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Germination conditions that require mitochondrial function in Saccharomyces cerevisiae: utilization of acetate and galactose.

Ascospores of Saccharomyces cerevisiae inherited at least one functioning mitochondrion as shown by their ability to germinate on nonfermentable carbon sources. After transfer to germination medium, the optical density of the culture at 600 nm decreased (phase-dark), reaching a minimum within 60 min in the presence of glucose and within 180 min after transfer to acetate medium; thereafter, the optical density increased. Budding cells first appeared 90 min after transfer to glucose and 150 min after transfer to acetate. Augmentation of respiratory components, respiratory activity, and macromolecular synthesis (except for DNA synthesis) started at about the same time on glucose and on acetate, although the highest values for all these processes were reached in the presence of glucose. Mitochondrial inhibitors which affected germination on acetate did not arrest germination on glucose. However, mitochondrial activity was required for germination on galactose in a strain carrying the mutated allele imp1 of the nucleomitochondrion-connecting gene IMP1.

Acetates↗

Induction of Ca2+-calmodulin signaling by hard-surface contact primes Colletotrichum gloeosporioides conidia to germinate and form appressoria.

Hard-surface contact primes the conidia of Colletotrichum gloeosporioides to respond to plant surface waxes and a fruit-ripening hormone, ethylene, to germinate and form the appressoria required for infection of the host. Our efforts to elucidate the molecular events in the early phase of the hard-surface contact found that EGTA (5 mM) and U73122 (16 nM), an inhibitor of phospholipase C, inhibited (50%) germination and appressorium formation. Measurements of calmodulin (CaM) transcripts with a CaM cDNA we cloned from C. gloeosporioides showed that CaM was induced by hard-surface contact maximally at 2 h and then declined; ethephon enhanced this induction. The CaM antagonist, compound 48/80, completely inhibited conidial germination and appressorium formation at a concentration of 3 microM, implying that CaM is involved in this process. A putative CaM kinase (CaMK) cDNA of C. gloeosporioides was cloned with transcripts from hard-surface-treated conidia. A selective inhibitor of CaMK, KN93 (20 microM), inhibited (50%) germination and appressorium formation, blocked melanization, and caused the formation of abnormal appressoria. Scytalone, an intermediate in melanin synthesis, reversed the inhibition of melanization but did not restore appressorium formation. The phosphorylation of 18- and 43-kDa proteins induced by hard-surface contact and ethephon was inhibited by the treatment with KN93. These results strongly suggest that hard-surface contact induces Ca2+-calmodulin signaling that primes the conidia to respond to host signals by germination and differentiation into appressoria.

Amino Acid Sequence↗

Mutations in the gerP locus of Bacillus subtilis and Bacillus cereus affect access of germinants to their targets in spores.

The gerP1 transposon insertion mutation of Bacillus cereus is responsible for a defect in the germination response of spores to both L-alanine and inosine. The mutant is blocked at an early stage, before loss of heat resistance or release of dipicolinate, and the efficiency of colony formation on nutrient agar from spores is reduced fivefold. The protein profiles of alkaline-extracted spore coats and the spore cortex composition are unchanged in the mutant. Permeabilization of gerP mutant spores by coat extraction procedures removes the block in early stages of germination, although a consequence of the permeabilization procedure in both wild type and mutant is that late germination events are not complete. The complete hexacistronic operon that includes the site of insertion has been cloned and sequenced. Four small proteins encoded by the operon (GerPA, GerPD, GerPB, and GerPF) are related in sequence. A homologous operon (yisH-yisC) can be found in the Bacillus subtilis genome sequence; null mutations in yisD and yisF, constructed by integrational inactivation, result in a mutant phenotype similar to that seen in B. cereus, though somewhat less extreme and equally repairable by spore permeabilization. Normal rates of germination, as estimated by loss of heat resistance, are also restored to a gerP mutant by the introduction of a cotE mutation, which renders the spore coats permeable to lysozyme. The B. subtilis operon is expressed solely during sporulation, and is sigma K-inducible. We hypothesize that the GerP proteins are important as morphogenetic or structural components of the Bacillus spore, with a role in the establishment of normal spore coat structure and/or permeability, and that failure to synthesize these proteins during spore formation limits the opportunity for small hydrophilic organic molecules, like alanine or inosine, to gain access to their normal target, the germination receptor, in the spore.

Alanine↗

Properties of spores of Bacillus subtilis blocked at an intermediate stage in spore germination.

Germination of mutant spores of Bacillus subtilis unable to degrade their cortex is accompanied by excretion of dipicolinic acid and uptake of some core water. However, compared to wild-type germinated spores in which the cortex has been degraded, the germinated mutant spores accumulated less core water, exhibited greatly reduced enzyme activity in the spore core, synthesized neither ATP nor reduced pyridine or flavin nucleotides, and had significantly higher resistance to heat and UV irradiation. We propose that the germinated spores in which the cortex has not been degraded represent an intermediate stage in spore germination, which we term stage I.

Bacillus subtilis↗

Identification of a new gene essential for germination of Bacillus subtilis spores with Ca2+-dipicolinate.

Bacillus subtilis spores can germinate with a 1:1 chelate of Ca(2+) and dipicolinic acid (DPA), a compound present at high levels in the spore core. Using a genetic screen to identify genes encoding proteins that are specifically involved in spore germination by Ca(2+)-DPA, three mutations were identified. One was in the gene encoding the cortex lytic enzyme, CwlJ, that was previously shown to be essential for spore germination by Ca(2+)-DPA. The other two were mapped to an open reading frame, ywdL, encoding a protein of unknown function. Analysis of ywdL expression showed that the gene is expressed during sporulation in the mother cell compartment of the sporulating cell and that its transcription is sigma(E) dependent. Functional characterization of YwdL demonstrated that it is a new spore coat protein that is essential for the presence of CwlJ in the spore coat. Assembly of YwdL itself into the spore coat is dependent on the coat morphogenetic proteins CotE and SpoIVA. However, other than lacking CwlJ, ywdL spores have no obvious defect in their spore coat. Because of the role for YwdL in a part of the spore germination process, we propose renaming ywdL as a spore germination gene, gerQ.

Amino Acid Sequence↗

Unusual ribosome particles occurring during spore germination.

Woese, Carl R. (Yale University, New Haven, Conn.). Unusual ribosome particles occurring during spore germination. J. Bacteriol. 82:695-701. 1961.-Spores of Bacillus subtilis germinated for varying amounts of time were disrupted under conditions of varying divalent ion concentration and analyzed for content of ribosomes by ultracentrifugal techniques. The picture of microsome development during germination is as follows: The spore contains only 50S and 68S particles. Early in germination, 25S and 35-8S particles increase in amount first. These particles seem to be formed in close association with the cell deoxyribonucleic acid. Their binding in the cell requires a chelating agent to release them maximally. Further, 25S and 35-8S particles are the only ones sensitive to ribonuclease and the only ones produced in the presence of chloramphenicol. The kinetics of their appearance, and their relative size, suggest them as precursors of 50S particles, which increase in amount shortly after appearance of 25S and 35S particles. It was found that 31S particles do not appear until later in germination, and their appearance signals a rise in 68S material, suggesting again the oft-reported relationship: one 31S + one 50S = one 68S. We have been unable to discern any precursors of the 31S material in these studies.

Bacillus subtilis↗

Effects of temperatures above the maximum for germination on the endospore of Bacillus cereus.

Knaysi, Georges (Cornell University, Ithaca, N.Y.). Effects of temperatures above the maximum for germination on the endospore of Bacillus cereus. J. Bacteriol. 87:1129-1136. 1964.-This is chiefly a study of heat activation and of the instability observed in spores of Bacillus cereus exposed to high temperatures. Temperatures up to 80 C for periods up to 6 hr were used. The spores were in colonies grown on collodion films, and were observed with the phase microscope, in dark contrast, for any evidence of change in their optical properties, the structure of their spodograms, and any other evidence of germination or growth. Exposure of the endospores to 80 C for 4 hr or more, whether in distilled water or in glucose broth, entirely eliminated stage II (i.e. the germcell stage, when the germinating spore begins to grow), and also tended to render the spores unstable and to produce uniformly dark spodograms. This instability involves some destructive processes and is not equivalent to normal entry into stage I (the initial stage). True heat-activation seemed to take place at a stage prior to the one which yielded uniformly dark spodograms. It was shown to consist of two factors: accumulation of germination-promoting components of the medium and activation of some spore components involved in germination. Activation of the medium components by preheating did not stimulate the spores of the strain employed. Unstable or activated spores can not be instantaneously stabilized, deactivated, or their changes arrested by exposure to acid buffers. It is concluded that the optimal temperature for activation, without danger of injury to the spore, in a given strain, is just above the maximum for stage I in that strain.

Bacillus cereus↗

Influence of exchangeable ions on germinability of bacterial spores.

Rode, L. J. (The University of Texas, Austin), and J. W. Foster. Influence of exchangeable ions on germinability of bacterial spores. J. Bacteriol. 91:1582-1588. 1966.-Native spores of Bacillus megaterium Texas, and H-spores produced by titration of native spores to pH 4 with mineral acid, did not germinate in a solution of alanine and inosine unless a strong electrolyte was present. Ca-spores prepared from either H-spores or native spores did germinate efficiently in the same solution without a strong electrolyte. Of several other bivalent cations tested, only strontium and barium could substitute for calcium in conditioning spores for subsequent germination in the absence of an electrolyte. Variable responses were obtained with different metal ion forms of 62 unidentified soil isolates and several stock species of Bacillus. Although the pattern of response was not uniform in all organisms, ions played a crucial role in the germinability of the great majority of strains tested.

Alanine↗

MSH2-dependent germinal CTG repeat expansions are produced continuously in spermatogonia from DM1 transgenic mice.

Myotonic dystrophy type 1 is a neuromuscular affection associated with the expansion of an unstable CTG repeat in the DM protein kinase gene. The disease is characterized by somatic tissue-specific mosaicism and very high intergenerational instability with a strong bias towards expansions. We used transgenic mice carrying more than 300 unstable CTG repeats within their large human genomic environment to investigate the dynamics of CTG repeat germinal mosaicism in males. Germinal mosaicism towards expansions was already present in spermatozoa at 7 weeks of age and continued to increase with age, suggesting that expansions are continuously produced throughout life. To determine the precise stage at which germinal expansions occur during spermatogenesis, we sorted and collected the different germ cell types produced during spermatogenesis from males of different ages and analyzed the CTG repeat mosaicism in each fraction. Strong mosaicisms towards expansions were already observed in spermatogonia before meiosis. In transgenic Msh2-deficient mice, germinal instability of the CTG repeats (only contractions) also occurs premeiotically. No significant difference in mosaicism was detected between spermatogonia and spermatozoa, arguing against continued expansions during postmeiotic stages. This indicates that germinal expansions are produced at the beginning of spermatogenesis, in spermatogonia, by a meiosis-independent mechanism involving MSH2.

Age Factors↗

Cystic degeneration of the telencephalic subependymal germinal layer in newborn infants.

Cystic lesions were found in the telencephalic germinal layer of 12 newborn babies. According to their location, the cysts could be divided into three groups: anterior, middle or thalamostriate, and posterior. The histological appearance of all cysts was essentially the same, but in three cases the germinal layer had a peculiar alveolar type of microcystic degeneration. A constant feature was the presence in the cyst wall of small white granulations composed of germinal cells and/or glial tissue. Cystic degeneration of the germinal layer was usually bilateral and sometimes quite extensive. After the involution of the germinal layer, these lesions are likely to persist as subependymal cysts, characterized by their specific location and the presence of glial granulations.

Brain Diseases↗

Microcycle conidiation in Penicillium urticae: an ultrastructural investigation of conidial germination and outgrowth.

A cultivation system has been developed for Penicillium urticae (NRRL 2159A) which yields 'microcycle' conidiation in submerged culture. Spherical growth of conidia was initiated by incubation at 37 degrees C in a growth-favoring medium. Transfer of these enlarged conidia to a nitrogen-poor medium at 35 degrees C resulted in synchronous germination and limited outgrowth followed by roughly synchronous conidiogenesis. An ultrastructural study of the germination stage indicated nuclear migration into the emerging germ tube whose new cell wall was an extension of the parent conidium's innermost cell wall layer. Septal formation at the neck of the germ tube followed. The septal pore was filled with particulate material and the septal membranes possessed unusual linear elements in their median hydrophobic zones. The germ tube, which possessed a smooth-surfaced plasma membrane, continued to elongate with periodic septum formation. The parent conidium and later the proximal germ tube showed progressive vacuolation and the cytoplasm became largely occupied by electron-translucent material. In older cells the septal pore was blocked by Woronin bodies. Compared with normal conidial germination this microcycle' germination is far more synchronous and the resultant germling is morphologically simpler. In ultrastructural terms, however, germination appears to be identical with that obtained at 28 degrees C.

Cell Wall↗

Purine metabolism and differential inhibition of spore germination in Phytophthora infestans.

An analysis of the effect of various purines and pyrimidines on the germination process in three different isolates of the late blight fungus, Phytophthora infestans, revealed increased rates of indirect germination in one isolate by adenine, hypoxanthine, and the riboside of dimethylaminopurine. This observation coupled with the capacity of sporangia of the race affected (1.2.3.4) for the uptake and interconversion of purines, as demonstrated by experiments with labelled purines under in vivo and in vitro conditions, pointed to hypoxanthine as a key intermediate in the purine metabolism directly associated with spore formation and development. This enhanced germination contrasted sharply with the almost complete arrest of indirect germination that occurred when sporangia were incubated with the purine analogue, benzimidazole, or with either of the respiratory inhibitors, sodium azide and 2,4-dinitrophenol. The pattern of differential inhibition exhibited by sporangia versus zoospores upon treatment with actinomycin D, 4-FLUOROURACIL, OR CYCLOHEXIMIDE INDICATED THat continued translation on preformed messenger RNA may be one essential requirement for the formation and release of zoospores, whereas their subsequent germination and development may depend upon renewed transcription as well.

Azides↗

Role of spore coats in the germinative response of Bacillus cereus to adenosine and its analogues.

Spores of the strain NCIB 8122 of Bacillus cereus have been depleted of coats by treatment with 0.1% sodium dodecyl sulfate--200 mM 2-mercaptoethanol--0.5 M NaCl (pH 9.6). The coat-depleted spores did not show any decrease in viability, heat resistance, refractility, dipicolinic acid content, or specific activities of several protoplastic enzymes. The germinative response of the coat-depleted spores to adenosine and several analogues thereof was found qualitatively similar to that obtained with intact spores. However, germination kinetics appeared to be affected by coat removal, since germination rate measured as loss of refractility was eight times slower even at inducer concentrations 10-fold higher than those required to promote optimal germination response of intact spores. Loss of heat resistance, on the other hand, was hardly affected by coat removal. These results suggest that, even though spore coats are not essential for the triggering reaction, they are required for a rapid evolution of the later events in the germination process.

Adenosine↗

Beagle pup germinal matrix maturation studies.

Intraventricular hemorrhage, or hemorrhage into the germinal matrix tissues of the developing brain, remains a common problem of preterm infants. The "risk period" for this insult is the first 3-4 postnatal days. We hypothesized that this risk period for hemorrhage is related to rapid perinatal maturation of the germinal matrix vasculature and employed the newborn beagle pup model for the study of this maturation. Newborn beagle pups (n = 30) were anesthetized and systemically perfused with buffered formalin; the brains were removed and prepared for immunohistochemical study. Sections stained with Bandeiraea lectin demonstrated that there was no difference in germinal matrix vessel density between postnatal days 1 and 4. Germinal matrix sections were also stained for antibodies to alpha-smooth muscle actin, collagen IV, collagen V, desmin, factor VIII-related antigen, fibronectin, glial fibrillary acidic protein, laminin, transferrin, and vimentin. Vasculature staining by alpha-smooth muscle actin was not noted until postnatal day 10, and differential staining was detected for antibodies to laminin and collagen V. Quantification of staining intensity by confocal microscopy demonstrated a significant increase in both extracellular matrix components at postnatal day 4 compared with day 1 (p less than 0.05 for both). These basement membrane proteins may add sufficient structural integrity to germinal matrix vessels to prevent capillary rupture and thus intraventricular hemorrhage.

Aging↗

The t(14;18) defines a unique subset of diffuse large B-cell lymphoma with a germinal center B-cell gene expression profile.

Recently we have identified subgroups of de novo primary diffuse large B-cell lymphoma (DLBCL) based on complementary DNA microarray-generated gene expression profiles. To correlate the gene expression profiles with cytogenetic abnormalities in these DLBCLs, we examined the occurrence of the t(14;18)(q32;q21) in the 2 distinctive subgroups of DLBCL: one with the germinal center B-cell gene expression signature and the other with the activated B cell-like gene expression signature. The t(14;18) was detected in 7 of 35 cases (20%). All 7 t(14;18)-positive cases had a germinal center B-cell gene expression profile, representing 35% of the cases in this subgroup, and 6 of these 7 cases had very similar gene expression profiles. The expression of bcl-2 and bcl-6 proteins was not significantly different between the t(14;18)-positive and -negative cases, whereas CD10 was detected only in the group with the germinal center B-cell expression profile, and CD10 was most frequently expressed in the t(14;18)-positive cases. This study supports the validity of subdividing DLBCL into 2 major subgroups by gene expression profiling, with the t(14;18) being an important event in the pathogenesis of a subset of DLBCL arising from germinal center B cells. CD10 protein expression is useful in identifying cases of DLBCL with a germinal center B-cell gene expression profile and is often expressed in cases with the t(14;18).

Chromosome Mapping↗

Phospholipase Cdelta regulates germination of Dictyostelium spores.

BACKGROUND: Many eukaryotes, including plants and fungi make spores that resist severe environmental stress. The micro-organism Dictyostelium contains a single phospholipase C gene (PLC); deletion of the gene has no effect on growth, cell movement and differentiation. In this report we show that PLC is essential to sense the environment of food-activated spores. RESULTS: Plc-null spores germinate at alkaline pH, reduced temperature or increased osmolarity, conditions at which the emerging amoebae can not grow. In contrast, food-activated wild-type spores return to dormancy till conditions in the environment allow growth. The analysis of inositol 1,4,5-trisphosphate (IP3) levels and the effect of added IP3 uncover an unexpected mechanism how PLC regulates spore germination: i) deletion of PLC induces the enhanced activity of an IP5 phosphatase leading to high IP3 levels in plc-null cells; ii) in wild-type spores unfavourable conditions inhibit PLC leading to a reduction of IP3 levels; addition of exogenous IP3 to wild-type spores induces germination at unfavourable conditions; iii) in plc-null spores IP3 levels remain high, also at unfavourable environmental conditions. CONCLUSIONS: The results imply that environmental conditions regulate PLC activity and that IP3 induces spore germination; the uncontrolled germination of plc-null spores is not due to a lack of PLC activity but to the constitutive activation of an alternative IP3-forming pathway.

Animals↗

Clonal heterogeneity in the germinal zone of the developing rat telencephalon.

A double-labeling technique, combining retroviral tagging of individual cell lines (one clone per brain hemisphere) with the simultaneous [3H]thymidine-labeling of dividing cells in S phase, was used to study proliferation characteristics of individual precursor cell lines in the germinal zone of the developing rat forebrain. The cortical germinal zone was found to be segregated into three spatially distinct horizontal populations of precursor cell lineages, which differed in cell cycle kinetics, amount of cell death, and synchronous versus asynchronous mode of proliferation. The striatal germinal zone demonstrated a similar heterogeneity in the cell cycle characteristics of proliferating clones, but did not show nearly as distinct a spatial segregation of these different populations. The results demonstrate the clonal heterogeneity among precursor populations in the telencephalon and the differential spatial organization of the cortical and the striatal germinal zones. This germinal zone heterogeneity may predict some of the differences found among cellular phenotypes in the adult forebrain.

Animals↗

Nucleolar activation and vacuolation in embryo radicle cells during early germination.

The activation of the nucleolus of primary root cells of Sinapis alba embryos during the first 72 h of germination was monitored by autoradiographic, ultrastructural and microstereological methods. Autoradiographs showed that within 48 h, the nucleolus progressively resumed the capacity to synthesize pre-rRNA molecules at a high rate. In quiescent embryos the nucleolus was small, compact and composed of mixed granular and fibrillar components. Within the first 6 h of germination a strong nucleolar vacuolation occurred, accompanied by a decrease in the volume of the nucleolus and a concomitant high loss of its ribonucleoproteins (RNPs). From 6 to 24 h, nucleolar vacuolation decreased to reach a stable level. During this last period the volume of the nucleolus increased by the accumulation of the fibrillar component resulting from a slow pre-rRNA processing. At 24 h the nucleolus presented a predominantly fibrillar texture. After 24 h, nucleolus growth continued but was due to the accumulation of the granular component, indicating that pre-rRNA processing occurred at a higher rate than during the first day of germination. From 48 h the nucleolus was composed of well-delineated granular and fibrillar areas. Dense nucleolus-associated chromatin as well as fibrillar centres were always observed during the whole period of observation. In addition, previous studies on the nucleolus of radicle cells of Zea mays embryo during early germination were completed by studying changes in the nucleolar volume and in the density of pre-ribosomal subunits of the granular component. On the basis of the data obtained with both species we suggest that a possible function for the nucleolar vacuoles is the increase in the nucleolus-nucleoplasm exchange interface in response to a rapid increase in the output of nucleolar RNPs. The nucleolar growth pattern during early germination is also discussed.

Autoradiography↗