A mitochondrial mutator system in maize

Terfestatin A (TrfA), terphenyl-beta-glucoside, was isolated from Streptomyces sp. F40 in a forward screen for compounds that inhibit the expression of auxin-inducible genes in Arabidopsis (Arabidopsis thaliana). TrfA specifically and competitively inhibited the expression of primary auxin-inducible genes in Arabidopsis roots, but did not affect the expression of genes regulated by other plant hormones such as abscisic acid and cytokinin. TrfA also blocked the auxin-enhanced degradation of auxin/indole-3-acetic acid (Aux/IAA) repressor proteins without affecting the auxin-stimulated interaction between Aux/IAAs and the F-box protein TIR1. TrfA treatment antagonized auxin responses in roots, including primary root inhibition, lateral root initiation, root hair promotion, and root gravitropism, but had only limited effects on shoot auxin responses. Taken together, these results indicate that TrfA acts as a modulator of Aux/IAA stability and thus provides a new tool for dissecting auxin signaling.     

Tests of 4 controlling-element systems of maize for mutator activity and their interaction with Mu mutator

The maize mutator system, Mu, behaves in a non-Mendelian manner that may be expected if it were an extremely active controlling-element system. To test this hypothesis, the maize controlling-element systems, a---dt---Dt, Ds---Ac (= MP), I---En, and r---cu---Fcu were tested for mutation activity. Ds---Ac and r---cu---Fcu tests were the only ones in which new mutants were induced, but at a frequency much lower than that found in Mu crosses. The mutation frequency in these controlling-element systems does not differ statistically from that found in control (Non-Mu) populations.

Tests also were made to determine if Mu will substitute for the regulatory element of any of the 4 conotrolling-element. All tests were negative, suggesting that, if Mu is a controlling-element system, it is a different one from those previously described.     

Genetic requirements and mutational specificity of the Escherichia coli SOS mutator activity.

To better understand the mechanisms of SOS mutagenesis in the bacterium Escherichia coli, we have undertaken a genetic analysis of the SOS mutator activity. The SOS mutator activity results from constitutive expression of the SOS system in strains carrying a constitutively activated RecA protein (RecA730). We show that the SOS mutator activity is not enhanced in strains containing deficiencies in the uvrABC nucleotide excision-repair system or the xth and nfo base excision-repair systems. Further, recA730-induced errors are shown to be corrected by the MutHLS-dependent mismatch-repair system as efficiently as the corresponding errors in the rec+ background. These results suggest that the SOS mutator activity does not reflect mutagenesis at so-called cryptic lesions but instead represents an amplification of normally occurring DNA polymerase errors. Analysis of the base-pair-substitution mutations induced by recA730 in a mismatch repair-deficient background shows that both transition and transversion errors are amplified, although the effect is much larger for transversions than for transitions. Analysis of the mutator effect in various dnaE strains, including dnaE antimutators, as well as in proofreading-deficient dnaQ (mutD) strains suggests that in recA730 strains, two types of replication errors occur in parallel: (i) normal replication errors that are subject to both exonucleolytic proofreading and dnaE antimutator effects and (ii) recA730-specific errors that are not susceptible to either proofreading or dnaE antimutator effects. The combined data are consistent with a model suggesting that in recA730 cells error-prone replication complexes are assembled at sites where DNA polymerization is temporarily stalled, most likely when a normal polymerase insertion error has created a poorly extendable terminal mismatch. The modified complex forces extension of the mismatch largely at the exclusion of proofreading and polymerase dissociation pathways. SOS mutagenesis targeted at replication-blocking DNA lesions likely proceeds in the same manner.     

Tests of two models for the transmission of the Mu mutator in maize

Molecular Genetics and Genomics - The mutator system Mu does not follow a typical Mendelian mode of transmission. In outcrosses in which 50 per cent mutator plants are expected, about 90 per cent...     

Genetic analysis of opaque2 modifier gene activity in maize endosperm

Modifier genes have been described that convert the soft endosperm of opaque2 mutants to a hard, vitreous phenotype. The mode of action and the components of the genetic system involved in this seed modification are poorly understood. We used genetic and biochemical analyses to investigate the number of opaque2 modifier genes, their mode of action and their relationship to the biochemical alterations in the modified endosperm. Using two inbred opaque2 lines, we showed that the activity of opaque2 modifier genes is influenced by the genetic background. Analysis of segregating progenies and recombinant inbred lines derived from crosses between opaque2 and modified opaque2 genotypes indicated two independent loci affecting seed opacity and density. Consistent association between endosperm modification and enhanced accumulation of the gamma-zein storage protein suggested that either this protein is directly involved in the process of seed modification, or else that a modifier gene could be tightly linked to the genes responsible for gamma-zein synthesis.     

DNA polymerase mu (Pol mu), homologous to TdT, could act as a DNA mutator in eukaryotic cells

A novel DNA polymerase has been identified in human cells. Human DNA polymerase mu (Pol mu), consisting of 494 amino acids, has 41% identity to terminal deoxynucleotidyltransferase (TdT). Human Pol mu, overproduced in Escherichia coli in a soluble form and purified to homogeneity, displays intrinsic terminal deoxynucleotidyltransferase activity and a strong preference for activating Mn(2+) ions. Interestingly, unlike TdT, the catalytic efficiency of polymerization carried out by Pol mu was enhanced by the presence of a template strand. Using activating Mg(2+) ions, template-enhanced polymerization was also template-directed, leading to the preferred insertion of complementary nucleotides, although with low discrimination values. In the presence of Mn(2+) ions, template-enhanced polymerization produced a random insertion of nucleotides. Northern-blotting and in situ analysis showed a preferential expression of Pol mu mRNA in peripheral lymphoid tissues. Moreover, a large proportion of the human expressed sequence tags corresponding to Pol mu, present in the databases, derived from germinal center B cells. Therefore, Pol mu is a good candidate to be the mutator polymerase responsible for somatic hyper- mutation of immunoglobulin genes.     

Tests of two models for the transmission of the Mu mutator in maize

Summary The mutator system Mu does not follow a typical Mendelian mode of transmission. In outcrosses in which 50 per cent mutator plants are expected, about 90 per cent are observed. Two possible models of transmission are tested. One assumes that Mu has segregation distortion activity such as has been described for the SD locus in Drosophila. The second model assumes an extra-chromosomal factor that is transmitted through the cytoplasm. No evidence of SD activity was found. Mutation frequencies in lines in which Mu was transmitted through the female were not greater than the frequencies observed when Mu was transmitted through the male; as might be expected on some models of cytoplasmic transmission. Thus, cytoplasmic transmission was not established. Other possible models of extrachromosomal inheritance that might not be detected by reciprocal crosses are discussed.     

Molecular cloning and tissue-specific expression of the mutator2 gene (mu2) in Drosophila melanogaster.

We present here the molecular cloning and characterization of the mutator2 (mu2) gene of Drosophila melanogaster together with further genetic analyses of its mutant phenotype. mu2 functions in oogenesis during meiotic recombination, during repair of radiation damage in mature oocytes, and in proliferating somatic cells, where mu2 mutations cause an increase in somatic recombination. Our data show that mu2 represents a novel component in the processing of double strand breaks (DSBs) in female meiosis. mu2 does not code for a DNA repair enzyme because mu2 mutants are not hypersensitive to DSB-inducing agents. We have mapped and cloned the mu2 gene and rescued the mu2 phenotype by germ-line transformation with genomic DNA fragments containing the mu2 gene. Sequencing its cDNA demonstrates that mu2 encodes a novel 139-kD protein, which is highly basic in the carboxy half and carries three nuclear localization signals and a helix-loop-helix domain. Consistent with the sex-specific mutant phenotype, the gene is expressed in ovaries but not in testes. During oogenesis its RNA is rapidly transported from the nurse cells into the oocyte where it accumulates specifically at the anterior margin. Expression is also prominent in diploid proliferating cells of larval somatic tissues. Our genetic and molecular data are consistent with the model that mu2 encodes a structural component of the oocyte nucleus. The MU2 protein may be involved in controlling chromatin structure and thus may influence the processing of DNA DSBs.     

Genetic structure and activity of the nitrate-reducers community in the rhizosphere of different cultivars of maize

In this study, the structure and activity of the nitrate-reducers community were analysed in bulk and rhizospheric soils from three different non-isogenic transgenic cultivars of maize (two Bacillus thuringiensis maize and one glyphosate-resistant maize) in a long-term field experiment. DNA was extracted from both rhizospheric and non-rhizospheric soil sampled at three different development stages of the plants and amplified using primers targeting the genes encoding the␣membrane-bound nitrate reductase (narG). Nitrate-reducers community structure was analysed by generating fingerprints and sequencing of narG clone libraries. The season seems to be the most important factor controlling the genetic structure of the nitrate-reducers community. Smaller differences in the narG fingerprints were also observed between bulk and rhizospheric soils suggesting that presence of maize roots was the second important factor affecting the structure of this functional community. Similarly, a rhizosphere effect was observed on the nitrate reductase activity with a 2–3-fold increased in the rhizospheric soil compared to the non-rhizospheric soil. However, for both structure and activity of the nitrate-reducers community, no effect of the maize cultivar was observed. This study suggests that the effect of the cultivar and/or of the agricultural practices associated with the cultivation of transgenic maize is not significant compared to the effect of other environmental factors.     

Defective kernel mutants of maize. I. Genetic and lethality studies

A planting of 3,919 M₁ kernels from normal ears crossed by EMS-treated pollen produced 3,461 M₁ plants and 3,172 selfed ears. These plants yielded 2,477 (72%) total heritable changes; the selfed ears yielded 2,457 (78%) recessive mutants, including 855 (27%) recessive kernel mutants and 8 (0.23%) viable dominant mutants. The ratio of recessive to dominant mutants was 201:1. The average mutation frequency for four known loci was three per 3,172 genomes analyzed. The estimated total number of loci mutated was 535 and the estimated number of kernel mutant loci mutated was 285. Among the 855 kernel mutants, 432 had a nonviable embryo, and 59 germinated but had a lethal seedling. A sample of 194 of the latter two types was tested for heritability, lethality, chromosome arm location and endosperm-embryo interaction between mutant and nonmutant tissues in special hyper-hypoploid combinations produced by manipulation of B-A translocations. The selected 194 mutants were characterized and catalogued according to endosperm phenotype and investigated to determine their effects on the morphology and development of the associated embryo. The possibility of rescuing some of the lethal mutants by covering the mutant embryo with a normal endosperm was investigated. Ninety of these 194 mutants were located on 17 of the 18 chromosome arms tested. Nineteen of the located mutants were examined to determine the effect of having a normal embryo in the same kernel with a mutant endosperm, and vice versa, as compared to the expression observed in kernels with both embryo and endosperm in a mutant condition. In the first situation, for three of the 19 mutants, the mutant endosperm was less extreme (the embryo helped); for seven cases, the mutant endosperm was more extreme (the embryo hindered); and for nine cases, there was no change. In the reverse situation, for four cases the normal endosperm helped the mutant embryo; for 14 cases there was no change and one case was inconclusive.     

Genetic mapping of the Isaac-CACTA transposon in maize

We constructed a genetic linkage map with Isaac-TD, SSR, and SNAP markers in a RIL population which had been derived from a cross of waxy corn (KW7) and dent corn (Mo17). A total of 368 markers, including 241 Isaac-TD, 121 SSR, and 6 SNAP markers, were assigned to 10 linkage groups, encompassing 1687.0 cM, with an average genetic distance of 4.6 cM between markers. SSR markers were utilized as chromosome anchors, in order to assign the Isaac-TD markers to the chromosomes, and the number of markers in each of the linkage groups ranged between 22 and 49. The majority of the Isaac-TD markers were determined to have been distributed throughout the ten maize chromosomes. In linkage analysis of the Isaac-TD markers with genes of agronomic interest, six genes related with maize kernel starch biosynthesis, ae1, bt2, sh1, sh2, su1, and wx1, were analyzed and shown that they were closely linked with either the Isaac-TD or SSR markers on chromosomes of 3, 4, 5, and 9. We observed and mapped segregation-distorted markers on chromosomes 1, 5, 6, 7, 8, and 10, where these markers were clustered. The Isaac-TD or SSR markers which were closely linked with starch synthesis genes may prove useful in marker-assisted breeding programs.     

Biochemical studies on the iojap mutant of maize

The white leaf tissue of seedlings of Zea mays L. affected by the recessive nuclear gene iojap shows no photosynthetic activity; it contains about 1.4% of carotenoid and less than 0.1% of chlorophyll a content of normal green tissue. Neither fraction I protein nor chloroplast adenosine triphosphatase (EC 3.6.1.4) (CF₁) is detectable. This confirms earlier observations that plastids of white sectors of iojap maize do not contain ribosomes. About 40% of the activity of phosphoenolpyruvate carboxylase (EC 4.1.1.31) in green leaves could be found in white leaves indicating that the phosphoenolpyruvate carboxylase EC 4.1.1.31 is made on cytoplasmic ribosomes. The oxygen consumption of iojap-affected leaves is decreased.     

Wider access to genotypic space facilitates loss of cooperation in a bacterial mutator

Understanding the ecological, evolutionary and genetic factors that affect the expression of cooperative behaviours is a topic of wide biological significance. On a practical level, this field of research is useful because many pathogenic microbes rely on the cooperative production of public goods (such as nutrient scavenging molecules, toxins and biofilm matrix components) in order to exploit their hosts. Understanding the evolutionary dynamics of cooperation is particularly relevant when considering long-term, chronic infections where there is significant potential for intra-host evolution. The impact of responses to non-social selection pressures on social evolution is arguably an under-examined area. In this paper, we consider how the evolution of a non-social trait--hypermutability--affects the cooperative production of iron-scavenging siderophores by the opportunistic human pathogen Pseudomonas aeruginosa. We confirm an earlier prediction that hypermutability accelerates the breakdown of cooperation due to increased sampling of genotypic space, allowing mutator lineages to generate non-cooperative genotypes with the ability to persist at high frequency and dominate populations. This may represent a novel cost of hypermutability.     

Genetic control of the UV-induced SOS mutator effect in single- and double-stranded DNA phages

The SOS hypothesis postulated that the mutator effect on undameged DNA that generates phage-untargeted mutagenesis (UTM) results directly from the mechanism of targeted mutagenesis. RecA protein, which stimulates the cleavage of both the LexA repressor and UmuD protein, and the UmuDC gene products are required for UV-induced targeted mutagenesis. The use of phage λ for analyzing UV-induced mutagenesis has permitted a distinction to be made between the mechanisms of targeted and untargeted mutagenesis, in that the two processes differ with respect to their genetic requirements for recA⁺ and umuDC⁺ genes. In this paper, we show thet (i) proficiency for excision repair is required for UTM in double-stranded DNA phage but not in single-stranded DNA phage; (ii) the umuC function, which is not required for UTM of the double-stranded DNA phage λ, is necessary for untargeted mutagenesis of the single-stranded DNA phages M13 and φX174; (iii) for both single-stranded and double-stranded DNA phage, UV irradiation of the host increases the level of recA730-induced UTM. Our results are also consistent with the interpretation that the expression of untargeted mutagenesis in phage λ and in M13 depends on the polymerase and to a lesser extent on the exonuclease 5′ → 3′, activities of Po1I. These results suggest that the involvement of the RecA and UmuDC proteins may be related to more than the presence of base damage in the DNA substrate.