Summtary. A new strain of Neuirospora crassa which exhibits a rhythm of conidiation when growing along an agar surface in a growth ttube is described. The rhythm has been shown to be circadian for it meets the following criteria: A) the period under constant environmental conditions in the dark is abolut 24 hours (22.7 hours at 250) ; B) the period is relativcly temperatuire-independent (Q, We have developed a system for the biochemical stuldy of a circadian rhythm employing a new strain of Neurospora crassa (timex)2 which satisfies both criteria mentioned above. Pittendrigh et al. (16) were the first to point ouit the advantages of working with Neutrospora, and Sussman, Dturkee, and Lowry (21), Berliner and Neuirath (4), and Bianchi (5) have uised Neuirospora to stuidy the rhythm expressed in the clock strains described by Sussman, Lowry, and Duirkee (11,22
liglit slhocks could shift the phase of the rhthlml Additional stu(iies oni tile effects of light have b)een purstied xvith the hope that the photor-eceptor could be identified and that tlie illechanislubs of the interaction betwveen the lighit receptor and tile system respoilsible for the circadian rhlythin in timex could be elucidated.Tlle advantages of this approacil were realized lollg ago and several inlvestigators ihave dleterm-iinled actioin spectra for the sliifting of plhase 1h lighlt in atteillpts to identi fy the pliotoreceptor llvolved. Elret (6) -hloxved tllat tiltravio-'et lig-lt -was effective ill slliftinll the plhase of tile niating-reactioll rx thlIll ill Para,wecillmz and tilat white light w as effective in phnotoreversal of tile phase slhifts. Tlie actioil spectrtllll for resetting the Gommaulax hi micscunce nilvtilI (7 )
Efforts to significantly perturb the timing mechanism, and thus the period, of the rhythm responsible for circadian conidiation in bd, a strain of Neurospora crassa, by altering the medium composition have been unsuccessful. Various salt solutions, sugars, and amino acids do, however, have pronounced effects on growth and conidiation, and thus on the expression and persistence of rhythmicity.Aeration August, 1970. perturb the postulated timing mechanism is to vary the growth medium. In general, perturbing the metabolism of an organism has little effect on its circadian rhythmicity (9), but changes in rhythm expression and period have been demonstrated in certain strains of Neurospora and other fungi by supplementing the growth medium with appropriate sugars and amino acids (3, 5, 10).The work reported here was initiated to determine if varying the growth medium would provide useful information about the mechanisms responsible for the bd rhythmicity. The experiments provide a better understanding of the band-gene role in Neurospora rhythmicity, and suggest that aeration is an important variable in the expression of Neurospora rhythmicity. The effects of aeration provide new insight into the occurrence of rhythmicity in Neurospora and the role of carbon dioxide in regulating conidiation of Neurospora cultures. MATERIALS AND METHODSStrains. A single-mutant strain, band (bd), which exhibits the timex phenotype on our standard glucose-arginine medium (16) was used throughout. This strain, MLS 41-4 (bd, a), was isolated after three backcrosses of the original bd (E7a) into the St. Lawrence wild-type, 74-OR 23-1A. The sources of the other wild-type and exotic strains used have been reported, as have the methods of stock maintenance and preservation (18).Media. The media contained Vogel's salts (22), 1.5% Difco Bacto-agar (unless otherwise stated), and the supplements listed. All percentage designations for media are weight per volume. The media were prepared, autoclaved, and dispensed (8 ml/tube) into 55-cm Pyrex growth-tubes stoppered with cotton. Heat-labile amino acids were filter sterilized and added to warm medium (45-50 C) before dispensing.Growth Conditions. Growth-tube cultures were inoculated, incubated 18 to 20 hr at room temperature in the light, and then placed in continuous darkness at 25 C. Scoring of rhythmicity and period calculations have been described previously (16, 18
The V4 region of the small subunit (18S) ribosomal RNA was examined in 72 different sequences representing a broad sample eukaryotic diversity. This domain is the most variable region of the 18S rRNA molecule and ranges in length from ca. 230 to over 500 bases. Based upon comparative analysis, secondary structural models were constructed for all sequences and the resulting generalized model shows that most organisms possess seven helices for this region. The protists and two insects show from one to as many as four helices in addition to the above seven. In this report, we summarize secondary structure information presented elsewhere for the V4 region, describe the general features for helical and apical regions, and identify signature sequences useful in helix identification. Our model generally agrees with other current concepts; however, we propose modifications or alternative structures for the start of the V4 region, the large protist inserts, and the sector that may possibly contain a pseudoknot.
The effect of flavin deficiency on blue light responses in Neurospora crassa was studied through the use of two riboflavin mutants (rib-i and rib-2). The photoresponses assayed were the, suppression of circadian conidiation, the phase shifting of the circadian conidiation rhythm, and the induction of carotenoid synthesis. Physiological responses to blue light are known in a large number oforganisms. Examples include phototropism in coleoptiles of monocots (1) and sporangiophores of fungi (2), induction of carotenoid synthesis in fungi (3), and entrainment of circadian pupal emergence in fruit flies (4). The photoreceptor pigments involved remain unidentified but most ofthe available evidence supports the hypothesis that a flavin is acting as the photoreceptor for many of these blue light responses. This evidence (reviewed in refs. 5 and 6) includes (i) the similarity between the absorption spectrum of riboflavin and the action spectra of both the blue light responses and light-induced absorbance changes involving a b-type cytochrome and (ii) the effects on blue light responses of inhibitors such as phenylacetic acid and potassium iodide which interact with flavins.Spectrophotometric studies with whole cells and cell fractions suggest that a primary photoreception event for the fungus Neurospora crassa is a flavin-mediated reduction of a b-type cytochrome (7,8). Experiments supporting cytochrome b involvement have been done with a cytoplasmic mutant, poky, which exhibits a low.cytochrome b content, a decrease in blue light-induced absorbance changes, and a decreased sensitivity to light for the suppression of circadian conidiation (9, 10).One approach to elucidation ofthe nature ofa photoreceptor is to use a system in which the photoreceptor levels can be perturbed. Ifthe photoreceptor is indeed a flavin, then a decrease in photoreceptor levels should occur in the case ofa generalized flavin deficiency and might result in a decrease of light sensitivity. To detect this effect, one needs a system having a blue light response that can be monitored quantitatively and in which a flavin deficiency can be specifically induced. Riboflavin-requiring mutants have been isolated in Neurospora (11,12)
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