A Maize Mutant with an Altered Vascular Pattern

A recessive maize mutant with disrupted seedling developmentwas isolated following transpositional mutagenesis with Mutator.This mutant, initially identified during germination on thebasis of abnormal growth of the scutellar node, was designatedlsn1 (l arge s cutellar n ode). The mutant seedling exhibitsan enlarged primary root with a longitudinal groove and multipleseparate root tips. The mutant root is shorter than normal,because of defective cell elongation, and lacks lateral roots.The mutant plant shows defective leaves and reduced internodeelongation. Histological analyses on primary root, shoot, scutellarnode and juvenile leaves revealed a series of defects, all relatedto an irregular differentiation of vascular elements. In addition,in situ hybridization of mutant leaves demonstrates an abnormalpattern of expression of Knotted-1, a marker of meristem function.The presence of multiple roots fused together can be interpretedas suppression of the negative control responsible for the differentiationof only one root primordium. Therefore, the data obtained onseedlings of lsn1 point to a relationship between meristem activity,vascular differentiation and auxin polar transport, and mayallow the identification of a gene which is active during embryogenesis.Copyright2000 Annals of Botany Company Auxin, defective seedling, maize, meristem activity, vascular differentiation, Zea mays.     

Taurocyamine-utilizing Mutant of Pseudomonas aeruginosa with Altered Induction of 3-Guanidinopropionate Amidinohydrolase

A mutant, strain M-6, capable of utilizing taurocyamine (2-guanidinoethanesulfonate) as a nitrogen source was isolated from the parent strain, Pseudomonas aeruginosa GB-4, a derivative of wild-type P. aeruginosa PAO1 lacking the ability to produce guanidinobutyrase (EC 3.5.3.7). 3-Guanidinopropionate amidinohydrolase (EC class 3.5.3), which acts slowly on taurocyamine, was induced effectively by only 3-guanidinopropionate in the parent strain, while the enzyme of strain M-6 was induced by taurocyanime, guanidinoacetate, 3-guanidinopropionate, 4-guanidinobutyrate, and guanidinosuccinate. Strain M-6 synthesized a slight amount of the enzyme constitutively. The enzyme partially purified from strain M-6 exhibited substrate specificity similar to that of the wild-type strain. The mutant could grow also on 4-guanidinobutyrate, unlike the parent strain. These results indicate that strain M-6 acquired the ability to grow on taurocyamine by virtue of a mutation at the regulatory gene for 3-guanidinopropionate amidinohydrolase, which led to alteration of the specificity of the regulatory protein.     

Recognition of Altered Deoxyribonucleic Acid in Recombination

Kinetics of inactivation of transduction by phage P1bt which had been treated with ultraviolet light (UV) or nitrous acid (NA) was examined. With Escherichia coli B/r (radiation-resistant), low doses of UV increased transduction frequency, but the frequency was exponentially inactivated by higher doses. Little initial stimulus was observed in strain B(s-1) (radiation-sensitive). The final rate of decay was the same as in B/r. The initial stimulus of transduction in B/r was probably a consequence of increased recombination resulting from dark repair. It was estimated that another nucleotide within 1000 nucleotide pairs had to be damaged by UV to prevent a given nucleotide from successful transduction. The NA dose response was the same for the two strains. An initial stimulus of transduction was followed by exponential decline. The UV-repair enzymes missing in B(s-1) were not required for repair of NA-induced damage to transducing or lytic phage DNA. Low recovery of new mutations in the transductants showed that mutagen-induced damage to transducing DNA was excluded from recombinant chromosomes. The few recovered mutants may have resulted from "normal" error in recombination.     

Altered Mitochondrial Respiration in a Chromosomal Mutant of Neurospora crassa

A mutant of Neurospora crassa (cni-1) has been isolated that has two pathways of mitochondrial respiration. One pathway is sensitive to cyanide and antimycin A, the other is sensitive only to salicyl hydroxamic acid. Respiration can proceed through either pathway and both pathways together in this mutant account for greater than 90% of all mitochondrial respiration. The cni-1 mutation segregates as a nuclear gene in crosses to other strains of Neurospora. Absorption spectra of isolated mitochondria from cni-1 show typical b- and c-type cytochromes but the absorption peaks corresponding to cytochrome aa(3) are not detectable. Extraction of soluble cytochrome c-546 from these mitochondria followed by reduction with ascorbate reveals a new absorption peak at 426 nm that is not present in wild-type mitochondria. This peak may be due to an altered cytochrome oxidase with abnormal spectral properties. Mitochondria from cni-1 have elevated levels of succinate-cytochrome c reductase but reduced levels of nicotinamide adenine dinucleotide reduced form cytochrome c reductase and of cyanide- and azide-sensitive cytochrome c oxidase. These studies suggest that the cni-1 mutation results in the abnormal assembly of cytochrome c oxidase so that the typical cytochrome aa(3) spectrum is lost and the enzyme activity is reduced. As a consequence of this alteration, a cyanide-insensitive respiratory pathway is elaborated by these mitochondria which may serve to stimulate adenosine 5'-triphosphate production via substrate level phosphorylation by glycolysis and the Krebs cycle.     

Altered Phytochrome Regulation of Greening in an aurea Mutant of Tomato

A brief pulse of red light accelerates chlorophyll accumulation upon subsequent transfer of dark-grown tomato (Lycopersicon esculentum) seedlings to continuous white light. Such potentiation of greening was compared in wild type and an aurea mutant W616. This mutant has been the subject of recent studies of phytochrome phototransduction; its dark-grown seedlings are deficient in phytochrome, and light-grown plants have yellow-green leaves. The rate of greening was slower in the mutant, but the extent (relative to the dark control) of potentiation by the red pulse was similar to that in the wild type. In the wild type, the fluence-response curve for potentiation of greening indicates substantial components in the VLF (very low fluence) and LF (low fluence) ranges. Far-red light could only partially reverse the effect of red. In the aurea mutant, only red light in the LF range was effective, and the effect of red was completely reversed by far-red light. When grown in total darkness, aurea seedlings are also deficient in photoconvertible PChl(ide). Upon transfer to white light, the aurea mutant was defective in both the abundance and light regulation of the light-harvesting chlorophyll a/b binding polypeptide(s) [LHC(II)]. The results are consistent with the VLF response in greening being mediated by phytochrome. Furthermore, the data support the hypothesis that light modulates LHC(II) levels through its control of the synthesis of both chlorophyll and its LHC(II) apoproteins. Some, but not all, aspects of the aurea phenotype can be accounted for by the deficiency in photoreception by phytochrome.     

Altered Axonal Transport of Cytoskeletal Proteins in the Mutant Diabetic Mouse

Polypeptides in the motor axons of the sciatic nerve in 120-day-old normal and diabetic mice C57BL/Ks (db/db) were labeled by injection of [35S]methionine into the ventral horn of the spinal cord. At 8, 15, and 25 days after the injection, the distribution of radiolabeled polypeptides along the sciatic nerve was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Four major radiolabeled polypeptides, tentatively identified as actin, tubulin, and the two lightest subunits of the neurofilament triplet, were studied in both diabetic and control mice. In the diabetic animals, the two polypeptides identified as actin and tubulin showed a reduction of average velocity of migration along the sciatic nerve, resulting in a higher fraction of radioactivity in the proximal part of the sciatic nerve, whereas the front of radioactivity (advancing at maximal velocity) moved at a normal rate. In contrast, both the average and maximal velocities of the two neurofilament subunits were slower in the diabetic mice than in the control mice. These results indicate that the axonal transport of the cytoskeletal proteins is differentially affected in the course of diabetic neuropathy, and may suggest that the impairment concerns mainly the proteins carried by the slowest component of axonal transport.     

Altered Tryptophan Metabolism in Mice with Herpes Simplex Virus Encephalitis: Increases in Spinal Cord Quinolinic Acid

Mice infected with the herpes simplex virus, type-1, developed a paralysis which was associated with increased levels of the neurotoxin quinolinic acid (QUIN). The largest increases in QUIN were observed in the spinal cord with much smaller changes in the rostral forebrain or serum. The time course for the paralysis coincided with the increase in spinal cord QUIN, a maximal 40-fold elevation, at 7–10 days post infection. The time course suggested that the increases in QUIN were due to its local synthesis. Consistent with this possibility, herpes virus-infected mice had increased activities of indoleamine 2,3-dioxygenase and kynurenine hydroxylase (two key enzymes in QUIN formation), when compared to non-infected controls. Since QUIN is formed by activated macro-phages, these new data are consistent with QUIN formation as part of the host response to a pathogen whose importance is discussed.     

Altered Nucleotide-Microtubule Coupling and Increased Mechanical Output by a Kinesin Mutant

Kinesin motors hydrolyze ATP to produce force and do work in the cell – how the motors do this is not fully understood, but is thought to depend on the coupling of ATP hydrolysis to microtubule binding by the motor. Transmittal of conformational changes from the microtubule- to the nucleotide-binding site has been proposed to involve the central β-sheet, which could undergo large structural changes important for force production. We show here that mutation of an invariant residue in loop L7 of the central β-sheet of the Drosophila kinesin-14 Ncd motor alters both nucleotide and microtubule binding, although the mutated residue is not present in either site. Mutants show weak-ADP/tight-microtubule binding, instead of tight-ADP/weak-microtubule binding like wild type – they hydrolyze ATP faster than wild type, move faster in motility assays, and assemble long spindles with greatly elongated poles, which are also produced by simulations of assembly with tighter microtubule binding and faster sliding. The mutated residue acts like a mechanochemical coupling element – it transmits changes between the microtubule-binding and active sites, and can switch the state of the motor, increasing mechanical output by the motor. One possibility, based on our findings, is that movements by the residue and the loop that contains it could bend or distort the central β-sheet, mediating free energy changes that lead to force production.     

Optimization of Protein Thermostability and Exploitation of Recognition Behavior to Engineer Altered Protein-DNA Recognition

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Altered Methionyl-tRNA Synthetase in a Spirulina platensis Mutant Resistant to Ethionine

Compared with the parental strain, a Spirulina platensis mutant that is resistant to ethionine incorporated methionine into protein at a reduced rate, whereas ethionine incorporation was practically nil. The methionyl-tRNA synthetase present in crude extracts from the resistant strain showed a reduced affinity for methionine and ethionine.     

A Genetic Basis for Altered Sexual Behavior in Mutant Female Mice

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Isolation and Partial Characterization of a Temperature-Sensitive Escherichia coli Mutant with Altered Glutaminyl-Transfer Ribonucleic Acid Synthetase

A temperature-sensitive mutant of Escherichia coli has been found in which the conditional growth is a result of a thermosensitive glutaminyl-transfer ribonucleic acid synthetase. The corresponding genetic locus glnS is cotransduced with lip. In a strain containing the mutationally altered glutaminyl-transfer ribonucleic acid synthetase, no derepression of the enzyme itself nor of glutamine synthetase was observed.     

Selection for Growth on 3-Nitrotoluene by 2-Nitrotoluene-Utilizing Acidovorax sp. Strain JS42 Identifies Nitroarene Dioxygenases with Altered Specificities

Acidovorax sp. strain JS42 uses 2-nitrotoluene as a sole source of carbon and energy. The first enzyme of the degradation pathway, 2-nitrotoluene 2,3-dioxygenase, adds both atoms of molecular oxygen to 2-nitrotoluene, forming nitrite and 3-methylcatechol. All three mononitrotoluene isomers serve as substrates for 2-nitrotoluene dioxygenase, but strain JS42 is unable to grow on 3- or 4-nitrotoluene. Using both long- and short-term selections, we obtained spontaneous mutants of strain JS42 that grew on 3-nitrotoluene. All of the strains obtained by short-term selection had mutations in the gene encoding the α subunit of 2-nitrotoluene dioxygenase that changed isoleucine 204 at the active site to valine. Those strains obtained by long-term selections had mutations that changed the same residue to valine, alanine, or threonine or changed the alanine at position 405, which is just outside the active site, to glycine. All of these changes altered the regiospecificity of the enzymes with 3-nitrotoluene such that 4-methylcatechol was the primary product rather than 3-methylcatechol. Kinetic analyses indicated that the evolved enzymes had enhanced affinities for 3-nitrotoluene and were more catalytically efficient with 3-nitrotoluene than the wild-type enzyme. In contrast, the corresponding amino acid substitutions in the closely related enzyme nitrobenzene 1,2-dioxygenase were detrimental to enzyme activity. When cloned genes encoding the evolved dioxygenases were introduced into a JS42 mutant lacking a functional dioxygenase, the strains acquired the ability to grow on 3-nitrotoluene but with significantly longer doubling times than the evolved strains, suggesting that additional beneficial mutations occurred elsewhere in the genome.     

The Drosophila Neurally Altered Carbohydrate Mutant Has a Defective Golgi GDP-fucose Transporter

Studying genetic disorders in model organisms can provide insights into heritable human diseases. The Drosophila neurally altered carbohydrate (nac) mutant is deficient for neural expression of the HRP epitope, which consists of N-glycans with core α1,3-linked fucose residues. Here, we show that a conserved serine residue in the Golgi GDP-fucose transporter (GFR) is substituted by leucine in nac(1) flies, which abolishes GDP-fucose transport in vivo and in vitro. This loss of function is due to a biochemical defect, not to destabilization or mistargeting of the mutant GFR protein. Mass spectrometry and HPLC analysis showed that nac(1) mutants lack not only core α1,3-linked, but also core α1,6-linked fucose residues on their N-glycans. Thus, the nac(1) Gfr mutation produces a previously unrecognized general defect in N-glycan core fucosylation. Transgenic expression of a wild-type Gfr gene restored the HRP epitope in neural tissues, directly demonstrating that the Gfr mutation is solely responsible for the neural HRP epitope deficiency in the nac(1) mutant. These results validate the Drosophila nac(1) mutant as a model for the human congenital disorder of glycosylation, CDG-IIc (also known as LAD-II), which is also the result of a GFR deficiency.     

Mutant Strain of Bradyrhizobium japonicum with Increased Symbiotic N2 Fixation Rates and Altered Mo Metabolism Properties

Mutant strains of Bradyrhizobium japonicum that required higher levels of molybdate than the wild-type strain for growth on NO₃-containing medium were obtained after transposon Tn5 mutagenesis of the wild-type strain. The mutant strains expressed more than fivefold-greater nitrate reductase activities in the range of 0.1 to 1.0 mM added molybdate compared with activities expressed upon incubation in non-Mo-supplemented medium, whereas the nitrate reductase activity of the wild-type strain (JH) was not markedly influenced by Mo supplementation. In free-living culture, mutant strains JH310 and JH359 expressed substantial nitrogenase activity, even in medium treated to remove molybdate, and nitrogenase activity was influenced little by Mo supplementation, whereas the wild-type strain required 100 nM added Mo for highest nitrogenase activity. Double-reciprocal plots of Mo uptake rates versus Mo concentration showed that both bacteroids and free-living cells of mutant strain JH359 had about the same affinity for Mo as did the parent strain. Bacteroids of both the mutants and the wild type also exhibited similar Mo accumulation rates over a 9-min period under very-low-Mo (4 nM) conditions. Nitrogenase activities for strain JH359 and for the wild-type strain in free-living culture were both strongly inhibited by tungsten; thus, the nitrogenase activities of both strains are probably the result of a “conventional” Mo-containing nitrogenase. Soybeans inoculated with strain JH359 and grown under either Mo-supplemented or Mo-deficient conditions had greater specific acetylene reduction rates and significantly greater plant fresh weight than those inoculated with the wild-type strain. Under Mo-deficient conditions, the acetylene reduction rates and plant fresh weights were up to 35 and 58% greater, respectively, for mutant-nodulated plants compared with wild-type-strain-nodulated plants.