Participation of bacteriophage T4 gene 41 in replication repair

The location of the non-essential T4 mutant uvs79, with defective replication repair, is described. After crosses with double mutants dispersed over the early region of T4, a linkage was observed with the double mutant am41 : am42. For more accurate location, crosses were made with single mutants. Uvs79 proved to be located between mutants amC23 and amN81 in gene 41, as shown by 3-point crosses. No genetic complementation with respect to multiplicity reactivation was found between amN81 and uvs79 after a co-infection of an su^? host. Apparently, mutant amN81 is disturbed as to replication repair and, owing to its lack of DNA synthesis, also in replication-dependent recombination repair. Consequently, the product of gene 41 has a function additional to its RNA-primer induction during replication of undamaged DNA. Presumably, the product of gene 41 induces RNA primers opposite DNA regions containing lesions. This capability is believed to be specifically affected by the uvs79 mutation.     

Nonsense mutants in the bacteriophage T4D v gene

Ten UV-sensitive mutants of T4D with the v phenotype were isolated. Of these ten mutants, two are amber and two opal. In UV curves and in photoreactivation and multiplicity reactivation (MR) experiments the nonsense mutants show the v phenotype in su⁻ hosts and almost the T4+ phenotype in su⁺ hosts. The mutations are located between rI and e and are alleles of v₁. In crosses with irradiated and non-irradiated phages the recombinant frequency is not reduced by uvs5.Amber uvs5 propagated in CR63 su⁺ is with B su⁻ just as sensitive to UV as uvs5 propagated in B su⁻, which permits the conclusion that the capsid of T4 phage particles does not contain the v gene product.In addition, four mutants with a relative UV sensitivity equal to that of T4x were isolated. These are discussed in the next paper³³.     

拟南芥乙烯突变体在干旱胁迫下的形态学差异

为了揭示乙烯在植物与环境相互作用过程中的生物学功能,以拟南芥（Arabidopsis thaliana）的ein2-5、ein3-1、EIN3ox、EIL1ox 4种乙烯突变体与Col-0野生型为材料,对比研究它们在干旱胁迫条件下的生长和形态学变化。研究发现,干旱胁迫导致莲座叶直径、叶片面积、花序、水势等指标发生显著变化,同时不同突变体的形态适应特点呈现显著差异。这些结果表明,乙烯积极参与了植物形态塑造过程,与植物的抗旱性具有紧密相关性。     

γ-Ray-sensitive mutants of Neurospora crassa with characteristics analogous to ataxia telagiectasia cell lines

Well characterized γ-ray sensitive mutants of the fungus Neurospora crassa have been screened for characteristics analogous to those of cell lines derived from humans with the genetic disease, ataxia telagiectasia (AT). Two Neurospora mutants, uvs-6 and mus-9, show the AT cell line characterteristics of γ-ray and bleomycin sensitivity, and little or no repression of DNA synthesis following treatment with these agents. Norman human or Neurospora cells show an extensive biphasic DNA synthesis repression (to 50% of control) and when DNA synthesis is analyzed by alkaline gradient centrifugation, repression of DNA synthesis by low doses of γ-radiation occurs primarily in low molecular weight (MW) DNA pieces in both organisms. In AT cells and the uvs-6 mutant, no repression of low or higher MW DNA is seen at low doses, while the mus-9 mutant shows little repression of high MW DNA, but an intermediate level of low MW synthesis. Both mutants have been shown previously to have an increased level of spontaneous chromosome instability as do AT lines. The uvs-6 and mus-9 mutations are known to be due to two different genes in two different epistatic groups. These results demonstrate that AT-like cellular characteristics can arise from defects in at least two and probably any of several genes, and that lower eukaryotes such as Neurospora can provide an inexpensive and useful model for AT while avoiding the problems inherent in using transformed cell lines.     

Ethylene signaling may be involved in the regulation of tocopherol biosynthesis in Arabidopsis thaliana

Tocopherol biosynthesis was investigated in ein3-1, etr1-1 and eto1-1 mutants of Arabidopsis thaliana, which show a defect in ethylene signaling, perception and over-produce ethylene, respectively. A mutation in the EIN3 gene delayed the water-stress related increase in α-tocopherol and caused a reduction in the levels of this antioxidant by ca. 30% compared to the wild type. In contrast to the wild type and ein3-1 mutants, both etr1-1 and eto1-1 mutants showed a sharp (up to 5-fold) increase in α-tocopherol levels during leaf aging. It is concluded that ethylene perception and signaling may be involved in the regulation of tocopherol biosynthesis during water stress and leaf aging.     

Oligomeric form of the light-harvesting chlorophyll a + b-protein complex CP II,phosphatidyldiacylglycerol, Δ3-trans-hexadecenoic acid and energy transfer in Chlamydomonas reinhardtii,wild type and mutants

Analyses of chlorophyll-protein complexes and of lipids were performed with the wild type of Chlamydomonas reinhardtii and three non-photosynthetic mutants: Fl 39, which was a ‘classical’ high-fluorescent Photosystem II (PS II)-lacking mutant, and mf 1 and mf 2, which lacked also functional PS II but were low-fluorescent and showed an abnormally predominant energy transfer from the main light-harvesting antenna towards Photosystem I. An oligomeric form of the chlorophyll a + b-protein complex CP II was clearly isolated from the wild type and the mutant Fl 39 but it was not detected in the mutants mf 1 and mf 2. The three mutants showed total lipid contents close to or greater than that of the wild type. Their phosphatidyldiacylglycerol (PG) contents, on a chlorophyll basis, were higher (Fl 39) or 1.4- (mf 1) and 2.0- (mf 2) times lower than that of the wild type. The fatty acid compositions of the wild type and of the mutant Fl 39 were comparable, showing about equal amounts of a C18 series and a C16 series which included the Δ3-trans-hexadecenoic acid (C16:1-trans). This C16:1-trans was not detected in the mutants mf 1 and mf 2 which contained the other fatty acids. These results indicate correlations between lack of C16:1-trans-containing PG, lack of an oligomeric form of CP II and an impaired mechanism of the regulation of excitation energy transfer from the main chlorophyll a + b antenna.     

Arsenic tolerance in a Chlamydomonas photosynthetic mutant is due to reduced arsenic uptake even in light conditions

Arsenate resistance has been used for screening for photosynthetic mutants of Chlamydomonas, since photosynthetic mutants, such as CC981 defective in phosphoribulokinase, were shown to have arsenate resistance. Also, another type of arsenate-resistant mutants, including AR3 that lacks a homolog of a phosphate (Pi) transporter, PTB1, has been isolated. We investigated the uptake of Pi and arsenate, and the gene expression of Pi transporters, which are involved in both Pi and arsenate transport, in mutants CC981 and AR3. In the wild type, both Pi and arsenate uptake were initially high, but were inactivated in the presence of arsenate with time, especially in the dark. In contrast, both mutants were shown to exhibit higher Pi uptake, but lower arsenate uptake than the wild type, regardless of the presence or absence of light. Then, the gene expression of Pi transporters in the cells used for the uptake measurements was investigated and compared between the mutants and the wild type. In CC981, the mRNA levels of PTA2 and PTA4 were higher, while those of PTB3 and PTB5 were lower, as compared with in the wild type. In AR3, those of PTA2 and PTB2 were higher, but that of PTB5 was lower than in the wild type. These findings suggest that the arsenate resistance shown by the mutants in light is due to reduction of arsenate uptake probably through the down-regulation of some Pi transporter expression, while the Pi uptake maintained even in the dark is possibly related to higher expression of other Pi transporter(s) than in the wild type.     

Events Surrounding the Early Development of Euglena Chloroplasts: 14. Biosynthesis of Cytochrome c-552 in Wild Type and Mutant Cells

Lack of a suitable assay has thwarted attempts to measure cytochrome c-552 in dark-grown wild type cells of Euglena gracilis var. bacillaris in mutants and in other situations where the concentrations are low. Purification methods are described based on electrofocusing which provide a cytochrome c-552 preparation homogeneous enough to elicit a single reactive antibody in rabbits; this antibody is then used as a specific and sensitive assay for cytochrome c-552. Dark-grown cells of wild type and of mutants O₁BS, O₂BX, G₁BU and P₁BXL (which make normal sized chloroplasts with abnormal internal structure in the light) have 0.02 to 0.1 × 10⁻¹¹ micromoles of cytochrome c-552 per cell, 10 to 150 times less than light-grown cells. Light-grown cells of these mutants and of wild type show a ratio of chlorophyll to cytochrome of about 300 (mole to mole). Cytochrome c-552 is undetectable in dark-grown Y₁BXD, Y₃BUD, and W₃₄ZUD which cannot carry plastid development beyond the proplastid in light; the light-grown cells of these mutants have levels of cytochrome similar to or lower than dark-grown wild type cells. Cytochrome c-552 is undetectable in light- and dark-grown mutants in which plastid DNA is undetectable (such as Y₂BUL, W₃BUL, W₈BHL, and W₁₀BSmL) consistent with the view, but not proving, that this molecule may be coded, at least in part, in plastid DNA. During light-induced chloroplast development in resting cells, cytochrome c-552 formation behaves in all respects like chlorophyll except that the dark-grown cells contain low amounts of the cytochrome c-552 but lack chlorophyll. Thus, both cytochrome c-552 and chlorophyll show the same lag period even when the length is changed by nutritional manipulation; preillumination largely eliminates the lag in the formation of both molecules, cycloheximide and streptomycin both inhibit the biosynthesis of chlorophyll and cytochrome c-552 in the same manner, and the formation of both during chloroplast development is strictly light-dependent. It is shown that chloroplasts isolated from Euglena by methods thought to give intact organelles, lack 95% of the cytochrome c-552; this and the loss of similar molecules may explain why these isolated chloroplasts are not photosynthetically active.     

Adaptation of Chlamydomonas reinhardtii High-CO(2)-Requiring Mutants to Limiting CO(2)

Photosynthetic characteristics of four high-CO₂-requiring mutants of Chlamydomonas reinhardtii were compared to those of wild type before and after a 24-hour exposure to limiting CO₂ concentrations. The four mutants represent two loci involved in the CO₂-concentrating system of this unicellular alga. All mutants had a lower photosynthetic affinity for inorganic carbon than did the wild type when grown at an elevated CO₂ concentration, indicating that the genetic lesion in each is expressed even at elevated CO₂ concentrations. Wild type and all four mutants exhibited adaptive responses to limiting CO₂ characteristic of the induction of the CO₂-concentrating system, resulting in an increased affinity for inorganic carbon only in wild type. Although other components of the CO₂-concentrating system were induced in these mutants, the defective component in each was sufficient to prevent any increase in the affinity for inorganic carbon. It was concluded that the genes corresponding to the ca-1 and pmp-1 loci exhibit at least partially constitutive expression and that all components of the CO₂-concentrating system may be required to significantly affect the photosynthetic affinity for inorganic carbon.     

BtubA-BtubB Heterodimer Is an Essential Intermediate in Protofilament Assembly

Background

BtubA and BtubB are two tubulin-like genes found in the bacterium Prosthecobacter. Our work and a previous crystal structure suggest that BtubB corresponds to α−tubulin and BtubA to β−tubulin. A 1∶1 mixture of the two proteins assembles into tubulin-like protofilaments, which further aggregate into pairs and bundles. The proteins also form a BtubA/B heterodimer, which appears to be a repeating subunit in the protofilament.

Methodology/Principal Findings

We have designed point mutations to disrupt the longitudinal interfaces bonding subunits into protofilaments. The mutants are in two classes, within dimers and between dimers. We have characterized one mutant of each class for BtubA and BtubB. When mixed 1∶1 with a wild type partner, none of the mutants were capable of assembly. An excess of between-dimer mutants could depolymerize preformed wild type polymers, while within-dimer mutants had no activity.

Conclusions

An essential first step in assembly of BtubA + BtubB is formation of a heterodimer. An excess of between-dimer mutants depolymerize wild type BtubA/B by sequestering the partner wild type subunit into inactive dimers. Within-dimer mutants cannot form dimers and have no activity.     

Recherche du cytochrome b-563 et du p700 chez trois mutants non photosynthétiques de Chlamydomonas reinhardti

Studies of cytochrome b-563 and P 700 in three non-photosynthetic mutants of Chlamydomonas reinhardAn investigation into the presence of cytochrome b-563 and of P700 in three non-photosynthetic mutants (Fl 5, Fl 9, Fl 15) of Chlamydomonas reinhardti was carried out. These three mutants exhibit several functional anomalies (described elsewhere), which indicate that the electron transport chain between the two photoreactions is blocked. In addition, Fl 5 is unable to carry out any reaction related to System I.Mutants Fl 9 and Fl 15 had less than 19% of the cytochrome b-563 content found in the wild type (which was about 0.27 mole per 100 moles chlorophyll); mutant Fl 5 had more than 80% of this content. The deficiencies (only traces) in bound cytochrome c-553, previously observed with mutants Fl 9 and Fl 15, but not Fl 5, were confirmed (in the wild type, there is about 0.20 mole bound cytochrome per 100 moles chlorophyll).Photosystem I particles, prepared from wild type and mutants Fl 9 and Fl 15 chloroplast fragments, had about 2 (Fl 9, Fl 15) and 3 (wild type) moles P700 per 100 moles chlorophyll. Mutant Fl 5 particles showed neither P700 spectroscopic characteristics nor photooxidation activity; their chlorophyll a/b ratio was lower by a factor of 2 and protein/chlorophyll ratio about 8 times higher than in the wild type particles. This mutant appears to lack P700.     

Three Classes of Abscisic Acid (ABA)-Insensitive Mutations of Arabidopsis Define Genes that Control Overlapping Subsets of ABA Responses

Wild type and three abscisic acid (ABA)-insensitive mutants of Arabidopsis (ABI1, ABI2, and ABI3) were compared for their ability to respond to ABA for a variety of ABA-inducible responses throughout the life cycle of the plants. The responses tested included effects on seedling growth, proline accumulation in seedlings, ABA-regulated protein synthesis in plantlets, and seed storage protein and lipid synthesis and accumulation. The abi1 and abi2 mutants showed reduced sensitivity to ABA for inhibition of seedling growth, induction of proline accumulation, and alterations in protein synthesis patterns during vegetative growth, but had wild type levels of storage reserves. In contrast, the abi3 mutant had wild type sensitivity for induction of proline accumulation and was only slightly less responsive to ABA with respect to effects on seedling growth and changes in patterns of protein synthesis. The major effects of this mutation were on seed development. Seeds of the abi3 mutant had two-thirds of the wild type level of storage protein and one-third the wild type level of eicosenoic acid, the major fatty acid component of storage lipids in wild type seeds. These results show that none of the abi mutants is insensitive for all ABA-inducible responses and that the abi3 effects are not seed-specific. Comparison of the degree of ABA sensitivity of monogenic mutant lines with that of digenic mutant lines carrying pairwise combinations of the abi mutations suggests that ABA responses in mature seeds are controlled by at least two parallel pathways.     

The Role of Mitogen-Activated Protein (MAP) Kinase Signaling Components in the Fungal Development,Stress Response and Virulence of the Fungal Cereal Pathogen Bipolaris sorokiniana

Mitogen-activated protein kinases (MAPKs) have been demonstrated to be involved in fungal development, sexual reproduction, pathogenicity and/or virulence in many filamentous plant pathogenic fungi, but genes for MAPKs in the fungal cereal pathogen Bipolaris sorokiniana have not been characterized. In this study, orthologues of three MAPK genes (CsSLT2, CsHOG1 and CsFUS3) and one MAPK kinase kinase (MAPKKK) gene (CsSTE11) were identified in the whole genome sequence of the B. sorokiniana isolate ND90Pr, and knockout mutants were generated for each of them. The ∆Csfus3 and ∆Csste11 mutants were defective in conidiation and formation of appressoria-like structures, showed hypersensitivity to oxidative stress and lost pathogenicity on non-wounded leaves of barley cv. Bowman. When inoculated on wounded leaves of Bowman, the ∆Csfus3 and ∆Csste11 mutants were reduced in virulence compared to the wild type. No morphological changes were observed in the ∆Cshog1 mutants in comparison with the wild type; however, they were slightly reduced in growth under oxidative stress and were hypersensitive to hyperosmotic stress. The ∆Cshog1 mutants formed normal appressoria-like structures but were reduced in virulence when inoculated on Bowman leaves. The ∆Csslt2 mutants produced more vegetative hyphae, had lighter pigmentation, were more sensitive to cell wall degrading enzymes, and were reduced in virulence on Bowman leaves, although they formed normal appressoria like the wild type. Root infection assays indicated that the ∆Cshog1 and ∆Csslt2 mutants were able to infect barley roots while the ∆Csfus3 and ∆Csste11 failed to cause any symptoms. However, no significant difference in virulence was observed for ∆Cshog1 mutants while ∆Csslt2 mutants showed significantly reduced virulence on barley roots in comparison with the wild type. Our results indicated that all of these MAPK and MAPKKK genes are involved in the regulation of fungal development under normal and stress conditions and required for full virulence on barley plants.     

Mutational and kinetic analysis of basic amino acid transport in the cyanobacterium Synechocystis sp. PCC 6803

Two nitrogen-deregulated mutants of Phanerochaete chrysosporium, der8-2 and der8-5, were isolated by subjecting wild type conidia to gamma irradiation, plating on Poly-R medium containing high levels of nitrogen, and identifying colonies that are able to decolorize Poly-R. The mutants showed high levels of ligninolytic activity (¹⁴C-synthetic lignin ¹⁴CO₂), and lignin peroxidase, manganese peroxidase and glucose oxidase activities in both low nitrogen (2.4 mM) and high nitrogen (24 mM) media. The wild type on the otherhand displayed these activities in low nitrogen medium but showed little or no activities in high nitrogen medium. Fast protein liquid chromatographic analyses showed that the wild type as well as the der mutants produce three major lignin peroxidase peaks (designated L1, L2 and L3) with lignin peroxidase activity in low nitrogen medium. Furthermore, in low nitrogen medium, mutant der8-5 produced up to fourfold greater lignin peroxidase activity than that produced by the wild type. In high nitrogen medium, the wild type produced no detectable lignin peroxidase peaks whereas the mutants produced peaks L1 and L2, but not L3, and a new lignin peroxidase protein peak designated LN. Mutants der8-2 and der8-5 also produced high levels of glucose oxidase, an enzyme known to be associated with secondary metabolism and an important source of H₂O₂ in ligninolytic cultures, both in low and high nitrogen media. In contrast, the wild type produced high levels of glucose oxidase in low nitrogen medium and only trace amounts of this enzyme in high nitrogen medium. The results of this study indicate that the der mutants are nitrogen-deregulated for the production of a set of secondary metabolic activities associated with lignin degradation such as lignin peroxidases, manganese peroxidases and glucose oxidase.     

Inactivation of genes,encoding tocopherol biosynthetic pathway enzymes,results in oxidative stress in outdoor grown Arabidopsis thaliana

Tocopherols (α-, β-, γ- and δ-tocopherols) represent a group of lipophilic antioxidants which are synthesized only by photosynthetic organisms. It is widely believed that protection of pigments and proteins of photosynthetic system and polyunsaturated fatty acids from oxidative damage caused by reactive oxygen species (ROS) is the main function of tocopherols. The wild type Columbia and two mutants of Arabidopsis thaliana with T-DNA insertions in tocopherol biosynthesis genes – tocopherol cyclase (vte1) and γ-tocopherol methyltransferase (vte4) – were analyzed after long-term outdoor growth. The concentration of total tocopherol was up to 12-fold higher in outdoor growing wild type and vte4 plant lines than in plants grown under laboratory conditions. The vte4 mutant plants had a lower concentration of chlorophylls and carotenoids, whereas the mutant plants had a higher level of total glutathione than of wild type. The activities of antioxidant enzymes superoxide dismutase (SOD, EC 1.15.1.1) and ascorbate oxidase (AO, EC 1.10.3.3) were lower in both mutants, whereas activities of catalase (EC 1.11.1.6) and ascorbate peroxidase (APx, EC 1.11.1.11) were lower only in vte1 mutant plants in comparison to wild type plants. However, the activity of guaiacol peroxidase (GuPx, EC 1.11.1.7) was higher in vte1 and vte4 mutants than that in wild type. Additionally, both mutant plant lines had higher concentration of protein carbonyl groups and oxidized glutathione compared to the wild type, indicating the development of oxidative stress. These results demonstrate in plants that tocopherols play a crucial role for growth of plants under outdoor conditions by preventing oxidation of cellular components.     

Transport defects as the physiological basis for eye color mutants of Drosophila melanogaster

Kynurenine-H ³ transport and conversion to 3-hydroxykynurenine were studied in organ culture using the Malpighian tubules and developing eyes from wild type and the eye color mutants w, st, 1td, ca, and cn of Drosophila melanogaster. Malpighian tubules from wild type have the ability to concentrate kynurenine and convert it to 3-hydroxykynurenine. The tubules from w, st, 1td, and ca are deficient in the ability to transport kynurenine, as are the eyes of the mutants w, st, and 1td. This defect in kynurenine transport provides a physiological explanation for the phenotypic properties of the mutants. The relationship of these measurements to previous observations on these eye color mutants is discussed and the transport defect hypothesis is consistently supported. We have concluded that several of the eye color mutants in Drosophila are transport mutants.