One way to do experiments on gene conversion?

Summary Competent cells of B. subtilis were transfected with heteroduplex SPP1 DNA, made by annealing complementary strands of wild type and 21 plaque type mutant DNAs. The frequencies of cells yielding mutant and wild type, only wild type, and only mutant phages were determined by single burst analyses of transfected cells. The data obtained reveal that an effective mechanism is operating in B. subtilis, which converts heterozygous to homozygous molecules prior to their replication. This correction mechanism is asymmetric with regard to the strand which is preferentially corrected in a given heteroduplex pair. The direction of asymmetry thus defined depends on the marker introduced into a particular heteroduplex. The efficiency of correction varies with the markers used and is correlated to the position of markers in the genetic map. From this correlation, the direction of replication of the SPP1 genome is deduced. The frequency distribution of wild type and mutant phages in cells yielding both genotypes indicates that both strands of the input DNA contribute equally to the production of progeny, i.e. DNA replication is symmetric.     

Changes in Thermostability of Photosystem Ⅱ and Leaf Lipid Composition of Rice Mutant with Deficiency of Light-harvesting Chlorophyll a/b Protein Complexes

We studied the difference in thermostability of photosystem Ⅱ （PSII） and leaf lipid composition between a T-DNA insertion mutant rice （Oryza sativa L.） VG28 and its wild type Zhonghuau. Native green gel and SDS-PAGE electrophoreses revealed that the mutant VG28 lacked all light-harvesting chlorophyll a/b protein complexes. Both the mutant and wild type were sensitive to high temperatures, and the maximal efficiency of PSII photochemistry （FJ Fm） and oxygen-evolving activity of PSII in leaves significantly decreased with increasing temperature. However, the PSII activity of the mutant was markedly more sensitive to high temperatures than that of the wild type. Lipid composition analysis showed that the mutant had less phosphatidylglycerol and sulfoquinovosyl diacylglycerol compared with the wild type. Fatty acid analysis revealed that the mutant had an obvious decrease in the content of 16：1t and a marked increase in the content of 18：3 compared with the wild type. The effects of lipid composition and unsaturation of membrane lipids on the thermostability of PSII are discussed.     

A PCR-based diagnostic tool for distinguishing grape skin color mutants

Berry skin color mutants are phenotypically different from their original cultivars, but they show identical molecular profile if analysed by using microsatellite markers. This work gives an easy, inexpensive and quick diagnostic tool to discriminate these somatic variants. We distinguished some grape (Vitis vinifera L.) skin color mutants from white to red or pink and from black to grey, pink or white and we investigated their molecular bases by single-strand conformational polymorphism (SSCP), single base primer extension and coding sequence analysis of anthocyanin biosynthetic enzyme genes and by polymerase chain reaction (PCR) analysis of VvmybA1 regulatory gene. Analyses of structural genes did not reveal polymorphisms between wild type and mutant cultivars but only among different varieties, whereas the study of VvmybA1 regulatory gene has given important outcomes for color mutants characterisation. The discrimination between white wild type and its derived colored mutant and between black wild type and white mutant has been obtained through a simple test of amplification for presence/absence. The discrimination between black wild type and less colored mutant has occurred through a quantitative result on agarose gel confirmed by real-time PCR analysis: the amount of functional allele in less colored somatic variants genome was about one-fourth of the correspondent quantity in original black cultivars genome.     

In vitro study of transgenic tobacco expressing Arabidopsis wild type and mutant acetohydroxyacid synthase genes

Summary Genes coding for the enzyme acetohydroxyacid synthase, often referred to as acetolactate synthase (AHAS, ALS; EC 4.1.3.18), from wild type Arabidopsis thaliana and a sulfonylurea-resistant mutant line GH50 (csrl-1; Haughn et al. 1988) were introduced in Nicotiana tabacum. Both genes were expressed at high levels with the 35S promoter. The csrl-1 gene conferred high levels of resistance to chlorsulfuron whereas the wild type gene did not. As selectable markers, chimaeric AHAS genes yielded transgenic plants on chlorsulfuron but at much lower efficiencies than with a chimaeric neomycin phosphotransferase gene on kanamycin (Sanders et al. 1987). Shoot differentiation from leaf discs was delayed on chlorsulfuron by 4–6 weeks. This study indicated a role for mutant AHAS genes in the genetic manipulation of herbicide resistance in transgenic plants but as selectable markers for plant cells undergoing differentiation no advantage over other genes was perceived.     

Transposon Tn5 as an identifiable marker in rhizobia: Survival and genetic stability of Tn5 mutant bean rhizobia under temperature stressed conditions in desert soils

Five transposon Tn5 insertion mutants of a beanRhizobium strain (Rhizobium leguminosarum b. v.phaseoli) were used in an ecological study to evaluate the extent to which transposon Tn5 was stable to serve as an identifiable marker in rhizobia under a high temperature stress condition in two Sonoran Desert soils. All the mutants possessed single chromosomal insertions of the transposon. In both soils, under the temperature stress conditions that were employed (40°C), both wild type and mutant populations possessing functional transposable elements declined rapidly. After 12 days, mutant cells, when screened using the Tn5 coded antibiotic resistance markers, were significantly less in number than when they were screened using only their intrinsic antibiotic resistance markers. There were no significant differences in numbers between the mutant cell population and the wild type when the mutant cells were screened using only the intrinsic antibiotic resistance markers. DNA-DNA hybridizations using a probe indicated neither deletion nor transposition of the transposable element. The results indicate that transposon DNA sequences are present within cells under high temperature stress conditions, but kanamycin/neomycin resistance is not expressed by some of these cells, suggesting that Tn5 undergoes a possible functional inactivation under these conditions. The possible implications of these findings are discussed.     

Isolation and Characterization of a Xanthophyll Aberrant Mutant of the Green Alga Nannochloropsis oculata

Novel mutants (xan1 and xan2) of the unicellular green alga Nannochloropsis oculata are impaired in xanthophyll biosynthesis, thereby producing aberrant levels of xanthophylls. High-performance liquid chromatography (HPLC) analysis revealed that the xan1 and xan2 mutants have double the violaxanthin (V) content, but have significantly decreased lutein content in their cells compared to the wild type. Furthermore, these mutants contain two to three times more zeaxanthin than the wild type under low light (LL) growth conditions. However, this xanthophyll aberration in N. oculata did not affect the normal growth and the major cellular chemical composition of the xan1 strain. The xanthophyll pool size of the LL-grown mutant was 1.8-fold greater than that of the wild type. Under high light (HL) growth conditions, V content was substantially decreased in both the mutant and wild types because of the epoxidation state of the xanthophylls. Under LL growth conditions, the deepoxidation states of the xanthophyll pool sizes were 0.1 and 1.2 in the wild type and the mutant, respectively. However, the deepoxidation states of the xanthophyll pool sizes were 0.78 in the wild type and 0.87 in the mutant under HL growth conditions. We observed that the level of one of the commercially important xanthophylls, zeaxanthin, was higher in the mutant than in the wild type under all culture conditions. This mutant is discussed in terms of its commercial value and potential utilization by the algal biotechnology industry for the production of zeaxanthin.     

<Emphasis Type="Italic">mesophyll cell defective1</Emphasis>, a mutation that disrupts leaf mesophyll differentiation in sunflower

Mutants with altered leaf morphology are useful as markers for the study of genetic systems and for probing the leaf differentiation process. One such mutant with deficient greening and altered development of the leaf mesophyll appeared in an inbred line of sunflower (Helianthus annuus L.). The objectives of the present study were to determine the inheritance of the mutant leaf trait and its morphological characterisation. The mutation, named mesophyll cell defective1 (mcd1), has pleiotropic effects and it is inherited as a monogenic recessive. The structure and tissue organization of mcd1 leaves are disrupted. In mcd1 leaves, the mesophyll has prominent intercellular spaces, and palisade and spongy tissues are not properly shaped. The mutant palisade cells also appear to be more vacuolated and with a reduced number of chloroplasts than the wild type leaves of equivalent developmental stage. The lamina thickness of mcd1 leaves is greatly variable and in some areas no mesophyll cells are present between the adaxial and abaxial epidermis. The leaf area of the mcd1 mutant is extremely reduced as well as the stem height. A deficient accumulation of photosynthetic pigments characterizes both cotyledons and leaves of the mutant. In mcd1 leaves, chlorophyll (Chl) fluorescence imaging evidences a spatial heterogeneity of leaf photosynthetic performance. Little black points, which correspond to photosystem II (PSII) maximum efficiency (F_v/F_m) values close to zero, characterize the mcd1 leaves. Similarly, the lightadapted quantum efficiency (Φ_PSII) values show a homogeneous distribution over wild type leaf lamina, while the damaged areas in mcd1 leaves, represented by yellow zones, are prominent. In conclusion, the loss of function of the MCD1 gene in Helianthus annuus is correlated with a variegated leaf phenotype characterized by a localized destruction of mesophyll morphogenesis and defeat of PSII activity.     

Ter mutation and susceptibility to phi X174 phage in E. coli K12.

The Ter-15 mutant derived from E. coli K12 W2252-11U^? RC^str (wild type I) is found to be sensitive to φx174 phage infection. Lipopolysaccharide extracted from this mutant inactivates the phage, and has core oligosaccharides identical in amounts to those in the lipopolysaccharide from wild type cells.In contrast, the Ter-21 mutant derived from E. coli K12 W2252-11U^? RC^rel (wild type II) is not sensitive to this phage infection, and its lipopolysaccharide does not inactivate the phage. Its lipopolysaccharide sugars are found to be D-glucose and D-ribose, thus differing from the lipopolysaccharide sugars of the wild type cells.     

Mutants ofPhycomyceswith Decreased Gallic Acid Content

Weinkove, D., Poyatos, J. A., Greiner, H., Oltra, E., Avalos, J., Fukshansky, L., Barrero, A. F., and Cerdá-Olmedo, E. 1998. Mutants ofPhycomyceswith decreased gallic acid content.Fungal Genetics and Biology25, 196–203. Most plants and some fungi accumulate phenols. Two hydroxybenzoic acids, gallic and protocatechuic acids, are abundant in the giant sporangiophores of the zygomycetePhycomyces blakesleeanus,much more so than in the basal mycelium or the culture medium. The actual concentrations vary with illumination, age of the culture, and composition of the medium. We devised a simple screening procedure to isolatehbamutants whose sporangiophores contained less gallic acid than the wild type. The most useful mutant had very low concentrations of hydroxybenzoic acids in the sporangiophores, but about the same as the wild type in the basal mycelium and the medium. The mutant was only slightly different from the wild type in growth and morphology. Mutant and wild-type sporangiophores grew away from ultraviolet C sources (260 nm) equally well. Contrary to previous conjectures, ultraviolet tropism does not depend on the ultraviolet absorption of gallic acid or other free hydroxybenzoic acids in the sporangiophore. Against expectations, phenols did not impair DNA extraction: sporangiophores produced better DNA preparations than basal mycelia and thehbamutant only slightly better than the wild type.     

Structural,molecular motions,and free-energy landscape of Leishmania sterol-14α-demethylase wild type and drug resistant mutant: a comparative molecular dynamics study

Sterol-14α-demethylase (CYP51) is an ergosterol pathway enzyme crucial for the survival of infectious Leishmania parasite. Recent high-throughput metabolomics and whole genome sequencing study revealed amphotericin B resistance in Leishmania is indeed due to mutation in CYP51. The residue of mutation (asparagine 176) is conserved across the kinetoplastidae and not in yeast or humans, portraying its functional significance. In order to understand the possible cause for the resistance, knowledge of structural changes due to mutation is of high importance. To shed light on the structural changes of wild and mutant CYP51, we conducted comparative molecular dynamics simulation study. The active site, substrate biding cavity, substrate channel entrance (SCE), and cavity involving the mutated site were studied based on basic parameters and large concerted molecular motions derived from essential dynamics analyses of 100 ns simulation. Results indicated that mutant CYP51 is stable and less compact than the wild type. Correspondingly, the solvent accessible surface area (SASA) of the mutant was found to be increased, especially in active site and cavities not involving the mutation site. Free-energy landscape analysis disclosed mutant to have a rich conformational diversity than wild type, with various free-energy conformations of mutant having SASA greater than wild type with SCE open. More residues were found to interact with the mutant CYP51 upon docking of substrate to both the wild and mutant CYP51. These results indicate that, relative to wild type, the N176I mutation of CYP51 in Leishmania mexicana could possibly favor increased substrate binding efficiency.     

Enhanced N2-fixing ability of a deletion mutant of arctic rhizobia with sainfoin (Onobrychis viciifolia)

Summary Mutagenesis provoked by exposure at elevated temperature of the cold-adapted, arctic Rhizobium strain N31 resulted in the generation of five deletion mutants, which exhibited loss of their smaller plasmid (200 kb), whereas the larger plasmid (> 500 kb) was still present in all mutants. Deletion mutants did not show differences from the wild type in the antibiotic resistance pattern, the carbohydrates and organic acids utilization, and the growth rate at low temperature. However, deletion mutants differed from the wild type and among themselves in the ex planta nitrogenase activity, the nodulation index, and the symbiotic effectiveness. The deletion mutant N31.6rif ^r showed higher nodulation index and exhibited higher nitrogenase activity and symbiotic efficiency than the other deletion mutants and the wild type. The process of deletion mutation resulted in the improvement of an arctic Rhizobium strain having an earlier and higher symbiotic nitrogen fixation efficiency than the wild type.     

Analysis of Some Barley Chlorophyll Mutants and Their Response to Temperature Stress

Six barley chlorophyll (Chl) mutants, viridis, flavoviridis, chlorina, xanhta, lutea, and albina, differed in the contents of Chl (a+b) and carotenoids (Cars). In accordance with their Chl-deficient phenotype, the Chl a and b and Car contents of mutants decreased from viridis to albina, only xantha had the same or even higher concentration of Cars as the wild type plant. The albina mutant completely lacked and xantha had a significantly reduced photosynthetic activity. We found quantitative differences in protein contents between wild type and mutant plants, with the lowest concentration per fresh mass in the albina mutant. Chl fluorescence analysis revealed that heat-treated barley leaves of both the wild type and Chl mutants had a lower photosystem 2 efficiency than the untreated ones. With ³⁵S-methionine labelling and SDS-PAGE we found that six to nine de novo synthetized proteins appeared after heat shock (2 h, 42 °C) in the wild type and Chl mutants. In albina the expression of heat shock proteins (HSPs) was reduced to 50 % of that in the wild type. Hence mainly albina mutants, with a completely destroyed proteosynthetic apparatus of the chloroplasts, are able to synthesize a small set of HSPs. The albina mutant is a very useful tool for the study of different gene expression of chloroplast and nuclear DNA.     

丙酮丁醇梭菌硫氧还蛋白系统在溶剂生产过程中的功能

[目的]探究丙酮丁醇梭菌硫氧还蛋白系统在生长和代谢过程中的功能.[方法]使用ClosTron系统对硫氧还蛋白系统中的硫氧还蛋白还原酶基因(trxB)进行插入失活,得到突变株,通过Southern杂交方法验证插入内含子的拷贝数;在基本培养基中进行分批发酵,比较并分析突变株的生长特点;通过pH控制,利用限磷的连续发酵方法使...     

Alterations by a defect in a rice G protein α subunit in probenazole and pathogen-induced responses

The rice dwarf1 (d1) mutant, which is deficient in an α subunit (Gα) of heterotrimeric G protein, was used to obtain specific evidence on the functions of Gα protein in defence signalling in rice. Using proteome analysis, a probenazole‐inducible protein (PBZ1) was detected in the cytosolic fraction of leaf blade of the wild type, but not the d1 mutant. After treatment with probenazol, PBZ1 reached maximal levels at 72 h in the wild type but 96 h in the d1 mutant. The induction of PBZ1 by probenazole treatment was inhibited by protein kinase inhibitors. A 48‐kDa putative mitogen‐activated protein kinase (MAPK) and a 55‐kDa putative Ca²⁺‐dependent protein kinase (CDPK) showed lower activities in the cytosolic fraction of the d1 mutant than that of the wild type. The activities of these protein kinases were enhanced at 24 h in the wild type and 48 h in the d1 mutant after probenazole treatment. Although the d1 mutant responded to the rice blast fungus similarly to the wild type, the d1 mutant developed rice blight symptoms earlier than the wild type when infected with Xoo. In addition, the blight symptoms were more severe on the mutant than on the wild type, and wilting was frequently observed in the d1 mutant. Furthermore, induction by the bacterial infection of the 48‐kDa putative MAPK and PBZ1 was delayed by 2 and 4 d, respectively, in the d1 mutant compared with the wild type. These results indicate that the Gα protein plays a role in the induction of PBZ1 and protein kinases by probenazole and Xoo, and suggest that the 48‐kDa putative MAPK may be involved in a signalling pathway for resistance to bacterial infection.     

Functional analysis of the PsbX protein by deletion of the corresponding gene in Synechocystis sp. PCC 6803

The psbX gene (sml0002) coding for a 4.1 kDa protein in Photosystem II of plants and cyanobacteria was deleted in both wild type and in a Photosystem I-less mutant of the cyanobacterium Synechocystis sp. PCC 6803. Polymerase chain reaction and sequencing analysis showed that the mutants had completely segregated. Deletion of the PsbX protein does not seem to influence growth rate, electron transport or water oxidation ability. Whereas a high light induction of the psbX mRNA could be observed in wild type, deletion of the gene did not lead to high light sensibility. Light saturation measurements and 77K fluorescence measurements indicated a minor disconnection of the antenna in the deletion mutant. Furthermore, fluorescence induction measurements as well as immuno-staining of the D1 protein showed that the amount of Photosystem II complexes in the mutants was reduced by 30%. Therefore, PsbX does not seem to be necessary for the Photosystem II electron transport, but directly or indirectly involved in the regulation of the amount of functionally active Photosystem II centres in Synechocystis sp. PCC 6803.     

Abscisic acid deficiency increases defence responses against Myzus persicae in Arabidopsis

Comparison of Arabidopsis thaliana (Arabidopsis) gene expression induced by Myzus persicae (green peach aphid) feeding, aphid saliva infiltration and abscisic acid (ABA) treatment showed a significant positive correlation. In particular, ABA‐regulated genes are over‐represented among genes that are induced by M. persicae saliva infiltration into Arabidopsis leaves. This suggests that the induction of ABA‐related gene expression could be an important component of the Arabidopsis–aphid interaction. Consistent with this hypothesis, M. persicae populations induced ABA production in wild‐type plants. Furthermore, aphid populations were smaller on Arabidopsis aba1‐1 mutants, which cannot synthesize ABA, and showed a significant preference for wild‐type plants compared with the mutant. Total free amino acids, which play an important role in aphid nutrition, were not altered in the aba1‐1 mutant line, but the levels of isoleucine (Ile) and tryptophan (Trp) were differentially affected by aphids in wild‐type and mutant plants. Recently, indole glucosinolates have been shown to promote aphid resistance in Arabidopsis. In this study, 4‐methoxyindol‐3‐ylmethylglucosinolate was more abundant in the aba1‐1 mutant than in wild‐type Arabidopsis, suggesting that the induction of ABA signals that decrease the accumulation of defence compounds may be beneficial for aphids.     

Photomorphogenic responses to UV radiation IV: a comparative study of UVB effects on growth and pigment accumulation in etiolated and de-etiolated hypocotyls of wild-type and aurea mutant of tomato Lycopersicon esculentum Mill)

ABSTRACT

The question of how de-etiolation of tomato seedling under continuous monochromatic yellow light exerts an influence on UV radiation-induced responses has been studied. Hypocotyl extension and the accumulation of anthocyanins and UV-absorbing compounds was compared in the aurea mutant of tomato and its isogenic wild type. The data of the present paper provide evidence that, during de-etiolation of tomato seedlings, yellow light exerts its effects over seedlings responsiveness to subsequent UV irradiation through several mechanisms: 1) a significant enhancement of shorter UVB wavelength efficiency in both the genotypes; 2) the abolition of UVA -blue light-induced accumulation of UV-absorbing compounds that does not involve pnyA; 3) the disappearance of UVA-blue light-induced hypocotyl growth inhibition that does not involve phyA; 4) higher anthocyanin accumulation rate in response to UV radiation mediated by phyA. Yellow light-induced growth inhibition and accumulation of UV-absorbing compound both mediated by phyA and present only in wild type tomato, appear to be completely separate from the action of UV radiation on the same responses.     

An integration-proficient int mutant of bacteriophage lambda

Summary We have isolated and characterized a novel int mutant of phage . This mutant promotes efficient recombination between the phage and bacterial attachment sites, but, unlike wild type, does not promote efficient recombination of any other pair of attachment sites tested in most conditions. IN particular, recombination between two phage or two prophage attachment sites is poor relative to the wild type frequency. We attribute this unusual phenotype to differences in the distribution of int protein binding sites among different attachment sites (Ross and Landy 1982, 1983). We suggest that int protein molecules bound to one of two recombining DNAs interact with empty sites or with bound proteins on the other, and that the mutant protein acts efficiently only if the distribution of protein binding sites within the two attachment sites is that of the attP-attB pair. Similar discrimination among attachment site pairs by wild type int protein may also modulate recombination frequencies.     

Role of sipA in the early stages of Salmonella typhimurium entry into epithelial cells

Salmonella virulence depends on an ability to invade host cells, which is in turn dependent on a type III protein secretion system encoded in Salmonella pathogenicity island 1 (SPI1). Several protein targets of the SPI1‐encoded secretion system are translocated into host cells, where they subvert cellular processes that contribute to bacterial invasion, actin rearrangement, membrane ruffling and other aspects of virulence. We examined the role of sipA (encoding the translocated protein SipA) and found that a sipA mutant was significantly less invasive in Madin–Darby canine kidney (MDCK) cells than in its parental strain at the earliest stages of infection (5 min). The invasion defect associated with sipA was no longer apparent after 15 min of infection. Confocal microscopy of F‐actin in tetramethyl rhodamine isothiocyanate (TRITC)–phalloidin‐stained MDCK cells revealed no difference in either the frequency or the morphology of membrane ruffles induced by wild‐type and sipA mutant strains of S. typhimurium. Time‐lapse phase‐contrast microscopy of membrane ruffle propagation in live cells confirmed that the sipA mutant induced membrane ruffles as efficiently as the wild‐type bacteria. These studies also revealed that, after ruffle propagation, individual sipA mutant S. typhimurium either invaded more slowly than wild‐type bacteria or failed to invade at all. Furthermore, although wild‐type S. typhimurium typically maintained a position central to the developing membrane ruffle, sipA mutant bacteria frequently moved initially to the periphery of the spreading ruffle and were sometimes observed to detach from it. A wild‐type pattern of invasion was restored to the sipA mutant after the introduction of sipA on a plasmid. Together, these data indicate that loss of sipA significantly decreases the efficiency of S. typhimurium invasion at the early stages of infection without affecting its ability to induce membrane ruffles. It thus appears that the secreted effector protein SipA promotes invasion by a previously unrecognized mechanism separate from the induction of membrane ruffling per se.     

Ion regulation in potassium-sensitive mutants of paramecium tetraurelia

Two recessive mutations of Paramecium tetraurelia confer sensitivity to potassium: While wild-type cells survive when up to 30 mM KCI is added to their growth medium, mutants cease to grow and die when levels of added KCl reach 20–25 mM. Similar sensitivities are seen to Rb⁺ and Cs⁺, but not to Na⁺. Swimming behavior of mutants is indistinguishable from wild type when place in stimulating solutions containing Na⁺, K⁺, or Ba²⁺. Behavioral adaptation to low levels of K⁺ also is indistiguishable from wild type. Flame photometry reveals that one mutant is unable to keep out K⁺ when that ion is at high levels in the medium, while the other mutant readily leaks K⁺ and Na⁺ when those ions are at low levels in the medium. Both mutants have markedly lower internal Na⁺ than does wild type. Problem with K⁺ permeability account for the sensitivity of the one mutant to elevated external K⁺, but the basis of sensitivity in the other mutant is unclear. These mutants expand the range of ion regulation mutants in Paramecium and demonstrate that lesions in cellular ion regulation in this organism need not result in changes in swimming behavior.