A lack of locomotor activity rhythms inDrosophila melanogaster larvae (Diptera: Drosophilidae)

We examined the locomotor activity ofDrosophila melanogaster for the existence of circadian rhythms, using the wild type and two mutants of theperiod (per) gene,per ^o andper ^s. This was accomplished using a newly described apparatus for the recording and measurement of larval path lengths over a 96-h test period. None of the larvae examined exhibited appreciable diel rhythms under cycling conditions of light or temperature. Larvae were also not rhythmic under free-running conditions. Our results suggest that theper gene does not influence an observable locomotor behavioral phenotype in the larval stage of development.     

Mechanism of suppression inDrosophila: Regulation of tryptophan oxygenase by thesu(s) + allele

The suppressor gene,su(s)², inDrosophila melanogaster restores the production of red and brown eye pigments for some purple and vermilion mutant alleles, respectively. We showed previously that the product of thesu(s)⁺ allele caused inhibition of the sepiapterin synthase A produced by the purple mutant but did not affect the wild-type enzyme. Suppression was accomplished by removingsu(s)⁺ from the genome. We now report that the tryptophan oxygenase, produced by suppressible vermilion alleles, is also inhibited by extracts fromsu(s)⁺ flies. The inhibition of the vermilion enzyme can be reduced or eliminated, respectively, by prior storage of the extract at 4 or –20°C or by boiling, whereas the wild-type enzyme is not affected by extracts ofsu(s)⁺ flies. Also, when the suppressible vermilion strain is raised on certain diets, brown eye pigment production occurs. This epigenetic suppression was reduced by the presence of an extra copy ofsu(s)⁺ in the genome. These data support a posttranslational mechanism for regulation of enzyme activity in which the activity of the mutant enzyme is reduced by the product of thesu(s)⁺ allele. How thesu(s)⁺ gene product can distinguish between the normal and the mutant forms of these two enzymes is discussed, along with other mechanisms for suppression that are currently under investigation.This work was supported in part by a grant from the KOSEF, Korea Science and Engineering Foundation, and the National Science Foundation under the U.S.-East Asia Cooperative Science Program as well as the Office of Health and Environmental Research, U.S. Department of Energy, under Contract DE-AC05-840R21400 with the Martin Marietta Energy Systems, Inc.     

Transfer of dye among salivary gland cells is not affected by genetic variations of the period clock gene in Drosophila melanogaster

Larval salivary gland cells of Drosophila melanogaster were injected with a fluorescent dye to assess strengths of intercellular communication among such cells, as influenced by mutations at the period locus and by a per transgene. This clock gene had been reported to increase the extent of dye transfer when mutated such that it shortens the period of biological rhythms; the previous study also showed that a per-null mutant decreased the strength of transfer among salivary gland cells. Our re-examination of this feature of larval physiology—in observer-blind analyses, using the per ^s and per ^o mutants as well as two per-normal strains—revealed no appreciable differences in extents of dye transfer among these four genotypes. These results are discussed in the context of emerging findings which suggest that the period gene's product controls pacemaker functioning as an intracellularly acting entity.     

The action of the Notch locus in Drosophila hydei

The phenotypic effects of different doses of the dominant, sex-linked mutant Notch (N) and its wildtype allele (N ⁺) were studied in Drosophila hydei, N being lethal in homozygous or hemizygous condition. Various dosage combinations were made by using N ⁺ N and N ⁺ N ⁺ attached-X chromosomes as well as X and Y N ⁺-duplication chromosomes (w ^mCoY, Xw^mCo,and DpCo ^Nt). The N mutant used, N ⁶⁸, is associated with a small inversion: In (I) N ⁶⁸.The wing phenotype was found to depend solely on the number of functional (N ⁺) alleles present, irrespective of the dose of N. Females with a single dose of N ⁺ are phenotypically Notch, females with three or four doses of N ⁺ show a Confluens wing phenotype. The latter occurs in varying degrees of expression which seem to be correlated with the relative amounts of sex-chromosomal heterochromatin present. In males the N ⁺ locus behaves as a dosage compensated locus either on the X or the Y chromosome.In the w ^mCo (w⁺N⁺) duplication, the w ⁺ locus shows variegation when placed over white, whereas N ⁺ placed over N ⁶⁸ does not. The former being situated closer to the heterochromatin in this aberration, this is consistent with the idea of gene inhibition by heterochromatin but at the same time would imply a very limited spreading effect.     

Circadian clock phenotypes of chromosome aberrations with a breakpoint at the per locus

Summary The circadian rhythm phenotypes of eight chromosome aberrations with a breakpoint in the region of the per locus (3B1-2) were analyzed. Two duplications and five deficiencies with a 3B1-2 breakpoint produce either a wild-type or an arrhythmic clock phenotype while one translocation with a 3B1-2 breakpoint, T(1;4)JC43, produces locomotor-activity rhythms with either very-long period (31–39 h), rhythms that grade into arrhythmicity, or completely arrhythmic phenotypes. This is a unique phenotype that had not previously been observed for mutants at the per locus. An extensive complementation analysis of 3B1-2 chromosome aberrations and per mutant alleles provided no compelling evidence for genetic complexity at the per locus. This is in contrast to the report of Young and Judd (1978). Analysis of both the locomotor-activity and eclosion phenotypes of 3B1-2 chromosome aberrations did not uncover differences in the genetic control of these two rhythms. The clock phenotypes of 3B1-2 chromosome aberrations, the three per mutant alleles, and per ⁺ duplications suggest that mutations at the per locus shorten, lengthen, or eliminate periodicity by respectively increasing, decreasing, or eliminating per activity.     

Restoration of enzyme activity by recessive missense suppressors in the fungus Coprinus.

Summary The acu-1 locus in Coprinus is the structural gene for acetyl-CoA synthetase. Five suppressor gene mutations, which suppress the acu-1,34 missense allele, were induced by mutagen treatment. All five suppressors were shown to have properties expected for tRNA structural gene mutations: they are recessive, they show a gene dosage effect in any doubly heterozygous combination of two sup ⁺ mutations and they are allele specific in action.Crosses between suppressed mutants established that at least four suppressor loci were represented. Doubly suppressed mutants derived from these crosses were used to show that the gene dosage effect is maintained when two sup ⁺ mutations are in cis as well as trans combinations in the two nuclei of the basidiomycete dikaryon.Extracts of the unsuppressed acu-1.34 mutant contained less than 2% of wild type acetyl-CoA synthetase activity whereas extracts of four of the five suppressor strains showed activities ranging from 28 to 37% of wild type. Only a slight increase in activity was detected in the fifth suppressor strain but this was associated with a temperature sensitive sup ⁺ phenotype. All five sup ⁺ mutations restored the ability of the acu-1.34 mutant to induce isocitrate lyase, an enzyme which, under the conditions of growth used, can only be induced when acetyl-CoA synthetase activity is present. Thus all five suppressors act to restore normal acu-1 protein function.     

The isolation and characterization of a mutant allele at a new X-linked locus,mex, affecting NADP+-dependent enzymes inDrosophila melanogaster

The isolation and characterization of mutant alleles in a regulatory gene affecting NADP⁺-dependent enzymes are described. The locus,mex, is at position 26.5 ± 0.74 on the X chromosome ofDrosophila melanogaster. The newly isolated mutant allele,mex ¹, is recessive to either themex allele found in Oregon-R wild-type individuals or that found in thecm v parental stock in which the new mutants were induced. Themex ¹ mutant allele is associated with statistically significant decreases in malic enzyme (ME) specific activity and ME specific immunologically cross-reacting material (ME-CRM) in newly emerged adult males. During this same developmental stage in males, the NADP⁺-dependent isocitrate dehydrogenase specific activity increases to statistically significant levels. Females of themex ¹ mutant strain show statistically significant elevated levels of the pentose phosphate shunt enzymes, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. Isoelectric focusing and thermolability comparisons of the active ME from mutant and control organisms indicate that the enzyme is the same. Developmental profiles ofmex ¹ and control strains indicate that this mutant allele differentially modulates the levels of ME enzymatic activity and ME-CRM during development. This work was supported by an Operating Grant from the Natural Sciences and Engineering Research Council of Canada to M.M.B.     

A Circadian Clock of Drosophila: Effects of Deuterium Oxide and Mutations at the period Locus

Mutations at the period (per) locus (1:1.3; 3B1-2) in Drosophila melanogaster lengthen (per^L), shorten (per⁵), or abolish (per°) overt circadian rhythmi-city. Deuterium oxide lengthens the free-running circadian period. We tested the effects of deuterium on three mutants of the per gene (per⁵ per^L, and per°) and wild-type Drosophila melanogaster (per⁺) to assess interactions. With increasing concentrations of deuterium, the free-running circadian period of locomotor activity rhythms increased. The dose-response was linear in all genotypes tested. With increasing dosages ofdeuterium, circadian rhythms became weaker as evidenced by the signal-to-noise ratio (SNR). Genotype and deuterium changed circadian period length independently and additively, showing no interaction. SNRs for all genotypes converged on a low level as deuterium concentration increased. Deuterium increased life span, except at high concentrations (40 and 50%).     

Genetic instability in Drosophila melanogaster: Transpositions of the white gene and their role in the phenotypic expression of the zeste gene

Summary Seven independent transpositions of the w ⁺ gene have been recovered as derivatives of two separate direct tandem duplications of the white locus. The transpositions map to discrete sites on both major autosomes. Five transpositions were employed to study the role of w ⁺ gene dos-age on zeste (z) gene expression. Each transposition generates a unique zeste phenotype; one transposition is not predictive for another. A functional allele of zeste, z ^77h, responds to w ⁺ gene dosage contrary to the z response.Supported by NIH grant GM22221     

ENTRAINMENT ELICITS PERIOD AFTEREFFECTS IN NEUROSPORA CRASSA

Circadian clocks continue to oscillate in constant conditions with their own period (τ) and entrain to a cyclic environment by adjusting their intrinsic period to that of the zeitgeber. When circadian clocks are released from entrained to constant conditions, the τ of their initial free-run often depends on the nature of the prior zeitgeber. These postentrainment effects on period (τ-aftereffects) have predominantly been reported for animals but, so far, not fungi. The authors therefore investigated τ aftereffects in the classic circadian model system Neurospora crassa. The standard laboratory strain frq⁺, the short-period mutant frq¹, and the long-period mutant frq⁷ were entrained to 11 different photoperiods in a 24-h day (2–22?h) and to zeitgebers with six different T (16–26?h), and then released to constant darkness. τ-Aftereffects in response to different photoperiods correlated weakly with prior photoperiod in frq⁺ and were unsystematic in both period mutant strains. Strength and direction of the τ-aftereffect in zeitgeber cycles with different T depended on their length and on the strain, showing a negative correlation with zeitgeber length in frq⁺ and positive correlations in frq¹ and frq⁷. It has been proposed that τ-aftereffects are based on interactions of oscillators within a cellular network. The present findings in Neurospora, which grows as a syncytium, suggest that τ-aftereffects also exist in circadian systems based on multioscillatory networks organized at the molecular level. (Author correspondence: roenneberg@lmu.de)     

Development of a homologous transformation system for Aspergillus niger based on the pyrG gene

Summary The development of a homologous transformation system for Aspergillus niger is described. The system is based on the use of an orotidine-5-phosphate decarboxylase deficient mutant (pyrG) and a vector, pAB4-1, which contains the functional A. niger pyrG gene as a selection marker. Transformation of the A. niger pyrG mutant with pAB4-1 resulted in the appearance of stable Pyr⁺ transformants at a frequency of 40 transformants per g of DNA. In 90% of these transformants integration had occurred at the resident pyrG locus, resulting either in replacement of the mutant allele by the wild-type allele (60%) or in insertion of one or two copies of the vector (40%). The A. niger pyrG mutant could also be transformed with the vector pDJB2 containing the pyr4 gene of Neurospora crassa, at a frequency of 2 transformants per g of DNA. Integration at the resident pyrG locus was not found with this vector. The vector pAB4-1 is also capable of transforming an Aspergillus nidulans pyrG mutant to Pyr⁺. The pyrG transformation system was used for the introduction of a non-selectable gene into A. niger.     

Cloning of the lkyB (tolB) gene of Escherichia coli K12 and characterization of its product

Summary The lkyB gene of Escherichia coli K12 has been cloned from the Clarke and Carbon colony bank by selecting a ColE1 plasmid conferring cholic acid resistance to lkyB mutants. The lkyB gene was localized on hybrid plasmid pJC778 by analysis of mutated plasmids generated by Tn5 insertions. Restriction analysis and complementation studies indicated that plasmid pJC778 carried genes nadA, lkyB and sucA which mapped at min 16.5; the lkyB ⁺ allele was dominant over the lkyB207 mutant allele. Analysis of cell envelope proteins from strains carrying plasmids pJC778 (lkyB ⁺), pJC2578 or pJC2579 (lkyB::Tn5), as well as plasmid-coded proteins in a maxicell system, made it likely that the lkyB gene product was a membrane protein of molecular weight 42,000.     

Phenotypic suppression of the Drosophila mitochondrial disease-like mutant tko by duplication of the mutant gene in its natural chromosomal context

A mutation in the Drosophila gene technical knockout (tko^25t), encoding mitoribosomal protein S12, phenocopies human mitochondrial disease. We isolated three spontaneous X-dominant suppressors of tko^25t (designated Weeble), exhibiting almost wild-type phenotype and containing overlapping segmental duplications including the mutant allele, plus a second mitoribosomal protein gene, mRpL14. Ectopic, expressed copies of tko^25t and mRpL14 conferred no phenotypic suppression. When placed over a null allele of tko, Weeble retained the mutant phenotype, even in the presence of additional transgenic copies of tko^25t. Increased mutant gene dosage can thus compensate the mutant phenotype, but only when located in its normal chromosomal context.     

Insights into the Molecular Mechanisms of Temperature Compensation from the Drosophila Period and Timeless Mutants

The relative constancy of the circadian period over a wide range of temperatures is a general property of circadian rhythms. Insights into the molecular mechanisms of temperature compensation are emerging from genetic and molecular genetic studies of the period (per) and timeless (tim) genes in Drosophila. These genes encode proteins that are thought to be part of a negative feedback cycle, which results in circadian oscillations of both per and tim mRNA, as well as a complex of the two proteins. Complex formation is temporally regulated and apparently necessary for nuclear localization of both per and tim proteins. While insights into the roles of per and tim in temperature compensation have been intriguing, they have also been somewhat perplexing. For instance, the interaction of wild-type per peptides is relatively insensitive to temperature in the yeast two-hybrid assay or in assays employing in-vitro-translated peptides, while the interaction of per^L mutant peptides is reduced at a high temperature. Apparently, the per^L mutation increases an intramolecular interaction between different parts of the per peptide in these assays, and this interaction reduces the amount of per homodimer. On the other hand, the same assays show that the intermolecular interaction between the per and tim peptides is reduced at a high temperature by the per^L mutation; this reduction does not require the competing intramolecular interaction. Despite this difference, in all of the experiments employing these assays the per^L mutation has rendered per-per and per-tim peptide interactions sensitive to high temperature, so it is likely that one or both of these reduced interactions contribute to the longer circadian periods at high temperature in per^L mutant flies. However, the tim^SL and per^S mutations, as well as deletion of the Thr-Gly repeats from per, affect temperature compensation but have not been shown to affect these molecular interactions of per and tim. Finally, a recent report of oscillating per and tim proteins in the cytoplasm (rather than the nuclei) of silk moth neurons may suggest an alternative mechanism for per and tim function in these cells. (Chronobiology International 14(5), 455–468, 1997)     

Molecular cloning of the phosphate (inorganic) transport (pit) gene of Escherichia coli K12. Identification of the pit+ gene product and physical mapping of the pit-gor region of the chromosome

Summary The pit ⁺ gene, encoding the phosphate (inorganic) transport system of Escherichia coli, was isolated from a library of E. coli genes inserted in the cosmid vector pHC79. A 25.5-kb chromosomal DNA fragment was shown also to carry the gor locus encoding glutathione oxidoreductase. Physical mapping placed the two genes about 10 kb apart, confirming bacteriophage P1 mapping of the 77-min region. Subcloning and deletion analysis indicated that the entire pit ⁺ gene was located within a 2.2-kb Sal1-Ava1 fragment. The pit ⁺ gene product was identified by SDS-polyacrylamide gel electrophoresis as a 39-kdal inner membrane protein by two methods: (i) ³⁵S-methionine-labelling of minicells carrying pit ⁺ plasmids or plasmids from which all or part of the pit ⁺ gene was deleted. (ii) Overproduction of the Pit protein using a thermoinducible runaway replication plasmid.Complementation of the pit-1 mutant allele using a unit-copy-number pit ⁺ plasmid indicated that the pit-1 mutation is recessive.Strains carrying a multicopy pit ⁺ plasmid show a 10-fold increase in the initial rate of phosphate uptake; however there is no change in the steady-state level of ³²Pi accumulation.Abbreviations kb kilobase-pairs - kdal kilodalton - Pi inorganic phosphate - G3P sn-glycerol-3-phosphate - LB Luria broth - Tc tetracycline - Cm chloramphenicol - Ap ampicillin - UV ultraviolet light - TE 10 mM Tris.HCl, pH 8.0, 1 mM EDTA - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid     

The recessive phenotype of forked can be uncovered by heat shock in Drosophila

Heat shock uncovers the recessive forked phenotype when heterozygotes between f^36a and wild-type are heated during sensitive periods in pupal development. We call the phenocopy of a mutant in such a heterozygote a heterocopy. The heterocopy in f^36a/+ is virtually identical to the mutant phenotype; however, bristles on different parts of the body are affected during different sensitive periods. We discuss the hypothesis that the heat shock acts by affecting expression of the wild-type gene product corresponding to the mutant gene. The sensitive period for heterocopy induction in a specific tissue is proposed to correspond to the normal time of gene expression for the forked gene product in a particular tissue.     

The gigas mutant in pea is deficient in the floral stimulus

Identification of a gene acting in the floral stimulus pathway should provide a basis for determining the identity of this elusive substance. Our tests indicate the Gi (gigas) gene in pea (Pisum sativum L.) acts in this manner. The gigas mutant was selected by Dl M. Vassiteva following gamma radiation of the late flowering, quantitative long day cultivar Virtus. The gigas trait showed single gene recessive inheritance and the mutant allele was symbolised gi consistent with our preliminary report. Gigas plants were later flowering than the initial line in all conditions tested and they showed an enhanced response to photoperiod and vernalisation. Unvernalised gigas plants did not flower under a 24-h photoperiod comprising 8 h of daylight and 16 h of weak (3μmol m^?2 s^?1) incandescent light and they took on a phenotype similar to the vegl (vegetative) mutant in pea. However, genetic tests showed the two mutants were not allelic. Three or four weeks vernalisation at 4^?C resulted in 100% flowering of gigas plants under the 24-h photoperiod. Applied gibberellin A₃ inhibited flowering in gigas plants given partial cold induction. Grafting studies showed the promotive effect of vernalisation occurred in the shoot. Grafting studies were also used to examine the physiological basis of delayed flowering in the gigas mutant. These studies indicated that gigas plants produced normal levels of flower inhibitor and they responded in a normal manner to the floral stimulus, Reciprocal grafts were made between the gigas mutant and the wild-type initial line. Under the 24-h photoperiod, either a wild-type root-stock with cotyledons or a wild-type shoot induced flowering in a gigas graft partner. However, under a 9-h photoperiod, flowering was only induced if the wild-type partner possessed both roots and a shoot. We conclude that gigas plants are deficient in the floral stimulus or a precursor which can be supplied across a graft union by a wild-type donor. Of the 12 major flowering genes known in pea, Gi is the first found to act on the synthesis pathway for the floral stimulus.     

Isolation of Hem3 mutants from Candida albicans by sequential gene disruption

Summary Molecular methods for directed mutagenesis in Candida albicans have relied on a combination of gene disruption by transformation to inactivate one allele and UV-induced mitotic recombination or point mutation to produce lesions in the second allele. An alternate method which uses two sequential gene disruptions was developed and used to construct a C. albicans mutant defective in a gene essential for synthesizing tetrapyrrole (uroporphyrinogen I synthase). The Candida gene was cloned from a random library by complementation of the hem3 mutation in Saccharomyces cerevisiae. The complementing region was limited to a 2.0 kb fragment by subcloning and a BglII site was determined to be within an essential region. Linear fragments containing either the Candida URA3 or LEU2 gene inserted into the BglII site were used to disrupt both alleles of a leu2, ura3 mutant by sequential transformation. Ura⁺, Leu⁺ heme-requiring strains were recovered and identified as hem3 mutants by Southern hybridization, transformation to heme independence by the cloned gene, and enzyme assays.     

The Na+/H+ antiporter of alkaliphilic Bacillus sp.

The Na⁺/H⁺ antiporter, which appears to predominantly contribute to the alkaliphily of Bacillus halodurans C-125, was studied in an alkali-sensitive mutant of this strain and a transformant with restored alkaliphily. The alkali-sensitive mutant, strain 38154, which has lost the ability to grow above pH 9.5, was found to lack electro-genic Na⁺/H⁺ antiport activity driven by ΔΨ (membrane potential, interior negative), and it showed defective regulation of intracellular pH under alkaline conditions. On the other hand, a transformant carrying a 2.0-kb DNA fragment from the parental genome that complemented this defect was able to maintain an intracellular pH lower than that of the external milieu, and it was found to have recovered the Na⁺/H⁺ antiport activity driven by ΔΨ. Sequence analyses found that a 5.1-kb DNA region contained four open reading frames (ORF-1 to ORF-4). Direct sequencing of the corresponding region in mutant 38154 revealed a G-to-A substitution, which resulted in an amino acid substitution from Gly-393 to Arg in the putative ORF-1 product. It has been recently found that a region homologous to the DNA fragment responsible for the alkaliphily of strain C-125 exists in the genomes of Bacillus subtilis, Sinorhizobium (Rhizobium) meliloti, and Staphylococcus aureus. These homologues are present as a cluster of seven ORFs in each case. The shaA gene product of B. subtilis shows significant similarity to the ORF-1 product of strain C-125. Disruption of the shaA gene resulted in a decrease in Na⁺/H⁺ antiport activity, and growth of the shaA-disrupted strain was impaired when the external Na⁺ concentration was increased. We conclude that the shaA gene encodes a Na⁺/H⁺ antiporter, which plays an important role in extrusion of cytotoxic Na⁺. Received: May 29, 2000 / Accepted: July 18, 2000     

Growth response of cotton to CO2 enrichment in differing light environments

Experiments were conducted to examine the growth responses of cotton (Gossypium hirsutum L. cv. Coker 315) to CO₂ enrichment under different light regimes. Plants were exposed to 350 or 700 μl l^?1 CO₂ and six light treatments differing in photosynthetic period length (8 or 16 h) and in photosynthetic photon flux density (PPFD) for 32 days of vegetative growth. Higher PPFD (1 100 μmol m^?2 s^?1) was provided by a combination of high intensity discharge and incandescent lamps (HID), and lower PPFD (550 μmol m^?2 s^?1) was provided by fluorescent and incandescent lamps (F) or HID and incandescent lamps with shade cloth (HID_s). Growth was generally much slower with the 8-h photosynthetic periods, but the growth stimulation by CO₂ enrichment was larger than with 16-h photosynthetic periods. After 28 to 32 days of treatment, the growth enhancement with CO₂ enrichment was 152 and 78% for 8- and 16-h photosynthetic periods, respectively, under HID; 100 and 77% in F, and 77 and 56% in HID_s. The higher PPFD of HID positively influenced the CO₂ effect only at the slower growth rate in the 8-h light period. The stimulation of leaf area expansion by CO₂ enrichment was also greater with the 8-h photosynthetic period for all light sources. These results, and others on net assimilation rate, shoot to root dry weight ratios and specific leaf weights, suggest that the growth response to CO₂ enrichment with the longer photosynthetic period was depressed by limiting factors, perhaps nutritional, in the growth environment. The results also show that extensive variability in CO₂ response can occur under light intensities which are often used in growth chamber experiments.