POLTERGEIST functions to regulate meristem development downstream of the CLAVATA loci

Mutations at the CLAVATA loci (CLV1, CLV2 and CLV3) result in the accumulation of undifferentiated cells at the shoot and floral meristems. We have isolated three mutant alleles of a novel locus, POLTERGEIST (POL), as suppressors of clv1, clv2 and clv3 phenotypes. All pol mutants were nearly indistinguishable from wild-type plants; however, pol mutations provided recessive, partial suppression of meristem defects in strong clv1 and clv3 mutants, and nearly complete suppression of weak clv1 mutants. pol mutations partially suppressed clv2 floral and pedicel defects in a dominant fashion, and almost completely suppressed clv2 phenotypes in a recessive manner. These observations, along with dominant interactions observed between the pol and wuschel (wus) mutations, indicate that POL functions as a critical regulator of meristem development downstream of the CLV loci and redundantly with WUS. Consistent with this, pol mutations do not suppress clv3 phenotypes by altering CLV1 receptor activation.     

Mutagen sensitivities and mutator effects of MMS-sensitive mutants in Neurospora

7 mus (mutagen-sensitive) mutants of Neurospora crassa, which are more sensitive to the toxic effects of MMS (methyl methanesulfonate) than wild-type, were investigated for cross-sensitivities to other mutagens and inhibitors. These mutants have recently been mapped in 5 new genes, mus-7 to mus-11, and mutant alleles from each gene were checked for their effects on mutation frequencies. It was found that mutants in 3 of these 5 genes showed radiation-induced mutation frequencies similar to wild-type. These included 2 alleles of the gene mus-10, which were cross-sensitive only to UV and were the only mutants that produced some viable ascospores in homozygous crosses. The mutant of the second gene, mus-8, was especially sensitive to UV and mitomycin C and produced slightly reduced frequencies of spontaneous mutation. In contrast, the mutant of the third gene, mus-7, was not UV-sensitive but showed some cross-sensitivity to X-rays; mus-7 was highly sensitive to MMS and also to histidine, which inhibits various repair-defective mutants at concentrations well below those that reduce wild-type growth. None of these mus resemble mutants previously found in Neurospora, nor do they conform clearly to mutant types identified in E. coli or yeast. On the other hand mutants in 2 further genes, mus-11, and especially 2 alleles of mus-9, are very similar to uvs-3 of Neurospora and generally resemble mutants that are considered to be defective in "error-prone" repair. They were UV- as well as X-ray-sensitive, and showed strong spontaneous mutator effects but almost no increase in recessive lethal frequencies in heterokaryons after UV-treatments.     

Two unlinked genes for the pyruvate dehydrogenase complex in Aspergillus nidulans.

The activity of the overall pyruvate dehydrogenase complex was found to be similar in extracts of Aspergillus nidulans after growth on either sucrose or acetate. Eight mutants lacking the activity of this complex were found among some 200 glycolytic mutants selected for their inability to grow on sucrose. The absence of pyruvate dehydrogenase complex activity was also confirmed for a mutant, g6 (pdhA1), isolated previously. Studies with the mutants supported the existence of two unlinked genes, pdhA and pdhB, controlling the function of the complex. In vivo and in vitro complementation between mutations at the two loci were shown by the ability of forced heterokaryons to grow on sucrose and by the restoration of overall pyruvate dehydrogenase complex activity in mixed cell-free extracts. The mutations were recessive to their wild-type alleles, and the pdhA and pdhB loci were assigned to linkage groups I and V, respectively.     

The Arabidopsis ELF3 gene regulates vegetative photomorphogenesis and the photoperiodic induction of flowering

Flowering in Arabidopsis thaliana is promoted by long-day (LD) photoperiods such that plants grown in LD flower earlier, and after the production of fewer leaves, than plants grown in short-day (SD) photoperiods. The early-flowering 3 ( elf 3) mutant of Arabidopsis , which is insensitive to photoperiod with regard to floral initiation has been characterized. elf 3 mutants are also altered in several aspects of vegetative photomorphogenesis, including hypocotyl elongation. When inhibition of hypocotyl elongation was measured, elf 3 mutant seedlings were less responsive than wild-type to all wavelengths of light, and most notably defective in blue and green light-mediated inhibition. When analyzed for the flowering-time phenotype, elf 3 was epistatic to mutant alleles of the blue-light receptor encoding gene, HY 4. However, when elf 3 mutants were made deficient for functional phytochrome by the introduction of hy 2 mutant alleles, the elf 3 hy 2 double mutants displayed the novel phenotype of flowering earlier than either single mutant while still exhibiting photoperiod insensitivity, indicating that a phytochrome-mediated pathway regulating floral initiation remains functional in elf 3 single mutants. In addition, the inflorescences of one allelic combination of elf 3 hy 2 double mutants form a terminal flower similar to the structure produced by tfl 1 single mutants. These results suggest that one of the signal transduction pathways controlling photoperiodism in Arabidopsis is regulated, at least in part, by photoreceptors other than phytochrome, and that the activity of the Arabidopsis inflorescence and floral meristem identity genes may be regulated by this same pathway.     

Salt-tolerant mutants in glycophytic salinity response (GSR) genes in <Emphasis Type="Italic">Catharanthus roseus</Emphasis>

The periwinkle Catharanthus roseus shares glycophytic properties of crop plants. To contribute towards an understanding of the glycophytic response to salinity, large populations of M(2) seeds having an origin in nitroso-methyl urea and ethyl methane sulphonate treatments were screened for germination with 250 mM of NaCl. Out of the nine mutant lines so recovered, which tolerated salt stress due to loss of the normal glycophytic salinity response ( GSR), the characteristics of six gsr mutants are reported here. All six, gsr-1 to gsr-6, differed from the wild-type in both seedling and adult-plant morphological characters beside being salt tolerant. The mutations in them were inherited as monogenic recessive alleles at the corresponding wild-type loci. The trans-complementation tests revealed that the gsr-1 to gsr-6 mutants specified six complementation groups. The mutant seedlings generally accumulated more proline and glycine betaine, constitutively, than the wild-type. The mutant plants transpired lower amounts of water and accumulated higher amounts of proline under drought stress. It was inferred that the products of the six GSR genes defined here are involved in the regulation of salt stress, as well as cell division, developmental and/or morphogenetic pathway(s), in C. roseus.     

The Effects of Mutagen-Sensitive Mutants of DROSOPHILA MELANOGASTER in Nonmutagenized Cells

The effects of 13 mutagen-sensitive (mus) mutants (representing seven loci) on mitotic chromosome stability in nonmutagenized cells have been examined genetically. To do this, mus-bearing flies heterozygous for the recessive somatic-cell marker, multiple wing hairs (mwh), were examined for increased frequencies of mwh clones in the wing blade. Mutants at the mus-103, mus-104 and mus-106 loci do not affect the frequency of mwh clones, while mus-101, mus-102, mus-105 and mus-109 alleles cause increases in the frequency of mwh clones. These data show that the wild-type alleles of latter four loci specify functions that are required for chromosome stability in nonmutagenized cells. Analysis of the size distribution of mwh clones produced by these mutants suggests that most chromosome instability caused by these mutants is the consequence of chromosome breakage; in the presence of mus-105 and mus-109 alleles a small fraction of the mwh clones are produced by an event (mitotic recombination, mutation, nondisjunction) that produces euploid clones. To inquire whether any of the extant alleles of the mus-101, mus-102, mus-105 and mus-109 loci might be leaky alleles of loci that carry out essential mitotic functions, chromosome stability in females homozygous for alleles of these loci has been compared to that of females carrying one dose of a mutant over a deficiency for that mus locus. These comparisons show that the extant alleles at the mus-101, mus-109 and mus-105 loci are all leaky mutants. It is suggested that all three of these loci may specify essential mitotic functions.     

SUPERWOMAN1 and DROOPING LEAF genes control floral organ identity in rice

We analyzed recessive mutants of two homeotic genes in rice, SUPERWOMAN1 (SPW1) and DROOPING LEAF (DL). The homeotic mutation spw1 transforms stamens and lodicules into carpels and palea-like organs, respectively. Two spw1 alleles, spw1-1 and spw1-2, show the same floral phenotype and did not affect vegetative development. We show that SPW1 is a rice APETALA3 homolog, OsMADS16. In contrast, two strong alleles of the dl locus, drooping leaf-superman1 (dl-sup1) and drooping leaf-superman2 (dl-sup2), cause the complete transformation of the gynoecium into stamens. In these strong mutants, many ectopic stamens are formed in the region where the gynoecium is produced in the wild-type flower and they are arranged in a non-whorled, alternate pattern. The intermediate allele dl-1 (T65), results in an increase in the number of stamens and stigmas, and carpels occasionally show staminoid characteristics. In the weakest mutant, dl-2, most of the flowers are normal. All four dl alleles cause midrib-less drooping leaves. The flower of the double mutant, spw1 dl-sup, produces incompletely differentiated organs indefinitely after palea-like organs are produced in the position where lodicules are formed in the wild-type flower. These incompletely differentiated organs are neither stamens nor carpels, but have partial floral identity. Based on genetic and molecular results, we postulate a model of stamen and carpel specification in rice, with DL as a novel gene controlling carpel identity and acting mutually and antagonistically to the class B gene, SPW1.     

Genetic characterization and floral development of female sterile and stubby head, two aposporous mutants of pearl millet

 Apomixis has never been reported in natural populations of pearl millet [Pennisetum glaucum (L.) R.Br.], although many wild relatives of pearl millet are obligate or facultative aposporous apomicts. Four-nucleate aposporous embryo sacs are formed from somatic cells of the nucellus that do not undergo meiosis. Two mutants of pearl millet, female sterile (fs) and stubby head, have two developmental characteristics in common: a significant reduction in head length compared with the wild-type and the formation of aposporous embryo sacs. Reproductive development in fs and stubby head mutants was examined in depth because of the potential for illuminating basic cellular or developmental factors that may function to alter embryo sac development. Genetic analysis of stubby head showed that this phenotype is conferred by genes at two loci linked in coupling within 29 cM. Crosses between fs and stubby head mutants showed that, despite the similarities in phenotypes, the mutations are at different loci. The mutants differ from wild-type in their inflorescence structure from the time of initiation of spikelet primordia through terminal differentiation of the ovule. Both mutations could be categorized as meristic, since a change in inflorescence branch or organ number was common and gynoecium development varied. We speculate that heterochronic development of the floral meristem and organ initiation/specification programs may be the underlying mechanism for phenotypic changes in these mutants throughout the floral phase. Received: 25 October 1996 / Accepted: 13 March 1997     

Sex and the Single Cell. I. on the Action of Major Loci Affecting Sex Determination in DROSOPHILA MELANOGASTER

Sex determination in Drosophila melanogaster is under the control of the X chromosome:autosome ratio and at least four major regulatory genes: transformer (tra), transformer-2 (tra-2), doublesex (dsx) and intersex (ix). Attention is focused here on the roles of these four loci in sex determination. By examining the sexual phenotype of clones of homozygous mutant cells produced by mitotic recombination in flies heterozygous for a given recessive sex-determination mutant, we have shown that the tra, tra-2 and dsx loci determine sex in a cell-autonomous manner. The effect of removing the wild-type allele of each locus (by mitotic recombination) at a number of times during development has been used to determine when the wild-type alleles of the tra, tra-2 and dsx loci have been transcribed sufficiently to support normal sexual development. The wild-type alleles of all three loci are needed into the early pupal period for normal sex determination in the cells that produce the sexually dimorphic (in pigmentation) cuticle of the fifth and sixth dorsal abdominal segments. tra⁺ and tra-2⁺ cease being needed shortly before the termination of cell division in the abdomen, whereas dsx⁺ is required at least until the end of division. By contrast, in the foreleg, the wild-type alleles of tra⁺ and tra-2⁺ have functioned sufficiently for normal sexual differentiation to occur by about 24 to 48 hours before pupariation, but dsx⁺ is required in the foreleg at least until pupariation.——A comparison of the phenotypes produced in mutant/deficiency and homozygous mutant-bearing flies shows that dsx, tra-2 and tra mutants result in a loss of wild-type function and probably represent null alleles at these genes.—All possible homozygous doublemutant combinations of ix, tra-2 and dsx have been constructed and reveal a clear pattern of epistasis: dsx > tra, tra-2 > ix. We conclude that these genes function in a single pathway that determines sex. The data suggest that these mutants are major regulatory loci that control the batteries of genes necessary for the development of many, and perhaps all, secondary sexual characteristics.—The striking similarities between the properties of these loci and those of the homeotic loci that determine segmental and subsegmental specialization during development suggest that the basic mechanisms of regulation are the same in the two situations. The phenotypes and interactions of these sex-determination mutants provide the basis for the model of how the wild-type alleles of these loci act together to effect normal sex determination. Implications of these observations for the function of other homeotic loci are discussed.     

Isolation of mutants with aberrant mitochondrial morphology from Arabidopsis thaliana

To identify genes related to plant mitochondrial morphology and dynamics, novel mutants with respect to mitochondrial morphology were isolated from an ethyl methane sulphonate (EMS)-mutated population of Arabidopsis thaliana. Mitochondria were visualized by transforming Arabidopsis with a gene for a fusion protein consisting of GFP and a mitochondria-targeting pre-sequence. From 19,000 M2 populations, 17 mutants were isolated by fluorescent microscopic observations. All mitochondria in these mutants were longer and/or larger than wild-type mitochondria. The approximate chromosomal loci of the mutations of seven mutants that grew well were determined. The mitochondrial phenotypes of six of the mutants were recessive but the mitochondrial phenotype of the seventh mutant was dominant. Chromosomal rough mapping of the seven mutants showed that the mutations occurred at four different loci. At least one of these loci was novel, i.e., it was different from loci of other known mitochondrial morphology mutants of Arabidopsis and different from loci of Arabidopsis homologues of yeast genes related to mitochondrial morphology.     

Five genetic loci involved in the synthesis of acidic exopolysaccharides are closely linked in the genome of Rhizobium sp strain NGR234

Summary R-prime plasmids were constructed from a derivative of Rhizobium strain NGR234 (ANU280) and were shown to contain overlapping genomic DNA segments involved in biosynthesis of exopolysaccharides (EPS). The R-primes originally constructed carried the mutant allele from Tn5-induced EPS-deficient (Exo^–) mutant ANU2811. This plasmid-located mutant allele was dominant to the corresponding wild-type allele as merodiploid strains were Exo^–. Exo⁺ revertants occurred at a low rate (1×10^-7) and these were shown to result from double reciprocal recombination events, which led to the isolation of R-prime plasmids carrying functional wild-type exo alleles. R-prime plasmids that carry overlapping segments of DNA from parental strain ANU280 complemented 28 of the 30 group 2 Exo^– mutants of strain ANU280. Complementation of these Exo^– mutants also restored their symbiotic abilities of effective nodulation. Subsequent in vivo recombination between the wild-type alleles located on the R-prime and the corresponding mutated allele on the genome, was used to generate a new family of R-primes, which carried mutations in the exo genes. The 30 group 2 Exo^– mutants were classified into 7 distinct genetic groups based upon complementation and physical mapping data. Five of the seven exo loci were gentically linked and located on a 15-kb region of DNA. Mutations at two loci were dominant only when the mutations were R-prime plasmid-located while a mutation at a second locus was cis-dominant to two other exo loci. At least five genes involved in the synthesis of acidic exopolysaccharide synthesis have been identified.     

Characterization of the Rice Floral Organ Number Mutant fon3

A spontaneous rice mutant named floral organ number 3 (fon3) had major mutations in floral organ numbers. Genetic analysis indicated thatfon3 acted as a single recessive gene. Microscopic observation showed that the number of floral organs infon3 increased centripetally. For example, the number of pistils was the more frequently increased than organs in the outer whorls. Homeotic conversion of lodicules and glumes into palea/lemma-like organs was observed in some flowers. Scanning electron microscopy observation showed that the size of flower meristems was maintained the same or similar until the lemma primordium started to differentiate, at which time the floral meristem became enlarged, suggesting abnormal development of the inner whorls of rice florets. The relationship offon3 with other similar rice mutants is discussed.     

Genetic Analysis of Long-Flagella Mutants of Chlamydomonas Reinhardtii

The length of the flagella of Chlamydomonas reinhardtii cells is tightly regulated; both short-flagella and long-flagella mutants have been described. This report characterizes ten long-flagella mutants, including five newly isolated mutants, to determine the number of different loci conferring this phenotype, and to study interactions of mutants at different loci. The mutants, each of which was recessive in heterozygous diploids with wild type, fall into three unlinked complementation groups. One of these defines a new gene, lf3, which maps near the centromere of linkage group I. The flagellar length distributions in populations of each mutant were broad, with the longest flagella measuring four times the length of the longest flagella seen on wild-type cells. Each of the ten mutants had defective flagellar regrowth after amputation. Some of the mutants showed no regrowth within the time required for wild-type cells to regenerate flagella completely. Other mutants had subpopulations with rapid regeneration kinetics, and subpopulations with no observable regeneration. The mutants were each crossed to wild type to form temporary quadriflagellate, dikaryon cells; in each case the long flagella were rapidly shortened in the presence of the wild-type cytoplasm, demonstrating that the mutants were recessive, and that length control could be exerted on already assembled flagella.     

Isolation and characterization of sexual sporulation mutants of Aspergillus nidulans.

For the genetic dissection of sexual sporulation in Aspergillus nidulans, we started a collection of ascosporeless mutants. After mutagenization of conidiospores with high doses of UV, we isolated 20 mutants with defects in ascospore formation. We crossed these mutants in two successive rounds with the wild-type strain. Eighteen of the 20 isolated mutants produced progeny with the original mutant phenotype in these crosses, and these mutants were further analyzed. All 18 analyzed mutations were recessive to wild type. We assigned them to 15 complementation groups, based on crosses between mutants. The mutants could be classified as follows according to their cytological phenotype: (1) no croziers, (2) arrest at prekaryogamy, (3) arrest in early meiotic prophase, (4) arrest in late meiotic prophase, (5) arrest in meiotic metaphase I, (6) defective postmeiotic mitosis and/or deliniation of ascospores, and (7) slow progression through the postmeiotic stages of ascospore formation. A large proportion of the mutants, namely 11 of 18, arrested in meiotic prophase or metaphase I. We discuss a possible approach for isolating the wild-type alleles of the genes that carry the sexual sporulation mutations.     

Characterization of the Rice Floral Organ Number Mutant fon3

A spontaneous rice mutant named floral organ number 3 (fon3) had major mutations in floral organ numbers. Genetic analysis indicated that fort3 acted as a single recessive gene. Microscopic observation showed that the number of floral organs infon3 increased centripetally. For example, the number of pistils was the more frequently increased than organs in the outer whorls. Homeotic conversion of lodicules and glumes into palea/lemma-like organs was observed in some flowers. Scanning electron microscopy observation showed that the size of flower meristems was maintained the same or similar until the lemma primordium started to differentiate, at which time the floral meristem became enlarged, suggesting abnormal development of the inner whorls of rice florets. The relationship of fort3 with other similar rice mutants is discussed.     

A Genetic Analysis of Suppressors of the Pf10 Mutation in Chlamydomonas Reinhardtii

A mutation at the PF10 locus of the unicellular green alga Chlamydomonas reinhardtii leads to abnormal cell motility. The asymmetric form of the ciliary beat stroke characteristic of wild-type flagella is modified by this mutation to a nearly symmetric beat. We report here that this abnormal motility is a conditional phenotype that depends on light intensity. In the absence of light or under low light intensities, the motility is more severely impaired than at higher light intensities. By UV mutagenesis we obtained 11 intragenic and 70 extragenic strains that show reversion of the pf10 motility phenotype observed in low light. The intragenic events reverted the motility phenotype of the pf10 mutation completely. The extragenic events define at least seven suppressor loci; these map to linkage groups IV, VII, IX, XI, XII and XVII. Suppressor mutations at two of the seven loci (LIS1 and LIS2) require light for their suppressor activity. Forty-eight of the 70 extragenic suppressors were examined in heterozygous diploid cells; 47 of these mutants were recessive to the wild-type allele and one mutant (bop5-1) was dominant to the wild-type allele. Complementation analysis of the 47 recessive mutants showed unusual patterns. Most mutants within a recombinationally defined group failed to complement one another, although there were pairs that showed intra-allelic complementation. Additionally, some of the mutants at each recombinationally defined locus failed to complement mutants at other loci. They define dominant enhancers of one another.     

Epistatic interactions between four rad loci in yeast

Haploid yeast strains carrying mutations in two or more of four ad genes were contrusted by tetrad dissection, and the UV survival of these strains was measured. It was found that (with one exception) double mutant strains were not significantly more sensitive than the most sensitive single mutants, for strains involving mutant loci rad 1, rad 3 and rad 4. The exception was the double mutant rad 1–5 rad 4-4, but another double mutant involving different alleles of the the same loci did not show an enhanced UV sensitivity. Triple and quadruple mutants also failed to show a significantly increased UV sensitivity with respect to the single mutants. The results indicate that all these four mutant loci confer UV sensitivity by the same mechanism, and it is suggested that the wild-type alleles mediate excision-repair of UV-induced DNA lesions. Enhanced sensitivity of the genotype rad 1–5 rad 4-4 is attributed to leakiness of these alleles.     

On the time for gene silencing at duplicate Loci

A simple approximate formula is found for the mean time for mutant genes to first fix at one or another of two duplicate loci. The fixation is a result of one-way mutation from the wild-type to the mutant alleles, but is retarded by strong selection against the doubly homozygous mutants. The two-dimensional diffusion that models the evolution of the mutant allele frequencies at the two loci is studied by means of a transformation to two univariate diffusions having different time scales. Most of the results, but not all, agree well with the numerical studies by previous authors. Some new simulation results are presented, which agree well with the analytical results and, therefore, cast doubt on some previous simulations.