Characterization of a Pseudomonas aeruginosa transposon insertion mutant with defective release of exoenzymes

A Pseudomonas aeruginosa transposon insertion mutant with defective release of several exoenzymes has been characterized. The Tn5-751 insertion mutation was located in the previously described xcp-1 locus at 0 min on the chromosomal map and caused several exoenzymes to remain in cell-bound form. At least one of the exoenzymes, elastase, was accumulated in the periplasmic space. The periplasmic elastase had the same Mr as the extracellular enzyme produced by the wild-type strain. The virulence of the mutant was comparable to that of wild-type strains in experimental burn infection in mice. The presence of an easily selectable antibiotic resistance marker in the xcp-1 locus offers the possibility of cloning the gene(s) involved in exoprotein secretion.     

Characterization of an Escherichia coli lysA insertion targeted mutant using phenotype arrays

The objective of this study was to investigate the effect of a lysine biosynthesis insertion mutation on the growth response and phenotype of Escherichia coli. The lysA gene encodes the last enzyme in the lysine biosynthetic pathway in most bacteria. This E. coli insertion mutant exhibited altered growth physiology and phenotype of the recipient E. coli. The constructed mutant could grow in the absence of lysine supplementation although the extent of growth after 7 h incubation in the presence of most lysine concentration was significantly (p<0.05) decreased compared to that observed with the parent E. coli strain. The mutant was also less able to utilize carbon and nitrogen substrates than the parent E. coli strain as determined by using phenotype arrays. These results suggest that the carbon and nitrogen phenotype profiles of E. coli when measured on phenotype arrays are altered after targeted insertion mutagenesis in the lysA gene. Creation of altered phenotypes may have potential for pharmaceutical and biotechnological applications of lysine E. coli metabolism.     

T—DNA插入水稻群体中卷叶突变体R1—A2的遗传分析

在根癌农杆菌介导的T-DNA(携带有除草剂Basta抗性基因bar和Ds因子)转化中花11水稻群体中,获得了一个叶片发生明显内卷的突变体Rl-A。经过连续三代的分离鉴定,获得突变体的纯合株(Rl-A2),并与中花11号进行杂交,在调查的36个F1植株中,全部表现为卷叶,并对Basta除草剂都表现为抗性。在852个F2单株中,卷叶为645株,正常叶207株,卷叶和正常叶的比例为3：1,其中,卷叶株均对Basta表现抗性,正常叶株均对Basta表现敏感,表明卷叶性状和Basta抗性存在着共分离关系。用扩增DS因子的引物,对F2中45个卷叶抗性株进行PCR鉴定,都获得预期长度的Ds因子片段,进一步表明在这些卷叶的植株中都有T-DNA的插入;而30个正常叶敏感株都不能检测到DS的特征片段。在以卷叶突变(Rl-A2)为回交亲本的F1B1植株中,全部植株表现卷叶;在以中花11号为回交亲本的F1B1植株中,卷叶和正常叶植株的分离比为1：1。上述结果表明该卷叶突变是个显性突变,受一个基因所控制,且该基因的突变与T-DNA的插入有关。     

Characterization of a novel high-tillering dwarf 3 mutant in rice

Tiller number and culm length are important components of plant architecture and determinate grain production in rice.A line SIL046,derived from an introgression lines population developed by an accession of common wild rice (Oryza rufipogon Griff.) and a high-yielding indica cultivar Guichao 2 (Oryza sativa L.).exhibits a higher tiller number and shorter culm length phenotype than the recipient parent Guichao 2 (GC2).Genetic analysis showed that the high-tillering dwarf phenotype was controlled by a novel single recessive gene,referred to as the high-tillering dwarf3 (htd3),which located within the genetic distance of 13.4 cM between SSR makers RM7003 and RM277 on chromosome 12.By means of fine-mapping strategy,we mapped HTD3 gene within the genetic distance of 2.5 cM and the physical distance of 3100 kb in the centromere of chromosome 12.Further identification of HTD3 gene would provide a new opportunity to uncover the molecular mechanism of the development of culm and tiller,two important components of yields in rice.     

Characterization of a zebra mutant of rice with increased susceptibility to light stress

The rice zebra mutant TCM248 is a single recessive mutant. This mutant develops transverse-striped leaves with green and white sectors under alternate light/dark growth conditions. Mutants that were grown under a higher light intensity during the light period showed a more intense striped phenotype. The white tissues contained abnormal chloroplasts with few internal membrane structures, while the green tissues in the mutants contained normal chloroplasts. The white tissue contained only trace amounts of Chls and carotenoids, and mRNA accumulation of nuclear genes encoding chloroplast proteins (rbcS, cab) was strongly suppressed compared to that in the wild type plants. A series of growth condition shift experiments demonstrated that the mutant displayed the striped phenotype only if it was exposed to the alternate light/dark growth conditions during a limited stage of early leaf development. These data suggest that the zebra gene is involved in the acquisition of photoprotective capacity of the plants and that this gene functions at an early stage of chloroplast differentiation.     

Characterization of a rice mutant having an increased susceptibility to light stress at high temperature

The effect of light irradiation at high temperature was investigated in a rice mutant, spl-2 , which is sensitive to solar radiation. Dead spots appeared on the mutant leaves when cultured at a high temperature (40°C) under strong white-light illumination (15 W m⁻²). A similar damage was also observed in the wild-type leaves under the same conditions when the plants were preincubated in the dark for one day. Preillumination with weak light (6 W m⁻²) lessened the irradiation-induced damage in the wild-type. These observations suggest that in rice plants the acclimatization to weak light has a protective effect against strong irradiation at high temperature, and the spl-2 locus participates in the mechanisms of the acclimatization. The action spectrum for the irradiation-induced damage at the high temperature (40°C) in the spl-2 leaves indicated that the maximum damage occurred at around 480 nm and, in a lesser extent, at around 680 nm. Activity of the O⁻₂₋ and H₂O₂₋scavenging enzymes in the spl−2 leaves were almost the same or somewhat higher than those in the wild-type after irradiation with strong white light (15 W m⁻²) at 40°C, whereas the content of ascorbic acid in the spl−2 decreased significantly compared with the wild type.     

Generation of transgenic rice lines with reduced contents of multiple potential allergens using a null mutant in combination with an RNA silencing method

Rice seed proteins are known to be a causative antigen in some patients with food allergy, especially cereal allergy, with clinical symptoms such as eczema and dermatitis. The α-amylase/trypsin inhibitors (14-16 kDa), α-globulin (26 kDa) and β-glyoxalase I (33 kDa) are regarded as major potential allergens of rice (Oryza sativa L.) seed based on specific recognition by serum IgE from allergy patients. In order to suppress the production of these major allergens in rice grains, a mutant in the 'Koshihikari' background lacking the 26 kDa allergen (GbN-1) was used as a host for RNA silencing. A binary vector harboring two RNA interference (RNAi) gene cassettes for suppression of 14-16 kDa and 33 kDa allergens driven by the 13 kDa and 10 kDa prolamin endosperm-specific promoters, respectively, was introduced into the GbN-1 genome by Agrobacterium-mediated transformation. In the most promising transgenic line, the content of the three potential allergens was remarkably reduced to a very faint level without a change in seed phenotype. IgE binding of 15 patients' sera to the transgenic rice seed mostly deficient in the three major allergens was on average only about 10% that of the control wild-type rice, suggesting that these three accounted for the great majority of rice seed causative allergens recognized by patients' IgE and that the sequential allergen deletion/reduction strategy works in the development of hypo-allergenic rice lines.     

Characterization and fine mapping of a shortened uppermost internode mutant in rice

Internode elongation is an important agronomic trait in rice that is associated with lodging, yield, flooding adaptation, and hybrid seed production. We identified a novel rice mutant line showing a shortened uppermost internode among the rice Ac/Ds insertional mutant population and named it shortened uppermost internode 4 (sui4). Interestingly, T1 plants of this line segregated into three phenotypes: extremely shortened uppermost internode-type, normal wild-type, and intermediate-type. The phenotypes of F1 plants and F2 plants from the cross of sui4 with its original variety, Dongjin, indicated that the SUI4 gene shows incomplete dominance or semidominance. Because the Ds genotypes did not co-segregate with the sui4 phenotypes, we performed mapping of this gene with 273 F2 plants from a cross between sui4 and Milyang23. Primary mapping revealed that the SUI4 locus was located between the S07012 and S07015 markers on rice chromosome 7. Further fine mapping with 36 F3 lines derived from F2 plants that have recombination in this region narrowed down the location of SUI4 to the 1.1-Mbp interval of RM1253–S07015.     

Isolation and characterization of a low phytic acid rice mutant reveals a mutation in the rice orthologue of maize MIK

Using a forward genetics approach, we isolated two independent low phytic acid (lpa) rice mutants, N15-186 and N15-375. Both mutants are caused by single gene, recessive non-lethal mutations, which result in approximately 75% (N15-186) and 43% (N15-375) reductions in seed phytic acid (inositol hexakisphosphate). High-performance liquid chromatography and GC–MS analysis of seed extracts from N15-186 indicated that, in addition to phytic acid, inositol monophosphate was significantly reduced whereas inorganic phosphorus and myo-inositol were greatly increased when compared with wild-type. The changes observed in N15-186 resemble those previously described for the maize lpa3 mutant. Analysis of N15-375 revealed changes similar to those observed in previously characterized rice lpa1 mutants (i.e. significant reduction in phytic acid and corresponding increase in inorganic phosphorus with little or no change in inositol phosphate intermediates or myo-inositol). Further genetic analysis of the N15-186 mutant indicated that the mutation, designated lpa N15-186, was located in a region on chromosome 3 between the microsatellite markers RM15875 and RM15907. The rice orthologue of maize lpa3, which encodes a myo-inositol kinase, is in this interval. Sequence analysis of the N15-186 allele of this orthologue (Os03g52760) revealed a single base pair change (C/G to T/A) in the first exon of the gene, which results in a nonsense mutation. Our results indicate that lpa N15-186 is a mutant allele of the rice myo-inositol kinase (OsMIK) gene. Identification and characterization of lpa mutants, such as N15-186, will facilitate studies on the regulation of phytic acid biosynthesis and accumulation and help address questions concerning the contribution of the inositol lipid-dependent and independent biosynthetic pathways to the production of seed phytic acid. The mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.     

Characterization of a rice chlorophyll-deficient mutant using the T-DNA gene-trap system

We have previously generated a large pool of T-DNA insertional lines in rice. In this study, we screened those T-DNA pools for rice mutants that had defective chlorophylls. Among the 1,995 lines examined in the T2 generation, 189 showed a chlorophyll-deficient phenotype that segregated as a single recessive locus. Among the mutants, 10 lines were beta-glucuronidase (GUS)-positive in the leaves. Line 9-07117 has a T-DNA insertion into the gene that is highly homologous to XANTHA-F in barley and CHLH in ARABIDOPSIS: This OsCHLH gene encodes the largest subunit of the rice Mg-chelatase, a key enzyme in the chlorophyll branch of the tetrapyrrole biosynthetic pathway. In the T2 and T3 generations, the chlorina mutant phenotypes are co-segregated with the T-DNA. We have identified two additional chlorina mutants that have a Tos17 insertion in the OsCHLH gene. Those phenotypes were co-segregated with Tos17 in the progeny. GUS assays and RNA blot analysis showed that expression of the OsCHLH gene is light inducible, while TEM analysis revealed that the thylakoid membrane of the mutant chloroplasts is underdeveloped. The chlorophyll content was very low in the OschlH mutants. This is the first report that T-DNA insertional mutagenesis can be used for functional analysis of rice genes.     

Characterization of a replicative DNA polymerase mutant with reduced fidelity and increased translesion synthesis capacity

Changing a highly conserved amino acid in motif A of any of the four yeast family B DNA polymerases, DNA polymerase alpha, delta, epsilon or zeta, results in yeast strains with elevated mutation rates. In order to better understand this phenotype, we have performed structure-function studies of homologous mutants of RB69 DNA polymerase (RB69 pol), a structural model for family B members. When Leu415 in RB69 pol is replaced with phenylalanine or glycine, the mutant polymerases retain high-catalytic efficiency for correct nucleotide incorporation, yet have increased error rates due to increased misinsertion, increased mismatch extension and inefficient proofreading. The Leu415Phe mutant also has increased dNTP insertion efficiency opposite a template 8-oxoG and opposite an abasic site. The 2.5 A crystal structure of a ternary complex of RB69 L415F pol with a correctly base-paired incoming dTTP reveals that the phenylalanine ring is accommodated within a cavity seen in the wild-type enzyme, without steric clash or major change in active site geometry, consistent with retention of high-catalytic efficiency for correct incorporation. In addition, slight structural differences were observed that could be relevant to the reduced fidelity of L415F RB69 pol.     

Characterization of a Schizosaccharomyces pombe morphological mutant altered in the galactomannan content

In a search for Schizosaccharomyces pombe mutants resistant to the antifungal agent papulacandin B, a morphological mutant was isolated. The mutant is round shaped in contrast to the rod shaped parental strain. This morphological defect segregated as a recessive Mendelian character and was not observed in other papulacandin B resistant mutants belonging to the same complementation group. The mutation mapped in the right arm of S. pombe chromosome III very close to pap1 marker. Mutant cell walls were more susceptible to alkali extraction and Novozyme degradation than those from the wild-type. A specific reduction in the cell wall galactomannan fraction was the only significant difference detected as compared to the wild-type strain. Levels of beta (1,3)-glucan and mannan synthases as well as other enzymic periplasmic mannoproteins were very similar in wild type and mutant strains.     

Characterization and genetic analysis of a light- and temperature-sensitive spotted-leaf mutant in rice

A rice spotted-leaf mutant was isolated from an ethane methyl sulfonate (EMS) -induced IR64 mutant bank. The mutant, designated as spl30 (spotted-leaf30), displayed normal green leaf color under shade but exhibited red-brown lesions under natural summer field conditions. Initiation of the lesions was induced by light and the symptom was enhanced at 33 (°) C relative to 26 (°) C. Histochemical staining did not show cell death around the red-brown lesions. Chlorophyll contents in the mutant were significantly lower than those of the wild type while the ratio of chlorophyll a/b remained the same, indicating that spl30 was impaired in biosynthesis or degradation of chlorophyll. Disease reaction patterns of the mutant to Xanthomonas oryzae pv. oryzae were largely unchanged to most races tested except for a few strains. Genetic analysis showed that the mutation was controlled by a single recessive gene, tentatively named spl30(t), which co-segregated with RM15380 on chromosome 3, and was delimited to a 94 kb region between RM15380 and RM15383. Spl30(t) is likely a novel rice spotted-leaf gene since no other similar genes have been identified near the chromosomal region. The genetic data and recombination populations provided in this study will enable further fine-mapping and cloning of the gene.     

水稻Ds插入淡绿叶突变体的鉴定和遗传分析

Ac/Ds插入突变是水稻基因功能鉴定的有力工具之一。文章从水稻中花11 Ds-T-DNA转化纯合体与Ac-T-DNA 转化纯合体的杂交群体中筛选到一个淡绿叶突变体。该突变体在三叶期由绿苗转为淡绿叶苗, 自然光照下突变体迅速焦枯, 但是在弱光照条件下, 突变体能缓慢生长至开花结实; 突变体光合作用特性研究表明该突变是典型的光抑制突变体。遗传分析表明该突变为Ds插入导致的隐性突变。     

Characterization of a respiratory mutant of Escherichia coli with reduced uptake of aminoglycoside antibiotics

A strain of Escherichia coli (NSW77) which is partially resistant to streptomycin was isolated by selecting for growth on plates supplemented with 12.5 μg/ml streptomycin, a concentration which completely inhibits growth of wild-type strains. The low-level resistance of the mutant appears to result from a reduced ability to accumulate streptomycin intracellularly. In addition, the mutant strain is unable to use succinate for growth because of a defective respiratory chain. Thus, membranes of the mutant strain were found to have approximately half the NADH and D-lactate oxidase activity of the parent strain. Succinate oxidase activity was reduced more drastically, to a level of 7% that of the parent strain. Moreover, membranes of the mutant were found to contain demethyl-menaquinone and, in place of ubiquinone, a structural analogue, 2-octaprenyl-3-methyl-6-methoxy-1,4 benzoquinone. The mutation responsible for both the Suc⁻ phenotype and partial resistance to streptomycin was found to be located near minute 15 on the bacterial chromosome. Both the biochemical and genetic evidence suggests that the mutation in strain NSW77 resides in the ubi F gene. Another previously characterized ubi F strain was also found to have a reduced capacity to take up an aminoglycoside antibiotic (gentamicin). These results suggest that the respiratory defects in ubi F strains are responsible for the reduced capacity of such strains to accumulate aminoglycosides.