Amber mutants of Salmonella-phage P22 in genes engaged in the establishment of lysogeny

Summary Phage mutants were isolated with amber mutations in genes necessary for establishment of lysogeny. These mutants form turbid plaques on su ⁺ strain 527R1 and clear plaques of different types on LT2. According to complementation tests, fourteen mutants fall in the c ₂ gene, four in the c ₃ gene but no amber mutants were found belonging to the c ₁ gene. Pulse labelling experiments to follow DNA synthesis after phage infection were done with the mutants classified by complementation tests. Furthermore the labelling experiments demonstrated that the nonleaky c ₃ amber mutants displayed the same DNA synthesis pattern as c ₁ missense mutants. Since these c ₃ amber mutants complement missense c ₁ mutants it is concluded that the c ₃ and c ₁ genes must act together for the first transient repression of DNA synthesis, i.e., seven minutes after infection. It is suggested that clear plaque forming c ₁ amber mutants cannot be isolated because of polarity leading to defectivity of lysogenic as well as of lytic functions.The majority of the experiments presented are a part of the dissertation of H. D. Dopatka at the University of Göttingen.     

Temperature sensitive radiosensitive mutants of the yeast Saccharomyces paradoxus

Summary The properties of four radiation sensitive ts mutants of the yeast Saccharomyces paradoxus, txs ₁, txs ₂, txs ₄and txs ₅,were studied. Genetic analysis proves the mutants to be non-allelic. The mutants are sensitive to X-rays and ultraviolet. Holding at 37°C in a complete medium but not in water sharply decreases the viability. With txs ₂this effect could also be observed in water. The mutants formed filaments and showed abnormal cytokinesis under restrictive conditions, this effect being the most pronounced with txs ₄.The frequency of mitotic recombiantion induced by X-rays and UV is much lower in the mutants (except txs ₂)as compared to the wild type. Restrictive temperature somewhat increased the mitotic recombination in the mutants (excepts txs ₁).Shift of the mutants into restrictive temperature leads to immediate, inhibition of DNA synthesis; the extent of this inhibition correlates with the levels of radiation and temperature sensitivity, both being higher in txs ₂and txs ₄.Elevated temperature also decreased RNA and protein synthesis in txs ₂.It is suggested that mutation txs ₂affects a function participating at the last stages in postirradiation repair of DNA, probably DNA ligase, txs ₄seemed to affect the process of nuclear division.     

Response of ad-2 mutants of Saccharomyces cerevisiae to carbon dioxide

Summary Confirmation that the ad-2 locus of yeast controls the carboxylation of aminoimidazole ribonucleotide (AIR) to 5-amino-4-imidazole carboxylate ribonucleotide (CAIR) is provided by the observation that 21 out of a sample of 113 ad-2 mutants were affected by CO₂. 19 of the mutants were stimulated by CO₂ and 2 were inhibited. The majority of the CO₂-stimulated mutants were confined to one section of the complementation map of the ad-2 locus.     

Identification of the primary lesion in a protoporphyrin accumulating mutant of Chlamydomonas reinhardtii

Summary A group of chlorophyll deficient mutants (br _s mutants) of Chlamydomonas accumulates protoporphyrin and has poorly developed chloroplast membrane systems (Wang et al. 1974). In order to determine whether a poorly developed chloroplast membrane system is the reason for, or the result of, the inability of the br _s mutants to metabolize protoporphyrin to chlorophyll, a second mutation was selected which restored chlorophyll synthesis in br _s mutants. One such double mutant (br _s-2 g-4) was analyzed. The double mutant br _s-2 g-4 has partially restored chlorophyll synthesis, but has defective photosystem II and photosystem I electron transport as well as abnormal chloroplast ultrastructure. Since these defects are not present in cells carrying only the g-4 mutation, they are presumed to be caused by the br _s-2 mutation. It is concluded that a defect in chloroplast membrane development resulting from the br _s-2 mutation causes an apparent defect in magnesium chelation by protoprophyrin. This is consistant with evidence that chlorophyll biosynthesis from magnesium protoporphyrin to chlorophyll takes place on the chloroplast membranes.     

A new class of clear mutants from coliphage 434 hy not complementing CII and C3 mutants for lysogenization

Summary Clear mutants which differ from regular C _I , C _II , CIIIand y mutants have been isolated from phage 434 hy. These mutants resemble C _I mutants in plaque and spot phenotype but efficiently complement C _I mutants for lysogenization. Like C _II mutants, they do not complement authentic C _II mutants for lysogenization but in contrast to C _II mutants they also fail to complement C _III mutants. They map between the lambda-434 non-homology region and Co ₁ (aC _II mutant). On account of this map position adjacent to C _II the mutants of the new type are called C _IIa . They arise from phage 434 hy with a frequency comparable to that of C _I and C _II mutants. Such mutants are also obtained from phage lambda but apparently not from phage b₅. C _IIa mutants would not fit into a picture of three independently acting cistrons C _I , C_II, and C _III . The hypothesis is presented that C _IIa and C _II mutants are in the same structural gene. Two possibilities are discussed that would account for the complementation patterns: 1. C _IIa mutants may block the expression of gene C _III in cis position; or 2. the products of genes C _II and C _III function through an oligomeric complex they form.     

Heterotrimeric G protein mediates ethylene‐induced stomatal closure via hydrogen peroxide synthesis in Arabidopsis

Heterotrimeric G proteins function as key players in hydrogen peroxide (H₂O₂) production in plant cells, but whether G proteins mediate ethylene‐induced H₂O₂ production and stomatal closure are not clear. Here, evidences are provided to show the Gα subunit GPA1 as a missing link between ethylene and H₂O₂ in guard cell ethylene signalling. In wild‐type leaves, ethylene‐triggered H₂O₂ synthesis and stomatal closure were dependent on activation of Gα. GPA1 mutants showed the defect of ethylene‐induced H₂O₂ production and stomatal closure, whereas wGα and cGα overexpression lines showed faster stomatal closure and H₂O₂ production in response to ethylene. Ethylene‐triggered H₂O₂ generation and stomatal closure were impaired in RAN1, ETR1, ERS1 and EIN4 mutants but not impaired in ETR2 and ERS2 mutants. Gα activator and H₂O₂ rescued the defect of RAN1 and EIN4 mutants or etr1‐3 in ethylene‐induced H₂O₂ production and stomatal closure, but only rescued the defect of ERS1 mutants or etr1‐1 and etr1‐9 in ethylene‐induced H₂O₂ production. Stomata of CTR1 mutants showed constitutive H₂O₂ production and stomatal closure, but which could be abolished by Gα inhibitor. Stomata of EIN2, EIN3 and ARR2 mutants did not close in responses to ethylene, Gα activator or H₂O₂, but do generate H₂O₂ following challenge of ethylene or Gα activator. The data indicate that Gα mediates ethylene‐induced stomatal closure via H₂O₂ production, and acts downstream of RAN1, ETR1, ERS1, EIN4 and CTR1 and upstream of EIN2, EIN3 and ARR2. The data also show that ETR1 and ERS1 mediate both ethylene and H₂O₂ signalling in guard cells.     

Storage protein characteristics of proline-requiring mutants of Zea mays (L.)

Summary Protein and amino acid composition of mature karnels from three allelic proline-requiring mutants in maize, pro _1-1, pro _1-2, and pro _1-3 were analyzed and compared to kernels of the stock A 188 containing the wild type allele. The amount of free proline was specifically reduced in the embryos of all three mutants, while in the endosperm such a reduction was only found for pro _1-2 and pro _1-3 Accumulation of the proline-rich zeins was strongly reduced in the mutants, but in contrast to opaque-2 the reduction affected all major zein polypeptides to the same extent, possibly as a consequence of the defective proline metabolism. Albumins and globulins as well as free amino acids were more abundant in the endosperms of the mutants than in the wild type. Analysis of the albumins and globulins by SDS-PAGE revealed specific increases as well as reductions of certain polypeptides in the endosperms and embryos of the mutants.     

Identification and characterization of plasmids in hydrogen uptake positive and hydrogen uptake negative strains ofRhizobium japonicum

Modifications were made of published procedures to allow routine isolation of plasmids fromRhizobium japonicum. The plasmid profiles of a series of H₂ uptake positive and H₂ uptake negative strains were compared. None of the strains ofR. japonicum with high H₂ uptake activities exhibited discernible plasmids, while most of the strains, with little or no H₂ uptake activity, showed plasmids with molecular weights ranging from approximately 49–290 x10⁶. An examination of H₂ uptake negative mutants derived from an H₂ uptake positive parent revealed two discernible plasmid bands in nonrevertible mutants but no detectable plasmids in revertible mutants or in the parent strain from which mutants were derived.     

Effects of hydrogen peroxide on the motility, catalase and superoxide dismutase of dam and/or seqA mutant of Salmonella typhimurium

In addition to their role in the virulence attenuation of Salmonella and other pathogens, dam or seqA genes increase the sensitivity towards hydrogen peroxide. The aim of our study is to investigate the effect of H₂O₂ on the motility, the catalase and superoxide dismutase activities of dam and/or seqA mutants of Salmonella typhimurium. Our findings showed significant differences of the effects of H₂O₂ on the motility between wild type strain and all of mutants. Hydrogen peroxide changes SOD isoenzyme profile of these mutants by disappearance of Fe-SOD. Concerning the catalase, an increase of its activity was observed in the wild type, dam and seqA mutant. However, H₂O₂ decreases the activity of this enzyme in the double mutant strain. We can suggest that the dam gene, together with seqA, play a protective role in the oxidative stress response of Salmonella typhimurium.     

Implications of paraquat and hydrogen peroxide-induced oxidative stress treatments on the GABA shunt pathway in Arabidopsis thaliana calmodulin mutants

Arabidopsis mutants with T-DNA insertion in seven calmodulin genes (CAM) were used to determine the specific role of CAM in the tolerance of plants to oxidative stress induced by paraquat and hydrogen peroxide (H₂O₂) treatments. Arabidopsis calmodulin mutants (cam) were screened for seedling growth, seed germination, induced oxidative damage, and levels of γ-aminobutyric acid (GABA) shunt metabolites. Only the cam5-4 and cam6-1 mutants exhibited an increased sensitivity to paraquat and H₂O₂ during seed germination and seedling growth. In response to treatments with 3 μM paraquat and 1 mM H₂O₂, only the cam5-4, cam6-1 mutants showed significant changes in malonaldehyde (MDA) levels in root and shoot tissues, with highly increased levels of MDA. In terms of the GABA shunt metabolites, GABA was significantly elevated in root and shoot tissues in response to the paraquat treatments in comparison to alanine and glutamate, while the levels of all shunt metabolites increased in root tissue but not in the shoot tissue following the H₂O₂ treatments. GABA, alanine and glutamate levels were significantly increased in root and shoot of the cam1, cam4, cam5-4, and cam6-1 mutants in response to paraquat (0.5, 1 and 3 μM), while they were increased only in the root tissue of the cam1, cam4, cam5-4, and cam6-1 mutants in response to H₂O₂ (200 and 500 μM, 1 mM). These data show that the cam5-4 and cam6-1 mutants were sensitive to the induced oxidative stress treatments in terms of seed germination, seedling growth, and oxidative damage. The accumulation of GABA shunt metabolites as a consequence of the induced oxidative stress treatments (paraquat and H₂O₂ treatments) suggests that the GABA shunt pathway and the accumulation of GABA metabolites may contribute in antioxidant machinery associated with reactive oxygen species and in the acquisition of tolerance in response to induced oxidative stress in Arabidopsis seedlings.     

Interaction of lead nitrate and cadmium chloride withEscherichia coli K-12 andSalmonella typhimurium global regulatory mutants

Summary To investigate the interactions of heavy metals with cells, a minimal medium for the growth of enteric bacteria using glycerol-2-phosphate as the sole phosphorus source was developed that avoided precipitation of Pb²⁺ with inorganic phosphate. Using this medium, spontaneous mutants ofEscherichia coli resistant to addition of Pb(NO₃)₂ were isolated. Thirty-five independent mutants all conferred a low level of resistance. Disk diffusion assays on solid medium were used to survey the response ofE. coli andSalmonella typhimurium mutants altered in global regulatory networks to Pb(NO₃)₂) and CdCl₂. Strains bearing mutations inoxyR andrpoH were the most hypersensitive to these compounds. Based upon the response of strains completely devoid of isozymes needed to inactivate reactive oxygen species, this hypersensitity to lead and cadmium is attributable to alteration in superoxide dismutase rather than catalase levels. Similar analysis of chaperonedefective mutants suggests that these metals damage proteins in vivo.     

Modification of symbiotic interaction of pea (Pisum sativum L.) andRhizobium leguminosarum by induced mutations

Summary In pea (Pisum sativum L.), mutants could be induced, modified in the symbiotic interaction withRhizobium leguminosarum. Among 250 M₂-families, two nodulation resistant mutants (K₅ and K₉) were obtained. In mutant K₅ the nodulation resistance was monogenic recessive and not Rhizobium strain specific. Out of 220 M₂-families one mutant nod₃ was found which could form nodules at high nitrate concentrations (15 mM KNO₃). This mutant nodulated abundantly with severalRhizobium strains, both in the absence and presence of nitrate. Probably as the result of a pleiotropic effect, its root morphology was also changed. Among 1800 M₂-families, five nitrate reductase deficient mutants were obtained and one of them (mutant E₁) was used to study the inhibitory effect of nitrate on nodulation and nitrogen fixation.The results of the present investigation show that pea mutants which are modified in their symbiosis withRhizobium leguminosarum, can readily be obtained. The significance of such mutants for fundamental studies of the legume-Rhizobium symbiosis and for applications in plant breeding is discussed.     

Slow Gating of Gap Junction Channels and Calmodulin

Certain COOH-terminus mutants of connexin32 (Cx32) were previously shown to form channels with unusual transjuctional voltage (V _j ) sensitivity when tested heterotypically in oocytes against Cx32 wild type. Junctional conductance (G _j ) slowly increased by severalfold or decreases to nearly zero with V _j positive or negative, respectively, at mutant side, and V _j positive at mutant side reversed CO₂-induced uncoupling. This suggested that the CO₂-sensitive gate might be a V _j -sensitive slow gate. Based on previous data for calmodulin (CaM) involvement in gap junction function, we have hypothesized that the slow gate could be a CaM-like pore plugging molecule (cork gating model). This study describes a similar behavior in heterotypic channels between Cx32 and each of four new Cx32 mutants modified in cytoplasmic-loop and/or COOH-terminus residues. The mutants are: ML/NN+3R/N, 3R/N, ML/NN and ML/EE; in these mutants, N or E replace M105 and L106, and N replace R215, R219 and R220. This study also reports that inhibition of CaM expression strongly reduces V _j and CO₂ sensitivities of two of the most effective mutants, suggesting a CaM role in slow and chemical gating. Received: 19 April 2000/Revised: 11 August 2000     

Disruption of <Emphasis Type="Italic">RCI2A</Emphasis> leads to over-accumulation of Na<Superscript>+</Superscript> and increased salt sensitivity in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> plants

For plant salt tolerance, it is important to regulate the uptake and accumulation of Na⁺ ions. The yeast pmp3 mutant which lacks PMP3 gene accumulates excess Na⁺ ions in the cell and shows increased Na⁺ sensitivity. Although the function of PMP3 is not fully understood, it is proposed that PMP3 contributes to the restriction of Na⁺ uptake and consequently salt tolerance in yeasts. In this paper, we have investigated whether the lack of RCI2A gene, homologous to PMP3 gene, causes a salt sensitive phenotype in Arabidopsis (Arabidopsis thaliana (L.) Heynh.) plants; and to thereby indicate the physiological role of RCI2A in higher plants. Two T-DNA insertional mutants of RCI2A were identified. Although the growth of rci2a mutants was comparable with that of wild type under normal conditions, high NaCl treatment caused increased accumulation of Na⁺ and more reduction of the growth of roots and shoots of rci2a mutants than that of wild type. Undifferentiated callus cultures regenerated from rci2a mutants also accumulated more Na⁺ than that from wild type under high NaCl treatment. Furthermore, when wild-type and rci2a plants were treated with NaCl, NaNO₃, Na₂SO₄, KCl, KNO₃, K₂SO₄ or LiCl, the rci2a mutants showed more reduction of shoot growth than wild type. Under treatments of tetramethylammonium chloride, CaCl₂, MgCl₂, mannitol or sorbitol, the growth reduction was comparable between wild-type and rci2a plants. These results suggested that RCI2A plays a role directly or indirectly for avoiding over-accumulation of excess Na⁺ and K⁺ ions in plants, and contributes to salt tolerance.     

Thermostability of photosynthesis in two new chlorophyllb-less rice mutants

Leaves of the two new chlorophyll b-less rice mutants VG28-1, VG30-5 and the wild type rice cv. Zhonghua 11 were subjected to temperatures 28, 36, 40, 44 and 48℃ in the dark for 30 min or gradually elevated temperature from 30℃ to 80℃ at 0.5℃/min. The thermostability of photosynthetic apparatus was estimated by the changes in chlorophyll fluorescence parameters, photosynthetic rate and pigment content, chloroplast ultrastructure and tissue location of H2O2 accumulation. There were different patterns of Fo-temperature curves between the Chl b-less mutants and the wild type plant, and the temperature of F_o rising threshold was shifted 3℃ lower in the Chl b-less mutants (48℃) than in the wild type (51℃). At temperature up to about 45℃, chloroplasts were swollen and thylakoid grana became misty accompanied with the complete loss of photosynthetic oxygen evolution in the two Chl b-less mutants, but chloroplast ultrastruc-ture in the wild type showed no obvious alteration. After 55℃ exposure, the disordered thylakoid and significant H₂O₂ accumulation in leaves were found in the two Chl _b-less mutants, whereas in the wild type plant, less H₂O₂ was accumulated and the swollen thylakoid still maintained a cer-tain extent of stacking. A large extent of the changes in qP, NPQ and F_v/F_m was consistent with the Pn decreasing rate in the Chl b-less mutants during high temperature treatment as compared with the wild type. The results indicated that the Chl b-less mutants showed a tendency for higher thermosensitivity, and loss of Chl b in LHC II could lead to less thermostability of PSII structure and function. Heat damage to photosynthetic apparatus might be partially attributed to the in-ternal oxidative stress produced at severely high temperature.     

Photosynthetic Characteristics of Two New Chlorophyll b-Less Rice Mutants

Two yellow rice mutants VG28-1 and VG30-5 were obtained during the tissue culture process from a rice plant (cv. Zhonghua No.11 japonica) with inserted maize Ds transposon element. Absorption spectra and pigment composition showed that two mutants had no chlorophyll (Chl) b and lower Chl a content in comparison to the wild type (WT). Net photosynthetic rate (P _N), total electron transport rate (J_F), photochemical quenching (q_p), quantum yield of PS2 dependent non-cyclic electron transport (_PS2), fraction of P_rate, and leaf area were lower but F_v/F_m and apparent quantum yield (AQY) remained at similar levels as in the WT plant. Xanthophyll cycle pool size (V+A+Z) on a Chl basis, and de-epoxidation state were enhanced in the mutants. The mutants had larger amounts of soluble protein and ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO), especially the small subunit of RuBPCO, than WT. The characteristics of two rice mutants differed somewhat from the other common Chl b-less mutants originating from mutagenic agent treatments.     

Heat tolerance in rice mutants is associated with reduced accumulation of reactive oxygen species

Four mutants induced by ethylmethane sulphonate (N22-H-dgl56, N22-H-dgl101, N22-H-dgl162 and N22-H-dgl219) with conspicuous dark green leaves were identified in the drought and heat-tolerant rice cultivar Nagina22 (N22), when screened under prolonged drought and heat conditions in field. During dark-induced senescence, these mutants maintained higher chlorophyll and carotenoid contents, and photochemical efficiency of photosystem 2 in comparison with N22. Following heat treatment, these mutants accumulated less reactive oxygen species (assayed by histochemical staining for H₂O₂ and superoxide radicals) and maintained higher chlorophyll content than N22.     

The induction of grain size and colour mutations in rice (Oryza sativa L.) by radioisotopes

Summary Seven mutants with changes in grain size and in pigmentation of various organs were isolated in the M ₂ and M ₃ generations of the Patnai variety of winter rice after treatment with ³² P and ³⁵ S. Some of these could be useful for breeding.The narrow grain mutant, isolated in the M ₃ generation after treatment with ³² P, was high-yielding and had fine grains and an increased number of long panicles.Two short grain mutants with changed husk colour were obtained in the M ₂ generation after treatment with ³² P. One was also early maturing.One purple apiculus and three purple husk mutants were isolated in the M ₂ generation after treatment with ³⁵ S. Some of these also had reduced plant height and short grains. The purple apiculus mutant bred true for stigma and apiculus colour, but segregated for leaf sheath colour. In purple husk mutants the segregation of mutant characteristics was unusual and there was manifestation of new characters.