susceptible to variable day-to-day changes of light. In contrast, flies with impaired peptidergic interclock signaling ( Pdf01 and han5304 PDF receptor mutants) eclosed mostly rhythmically with normal gate sizes, similar to wild-type controls. Our results suggest that the presence of natural zeitgebers is not sufficient, and a functional molecular clock is required to induce stable temporal eclosion patterns in flies under temperate conditions with considerable day-to-day variation in light intensity and temperature. Temperate zeitgebers are, however, sufficient to functionally rescue a loss of PDF-mediated clock-internal and -output signaling

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Natural Zeitgebers cannot compensate for the loss of a functional circadian clock in timing of a vital behaviour in Drosophila

10.1101/2019.12.22.886309 ◽

2019 ◽

Cited By ~ 2

Author(s):

Franziska Ruf ◽

Oliver Mitesser ◽

Simon Tii Mungwa ◽

Melanie Horn ◽

Dirk Rieger ◽

...

Keyword(s):

Molecular Clock ◽

Fruit Fly ◽

Diel Activity ◽

Laboratory Studies ◽

Natural Conditions ◽

Clock Mutant ◽

Full Complement ◽

Central Clock ◽

The Right

AbstractThe adaptive significance of adjusting behavioural activities to the right time of the day is intuitive. Laboratory studies have implicated an important role of circadian clocks in behavioural timing and rhythmicity. Yet, recent studies on clock-mutant animals questioned this importance under more naturalistic settings, as various clock mutants showed nearly normal diel activity rhythms under semi-natural Zeitgeber conditions.We here report evidence that proper timing of eclosion, a vital behaviour of the fruit fly Drosophila melanogaster, requires a functional molecular clock even under quasi-natural conditions. In contrast to wildtype flies, period01 mutants with a defective molecular clock eclose mostly arrhythmically in a temperate environment even in the presence of a full complement of abiotic Zeitgebers. Moreover, period01 mutants eclose during a much larger portion of the day, and peak eclosion time becomes more susceptible to variable day-to-day changes of light and temperature. Under the same conditions, flies with impaired peptidergic inter-clock signalling (pdf01 and han5304 mutants) stayed largely rhythmic with normal gate sizes. Our results suggest that the presence of natural Zeitgebers can mitigate a loss of peptide-mediated phasing between central clock neuron groups, but cannot substitute for the lack of a functional molecular clock under natural temperate conditions.

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Examination of the Catalytic Role of the Axial Cystine Ligand in the Co-Type Nitrile Hydratase from Pseudonocardia thermophila JCM 3095

Catalysts ◽

10.3390/catal11111381 ◽

2021 ◽

Vol 11 (11) ◽

pp. 1381

Author(s):

Irene R. A. M. Ogutu ◽

Martin St. Maurice ◽

Brian Bennett ◽

Richard C. Holz

Keyword(s):

Nitrile Hydratase ◽

Bond Formation ◽

Disulfide Bond Formation ◽

Protein Species ◽

Wild Type ◽

Cobalt Ions ◽

X Ray ◽

Full Complement ◽

Catalytic Role

The strictly conserved αSer162 residue in the Co-type nitrile hydratase from Pseudonocardia thermophila JCM 3095 (PtNHase), which forms a hydrogen bond to the axial αCys108-S atom, was mutated into an Ala residue. The αSer162Ala yielded two different protein species: one was the apoform (αSerA) that exhibited no observable activity, and the second (αSerB) contained its full complement of cobalt ions and was active with a kcat value of 63 ± 3 s−1 towards acrylonitrile at pH 7.5. The X-ray crystal structure of αSerA was determined at 1.85 Å resolution and contained no detectable cobalt per α2β2 heterotetramer. The axial αCys108 ligand itself was also mutated into Ser, Met, and His ligands. All three of these αCys108 mutant enzymes contained only half of the cobalt complement of wild-type PtNHase, but were able to hydrate acrylonitrile with kcat values of 120 ± 6, 29 ± 3, and 14 ± 1 s−1 for the αCys108His, Ser, and Met mutant enzymes, respectively. As all three of these mutant enzymes are catalytically competent, these data provide the first experimental evidence that transient disulfide bond formation is not catalytically essential for NHases.

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Diurnal Amplitudes of Arterial Pressure and Heart Rate Are Dampened in Clock Mutant Mice and Adrenalectomized Mice

Endocrinology ◽

10.1210/en.2007-1714 ◽

2008 ◽

Vol 149 (7) ◽

pp. 3576-3580 ◽

Cited By ~ 38

Author(s):

Hiroyoshi Sei ◽

Katsutaka Oishi ◽

Sachiko Chikahisa ◽

Kazuyoshi Kitaoka ◽

Eiji Takeda ◽

...

Keyword(s):

Heart Rate ◽

Arterial Pressure ◽

Clock Gene ◽

Clock Genes ◽

Plasma Aldosterone ◽

Mutant Mice ◽

Wild Type ◽

Clock Mutant ◽

Wild Type Control

Arterial pressure (AP), heart rate (HR), and cardiovascular diseases, including ischemic heart attack and cerebrovascular accident, show diurnal variation. Evidence that circadian-related genes contribute to cardiovascular control has been accumulated. In this study, we measured the AP and HR of Clock mutant mice on the Jcl/ICR background to determine the role of the Clock gene in cardiovascular function. Mice with mutated Clock genes had a dampened diurnal rhythm of AP and HR, compared with wild-type control mice, and this difference disappeared after adrenalectomy. The diurnal acrophase in both mean arterial pressure and HR was delayed significantly in Clock mutant mice, compared with wild-type mice, and this difference remained after adrenalectomy. Clock mutant mice had a lower concentration of plasma aldosterone, compared with wild-type mice. Our data suggest that the adrenal gland is involved in the diurnal amplitude, but not the acrophase, of AP and HR, and that the function of the Clock gene may be related to the nondipping type of AP elevation.

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The role of heat-shock proteins in thermotolerance

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.1993.0026 ◽

1993 ◽

Vol 339 (1289) ◽

pp. 279-286 ◽

Cited By ~ 123

Keyword(s):

Heat Shock ◽

Heat Shock Proteins ◽

High Temperatures ◽

Fruit Fly ◽

Yeast Cells ◽

Major Protein ◽

Wild Type ◽

Hsp 70 ◽

Shock Proteins

The role of heat-shock proteins (hsps) in thermotolerance was examined in the budding yeast Saccharomyces cerevisiae and in the fruit fly Drosophila melanogaster . In yeast cells, the major protein responsible for thermotolerance is hsp 100. In cells carrying mutations in the hsp 100 gene, HSP 104 , growth is normal at both high and low temperatures, but the ability of cells to survive extreme temperatures is severely impaired. The loss of thermotolerance is apparently due to the absence of the hsp 104 protein itself because, with the exception of the hsp 104 protein, no differences in protein profiles were observed between mutant and wild-type cells. Aggregates found in mutant cells at high temperatures suggest that the cause of death may be the accumulation of denatured proteins. No differences in the rates of protein degradation were observed between mutant and wild-type cells. This, and genetic analysis of cells carrying multiple hsp 70 and hsp 104 mutations, suggests that the primary function of hsp 104 is to rescue proteins from denaturation rather than to degrade them once they have been denatured. Drosophila cells do not produce a protein in the hsp 100 class in response to high temperatures. In this organism, hsp 70 appears to be the primary protein involved in thermotolerance. Thus, the relative importance of different hsps in thermotolerance changes from organism to organism.

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Mutations affecting the formation of the notochord in the zebrafish, Danio rerio

Development ◽

10.1242/dev.123.1.103 ◽

1996 ◽

Vol 123 (1) ◽

pp. 103-115 ◽

Cited By ~ 38

Author(s):

J. Odenthal ◽

P. Haffter ◽

E. Vogelsang ◽

M. Brand ◽

F.J. van Eeden ◽

...

Keyword(s):

Sonic Hedgehog ◽

Large Scale ◽

Floor Plate ◽

Precursor Cells ◽

Wild Type ◽

Plate Formation ◽

Notochord Cells ◽

The Right ◽

Adaxial Cells

In a large scale screen for mutants with defects in the embryonic development of the zebrafish we identified mutations in four genes, floating head (flh), momo (mom), no tail (ntl), and doc, that are required for early notochord formation. Mutations in flh and ntl have been described previously, while mom and doc are newly identified genes. Mutant mom embryos lack a notochord in the trunk, and trunk somites from the right and left side of the embryo fuse underneath the neural tube. In this respect mom appears similar to flh. In contrast, notochord precursor cells are present in both ntl and doc embryos. In order to gain a greater understanding of the phenotypes, we have analysed the expression of several axial mesoderm markers in mutant embryos of all four genes. In flh and mom, Ntl expression is normal in the germ ring and tailbud, while the expression of Ntl and other notochord markers in the axial mesodermal region is disrupted. Ntl expression is normal in doc embryos until early somitic stages, when there is a reduction in expression which is first seen in anterior regions of the embryo. This suggests a function for doc in the maintenance of ntl expression. Other notochord markers such as twist, sonic hedgehog and axial are not expressed in the axial mesoderm of ntl embryos, their expression parallels the expression of ntl in the axial mesoderm of mutant doc, flh and mom embryos, indicating that ntl is required for the expression of these markers. The role of doc in the expression of the notochord markers appears indirect via ntl. Floor plate formation is disrupted in most regions in flh and mom mutant embryos but is present in mutant ntl and doc embryos. In mutant embryos with strong ntl alleles the band of cells expressing floor plate markers is broadened. A similar broadening is also observed in the axial mesoderm underlying the floor plate of ntl embryos, suggesting a direct involvement of the notochord precursor cells in floor plate induction. Mutations in all of these four genes result in embryos lacking a horizontal myoseptum and muscle pioneer cells, both of which are thought to be induced by the notochord. These somite defects can be traced back to an impairment of the specification of the adaxial cells during early stages of development. Transplantation of wild-type cells into mutant doc embryos reveals that wild-type notochord cells are sufficient to induce horizontal myoseptum formation in the flanking mutant tissue. Thus doc, like flh and ntl, acts cell autonomously in the notochord. In addition to the four mutants with defects in early notochord formation, we have isolated 84 mutants, defining at least 15 genes, with defects in later stages of notochord development. These are listed in an appendix to this study.

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Role of the Agrobacterium tumefaciens VirD2 Protein in T-DNA Transfer and Integration

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.1998.11.7.668 ◽

1998 ◽

Vol 11 (7) ◽

pp. 668-683 ◽

Cited By ~ 75

Author(s):

Kirankumar S. Mysore ◽

Burgund Bassuner ◽

Xiao-bing Deng ◽

Nune S. Darbinian ◽

Andrei Motchoulski ◽

...

Keyword(s):

Agrobacterium Tumefaciens ◽

Fusion Protein ◽

Binary Vector ◽

Type A ◽

Blue Staining ◽

Wild Type ◽

Dna Integration ◽

Right Border ◽

The Right

VirD2 is one of the key Agrobacterium tumefaciens proteins involved in T-DNA processing and transfer. In addition to its endonuclease domain, VirD2 contains a bipartite C-terminal nuclear localization sequence (NLS) and a conserved region called ω that is important for virulence. Previous results from our laboratory indicated that the C-terminal, bipartite NLS and the ω region are not essential for nuclear uptake of T-DNA, and further suggested that the ω domain may be required for efficient integration of T-DNA into the plant genome. In this study, we took two approaches to investigate the importance of the ω domain in T-DNA integration. Using the first approach, we constructed a T-DNA binary vector containing a promoterless gusA-intron gene just inside the right T-DNA border. The expression of β-glucuronidase (GUS) activity in plant cells transformed by this T-DNA would indicate that the T-DNA integrated downstream of a plant promoter. Approximately 0.4% of the tobacco cell clusters infected by a wild-type A. tumefaciens strain harboring this vector stained blue with 5-bromo-4-chloro-3-indolyl β-d-glucuronic acid (X-gluc). However, using an ω-mutant A. tumefaciens strain harboring the same binary vector, we did not detect any blue staining. Using the second approach, we directly demonstrated that more T-DNA is integrated into high-molecular-weight plant DNA after infection of Arabidopsis thaliana cells with a wild-type A. tumefaciens strain than with a strain containing a VirD2 ω deletion/substitution. Taken together, these data indicate that the VirD2 ω domain is important for efficient T-DNA integration. To determine whether the use of the T-DNA right border is altered in those few tumors generated by A. tumefaciens strains harboring the ω mutation, we analyzed DNA extracted from these tumors. Our data indicate that the right border was used to integrate the T-DNA in a similar manner regardless of whether the VirD2 protein encoded by the inciting A. tumefaciens was wild-type or contained an ω mutation. In addition, a mutant VirD2 protein lacking the ω domain was as least as active in cleaving a T-DNA border in vitro as was the wild-type protein. Finally, we investigated the role of various amino acids of the ω and bipartite NLS domains in the targeting of a GUS-VirD2 fusion protein to the nucleus of electroporated tobacco protoplasts. Deletion of the ω domain, or mutation of the 10-amino-acid region between the two components of the bipartite NLS, had little effect upon the nuclear targeting of the GUS-VirD2 fusion protein. Mutation of both components of the NLS reduced, but did not eliminate, targeting of the fusion protein to the nucleus.

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Most sleep does not serve a vital function: Evidence from Drosophila melanogaster

Science Advances ◽

10.1126/sciadv.aau9253 ◽

2019 ◽

Vol 5 (2) ◽

pp. eaau9253 ◽

Cited By ~ 14

Author(s):

Quentin Geissmann ◽

Esteban J. Beckwith ◽

Giorgio F. Gilestro

Keyword(s):

Drosophila Melanogaster ◽

Fruit Fly ◽

Video Tracking ◽

Sleep Restriction ◽

Wild Type ◽

Vital Function ◽

High Throughput Analysis ◽

New Perspective ◽

Open Question

Sleep appears to be a universally conserved phenomenon among the animal kingdom, but whether this notable evolutionary conservation underlies a basic vital function is still an open question. Using a machine learning–based video-tracking technology, we conducted a detailed high-throughput analysis of sleep in the fruit fly Drosophila melanogaster, coupled with a lifelong chronic and specific sleep restriction. Our results show that some wild-type flies are virtually sleepless in baseline conditions and that complete, forced sleep restriction is not necessarily a lethal treatment in wild-type D. melanogaster. We also show that circadian drive, and not homeostatic regulation, is the main contributor to sleep pressure in flies. These results offer a new perspective on the biological role of sleep in Drosophila and, potentially, in other species.

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