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J C Dunlap

Publications and source records attributed to J C Dunlap.

At least 55 records · Page 3Linked to original sources
PubMed1994

Negative feedback defining a circadian clock: autoregulation of the clock gene frequency.

The frequency (frq) locus of Neurospora crassa was originally identified in searches for loci encoding components of the circadian clock. The frq gene is now shown to encode a central component in a molecular feedback loop in which the product of frq negatively regulated its own transcript, which resulted in a daily oscillation in the amount of frq transcript. Rhythmic messenger RNA expression was essential for overt rhythmicity in the organism and no amount of constitutive expression rescued normal rhythmicity in frq loss-of-function mutants. Step reductions in the amount of FRQ-encoding transcript set the clock to a specific and predicted phase. These results establish frq as encoding a central component in a circadian oscillator.

Base Sequence↗
PubMed1994

An efficient method for gene disruption in Neurospora crassa.

The frequency with which transforming DNA undergoes homologous recombination at a chromosomal site can be quite low in some fungal systems. In such cases, strategies for gene disruption or gene replacement must either select against ectopic integration events or provide easy screening to identify homologous site, double-crossover insertion events. A protocol is presented for efficient isolation of Neurospora crassa strains carrying a definitive null allele in a target gene. The protocol relies on the presence of a selectable marker flanking a disrupted plasmid-borne copy of the gene, and in the case presented led to a seven-fold enrichment for putative homologous site replacement events. In addition, a polymerase chain reaction assay is utilized for rapid identification of homologous recombinants among the remaining candidates. This protocol was used to identify 3 isolates, out of 129 primary transformants, which have a disruption in the Neurospora ccg-1 gene. The method should be applicable to a variety of fungal systems in which two selectable markers can be expressed, including those in which homologous recombination rates are too low to allow easy identification of homologous site insertions by the more traditional molecular method of Southern analysis. In addition to disrupting target genes for the purpose of generating null mutations, this method is useful for the targeting of reporter gene fusions to a native chromosomal site for the purpose of studying gene regulation.

Base Sequence↗
PubMed1993

The interplay of light and the circadian clock. Independent dual regulation of clock-controlled gene ccg-2(eas).

Ambient light is the major agent mediating entrainment of circadian rhythms and is also a major factor influencing development and morphogenesis. We show that in Neurospora crassa the expression of clock-controlled gene 2 (ccg-2), a gene under the control of the circadian clock and allelic to the developmental gene easy wettable (eas), is regulated by light in wild-type strains. Light elicits a direct and important physiological effect on ccg-2(eas) expression as demonstrated using several mutant Neurospora strains. In white collar mutants (wc-1 and wc-2) that are "blind" to blue light, ccg-2(eas) mRNA shows no variation following illumination with saturating light. By contrast, ccg-2(eas) mRNA is photoinduced in clock-null strains such as frequency (bd;frq). The results in the clock mutants show that an intact circadian oscillator is not required for light induction of ccg-2(eas). Thus, ccg-2(eas) is subject to a dual regulation that involves separable regulation by light and circadian rhythm.

Alleles↗
PubMed1992

The Neurospora circadian clock-controlled gene, ccg-2, is allelic to eas and encodes a fungal hydrophobin required for formation of the conidial rodlet layer.

The Neurospora crassa clock-controlled gene (ccg-2) is transcriptionally activated by the circadian clock in a time-of-day-specific manner. Transcript and sequence analyses of ccg-2 reveal that the predicted Ccg-2 polypeptide bears significant similarity to a class of low-molecular-weight, cysteine-rich, hydrophobic proteins (hydrophobins), first identified in Schizophyllum, and including the product of the developmentally regulated Aspergillus gene, rodletless, required for spore surface rodlets. Allelism between ccg-2 and easily wettable (eas) (one of the first developmental genetic loci identified in Neurospora) was predicted on the basis of this similarity, their close genetic linkage, and previous findings demonstrating that eas mutants lack rodlets. In this study allelism is confirmed experimentally by showing that (1) transformation of an eas mutant strain with ccg-2 DNA results in phenotypic complementation, including restoration of surface rodlets, (2) inactivation of the ccg-2 gene, by RIP, results in an eas phenotype including loss of rodlet fascicles, and (3) the original eas strain has dramatically reduced levels of ccg-2 mRNA. Thus, the clock-controlled ccg-2 gene encodes an integral component of fungal asexual spores important for spore dispersal. The dramatic reduction of ccg-2 expression in the eas mutant has no apparent effect on the normal operation of the circadian clock, confirming that there is no feedback of this clock output on the oscillator itself. These data, in conjunction with the previous observation that ccg-2 is light induced, serve to focus attention on the dual interacting role of light and the circadian clock in the regulation of fungal spore development.

Alleles↗
PubMed1992

Molecular analysis of the Neurospora clock: cloning and characterization of the frequency and period-4 genes.

Genetic analysis of Neurospora crassa has identified many mutants that affect the biological clock. In this article we review the cloning of two of these genes, frq and prd-4. Both genes were isolated using a chromosome walk technique. Subcloning experiments and subsequent Northern analysis of frq implicate the importance of two transcripts that emanate from this locus. In preliminary data, no protein-coding region is evident in the smaller transcript; the larger transcript contains a 962-amino acid open reading frame. The open reading frame shows limited homology to per, a clock gene identified in Drosophila. Sequence analysis of all existing frq alleles suggests that the defect in each case lies within the open reading frame. Successful cloning of the prd-4 gene required walking a distance of greater than 40 kb. A physical map of this region has been constructed using restriction analysis. The dominance-recessive relationship of prd-4 and prd-4+ was established by examining the period lengths of strains harboring a wide range of prd-4/prd-4+ nuclear ratios.

Amino Acid Sequence↗
PubMed1991

Neurospora crassa clock-controlled genes are regulated at the level of transcription.

Although an extensive number of biological processes are under the daily control of the circadian biological clock, little is known about how the clock maintains its regulatory networks within a cell. An important aspect of this temporal control is the daily control of gene expression. Previously we identified two morning-specific genes that are regulated by the clock through daily control of gene expression (J. Loros, S. Denome, and J.C. Dunlap, Science 243:385-388, 1989). We have now introduced a method for transcriptional analysis in Neurospora crassa and used this nuclear run-on procedure to show that regulation of mRNA abundance for these two morning-specific genes occurs at the level of transcription. This transcriptional regulation by the circadian clock provides a basis for isolating circadian rhythm mutants.

Amanitins↗
PubMed1990

Closely watched clocks: molecular analysis of circadian rhythms in Neurospora and Drosophila.

Circadian rhythms represent a type of cellular regulation common to most eukaryotes. Analysis of the genetic basis of this phenomenon is beginning to provide information about how clocks function at the molecular level. Surprisingly, the first two cloned 'clock genes', one from a fruit fly and one from a fungus, share some common characteristics both genetically and in the nature of the proteins they encode. In related work, the recent identification and molecular analysis of clock-controlled genes is revealing how biological clocks control gene expression, and may pave the way for the isolation of novel 'clock genes' in the future.

Animals↗
PubMed1989

The Neurospora clock gene frequency shares a sequence element with the Drosophila clock gene period.

The isolation and characterization of single gene mutations affecting the circadian biological clocks of several organisms has left little doubt that circadian rhythms can be subjected to classical genetical analysis. Many of these mutations occur at the same few genetic loci (frequency (frq) in the fungus Neurospora, and period (per) in fruit fly Drosophila); these loci represent the best studied clock-affecting genes known. Mutant strains are usually affected in more than one basic clock property, suggesting an inter-relatedness at the molecular level among these basic properties that would not have been predicted a priori. The extensive background information available concerning the frq locus provides a basis for the molecular dissection of the Neurospora circadian clock--the most minimal circadian system thus far described. We report here the cloning and analysis of the frq locus and show it to be larger and more complex than would have been predicted from the available genetic data. Complete rescue of all of the pleiotropic mutant phenotypes of the recessive frq allele requires transformation with a 7.7-kilobase (kb) region of DNA encoding at least two transcripts. Sequence analysis of this region has allowed the identification of a common element between frq and per which, given the background similarities in their classical genetic characteristics, suggests the possibility of a common element in the clock mechanisms of these two organisms.

Animals↗
PubMed1989

Molecular cloning of genes under control of the circadian clock in Neurospora.

To investigate the regulation of messenger RNA abundance by circadian clocks, genomic and complementary DNA libraries were screened with complementary DNA probes enriched, by means of sequential rounds of subtractive hybridization, for sequences complementary to transcripts specific to either early morning or early evening cultures of Neurospora. Only two morning-specific genes were identified through this protocol. RNA blot analysis verified that the abundance of the transcripts arising from these genes oscillates with a period of 21.5 hours in a clock wild-type strain and 29 hours in the long-period clock mutant strain frq7. Genetic mapping through the use of restriction fragment length polymorphisms shows the two genes, ccg-1 and ccg-2, to be unlinked. These data provide a view of the extent of clock control of gene expression.

Chromosome Mapping↗
PubMed1988

On the role of protein synthesis in the circadian clock of Neurospora crassa.

Inhibitors of protein synthesis reset the biological clocks of many organisms. This has been interpreted to mean either that the synthesis per se of proteins is a step in the oscillatory feedback loop or merely that certain unstable protein(s) are required at certain times of the cycle to complete the feedback loop. We report here that Neurospora strains bearing the clock mutation frq-7 are relatively insensitive to the resetting action of the protein-synthesis-inhibitor cycloheximide. Protein synthesis itself in this mutant is inhibited by the drug to the same extent as in wild type. Since the clock of frq-7 continues to run relatively unimpeded even in the virtual absence of protein synthesis, it is unlikely that synthesis per se can be a part of the feedback cycle. Rather, we suggest that for normal operation of the Neurospora clock, certain protein(s) with a high turnover rate are required daily and, thus, must be resynthesized each day (at least) during discrete times in the cycle. The frq-7 mutation simultaneously alters several distinct clock characteristics--period length, temperature compensation, and resetting by cycloheximide. A model is presented to unify these observations.

Circadian Rhythm↗
PubMed1982

Inhibitors of protein synthesis on 80S ribosomes phase shift the Gonyaulax clock.

One-hour pulses of anisomycin (0.3 microM), streptimidone (30 microM) and cycloheximide (5 microM) caused strong phase-shifts (either advances or delays, of up to 12 h) in the circadian rhythm of the bioluminescence glow in the marine photosynthetic dinoflagellate, Gonyaulax polyedra. Similar pulses of emetine (0.1-100 microM) caused small (less than 4 h) phase shifts. Drug pulses have quantitatively different effects when applied at different phases of the circadian cycle, thus giving rise to 'phase response curves' (PRC's). The results lend additional support to the generalization, based on results from several different organisms, that 80s ribosome protein synthesizing system is of key importance in the mechanism responsible for circadian rhythms.

Animals↗
PubMed1981

The biological clock in Gonyaulax controls luciferase activity by regulating turnover.

Luciferase activity in cell-free extracts of the bioluminescent marine dinoflagellate Gonyaulax polyedra undergoes a cyclic daily change such that activities of extracts made in the middle of the night phase may be 10 times greater than in extracts of day phase cells. These cyclic changes continue under constant conditions, in a manner indicative of control by an endogenous circadian cellular mechanism. This paper describes the purification and properties of the higher molecular weight unproteolyzed luciferase from both day and night phase cells. Comparisons of the two preparations with respect to several physicochemical, enzymatic, and immunological criteria were made in order to establish the basis for the activity difference; no differences between day and night species were found. A given amount of antiluciferase inactivated the same amount of luciferase activity in both day and night extracts; their specific activities are therefore the same. These data strongly suggest that the luciferase is the same polypeptide in day and night extracts, and that such extracts contain different amounts of the enzyme. We therefore postulate that the circadian rhythm of luciferase activity is a result of biological clock-controlled synthesis and/or degradation of the luciferase polypeptide.

Amino Acids↗
PubMed1981

Biochemistry of dinoflagellate bioluminescence: purification and characterization of dinoflagellate luciferin from Pyrocystis lunula.

Bioluminescence in all dinoflagellate species studied to date is produced by the luciferase-catalyzed oxidation of a newly elucidated type of luciferin, hypothesized to have a substituted polypyrrole-type structure. This paper presents the purification and characterization of the luciferin from Pyrocystis lunula along with evidence that it is a polypyrrole-type molecule. Luciferin is extremely labile at low pH, at high salt concentration, and to O2, so, where possible, the purification steps were carried out in the presence of a buffered reducing agent and under argon. Purified luciferin is soluble in water and polar organic solvents. It is yellow (lambda max 245 and 390 nm with a shoulder at 290 nm in neutral or basic aqueous solution) and displays a strong blue fluorescence (lambda max for excitation at 390 nm, for emission at 474 nm) that closely matches the bioluminescence emission spectrum [Bode, V. C., & Hastings, J. W. (1963) Arch. Biochem. Biophys. 103, 488--499]. Autoxidation leads to concomitant decreases in the 390-nm absorbance, 474-nm fluorescence, and biological activity; similar changes occurred with oxidation by K3Fe(CN)6, thus allowing a quantitation of luciferin by titration. Luciferin has a molecular weight between 500 and 600, displays positive Ehrlich and Schlesinger reactions, and yields on acid chromate oxidation fragments apparently resembling substituted maleimides; these data support the proposal that dinoflagellate luciferin contains a substituted polypyrrole of the bile pigment type.

Animals↗
PubMed1980

Crossreactivity between the light-emitting systems of distantly related organisms: Novel type of light-emitting compound.

Dinoflagellate luciferin has been found to crossreact and emit light with euphausid photoprotein; and euphausid fluorescent substance gives luminescence with dinoflagellate luciferase. Luciferin and the fluorescent substance, both highly unstable and fluorescent compounds, are biochemically similar but not identical. Preliminary spectral and chemical data suggest that both compounds contain an open-chain polypyrrole structure, novel among compounds so far known to be involved in light emission in any biological system. The dinoflagellates and euphausids are phylogenetically distant; the possibility that the latter obtain the molecule nutritionally from the former is suggested.

Journal Article↗
PubMed1977

Comparison of the biosynthetic and biodegradative ornithine decarboxylases of Escherichia coli.

Biosynthetic ornithine decarboxylase was purified 4300-fold from Escherichia coli to a purity of approximately 85% as judged by polyacrylamide gel electrophoresis. The enzyme showed hyperbolic kinetics with a Km of 5.6 mM for ornithine and 1.0 micronM for pyridoxal phosphate and it was competitively inhibited by putrescine and spermidine. The biosynthetic decarboxylase was compared with the biodegradative ornithine decarboxylase [Applebaum, D., et al. (1975), Biochemistry 14, 3675]. Both enzymes were dimers of 80 000-82 000 molecular weight and exhibited similar kinetic properties. However, they differed significantly in other respects. The pH optimum of the biosynthetic enzyme was 8.1, compared with 6.9 for the biodegradative. Both enzymes were activated by nucleotides, but with different specificity. Antibody to the purified biodegradative ornithine decarboxylase did not cross-react with the biosynthetic enzyme. The evolutionary relationship of these two decarboxylases to the other amino acid decarboxylases of E. coli is discussed.

Carboxy-Lyases↗