An overview of the secondary structure of the V4 region of eukaryotic small-subunit ribosomal RNA.

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.

Modification of Blue Light Photoresponses by Riboflavin Analogs in Neurospora crassa.

The effect of riboflavin analogs on blue light responses in a riboflavin mutant of Neurospora crassa was studied. The analogs 1-deazariboflavin and roseoflavin, which have red-shifted absorption, acted as photoreceptors for the photosuppression and phase shifting of circadian conidiation by 540 nm light, but were ineffective as photoreceptors for the induction of carotenoid synthesis. These results provide addtional evidence implicating a flavin photoreceptor for at least two blue light responses of Neurospora.

Isolation and characterization of light-insensitive mutants of Neurospora crassa.

As part of a genetic analysis of blue light photoreception in Neurospora, three mutants were isolated that do not exhibit photosuppression of circadian conidiation, i.e., they show periodic conidiation in constant light. The mutations have been given the designations lis-1, lis-2 and lis-3 ("light insensitive"). The three mutations segregate as single nuclear genes, are nonallelic and are recessive to wild type in heterokaryon tests. The linkage groups of the mutations are as follows: lis-1, I; lis-2, VI; and lis-3, V. The light -insensitive phenotype of the mutants is restricted to the photosuppression response; other responses such as photoinduced phase shifting of the conidiation rhythm and photoinduced carotenogenesis are not altered. The physiological or biochemical defects of the mutants have not been established, but they are not similar to previous reported cases (i.e., rib and poky) in which a reduction in light sensitivity has been observed.

Photoreception in Neurospora crassa: correlation of reduced light sensitivity with flavin deficiency.

The effect of flavin deficiency on blue light responses in Neurospora crassa was studied through the use of two riboflavin mutants (rib-1 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. Flavin deficiency was induced in the rib-1 mutant by restrictive growth temperatures and in the rib-2 mutant by low levels of supplemental riboflavin. At 26 degrees C, a semirestrictive growth temperature, the rib-1 mutant is about 1/80th as sensitive to light for the photosuppression of circadian conidiation. Flavin deficiency in the rib-1 and rib-2 strains was effective in reducing the photosensitivity for phase shifting and carotenogenesis to about 1/16th and 1/4th of normal, respectively. Experiments with permissive temperatures, riboflavin supplementation, and revertants at the rib locus all indicated that the effects on light sensitivity were due solely to the presence of the rib mutations. These results provide evidence that one or more flavin photoreceptors are involved in the blue light responses of Neurospora.

Effects of temperature perturbations on circadian conidiation in neurospora.

Studies on the circadian rhythm of conidiation in the bd strain of Neurospora crassa Shear and Dodge have shown that temperature step-up and step-down perturbations produce phase advances and delays, respectively. Pulse-up and pulse-down treatments lead to both phase advances and delays. The resulting phase shifts can be very large, and few to no transients are observed.Small amplitude temperature cycles are capable of entraining the circadian rhythm, and holding bd at low temperatures appears to stop the circadian oscillator late in the subjective night (circadian time 2200). Aspects of the temperature responses that are somewhat unusual include the high sensivity, the lack of transients, and the phase at which the oscillator stops under low temperatures.

Regulation of the Photosynthesis Rhythm in Euglena gracilis: II. Involvement of Electron Flow through Both Photosystems.

Rhythmic changes in the light reactions of Euglena gracilis have been found which help to explain the basic reactions effected in the circadian rhythm of O(2) evolution. Diurnal changes in the slope of light intensity plots indicated that the maximal rate of photosynthesis changed throughout the circadian cycle. No evidence was obtained consistent with the premise that changes in chlorophyll content, as measured by total chlorophyll or chlorophyll a/b ratio, or photosynthetic unit size are responsible for this rhythim.The rate of light-induced electron flow through the entire electron chain (H(2)O to methyl viologen) was rhythmic both in whole cells and in isolated chloroplasts, and the highest rate of electron flow coincided with the highest rate of O(2) evolution. The individual activities of photosystem I (reduced from 2,6-dichlorophenol-indophenol to methyl viologen) and photosystem II (H(2)O to 2,6-dichlorophenol-indophenol) did not, however, change significantly with time of day, suggesting that the coordination of the two photosystems may be the site of circadian control. Evidence consistent with this concept was obtained from studies of low temperature emission from systems I and II following preillumination with system I or II light.

Regulation of the Photosynthesis Rhythm in Euglena gracilis: I. Carbonic Anhydrase and Glyceraldehyde-3-Phosphate Dehydrogenase Do Not Regulate the Photosynthesis Rhythm.

A circadian rhythm of O(2) evolution has been found in Euglena gracilis, Klebs strain Z. The rhythm persists for at least 5 days in constant dim light and temperature, but damps out in constant bright light. The phase of this rhythm can be shifted by a pulse of bright light and the period length is not changed over a 10 C span of growth temperature.The O(2) evolution rhythm is found in both logarithmic and stationary phase cultures, but CO(2) uptake is clearly rhythmic only in stationary phase cultures.The activity of glyceraldehyde-3-phosphate dehydrogenase was not rhythmic as previously reported (Walther and Edmunds [1973] Plant Physiol. 51: 250-258). Carbonic anhydrase activity was rhythmic when the cultures were maintained under a light-dark cycle with the highest enzyme activity coinciding with the fastest rate of O(2) evolution. However, the rhythm in carbonic anhydrase activity disappeared under constant conditions. Changes in the activities of these two enzymes are therefore not responsible for the rhythmic changes in photosynthetic capacity.

Inactivation of carotenoid-producing and albino strains of Neurospora crassa by visible light, blacklight, and ultraviolet radiation.

Suspensions of Neurospora crassa conidia were inactivated by blacklight (BL) radiation (300 to 425 nm) in the absence of exogenous photosensitizing compounds. Carotenoid-containing wild-type conidia were less sensitive to BL radiation than albino conidia, showing a dose enhancement factor (DEF) of 1.2 for dose levels resulting in less than 10% survival. The same strains were about equally sensitive to shortwave ultraviolet (UV) inactivation. The kinetics of BL inactivation are similar to those of photodynamic inactivation by visible light in the presence of a photosensitizing dye (methylene blue). Only limited inactivation by visible light in the absence of exogenous photosensitizers was observed. BL and UV inactivations are probably caused by different mechanisms since wild-type conidia are only slightly more resistant to BL radiation (DEF = 1.2 at 1.0% survival) than are conidia from a UV-sensitive strain (upr-1, uvs-3). The BL-induced lethal lesions are probably no cyclobutyl pyrimidine dimers since BL-inactivated Haemophilus influenzae transforming deoxyribonucleic acid is not photoreactivated by N. crassa wild-type enzyme extracts, whereas UV-inactivated transforming deoxyribonucleic acid is photoreactivable with this treatment.

An altered invertase in the cot-2 mutant of Neurospora crassa.

Because the cot-2 and inv loci of Neurospora crassa are closely linked, the invertase from the morphological mutant, cot-2, was examined. The cot-2 strains produce an invertase with altered heat sensitivity, Km, and ratio of heavy to light forms. The cellular localization of cot-2 invertase is different from that of the wild type. There were no observable changes in the energy of activation or the pH optimum of cot-2 invertase, and some of the differences detected were not apparent under culture conditions that promoted wild-type growth. Since recombination (about 5 percent) occurred between cot-2 and inv and culture conditions affected the enzyme characteristics, we suggest cot-2 determines, in part, the carbohydrate composition of the enzyme.

Rhythms of enzyme activity associated with circadian conidiation in Neurospora crassa.

The mycelial growth front of the band strain of Neurospora grown on a solid surface exhibits a circadian rhythm of conidiation. Enzyme assays on extracts from that mycelium have shown that the activities of 6 of 13 enzymes (nicotinamide adenine dinucleotide nucleosidase, isocitrate lyase, citrate synthase, glyceraldehydephosphate dehydrogenase, phosphogluconate dehydrogenase, and glucose-6-phosphate dehydrogenase) and soluble-protein content oscillate with the visible morphological change. The rhythmic enzymes associated with the Krebs and glyoxylate cycles are more active during conidiogenesis, whereas the activities of the rhythmic enzymes of glycolysis and the hexose monophosphate shunt are reduced during that phase. The absence of enzyme oscillations in wild-type and fluffy strains which do not form conidia under the conditions employed suggests that the enzyme fluctuations are associated with conidiogenesis itself. Oscillations of enzyme activity as a function of time are restricted to the growth front. A permanent record of rhythmicity associated with conidial and nonconidial regions does, however, exist in the mycelial mat behind the growth front. The activities of three enzymes (nicotinamide adenine dinucleotide nucleosidase, glucose-6-phosphate dehydrogenase, and phosphogluconate dehydrogenase) are not directly influenced by CO(2) concentration, but are correlated with the prescence or absence of conidiation which is controlled by CO(2) concentration. In contrast, citrate synthase and malate dehydrogenase activities are correlated with changes in CO(2) concentration.

Circadian rhythms of nucleic acid metabolism in Neurospora crassa.

Wild-type, band, and fluffy strains of Neurospora crassa exhibit circadian rhythms of ribonucleic acid and deoxyribonucleic acid content in the growth-front hyphae of cultures grown on a solid medium. There is also a rhythm of (3)H-uridine incorporation into the nucleic acids of the band strain. Maximum incorporation precedes the peaks of nucleic acid content which occur during conidiation. As cultures age, ribonucleic acid content decreases rapidly and deoxyribonucleic acid content decreases gradually in standing, shake, and bubble cultures. A reduction of ribonuclease activity with age is also noted in standing and shake cultures. The nucleic acid content, nuclease activity, and changes associated with age vary with the culture conditions.

A Failure to Detect an Influence of Magnetic Fields on the Growth Rate and Circadian Rhythm of Neurospora crassa.

Low strength magnetic fields, 6.36 and 32.25 gauss, were found to have no effect, with one questionable exception, on the circadian rhythm and growth rate of Neurospora crassa. This was true whether the fields were continuous, pulsed 20 minutes daily, or on a 12: 12, on-off cycle.

Effects of medium composition and carbon dioxide on circadian conidiation in neurospora.

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 stimulates conidiation in growth-tube cultures, thereby allowing experiments which demonstrate that nearly all strains and species of Neurospora are capable of circadian conidiation. These results extend to Neurospora the generalization that physiological and developmental regulation in all eukaryotic organisms has a circadian component. Aeration also increases the persistence of circadian conidiation suggesting that the damping of rhythmicity previously observed on certain media represents the cessation of conidiation, rather than the stopping of a timing mechanism.Aeration is apparently effective in that it maintains CO(2) levels in growth-tube cultures below a critical concentration. Carbon dioxide was shown to inhibit conidiation in both wildtype and bd strains, with the latter being about 200 times more resistant than the former.

Genetic determinants of circadian rhythmicity in Neurospora.

Timex, a strain of Neurospora crassa which exhibits a circadian rhythm of conidia formation in growth-tube cultures, has been found to differ from wild-type strains by two genes. One gene, inv, is responsible for an invertase deficiency, whereas the second gene, bd, is of unknown function. Both genes map independently from other genes known to induce Neurospora rhythmicity. The inv gene is not essential for the timex phenotype because bd strains express that phenotype on certain media. Although inv strains do exhibit some rhythmicity of their own, the rhythmicity apparently is not a direct result of the invertase deficiency, since there is no correlation between invertase level and rhymicity in 29 strains tested. Of the 29 strains tested, 20 exhibited some rhythmicity in growth-tube cultures, suggesting that morphological manifestations of rhythmicity in Neurospora may result from the function or the loss of function of numerous genes, or both. There was no correlation in these strains between rhythmicity and (i) genetic background; (ii) geographical origin; or (iii) nutritional requirements.

Gene-enzyme relationships in Neurospora invertase.

A spontaneous, single-gene mutation responsible for a total lack of invertase activity in Neurospora crassa is described. The mutation is believed to lie in the structural gene for invertase, since an immunologically cross-reacting protein is made by the mutant strain. In addition, there was no evidence for a defect in regulation of invertase activity or synthesis by the following criteria. (i) The invertaseless condition was recessive in heterokaryons; (ii) no invertase inhibitor was found in mutant extracts by mixing experiments; and (iii) none of the several sugars able to induce activity in wild-type strains was able to induce activity in the mutant strain. It was also discovered that most of the wild-type enzyme (55 to 75%) cannot be washed free from the rapidly sedimenting cell debris. This finding provided additional support for the hypothesis that Neurospora invertase is located within or about the cell wall.