Genetic Analysis of Mutants with a Reduced Ca2+-Dependent K+ Current in PARAMECIUM TETRAURELIA

Two mutants of Paramecium tetraurelia with greatly reduced Ca2+-dependent K+ currents have been isolated and genetically analyzed. These mutants, designated pantophobiac, give much stronger behavioral responses to all stimuli than do wild-type cells. Under voltage clamp, the Ca2+-dependent K+ current is almost completely eliminated in these mutants, whereas the Ca2+ current is normal. The two mutants, pntA and pntB, are recessive and unlinked to each other. pntA is not allelic to several other ion-channel mutants of P. tetraurelia. The microinjection of a high-speed supernatant fraction of wild-type cytoplasm into either pantophobiac mutant caused a temporary restoration to the wild-type phenotype.     

Genetic Analysis of Mating-Type Differentiation in PARAMECIUM TETRAURELIA

Whereas each of the two complementary mating types, O and E, of Paramecium tetraulrelia normally shows cytoplasmic inheritance, an abnormal heredity of mating type was observed in the progeny of crosses between two stocks of different geographical origin of Paramecium tetraurelia (stock 51 and stock 32). The modified pattern of mating-type inheritance was shown to result from the interaction of the two wild-type alleles at the locus mtD (mtD51 and mtD32), leading to a new differentiated state O*, different from the normal O and E states observed in both stock 51 and stock 32 cells. The genetic analysis of O* clones showed that the O* phenotype involves both a new heritable cytoplasmic state and possibly a nuclear change which can be transmitted through conjugation and segregates in a Mendelian fashion. All the data can be interpreted if the assumption is made that mating-type determination is achieved only by the commitment or noncommitment to the expression of mating-type E , and that this commitment may simply reflect the activation or nonactivation of the locus mtD, under the influence of one or two "cytoplasmic factors" including the product of the gene mtD itself.     

Genetic Analysis of the Relationships between the Cell Surface and the Nuclei in PARAMECIUM TETRAURELIA

In Paramecium tetraurelia, a number of mutations have been shown to affect simultaneously cortical organization (attachment of trichocysts to the cortex) and nuclear division (Ruiz et al. 1976). In order to analyze the genetic and physiological basis of this correlation, we have isolated new mutations affecting the properties of the trichocysts and studied their genetic relationships with other previously known mutations. Of 24 to 28 loci controlling the biogenesis and properties of the trichocysts, mutations only in the 16 to 20 loci that control trichocyst attachment to the cortex result in nuclear defects. Cytological observations show that all of these mutants display the same set of nuclear abnormalities: in particular, rounded shape of the resting macronucleus, mispositioning and defective elongation of the dividing macronucleus and unequal repartition of the macro- and micronuclei. This common syndrome is independent of both the mutagenic origin and the mutated locus. Furthermore, by microinjection, it is possible to localize the site of action of the mutations in either the trichocyst compartment or the nontrichocyst compartment. It was found by this technique that the nuclear syndrome is also independent of the site of action of the mutation. All the genetic and physiological data support the conclusion that the nuclear defects are the consequence of the lack of trichocyst attachment to the cortex: in wild-type cells, trichocyst attachment would induce a membranar or perimembranar state necessary for correct nuclear positioning during cell division. In the absence of trichocyst attachment, the cortical control of nuclear division would be abolished. The possible involvement of cytoskeletal links between surface and nuclei is discussed.     

Isolation and Characterization of Rifampin-Resistant and Streptolydigin-Resistant Mutants of Bacillus subtilis with Altered Sporulation Properties

Mutants of Bacillus subtilis with altered deoxyribonucleic-dependent ribonucleic acid polymerase activity have been isolated and characterized. These mutants, selected as strains resistant to rifampin or streptolydigin, demonstrate drug-resistant in vitro ribonucleic acid synthesis. Sporeforming ability and support of phage infection are altered in many of the mutants. Mutations to rifampin and streptolydigin resistance have been located on the B. subtilis chromosome and ordered relative to the markers cysA14 and str.     

Isolation and Genetic Analysis of Arabidopsis Mutants with Low-K+ Tolerance

Ethyl methane-sulfonate (EMS)-mutagenized Arabidopsis M2 populations were screened in low-K+ medium using the root-bending assay. Forty-two putative low-K+-tolerant ( lkt ) mutants were selected from 150?000 tested M2 seedlings, and two of these mutants maintained their low-K+-tolerant phenotype in their M3 generations, respectively. Genetic analysis showed that either one of these two mutants has a monogenic recessive mutation in a nuclear gene, and that the two mutations in two independent mutants are allelic to each other.     

Acid Phosphatase Mutants in Chlamydomonas: Isolation and Characterization by Biochemical, Electrophoretic and Genetic Analysis

In order to isolate acid phosphatase mutants in the green alga Chlamydomonas reinhardi, a staining method for detecting the enzyme activity in colonies has been developed. The occurrence of more than one acid phosphatase brought about some difficulty in the selection of mutants. We have, however, found an original method of selection based on the differential heat sensitivity of the enzymes. After treatment of the wild-type strain with N-methyl-N'-nitro-N-nitrosoguanidine, two types of mutants were recovered, then analyzed by biochemical and electrophoretic methods. In the first class of mutants (P(1), P(2), P(3),...) a heat-stable acid phosphatase bound to cellular debris of the crude extract was missing. The mutant P(a), representing the second class of mutations, was lacking a soluble heat-sensitive enzyme. These mutations were genetically different and exhibited mendelian inheritance.     

川楝素对K+、Ca2+通道活动及细胞内Ca2+浓度的调控

川楝素是我国学者从驱蛔中药中分离、鉴定的一个三萜化合物,已证明具选择地影响神经递质释放,有效地对抗肉毒中毒,促进细胞分化、凋亡,抑制肿瘤增殖,抑制昆虫发育和取食,影响K 、Ca2 通道活动等多种生物效应.综述了证明川楝素抑制多种K 通道,选择地易化L型Ca2 通道和进而升高胞内Ca 浓度的研究资料,并对川楝素产生这些生物效应的机制进行了讨论.     

Isolation and Characterization of Pediocin AcH Chimeric Protein Mutants with Altered Bactericidal Activity

A collection of pediocin AcH amino acid substitution mutants was generated by PCR random mutagenesis of DNA encoding the bacteriocin. Mutants were isolated by cloning mutagenized DNA into an Escherichia coli malE plasmid that directs the secretion of maltose binding protein-pediocin AcH chimeric proteins and by screening transformant colonies for bactericidal activity against Lactobacillus plantarum NCDO955 (K. W. Miller, R. Schamber, Y. Chen, and B. Ray, 1998. Appl. Environ. Microbiol. 64:14–20, 1998). In all, 17 substitution mutants were isolated at 14 of the 44 amino acids of pediocin AcH. Seven mutants (N5K, C9R, C14S, C14Y, G37E, G37R, and C44W) were completely inactive against the pediocin AcH-sensitive strains L. plantarum NCDO955, Listeria innocua Lin11, Enterococcus faecalis M1, Pediococcus acidilactici LB42, and Leuconostoc mesenteroides Ly. A C24S substitution mutant constructed by other means also was inactive against these bacteria. Nine other mutants (K1N, W18R, I26T, M31T, A34D, N41K, H42L, K43N, and K43E) retained from <1% to ~60% of wild-type activity when assayed against L. innocua Lin11. One mutant, K11E, displayed ~2.8-fold-higher activity against this indicator. About one half of the mutations mapped to amino acids that are conserved in the pediocin-like family of bacteriocins. All four cysteines were found to be required for activity, although only C9 and C14 are conserved among pediocin-like bacteriocins. Several basic amino acids as well as nonpolar amino acids located within the hydrophobic C-terminal region also were found to be important. The mutations are discussed in the context of structural models that have been proposed for the bacteriocin.     

Isolation and Characterization of Escherichia Coli Mutants with Altered Rates of Deletion Formation

Using site-specific mutagenesis in vitro we constructed a genetic system to detect mutants with altered rates of deletion formation between short repeated sequences in Escherichia coli. After in vivo mutagenesis with chemical mutagens and transposons, the system allowed the identification of mutants with either increased or decreased deletion frequencies. One mutational locus, termed mutR, that results in an increase in deletion formation, was studied in detail. The mutR gene maps at 38.5 min on the E. coli genetic map. Since the precise excision of many transposable elements is also mediated at short repeated sequences, we investigated the effects of the mutant alleles, as well as recA, on precise excision of the transposon Tn9. Neither mutR nor recA affect precise excision of the transposon Tn9, from three different insertions in lacI, whereas these alleles do affect other spontaneous deletions in the same system. These results indicate that deletion events leading to precise excision occur principally via a different pathway than other random spontaneous deletions. It is suggested that, whereas precise excision occurs predominantly via a pathway involving replication enzymes (for instance template strand slippage), deletions on an F'factor are stimulated by recombination enzymes.     

Genetic Analysis of Mating Type Differentiation in PARAMECIUM TETRAURELIA. II. Role of the Micronuclei in Mating-Type Determination

The two complementary mating types, O and E, of Paramecium tetraurelia are normally inherited cytoplasmically. This property has generally been interpreted to indicate the presence of cytoplasmic factors that determine macronuclear differentiation towards O or E. In these macronuclear-cytoplasmic interactions, the micronuclei were held to be unbiased and the determination to be established in the course of macronuclear development. In order to ascertain whether the micronuclei were actually neutral, amicronucleate clones were needed and a method to produce them was developed. In crosses between amicronucleate clones and normal micronucleate clones, we have observed regular deviations from cytoplasmic inheritance: the commonest deviation is that most O amicronucleate cells become E when they receive a micronucleus from an E partner. The data can be interpreted by assuming that the micronuclei are predetermined and that the apparent "cytoplasmic" inheritance of the two mating types is due, in E cells, to E-determining factors present in the cytoplasm and in the nucleus; and, in O cells, to O-determining factors present only or mainly in the nucleus.     

Characterization of the Ca2+-Gated and Voltage-Dependent K+-Channel Slo-1 of Nematodes and Its Interaction with Emodepside

The cyclooctadepsipeptide emodepside and its parent compound PF1022A are broad-spectrum nematicidal drugs which are able to eliminate nematodes resistant to other anthelmintics. The mode of action of cyclooctadepsipeptides is only partially understood, but involves the latrophilin Lat-1 receptor and the voltage- and calcium-activated potassium channel Slo-1. Genetic evidence suggests that emodepside exerts its anthelmintic activity predominantly through Slo-1. Indeed, slo-1 deficient Caenorhabditis elegans strains are completely emodepside resistant. However, direct effects of emodepside on Slo-1 have not been reported and these channels have only been characterized for C. elegans and related Strongylida. Molecular and bioinformatic analyses identified full-length Slo-1 cDNAs of Ascaris suum, Parascaris equorum, Toxocara canis, Dirofilaria immitis, Brugia malayi, Onchocerca gutturosa and Strongyloides ratti. Two paralogs were identified in the trichocephalids Trichuris muris, Trichuris suis and Trichinella spiralis. Several splice variants encoding truncated channels were identified in Trichuris spp. Slo-1 channels of trichocephalids form a monophyletic group, showing that duplication occurred after the divergence of Enoplea and Chromadorea. To explore the function of a representative protein, C. elegans Slo-1a was expressed in Xenopus laevis oocytes and studied in electrophysiological (voltage-clamp) experiments. Incubation of oocytes with 1-10 µM emodepside caused significantly increased currents over a wide range of step potentials in the absence of experimentally increased intracellular Ca²⁺, suggesting that emodepside directly opens C. elegans Slo-1a. Emodepside wash-out did not reverse the effect and the Slo-1 inhibitor verruculogen was only effective when applied before, but not after, emodepside. The identification of several splice variants and paralogs in some parasitic nematodes suggests that there are substantial differences in channel properties among species. Most importantly, this study showed for the first time that emodepside directly opens a Slo-1 channel, significantly improving the understanding of the mode of action of this drug class.     

Isolation and Characterization of Staphylococcus aureus Mutants Which Form Altered Cell Clusters

Staphylococcus aureus FDA 209P produces two extracellular bacteriolytic enzymes, 51-kDa endo-β-N-acetylglucosaminidase (GL) and 62-kDa N-acetylmuramyl-l -alanine amidase (AM), both of which can disperse cell clusters. To characterize the physiological roles of these enzymes in vivo, mutants with altered autolysin activity were isolated, and their degree of cluster formation in broth culture was assessed. Bacteriolytic activities of GL and AM, produced and secreted from these mutants into the culture fluid and detected with activity gels, coincided well with the degree of cluster formation of the mutants. The mutants with little or no enzyme activity grew in clusters, whereas those with high activity grew as well-separated cocci, suggesting that these enzymes are involved in cell separation of S. aureus in vivo.     

拟南芥耐低钾突变体的筛选及遗传分析

利用乙酰甲基磺酸（ＥＭＳ）诱变方法,以幼苗根在重力作用下的弯曲生长为指标、筛选得到了拟南芥（Ａrabidopsis thaliana)耐低钾突变体。经过对突变体杂交后代的遗传分析证明,其中两株突变体的耐低钾性状为隐性单基因突变所致。鉴定、分离与植物耐低钾性状连锁的基因将有可能与对培育钾高效作物品种有重要意义。     

Isolation and Some Properties of Cell Envelope Altered Mutants of Escherichia coli

Mutants of Escherichia coli which have a defect in their permeability barrier were selected. The technique used was to employ a strain of E. coli having a deletion in the gene for lactose permease and to select for mutants which can grow on lactose at 40 C. Twenty such mutants were isolated and six of these were found to be more sensitive to actinomycin D, sodium deoxycholate, and sodium dodecyl sulfate than was the parental strain. They were also more sensitive to the antibiotics vancomycin and bacitracin, which inhibit peptidoglycan biosynthesis. These mutants were no more sensitive to several different colicins or phages than was the wild-type strain. One of the mutants selected by this technique has an abnormal morphology when grown on certain carbon sources in minimal medium, and this mutant is more extensively studied in the accompanying paper.     

Isolation and Partial Characterization of Escherichia coli Mutants with Altered Glycyl Transfer Ribonucleic Acid Synthetases

Isolates with mutations in glyS, the structural gene for glycyl-transfer ribonucleic acid (tRNA) synthetase (GRS) in Escherichia coli, are frequently found among glycine auxotrophs. Extracts of glyS mutants have altered GRS activities. The mutants grow with normal growth rates in minimal media when high levels of glycine are provided. No other metabolite of a variety tested is capable of restoring normal growth. The glyS mutants fail to make ribonucleic acid (RNA) when depleted of exogenous glycine in strains which are RC(str) but do so when the cells are RC(rel). In contrast, biosynthetic mutants which are unable to synthesize glycine (glyA mutants) do not make RNA when deprived of glycine even if they are RC(rel); in this case, RNA is synthesized upon glycine deprivation only when the nucleic acid precursors made from glycine are provided in the medium. The level of serine transhydroxymethylase is unaltered in extracts of any of the glyS mutants, even though the level of charged tRNA(Gly) is at least 20-fold lower than that found in a prototrophic parent; this indicates that, if there is control over the synthesis of serine transhydroxymethylase, it is not modified by reduced levels of charging of the major species of tRNA(Gly).     

Altered Ca2+ signaling in enamelopathies

Biomineralization requires the controlled movement of ions across cell barriers to reach the sites of crystal growth. Mineral precipitation occurs in aqueous phases as fluids become supersaturated with specific ionic compositions. In the biological world, biomineralization is dominated by the presence of calcium (Ca²⁺) in crystal lattices. Ca²⁺ channels are intrinsic modulators of this process, facilitating the availability of Ca²⁺ within cells in a tightly regulated manner in time and space. Unequivocally, the most mineralized tissue produced by vertebrates, past and present, is dental enamel. With some of the longest carbonated hydroxyapatite (Hap) crystals known, dental enamel formation is fully coordinated by specialized epithelial cells of ectodermal origin known as ameloblasts. These cells form enamel in two main developmental stages: a) secretory; and b) maturation. The secretory stage is marked by volumetric growth of the tissue with limited mineralization, and the opposite is found in the maturation stage, as enamel crystals expand in width concomitant with increased ion transport. Disruptions in the formation and/or mineralization stages result, in most cases, in permanent alterations in the crystal assembly. This introduces weaknesses in the material properties affecting enamel's hardness and durability, thus limiting its efficacy as a biting, chewing tool and increasing the possibility of pathology. Here, we briefly review enamel development and discuss key properties of ameloblasts and their Ca²⁺-handling machinery, and how alterations in this toolkit result in enamelopathies.