Use of Constructed Double Mutants for Determining the Temporal Order of Expression of Sporulation Genes in Bacillus subtilis

Double mutants containing two Spo mutations concerned with different stages of sporulation were constructed. In these, the phenotype that is exhibited is that of the earlier sporulation block. The same procedure was applied to sporulation mutants damaged in the same stage of development. The results provide a basis for placing in a temporal order different mutations concerned in stage II and stage IV of spore development. In general, the order indicated by the phenotypes of the double mutants is in agreement with the order derived on biochemical grounds. Double oligosporogenous mutants have also been constructed. Their sporulation incidence is roughly equal to the product of the incidences of the parent strains, idicating that separate factors are involved in overcoming each oligosporogenous block. The number of dependent sequential steps in sporulation is estimated as not less than about 12.     

Sporulation in Bacillus subtilis. Correlation of biochemical events with morphological changes in asporogeneous mutants

A comparison was made of morphological changes and successive, mainly biochemical, marker events for sporulation in 14 asporogenous mutants. The morphological and biochemical sequences are linked so that arrested development in one is accompanied by corresponding effects in the other. Thus mutants that fail to produce both protease and antibiotic do not progress beyond stage 0, formation of alkaline phosphatase appears to be associated with the transition from stage II to stage III and glucose dehydrogenase with that from stage III to stage IV. Stage II mutants may produce ;pygmy' cells or other bizarre cell-division forms. The biochemical sequence is dependent in the sense that if the occurrence of any one event is blocked that of all the succeeding events is also blocked. This has implications for biochemical models that have been proposed to explain the temporal sequence observed in spore development.     

The genetics of conidiophore pigmentation in Aspergillus nidulans

The grey-brown pigmentation of Aspergillus nidulans conidiophores depends on the functions of two 'ivory' loci. ivoB codes for a developmental specific phenol oxidase, and mutants accumulate its substrate N-acetyl-6-hydroxytryptophan. ivoA mutants are unable to make this substrate. ygA mutants are also poorly pigmented, and extracts require copper salts to activate both the phenol oxidase and conidial laccase. ivoA and ivoB mutants partially suppress the spore colour phenotype of ygA mutants. Comparisons of morphology, phenol oxidase and substrate accumulation in morphological mutants at the brlA locus suggest that the brlA protein regulates ivoA, ivoB and morphogenetic loci independently. The medA locus, which also affects morphology and pigmentation, may code for a modifier of brlA function. abaA mutants which are blocked at a later stage of development than brlA or medA mutants have low phenol oxidase levels, implying that by this stage of development the activity of the ivoB locus is declining.     

Genetic dissection of the initiation of the infection process and nodule tissue development in the Rhizobium-pea (Pisum sativum L.) symbiosis

Twelve non-nodulating pea (Pisum sativum L.) mutants were studied to identify the blocks in nodule tissue development. In nine, the reason for the lack of infection thread (IT) development was studied; this had been characterized previously in the other three mutants. With respect to IT development, mutants in gene sym7 are interrupted at the stage of colonization of the pocket in the curled root hair (Crh- phenotype), mutants in genes sym37 and sym38 are blocked at the stage of IT growth in the root hair cell (Ith- phenotype) and mutants in gene sym34 at the stage of IT growth inside root cortex cells (Itr- phenotype). With respect to nodule tissue development, mutants in genes sym7, sym14 and sym35 were shown to be blocked at the stage of cortical cell divisions (Ccd- phenotype), mutants in gene sym34 are halted at the stage of nodule primordium (NP) development (Npd- phenotype) and mutants in genes sym37 and sym38 are arrested at the stage of nodule meristem development (Nmd- phenotype). Thus, the sequential functioning of the genes Sym37, Sym38 and the gene Sym34 apparently differs in the infection process and during nodule tissue development. Based on these data, a scheme is suggested for the sequential functioning of early pea symbiotic genes in the two developmental processes: infection and nodule tissue formation.     

Synthesis of nitrogenase in mutants of the cyanobacterium Anabaena sp. strain PCC 7120 affected in heterocyst development or metabolism.

Mutants of Anabaena sp. strain PCC 7120 that are incapable of sustained growth with air as the sole source of nitrogen were generated by using Tn5-derived transposons. Nitrogenase was expressed only in mutants that showed obvious morphological signs of heterocyst differentiation. Even under rigorously anaerobic conditions, nitrogenase was not synthesized in filaments that were unable to develop heterocysts. These results suggest that competence to synthesize nitrogenase requires a process that leads to an early stage of visible heterocyst development and are consistent with the idea that synthesis of nitrogenase is under developmental control (J. Elhai and C. P. Wolk, EMBO J. 9:3379-3388, 1990). We isolated mutants in which differentiation was arrested at an intermediate stage of heterocyst formation, suggesting that differentiation proceeds in stages; those mutants, as well as mutants with aberrant heterocyst envelopes and a mutant with defective respiration, expressed active nitrogenase under anaerobic conditions only. These results support the idea that the heterocyst envelope and heterocyst respiration are required for protection of nitrogenase from inactivation by oxygen. In the presence of air, such mutants contained less nitrogenase than under anaerobic conditions, and the Fe-protein was present in a posttranslationally modified inactive form. We conclude that internal partial oxygen pressure sufficient to inactivate nitrogenase is insufficient to repress synthesis of the enzyme completely. Among mutants with an apparently intact heterocyst envelope and normal respiration, three had virtually undetectable levels of dinitrogenase reductase under all conditions employed. However, three others expressed oxygen-sensitive nitrogenase activity, suggesting that respiration and barrier to diffusion of gases may not suffice for oxygen protection of nitrogenase in these mutants; two of these mutants reduced acetylene to ethylene and ethane.     

Late embryo-defective mutants of Arabidopsis

The embryo-defective (emb) mutants of Arabidopsis constitute a large and diverse group of mutants disrupted in a broad range of embryonic processes, including morphogonesis, cell differentiation, and maturation programs. This report describes a subset of these mutants, the late embryo defectives, which develop beyond the globular stage of embryogenesis but fail to complete normal morphogenesis. A representative sample of 12 late mutants was chosen for this study, patterns of morphogenesis were characterized, the germination potential of mutant seeds was investigated, and additional mutant alleles within the collection were identified. Morphological defects in mutant embryos became apparent during the heart stage of development, when embryos normally begin the rapid cell division and expansion required for the completion of morphogenesis. Despite their morphological abnormalities, mutant embryos often germinated from dry seed, demonstrating that genetic programs required for the establishment of desiccation tolerance remained intact. Mutant seedlings displayed a wide range of developmental abnormalities, including altered morphology, lack of pigmentation, dwarfism, and disorganized vegetative growth. One late mutant was found to be allelic to an early embryo defective that arrests at the globular stage. These results suggest that a number of late EMB genes encode basic cellular and metabolic functions needed for cell division, enlargement, and embryonic growth. The rapid growth and metabolic changes that occur at the heart stage may present a barrier to normal development in the late mutants, resulting in altered embryo morphology and other developmental defects. It is proposed that many Arabidopsis mutants with abnormal embryo and seedling morphology are not defective in the regulation of pattern formation or morphogenesis, but rather in fundamental physiological and cellular processes required for the completion of normal growth and development. © 1995 Wiley-Liss, Inc.     

Daf-2, Daf-16 and Daf-23: Genetically Interacting Genes Controlling Dauer Formation in Caenorhabditis Elegans

Under conditions of high population density and low food, Caenorhabditis elegans forms an alternative third larval stage, called the dauer stage, which is resistant to desiccation and harsh environments. Genetic analysis of some dauer constitutive (Daf-c) and dauer defective (Daf-d) mutants has revealed a complex pathway that is likely to function in particular neurons and/or responding tissues. Here we analyze the genetic interactions between three genes which comprise a branch of the dauer formation pathway that acts in parallel to or downstream of the other branches of the pathway, the Daf-c genes daf-2 and daf-23 and the Daf-d gene daf-16. Unlike mutations in other Daf-c genes, mutations in both daf-2 and daf-23 cause non-conditional arrest at the dauer stage. Our epistasis analysis suggests that daf-2 and daf-23 are functioning at a similar point in the dauer pathway. First, mutations in daf-2 and daf-23 are epistatic to mutations in the same set of Daf-d genes. Second, daf-2 and daf-23 mutants are suppressed by mutations in daf-16. Mutations in daf-16 do not suppress any of the other Daf-c mutants as efficiently as they suppress daf-2 and daf-23 mutants. Third, double mutants between either daf-2 or daf-23 and several other daf-d mutants exhibit an unusual interaction. Based on these results, we present a model for the function of daf-2, daf-23 and daf-16 in dauer formation.     

Sporulation and Enterotoxin Production by Mutants of Clostridium perfringens

The ability of Clostridium perfringens type A to produce an enterotoxin active in human food poisoning has been shown to be directly related to the ability of the organism to sporulate. Enterotoxin was produced only in a sporulation medium and not in a growth medium in which sporulation was repressed. Mutants with an altered ability to sporulate were isolated from an sp(+) ent(+) strain either as spontaneous mutants or after mutagenesis with acridine orange or nitrosoguanidine. All sp(0) (-) mutants were ent(-). Except for one isolate, these mutants were not disturbed in other toxic functions characteristic of the wild type and unrelated to sporulation. A total of four of seven osp(0) mutants retained the ability to produce detectable levels of enterotoxin. None of the ent(-) mutants produced gene products serologically homologous to enterotoxin. A total of three sp(-) mutants, blocked at intermediate stages of sporulation, produced enterotoxin. Of these mutants, one was blocked at stage III, one probably at late stage IV, and one probably at stage V. A total of three sp(+) revertants isolated from an sp(-) ent(-) mutant regained not only the ability to sporulate but also the ability to produce enterotoxin. The enterotoxin appears to be a sporulation-specific gene product; however, the function of the enterotoxin in sporulation is unknown.     

Ribonucleic acid polymerase mutation affecting glutamate synthase activity in and sporulation of Bacillus subtilis.

Spore formation of 15 rifampin-resistant (Rifr) mutants of Bacillus subtilis strain 168 was examined. As a pleiotropic effect of a Rifr mutation, glutamate synthase activity was lost in these mutants. Twelve of the 15 mutants examined formed as many spores as the parent, but the remaining 3 formed significantly fewer (1%) spores. One of the latter mutants characterized further (RF301) was blocked in its sporulation process at stage 0. Thus, it was concluded that a certain modification of ribonucleic acid polymerase may affect specifically the gene expression of glutamate synthase and also the sporulation process at the initial stage.     

Selection of mouse neuroblastoma cell-specific polyoma virus mutants with stage differentiative advantages of replication.

Two mouse neuroblastoma cell lines were analyzed for their permissivity for polyoma virus growth. One (N18) is fully permissive for polyoma replication, the other (41A3) shows limited permissivity and the viral genome persists, without noticeable cell death. Virus persistence does not seem to alter the cells' ability to differentiate in vitro and leads to selection of viral mutants altered in the untranscribed regulatory region of the genome. The mutant types obtained appear to be related to the degree of host cell differentiation. Nucleotide sequence analysis of the restriction fragment covering the regulatory region shows that duplications are present in all mutants, while deletions in the non-duplicated segment are only present in mutants selected from less differentiated cells. These alterations involve both domains of the regulatory region that are considered to be essential for DNA replication and for enhancer activity. Mixed infections with polyoma wild type show that the selected mutants have cis-advantage in replication in neuroblastoma cells and not in 3T6 cells. Mutants carrying the deletion in the non-duplicated segment of the enhancer show a selective advantage in replication over the undeleted one in mixed infection. This advantage is much stronger in neuroblastoma cells in suspension (less-differentiated stage) than in monolayer cells (more-differentiated stage). An interpretation of the overall structure of the regulatory enhancer region, based on the observed differences between the mutants selected at different stages of differentiation in neuroblastoma and previously described mutants selected in undifferentiated cells, is discussed.     

Isolation and characterization of perithecial development mutants in neurospora

The isolation and characterization of mutants that block perithecial development in Neurospora crassa are described. Several classes of mutants have been isolated after UV mutagenesis, and those that block perithecial development when used as the female (protoperithecial) component of a cross have been further characterized. These mutants fall into 29 complementation groups. Twelve of the 33 mutants block development at the protoperithecial stage; no other clustering of block points is observed. Many of the mutants show an altered vegetative growth rate as well; in several mutants this lower growth rate cosegregates with the female sterile phenotype. Only one mutant also blocks development of the perithecium when used as the conidial parent. None of the mutants are temperature sensitive; two can be suppressed by growth on a complete crossing medium. There is no indication that the mutants are at or in the mating-type locus, nor are any of the mutants mating-type specific. Genetic mosaics have been formed using mixtures of mutant and marked wild-type nuclei; no mutants are cell autonomous by this criterion. The significance of these results in terms of "developmental" mutants isolated in other organisms and in relation to models of eukaryotic development is discussed.     

Herpes simplex virus-1-specific proteins are involved in alteration of polyphosphoinositide metabolism in baby-hamster kidney cells.

Herpes simplex virus type 1 (HSV-1) induces altered phosphoinositide metabolism in baby hamster kidney (BHK) cells, measured as incorporation of [3H]inositol or [32P]Pi [Langeland, Haarr & Holmsen (1986) Biochem. J. 237, 707-712]. We now report that this response in the inositol phospholipids is dependent on virus-specific proteins synthesized in the beta (early) stage of virus protein synthesis. This was demonstrated both by resistance to the inhibitory effect of cycloheximide after this stage of infection, and by the use of temperature-sensitive (ts) mutants of HSV-1; ts mutants in which protein synthesis was blocked so that only the alpha proteins were expressed showed a PIP2/PIP (phosphatidylinositol 4,5-bisphosphate/phosphatidylinositol 4-monophosphate) ratio similar to uninfected cells, while ts mutants which were defective in protein synthesis at a late beta stage or later showed increased PIP2/PIP ratios similar to cells infected by wild type HSV-1.     

Maintenance of neuronal positions in organized ganglia by SAX-7, a Caenorhabditis elegans homologue of L1

The L1 family of cell adhesion molecules is predominantly expressed in the nervous system. Mutations in human L1 cause neuronal diseases such as HSAS, MASA, and SPG1. Here we show that sax-7 gene encodes an L1 homologue in Caenorhabditis elegans. In sax-7 mutants, the organization of ganglia and positioning of neurons are abnormal in the adult stage, but these abnormalities are not observed in early larval stage. Misplacement of neurons in sax-7 mutants is triggered by mechanical force linked to body movement. Short and long forms of SAX-7 exhibited strong and weak homophilic adhesion activities in in vitro aggregation assay, respectively, which correlated with their different activities in vivo. SAX-7 was localized on plasma membranes of neurons in vivo. Expression of SAX-7 only in a single neuron in sax-7 mutants cell-autonomously restored its normal neuronal position. Expression of SAX-7 in two different head neurons in sax-7 mutants led to the forced attachment of these neurons. We propose that both homophilic and heterophilic interactions of SAX-7 are essential for maintenance of neuronal positions in organized ganglia.     

The role of natural selection in evolution

To evaluate properly a role of natural selection, its effect should be considered in relation to different phases of the evolutionary cycle postulated earlier by the author. At the first stage of the cycle natural selection is directed towards organism's persistence to detrimental external factors and leads to an increased fitness (that is viability and fecundity) in every generation. At the next stage of the cycle natural selection occurs under conditions of intraspecific competition and is directed towards a more efficient utilization of food resources. At this stage natural selection leads to formation and divergence of intraspecific races and is carried out by "single" selection actions occurring now and then and consisting of the survival of rare mutants with an altered ecological potential. Such a strict selection for certain mutants occurs again during the periods of acute competition for food, the selected mutants being characterized by a decrease of fitness, the latter to have been restored by means of the "ordinary" selection within the intervals between crises. According to the model suggested, homozygotes for "detrimental" recessive alleles could be selected in diploids, as the mutants mentioned with altered ecological potential. At the end of the cycle, there is a kind of selection for hybrids in which ecological potential of specialized intraspecific races is combined. The genetic drift is considered as an inevitable consequence of the postulated mechanism of natural selection.     

Decadent sporulation mutants of Bacillus subtilis.

In decadent sporulation mutants, sporulating populations are heterogeneous: the cells reach successive chemical and physical resistances with progressively decreasing frequencies. Each decadent mutant can be characterized by the shape and slope of the curve describing the frequency of cells resistant to various agents ('the resistance spectrum'). In some mutants the resistance spectrum decreases progressively from xylene resistance to heat resistance; in other mutants it decreases rapidly between octanol resistance and chloroform resistance. Electron microscopy showed that in two mutants the majority of the cells are blocked at stages III and IV; the number of cells that develop further to reach successive morphological stages falls off progressively. In two other mutants most cells reach stage V. Cortexless spores are also frequent. One of the decadent mutations, SpoL1, was localized between aroD and acf. The phenotype of decadent mutants is discussed in terms of sequential gene activation.     

Genetic interaction of an origin recognition complex subunit and the Polycomb group gene MEDEA during seed development

The eukaryotic origin recognition complex (ORC) is made up of six subunits and functions in nuclear DNA replication, chromatin structure, and gene silencing in both fungi and metazoans. We demonstrate that disruption of a plant ORC subunit homolog, AtORC2 of Arabidopsis (Arabidopsis thaliana), causes a zygotic lethal mutant phenotype (orc2). Seeds of orc2 abort early, typically producing embryos with up to eight cells. Nuclear division in the endosperm is arrested at an earlier developmental stage: only approximately four nuclei are detected in orc2 endosperm. The endosperm nuclei in orc2 are dramatically enlarged, a phenotype that is most similar to class B titan mutants, which include mutants in structural maintenance of chromosomes (SMC) cohesins. The highest levels of ORC2 gene expression were found in preglobular embryos, coinciding with the stage at which homozygous orc2 mutant seeds arrest. The homologs of the other five Arabidopsis ORC subunits are also expressed at this developmental stage. The orc2 mutant phenotype is partly suppressed by a mutation in the Polycomb group gene MEDEA. In double mutants between orc2 and medea (mea), orc2 homozygotes arrest later with a phenotype intermediate between those of mea and orc2 single mutants. Either alterations in chromatin structure or the release of cell cycle checkpoints by the mea mutation may allow more cell and nuclear divisions to occur in orc2 homozygous seeds.     

The bld mutants of Streptomyces coelicolor are defective in the regulation of carbon utilization, morphogenesis and cell–cell signalling

Mutants of Streptomyces coelicolor blocked at the earliest visible stage of morphological differentiation are called bld mutants. These mutants fail to form aerial hyphae on rich medium and most are defective in antibiotic production. One striking feature of these mutants is that, with the exception of bldB , their morphological defect is carbon-source dependent. In our investigation of catabolite control in Streptomyces , we identified mutants that were resistant to glucose repression and were also bld . The existence of these new bld mutants led us to examine the catabolite control phenotype of the previously described bld mutants which were not known to contain defects in carbon regulation. We report here that all of the characterized bld mutants of S. coelicolor are defective in the regulation of galP1 , and that at least one of the bld mutants, bldB , is globally deregulated for carbon utilization. Complementation of the morphological defect of bldA and bldB mutants with a cloned copy of the wild-type bld gene simultaneously restored normal regulation of galP1 , indicating that both aspects of the mutant phenotype are caused by the same lesion. We suggest a new interpretation for the role of the bld genes in development in Streptomyces. We suggest that the primary defect in bld mutants is in the regulation of carbon utilization, not specifically in the activation of genes whose products regulate the development pathway as previously suggested. We speculate that the inability of bld mutants to initiate morphogenesis is a secondary consequence of their inability to sense and/or signal starvation.