Genes expressed during conidiation in Neurospora crassa: molecular characterization of con-13

Asexual development in Neurospora crassa proceeds through a series of discrete morphological stages that culminate in the production of dormant spores called conidia. Changes in the pattern of gene expression parallel the morphological transformations associated with conidiation. As a prerequisite to the analysis of developmental gene expression in N. crassa, several genes of unknown function that are preferentially expressed during conidiation were isolated [Berlin and Yanofsky, Mol. Cell. Biol. 5 (1985) 849-855]. The molecular structure and nucleotide sequence of one of these genes, designated con-13, is presented. The con-13 gene specifies a relatively rare 1.35-kb message which is first detected about 8 h following the induction of conidiation. Sequence analysis of both cDNA and genomic clones indicates that the con-13 gene consists of three exons divided by two small introns. It encodes a polypeptide of 340 amino acid residues (37.1 kDa). The Con-13 protein is weakly acidic and hydrophilic. A comparison of the regions upstream from the con-8, con-10, and con-13 genes revealed several short sequence motifs which may be important in developmental gene regulation.     

Temperature-shift analysis of conidial development in Aspergillus nidulans

Temperature-shift experiments have been performed on spore-originated colonies of 11 thermosensitive aconidial mutants of Aspergillus nidulans in order to determine the latest time of shift to the restrictive temperature that prevents the initiation of conidiation. This time defines the beginning of the thermosensitive period (TSP) of the mutant. Eight of the mutants have TSPs that begin in the 7-hour period (32–39 hr) just prior to the first appearance of conidia-bearing structures, while 3 of the mutants have TSPs that begin later and very close to the time of onset of conidiation (45 hr). Thus no mutant of the set has a TSP that begins during the first 32 hr of vegetative growth of spore-originated colonies. For all mutants, an upshift performed after the beginning of the TSP allows initiation of conidiation at close to the normal time and at the normal rate, but results in an abrupt cessation of conidiation at some fixed time after upshift, characteristic of the mutant. The mutant whose TSP begins the earliest (aco-49) is exceptional in that, if conidiation is suppressed by growth of colonies in submerged culture, this mutant becomes thermoinsensitive during vegetative submerged growth; in contrast, the remaining 10 mutants become thermoinsensitive only after the suppressive condition has been relieved. We discuss the possibility that this exceptional mutant is defective in a function required for initiation of the process that ultimately results in the formation of conidia.     

Identification of genes up-regulated during conidiation of Fusarium oxysporum through expressed sequence tag analysis

Fusarium oxysporum produces three kinds of asexual spores, microconidia, macroconidia, and chlamydospores. F. oxysporum produces microconidia and macroconidia in carboxymethyl cellulose-added liquid medium (CMCLM) and exhibits vegetative growth without conidiation in complete liquid medium (CLM). The cDNA libraries were constructed using mRNAs from CLM and CMCLM cultures. A total of 1288 and 1353 clones from CLM (vegetative growth) and CMCLM (conidiation) libraries, respectively, were sequenced, and 641 and 626 unique genes were identified. Of these unique genes, only 130 ( approximately 20%) were common in the two libraries, indicating different patterns of gene expression during vegetative growth and conidiation. The expression levels of 496 CMCLM-specific genes were compared during vegetative growth and conidiation by cDNA dot-blot differential hybridization and real-time quantitative PCR analyses, and 42 genes were identified to display >5-fold increases in mRNA abundance during conidiation. These genes provide ideal candidates for further studies directed at understanding fungal conidiogenesis and its molecular regulation.     

Synthesis of the major adult cuticle proteins of Drosophila melanogaster during hypoderm differentiation

Differentiating imaginal hypodermal cells of Drosophila melanogaster form adult cuticle during the second half of the pupal stage (about 40 to 93 hr postpupariation). A group of proteins with molecular weights of 23,000, 20,000, and 14,000 is identified as putative major wing cuticle proteins with the following biological properties: These proteins are abundant components of cuticle and are major synthetic products of cuticle-secreting hypodermal cells. They are leucine-rich and methionine-free and are the most prominent proteins of this type synthesized by wing hypoderm at 65 hr, during the period of procuticle formation. Electron microscopic autoradiography shows that leucine-rich, methionine-free proteins specifically localize to the apical cell surface and newly secreted cuticle of 65-hr wing cells. This strongly suggests the export of these proteins to the cuticle. Lastly, these proteins undergo a reduction in extractability just after eclosion, during the period of cuticle protein crosslinking (sclerotization). The synthesis of these major hypoderm proteins is temporally regulated in development. In wing cells, the 14-kDa proteins are synthesized first, from 53 to 78 hr, and the 20- and 23-kDa proteins are synthesized from 63 to 93 hr. The pattern of synthesis for these proteins is similar in abdominal cells but delayed by 6 to 10 hr. Two-dimensional gel electrophoresis shows that each of the 23-, 20-, and 14-kDa size classes contains at least two component polypeptides. Patterns of protein synthesis in cells of the imaginal hypodermis are regulated in a precise temporal sequence during the production of adult cuticle. Their study yields a useful system for the analysis of molecular events in gene control and cell differentiation.     

Mitomycin C induced leaf mosaicism in Glycine max (L.) Merrill in relation to the post-germination age of the seed

Summary The frequency of mitomycin C induced somatic crossing over in variety L65-1237 of Glycine max is shown to be dependent upon the (physiological) age of the seed during post germination period. Effect of mitomycin C during the first four hr of germination is significantly lower than during later periods. This increase in the frequency of somatic crossing over is observed up to about 20–24 hr and is then followed by a decrease. These changes did not appear to be related to the onset and pattern of synthesis of DNA or/and proteins in the embryonic tissues. However, mitomycin C is effective even when no DNA synthesis is going on.     

Study of newly synthesized proteins during bovine oocyte maturation in vitro using image analysis of two-dimensional gel electrophoresis

To investigate protein synthesis during bovine oocyte maturation in vitro, oocytes were put in culture with 35S-methionine for 4 hr periods from time zero to 28 hr. Pools of 10 oocytes were then prepared for two-dimensional gel electrophoresis (2-DE). For each time interval, three gels were obtained, digitalized, and analyzed to detect proteins. Then, the gel containing the most proteins was chosen as the reference gel and compared with the others. An averaged gel was created with proteins present in at least two gels of the three. Our results indicate that the rate of protein synthesis is higher at the beginning of maturation until the appearance of metaphase I (MI, 8-12 hr) and then it decreases and stays relatively constant. Percentages of initial proteins (0-4 hr) and remaining present during the progression decrease progressively from 100% to 53%. In contrast, when we compare proteins synthesized from the 4 to 8 hr period with proteins from the 8 to 12 or the 12 to 16 hr intervals, percentages of overall protein matching are stable with values of 81 and 79%, respectively. Comparison of proteins from 20 to 24 hr with proteins from 16 to 20 or 24 to 28 hr intervals also gives stable percentages of overall protein matching with values of 83 and 84%, respectively. Furthermore, a higher number of new proteins is observed at 4-8 hr (n=130) and 16-20 hr (n=136) of maturation. Thus, three major patterns of protein synthesis were observed during bovine oocyte maturation in vitro: one at the beginning of maturation (0-4 hr), another one in the middle (4-16 hr), and the last one after the completion of MI stage (16-28 hr).     

Mutation of the Cys-9 Gene, Which Encodes Thioredoxin Reductase, Affects the Circadian Conidiation Rhythm in Neurospora Crassa

Ten cysteine auxotrophs of Neurospora crassa were examined with regard to the period lengths of their circadian conidiation rhythms. One of the these cysteine auxotrophs, cys-9, showed dramatic changes in the circadian conidiation rhythm. At 10 μM methionine, the cys-9 mutant had a period length that was 5 hr shorter than that of the wild-type strain during the first 3 days after transfer to continuous darkness. At this concentration of methionine, the period length was unstable after the fourth day and varied widely from 11 to 31 hr. In contrast, other cysteine auxotrophs did not show such instability of the period length at any of the concentrations of methionine tested. Furthermore, only the cys-9 mutant exhibited partial loss of the capacity for temperature compensation of the period length. With regard to cold-induced phase-shifting of the circadian conidiation rhythm, the cys-9 mutant was more sensitive than the wild-type strain to low temperature. The cys-9(+) gene was cloned and was found to encode NADPH-dependent thioredoxin reductase. These results indicate that mutation of the gene for thioredoxin reductase results in abnormal expression of the circadian conidiation rhythm in N. crassa.     

Temporal distinctions in the synthesis and accumulation of proteins by oocytes and cumulus cells during maturation in vitro of bovine oocytes

Successful in vitro maturation (IVM) of bovine oocytes requires continual and/or episodic protein synthesis by cumulus-oocyte complexes. This study was designed to expose time-dependent changes in protein synthesis and accumulation by bovine oocytes and cumulus cells during routine IVM. Silver staining after sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated little if any change in protein species present or their relative contents in oocytes during IVM; one notable exception, however, was the gradual accumulation of a 39-kDa polypeptide between 4–24 hr of maturation culture. Cumulus cells, on the other hand, exhibited no qualitative differences during the period examined, but total protein content did increase during IVM. Metabolic labeling with [³⁵S]-methionine, however, demonstrated changes in protein synthesis, both quantitative and qualitative, by both cell types. Oocytes exhibited a steady or slightly increasing rate of synthesis during the first 12 hr of IVM; thereafter, protein synthesis declined to about 10% of the initial rate by 40 hr in culture. In contrast, protein synthesis in cumulus cells was relatively constant during the first 24 hr. Of greater interest is the demonstration that the synthesis of at least seven oocyte-specific and five cumulus-specific proteins was stage-dependent during maturation. These results indicate that maturation of bovine oocytes is associated with the synthesis of several distinct and temporally expressed proteins which may play roles in the highly ordered sequence of events that culminates in oocyte maturation. © 1996 Wiley-Liss, Inc.     

Program of early development in the mammal: changes in patterns and absolute rates of tubulin and total protein synthesis during oogenesis and early embryogenesis in the mouse.

The absolute rates of total protein synthesis and tubulin synthesis during oogenesis and early embryogenesis in the mouse have been determined by measuring specific activities of the endogenous methionine pool and rates of incorporation of [³⁵S]methionine into total protein and tubulin. The absolute rate of protein synthesis decreases from 43 to 33 pg/hr/oocyte during meiotic maturation, while the size of the endogenous methionine pool remains essentially unchanged at 65 fmole/oocyte (R. M. Schultz, M. J. LaMarca, and P. M. Wassarman, 1978, Proc. Nat. Acad. Sci. USA,75, 4160). The one-cell mouse embryo synthesizes protein at a rate of 45 pg/hr/embryo, so that fertilization is accompanied by about a 40% increase in the absolute rate of total protein synthesis. The eight-cell compacted embryo synthesizes protein at the rate of 51 pg/hr/embryo. The size of the endogenous methionine pool increases dramatically during early embryogenesis, from 74 fmole in the unfertilized ovum to 137 and 222 fmole in the one-cell embryo and eight-cell compacted embryo, respectively. Tubulin is one of the major proteins synthesized by the mouse oocyte and embryo since the absolute rate of tubulin synthesis is, on the average, 1.3% that of total protein synthesis. The absolute rate of tubulin synthesis decreases from 0.61 to 0.36 pg/hr/oocyte during meiotic maturation and then increases to 0.60 pg/hr/embryo in the one-cell embryo and to 0.66 pg/hr/embryo in the eight-cell compacted embryo. During meiotic maturation and early embryogenesis the direction and magnitude of changes in the rate of tubulin synthesis closely parallel those of total protein synthesis. Although equimolar amounts of tubulin subunits are present in microtubules, the ratio of the absolute rate of synthesis of the β subunit to that of the α subunit is about 2.0 throughout meiotic maturation and early embryogenesis.High-resolution two-dimensional gel electrophoretic analysis of [³⁵S]methionine-labeled proteins reveals that many of the newly synthesized proteins that first appear during meiotic maturation of the oocyte continue to be synthesized in the one-cell embryo. Nearly all of the proteins synthesized in the one-cell embryo are also synthesized in the unfertilized ovum, although some changes in the pattern of protein synthesis are associated with fertilization. Therefore, the developmental program for early embryogenesis in the mouse appears to be activated during meiotic maturation of the oocyte. These results are compared with those obtained using oocytes and embryos from nonmammalian animal species.     

Developmental changes in messenger RNAs and protein synthesis in Dictyostelium discoideum

The pattern of proteins synthesized at different stages of differentiation of the slime mold Dictyostelium discoideum was studied by two-dimensional polyacrylamide gel electrophoresis. Of the approximately 400 proteins detected during growth and/or development, synthesis of most continued throughout differentiation. Approximately 100 proteins show changes in their relative rates of synthesis. During the transition from growth to interphase, the major change observed is reduction in the relative rate of synthesis of about 8 proteins. Few further changes are noticeable until the stage of late cell aggregation, when production of about 40 new proteins begins and synthesis of about 10 is reduced considerably. Thereafter, there are few changes in the pattern of protein synthesis. Major changes in the relative rates of synthesis of a number of proteins are found during culmination, but few culmination-specific proteins are observed. In an attempt to understand the molecular basis for these changes, mRNA was isolated from different stages of differentiation and translated in an improved wheat germ cell-free system; the products were resolved on two-dimensional gels. The ratio of total translatable mRNA to total cellular RNA is constant throughout growth and differentiation. Messenger RNAs for many, but not all, developmentally regulated proteins can be identified by translation in cell-free systems. Actin is the major protein synthesized by vegetative cells and by early differentiating cells. The threefold increase in the relative rate of synthesis of actin during the first 2 hr of differentiation and the decrease which occurs thereafter can be accounted for by parallel changes in the amount of translatable actin mRNA. Most of the changes in the pattern of protein synthesis which occur during the late aggregation and culmination stages can also be accounted for by parallel increases or decreases in the amounts of translatable mRNAs encoding these proteins. It is concluded that mRNAs do not appear in a translatable form before synthesis of the homologous protein begins, and that regulation of protein synthesis during development is primarily at the levels of production or destruction of mRNA.     

Regulation and Self-Inhibition of Microsporum gypseum Macroconidia Germination

Germination of Microsporum gypseum macroconidia was accompanied by the release of alkaline protease, calcium ions, and inorganic phosphate into the germination fluid. The rate of germination was greatest during the first 2 hr, decreasing thereafter. This decrease in rate was accompanied by a decrease in protease activity, which was caused by an interaction of the enzyme with the inorganic phosphate released from the spores and accumulated in the germination medium after 2 hr. Germination of high spore densities was regulated by the ratio of released phosphate to protease protein, resulting in a constant percentage of germination at both high and low spore densities. A germination-defective mutant strain failed to germinate normally and released excessively high concentrations of phosphate into the germination medium during the initial 2 hr of incubation. Addition of calcium ions to germination mutant macroconidia stabilized spore morphology, prevented protease inactivation, and allowed normal germ-tube outgrowth. The germination of macroconidia appears to be regulated by the release of phosphate ions, which then inhibit the alkaline protease.     

Changing rates of histone mRNA synthesis and turnover in Drosophila embryos

The rates of synthesis and turnover of histone mRNA in Drosophila embryos were determined by hybridization of in vivo and in vitro labeled embryonic RNA to Drosophila histone DNA of the recombinant plasmid cDm500. There is a large store of maternal histone mRNA, equivalent to at least 7 X 10(7) copies of each of the five classes of histone mRNA per embryo. Embryonic synthesis of histone mRNA begins at 90 min after oviposition, making the histone genes among the first to be transcribed by embryonic nuclei. Embryonic histone mRNA accumulates rapidly during the blastoderm and gastrula stages. The peak in the rate of histone mRNA synthesis per embryo coincides with the peak in the rate of DNA synthesis per embryo, which occurs at 6 hr after oviposition. After 6 hr, as the rate of DNA synthesis per embryo decreases, the rate of histone mRNA synthesis and the total mass of histone mRNA per embryo both drop sharply. The rate of histone mRNA synthesis per gene falls more than 60 fold in the first 13 hr after oviposition, from 1.3 -2.5 copies per gene-min at 2 hr to 0.02-0.03 copies per gene-min at 13 hr. From measurements of the mass of histone mRNA per embryo and of the rate of accumulation of newly synthesized histone mRNA at a number of stages of early embryogenesis we determined that the cytoplasmic half-life of histone mRNA decreases approximately 7 fold during early Drosophila development, from 2.3 hr at blastoderm to 20 min by the end of gastrulation. Thus the level of expression of histone genes in Drosophila development is controlled not only by the size of the maternal mRNA pool and changes in the rate of histone mRNA synthesis, but also by changes in the rate of histone mRNA turnover.     

The fluffy gene of Neurospora crassa is necessary and sufficient to induce conidiophore development

The fl (fluffy) gene of Neurospora crassa encodes a binuclear zinc cluster protein that regulates the production of asexual spores called macroconidia. Two other genes, acon-2 and acon-3, play major roles in controlling development. fl is induced specifically in differentiating tissue during conidiation and acon-2 plays a role in this induction. We examined the function of fl by manipulating its level of expression in wild-type and developmental mutant strains. Increasing expression of fl from a heterologous promoter in a wild-type genetic background is sufficient to induce conidiophore development. Elevated expression of fl leads to induction of development of the acon-2 mutant in nitrogen-starved cultures, but does not bypass the conidiation defect of the acon-3 mutant. These findings indicate that fl acts downstream of acon-2 and upstream of acon-3 in regulating gene expression during development. The eas, con-6, and con-10 genes are induced at different times during development. Morphological changes induced by artificially elevated fl expression in the absence of environmental cues were correlated with increased expression of eas, but not con-6 or con-10. Thus, although inappropriate expression of fl in vegetative hyphae is sufficient to induce conidial morphogenesis, complete reconstitution of development leading to the formation of mature conidia may require environmental signals to regulate fl activity and/or appropriate induction of fl expression in the developing conidiophore.     

EFFECTS OF CHLORAMPHENICOL ON CHLOROPLAST AND MITOCHONDRIAL ULTRASTRUCTURE IN OCHROMONAS DANICA

The effect of chloramphenicol (CAP) on cell division and organelle ultrastructure was studied during light-induced chloroplast development in the Chrysophyte alga, Ochromonas danica. Since the growth rate of the CAP-treated cells is the same as that of the control cells for the first 12 hr in the light, CAP is presumed to be acting during that interval solely by inhibiting protein synthesis on chloroplast and mitochondrial ribosomes. CAP markedly inhibits chloroplast growth and differentiation. During the first 12 hr in the light, chlorophyll synthesis is inhibited by 93%, the formation of new thylakoid membranes is reduced by 91%, and the synthesis of chloroplast ribosomes is inhibited by 81%. Other chloroplast-associated abnormalities which occur during the first 12 hr and become more pronounced with extended CAP treatment are the presence of prolamellar bodies and of abnormal stacks of thylakoids, the proliferation of the perinuclear reticulum, and the accumulation of dense granular material between the chloroplast envelope and the chloroplast endoplasmic reticulum. CAP also causes a progressive loss of the mitochondrial cristae, which is paralleled by a decline in the growth rate of the cells, but it has no effect on the synthesis of mitochondrial ribosomes. We postulate that one or more chloroplast ribosomal proteins are synthesized on chloroplast ribosomes, whereas mitochondrial ribosomal proteins are synthesized on cytoplasmic ribosomes.     

Effect of cycloheximide or puromycin on induction of thermotolerance by sodium arsenite in Chinese hamster ovary cells: involvement of heat shock proteins

After sodium arsenite (100 microM) treatment, the synthesis of three major heat shock protein families (HSPs; Mr = 110,000, 87,000, and 70,000), as studied with one-dimensional gels, was enhanced twofold relative to that of unheated cells. The increase of unique HSPs, if studied with two-dimensional gels, would probably be much greater. In parallel, thermotolerance was observed as a 100,000-fold increase in survival from 10(-6) to 10(-1) after 4 hr at 43 degrees C, and as a thermotolerance ratio (TTR) of 2-3 at 10(-3) isosurvival for heating at 45.5 degrees C. Cycloheximide (CHM: 10 micrograms/ml) or puromycin (PUR: 100 micrograms/ml), which inhibited total protein synthesis and HSP synthesis by 95%, completely suppressed the development of thermotolerance when either drug was added after sodium arsenite treatment and removed prior to the subsequent heat treatment. Therefore, thermotolerance induced by arsenite treatment correlated with an increase in newly synthesized HSPs. However, with or without arsenite treatment, CHM or PUR added 2-6 hr before heating and left on during heating caused a 10,000-100,000-fold enhancement of survival when cells were heated at 43 degrees C for 4 hr, even though very little synthesis of heat shock proteins occurred. Moreover, these cells manifesting resistance to heating at 43 degrees C after CHM treatment were much different than those manifesting resistance to 43 degrees C after arsenite treatment. Arsenite-treated cells showed a great deal of thermotolerance (TTR of about 10) when they were heated at 45 degrees C after 5 hr of heating at 43 degrees C, compared with less thermotolerance (TTR of about 2) for the CHM-treated cells heated at 45 degrees C after 5 hr of heating at 43 degrees C. Therefore, there are two different phenomena. The first is thermotolerance after arsenite treatment (observed at 43 degrees C or 45.5 degrees C) that apparently requires synthesis of HSPs. The second is resistance to heat after CHM or PUR treatment before and during heating (observed at 43 degrees C with little resistance at 45.5 degrees C) that apparently does not require synthesis of HSPs. This phenomenon not requiring the synthesis of HSPs also was observed by the large increase in thermotolerance to 45 degrees C caused by heating at 43 degrees C, with or without CHM, after cells were incubated for 6 hr following arsenite pretreatment. For both phenomena, a model based on synthesis and redistribution of HSPs is presented.