Genetic analysis of two tomato mutants affected in the regulation of iron metabolism

Iron is one of the most important micronutrients for plants. Like other organisms, plants have developed active mechanisms for the acquisition of sufficient iron from the soil. Nevertheless, very little is known about the genetic mechanisms that control the active uptake. In tomato, two spontaneously derived mutants are available, which are defective in key steps that control this process. The recessive mutationchloronerva (chln) affects a gene which controls the synthesis of the non-protein amino acid nicotianamine (NA), a key component in the iron physiology of plants. The root system of the recessive mutantfer is unable to induce any of the characteristic responses to iron deficiency and iron uptake is thus completely blocked. We present a characterization of the double mutant, showing that thefer gene is epistatic over thechln gene and thus very likely to be one of the major genetic elements controlling iron physiology in tomato. In order to gain access to these two genes at the molecular level, both mutants were precisely mapped onto the high density RFLP map of tomato. Thechln gene is located on chromosome 1 and thefer gene is on chromosome 6 of tomato. Using this high-resolution map, a chromosome walk has been started to isolate thefer gene by map-based cloning. The isolation of thefer gene will provide new insights into the molecular mechanisms of iron uptake control in plants.     

Arginine and proline genes ofAspergillus niger

Summary Aspergillus niger mutants defective in arginine or proline biosynthesis have been isolated and 12 genetic loci were identified. Mutation was induced by low doses UV, and mutants were isolated after filtration enrichment. The mutants were classified according to their phenotype in growth tests and were further characterized in complementation tests. The arginine auxotrophic mutants represent nine complementation groups. Three additional complementation groups were found for mutants that could grow on proline (two of them on arginine too). Linkage group analysis was done in somatic diploids obtained from a mutant and a master strain with genetic markers on six chromosomes. Thearg genes belong to six different linkage groups and thepro genes to two. Onearg-mutant could be complemented by transformation with theA. nidulans arg B ⁺ gene, and thisA. niger gene thus appeared to be homologous to theA. nidulans arg B. We isolated anA. niger strain with theargB gene tightly linked with thenicA1 marker. This strain is very suitable as acceptor for transformation with anargB-plasmid, because transformants with inserts on the homologous site can be recognized and analyzed genetically using thenicA1 marker gene.     

Identification of two new genes,mukE andmukF, involved in chromosome partitioning inEscherichia coli

We have previously reported that the MukB protein is essential for chromosome partitioning inEscherichia coli and thatmukB mutants produce anucleate cells and are temperature-sensitive for colony formation. ThemukB gene maps at 21 min on theE. coli chromosome andsmtA-mukF-mukE-mukB genes might comprise an operon, which is transcribed in a clockwise direction. Here, we report thatmukF andmukE null mutants are both temperature-sensitive for colony formation and produce anucleate cells even at the permissive temperature. These phenotypes are the same as those observed in themukB null mutant. The primary sequence of MukF includes a leucine zipper structure and an acidic domain. Mutational analysis revealed that both are required for MukF function. When the MukF protein was overproduced in the wild-type strain, anucleate cells were produced. In contrast, overproduction of either MukE or MukB did not cause the defect. In null mutants for themukF, mukE, andmukB genes, the synchronous initiation of chromosome replication was not affected. The mini-F plasmid was as stably maintained in these mutants as in the wild-type strain. These results indicate that the MukF, MukE, and MukB proteins are involved in the chromosome partitioning steps, but are not required for mini-F plasmid partitioning.     

Comparing mutants,selective breeding,and transgenics in the dissection of aging processes ofCaenorhabditis elegans

The genetic analysis of aging processes has matured in the last ten years with reports that long-lived strains of both fruit flies and nematodes have been developed. Several attempts to identify mutants in the fruit fly with increased longevity have failed and the reasons for these failures are analyzed. A major problem in obligate sexual species, such as the fruit fly, is the presence of inbreeding depression that makes the analysis of life-history traits in homozygotes very difficult. Nevertheless, several successful genetic analyses of aging inDrosophila suggest that with careful design, fruitful analysis of induced mutants affecting life span is possible. In the nematodeCaenorhabditis elegans, mutations in theage-1 gene result in a life extension of some 70%; thusage-1 clearly specifies a process involved in organismic senescence. This gene maps to chromosome II, well separated from a locus (fer-15) which is responsible for a large fertility deficit in the original stocks. There is no trade-off between either rate of development or fertility versus life span associated with theage-1 mutation. Transgenic analyses confirm that the fertility deficit can be corrected by a wild-typefer-15 transformant (transgene); however, the life span of these transformed stocks is affected by the transgenic array in an unpredictable fashion. The molecular nature of theage-1 gene remains unknown and we continue in our efforts to clone the gene.     

A pH-regulated promoter for the expression of recombinant proteins inEscherichia coli

Summary A series of plasmid vectors have been developed which allows control of recombinant gene expression by altering the pH of the growth medium. Expression is controlled by the regulatory region of thecadA gene ofE. coli. Experiments using -galactosidase as the expressed gene have resulted in an induced expression of up to 60-fold when the pH of the growth medium is lowered from pH 7 to 5.5. Expression can also be induced by switching from aerobic to anaerobic growth environment. The pH and anaerobic effects are additive boosting the expression level of -galactosidase to a dramatic value of 200 fold. Finally, the pH-induction effect is fully reversible, a unique property which allows continuous control of gene expression using exiting pH monitoring and control equipment.     

Cloning and sequencing of mutantpsbB genes of the cyanobacteriumSynechocystis PCC 6803

Ten strains from a collection of mutants ofSynechocystis 6803 defective in Photosystem II (PS II) function were transformed with chromosomal DNA of wild-type and mutant cells. Cross hybridization data allowed to identify four groups of PS II-mutants. Highly efficient transformation was observed between different mutant groups, but not within the groups. Restoration of photosynthetic activity of the mutant cells was also achieved by transformation with different parts of a 5.6 kbBam HI fragment of wild typeSynechocystis DNA containing thepsbB gene. Each group of mutants was transformed to photoautotrophic growth by specific subfragments of thepsbB gene. DNA fragments of four selected mutant strains hybridizing with thepsbB gene were isolated and sequenced. The mutations were identified as a single nucleotide insertion or substitution leading to stop codon formation in two of the mutants, as a deletion of 12 nucleotides, or as a nucleotide substitution resulting in an amino acid substitution in the other two mutants. Deletion of 12 nucleotides in mutant strain PMB1 and stop codon formation in strain NF16 affect membrane-spanning regions of the gene product, the CP 47 protein.     

Molecular cloning of mei-41, a gene that influences both somatic and germline chromosome metabolism of Drosophila melanogaster

The mei-41 gene of Drosophila melanogaster plays an essential role in meiosis, in the maintenance of somatic chromosome stability, in postreplication repair and in DNA double-strand break repair. This gene has been cytogenetically localized to polytene chromosome bands 14C4-6 using available chromosomal aberrations. About 60 kb of DNA sequence has been isolated following a bidirectional chromosomal walk that extends over the cytogenetic interval 14C1-6. The breakpoints of chromosomal aberrations identified within that walk establish that the entire mei-41 gene has been cloned. Two independently derived mei-41 mutants have been shown to carry P insertions within a single 2.2 kb fragment of the walk. Since revertants of those mutants have lost the P element sequences, an essential region of the mei-41 gene is present in that fragment. A 10.5 kb genomic fragment that spans the P insertion sites has been found to restore methyl methanesulfonate resistance and female fertility of the mei-41 ^D3 mutants. The results demonstrate that all the sequences required for the proper expression of the mei-41 gene are present on this genomic fragment. This study provides the foundation for molecular analysis of a function that is essential for chromosome stability in both the germline and somatic cells.This Paper is dedicated to the memory of Professor James B. Boyd     

Molecular genetic analysis of theripening-inhibitor andnon-ripening loci of tomato: A first step in genetic map-based cloning of fruit ripening genes

Ripening represents a complex developmental process unique to plants. We are using tomato fruit ripening mutants as tools to understand the regulatory components that control and coordinate the physiological and biochemical changes which collectively confer the ripe phenotype. We have genetically characterized two loci which result in significant inhibition of the ripening process in tomato,ripening-inhibitor (rin), andnon-ripening (nor), as a first step toward isolating genes likely to encode key regulators of this developmental process. A combination of pooled-sample mapping as well as classical restriction fragment length polymorphism (RFLP) analysis has permitted the construction of high-density genetic maps for the regions of chromosomes 5 and 10 spanning therin andnor loci, respectively. To assess the feasibility of initiating a chromosome walk, physical mapping of high molecular weight genomic DNA has been employed to estimate the relationship between physical distance (in kb) and genetic distance (in cM) around the targeted loci. Based on this analysis, the relationship in the region spanning therin locus is estimated to be 200–300 kb/cM, while thenor locus region ratio is approximately 200 kb/1 cM. Using RFLP markers tightly linked torin andnor, chromosome walks have been initiated to both loci in a yeast artificial chromosome (YAC) library of tomato genomic DNA. We have isolated and characterized several YAC clones linked to each of the targeted ripening loci and present genetic evidence that at least one YAC clone contains thenot locus.     

IgG-specific helper activity of t lymphocytes from mice lacking theIr-IgG gene

Cooperative interactions between T and B cells from the congenic inbred mouse strains B10.A(2R) and B10.A(4R) in antibody responses controlled byIr genes have been studied. Within theI region of the MHC, these strains share only theI-A subregion. TheIr gene controlling responsiveness to IgA maps in theI-A subregion, both strains being responders to IgA. T cells from 2R mice collaborate effectively with B cells from 2R or 4R mice for antihapten antibody responses to DNP-IgA. TheIr gene controlling responses to IgG maps in theI-B subregion, and 2R mice are nonresponders for this antigen. Nevertheless, 2R T cells primed with IgG can help responder (4R) B cells -but not syngeneic nonresponder (2R) B cells -in responding to DNP-IgG. These results indicate that mice lacking theIr-IgG gene nonetheless may develop helper T lymphocytes specific for myeloma proteins. In addition, they indicate that cells from congenic mice sharing only theA subregion of theI region can collaborate efficiently.     

Mapping of a gene causing resistance to chlorate inSalmonella typhimurium

Chlorate-resistant mutants ofS. typhimurium LT2 and LT7 and ofS. abony have been isolated, which are deficient in the biosynthesis of nicotinic acid and thiamin and in the fermentation of inositol. These mutants could be divided into 5 groups. The most likely gene order isnicB-chlG-thiB-inlB. This segment is transferred early in conjugation experiments with Hfr H2 and Hfr B2 as donors. In time-of-entry experiments with Hfr B2 as donor the segment entered about 3 minutes afterpur C. Consequently this segment maps in the 79- to 82-minutes region of the genetic map. From recombinant analysis of nic⁺ recombinants obtained in a four-point cross between Hfr B2 and ahis carBpur C del (nic chl G) acceptor the incorporation frequency of the transferred donor fragment was calculated to be about 0.41. The number of crossing-over events per minute length of the chromosome was about the same as in similar crosses betweenE. coli Hfr and F^–. However, between thenic and thepur C markers it was much higher; it may therefore be inferred that there is a higher probability for a crossing-over event in the regions adjacent to the region that is deleted in the recipient.In crosses betweenS. abony Hfr H2 del (nic thi inl chl) and F^– strains no recombinants were observed which have obtained the deletion from the donor. Nearly all auxotrophic or nic⁺ recombinants obtained in a cross between Hfr B2 and a F^– del (nicBthiBchlGinlB) strain have inherited all markers of the donor, which are present in the deletion of the recipient.     

Duplication of theDR3Gene on Human Chromosome 1p36 and Its Deletion in Human Neuroblastoma

The humanDR3gene, whose product is also known as Wsl-1/APO-3/TRAMP/LARD, encodes a tumor necrosis factor-related receptor that is expressed primarily on the surface of thymocytes and lymphocytes. DR3 is capable of inducing both NF-κB activation and apoptosis when overexpressed in mammalian cells, although its ligand has not yet been identified. We report here that theDR3gene locus is tandemly duplicated on human chromosome band 1p36.2–p36.3 and that these genes are hemizygously deleted and/or translocated to another chromosome in neuroblastoma (NB) cell lines with amplifiedMYCN.Duplication of at least a portion of theDR3gene, including the extracellular and transmembrane regions but not the cytoplasmic domain, was demonstrated by both fluorescencein situhybridization and genomic Southern blotting. In most NB cell lines, both theDR3and theDR3Lsequences are simultaneously deleted and/or translocated to another chromosome. Finally, DR3/Wsl-1 protein expression is quite variable among these NB cell lines, with very low or undetectable levels in 7 of 17 NB cell lines.     

Physical mapping of a rice lesion mimic gene,<Emphasis Type="Italic"> Spl1</Emphasis> , to a 70-kb segment of rice chromosome 12

The rice lesion mimic mutant spotted leaf 1 ( spl1) was first identified in the rice ( Oryza sativa) cultivar Asahi in 1965. This mutant displayed spontaneous disease-like lesions in the absence of any pathogen, and was found to confer resistance to multiple isolates of rice blast. We employed a map-based cloning strategy to localize the Spl1 gene. A total of ten cleaved amplified polymorphic sequence (CAPS) markers linked to the Spl1 gene were identified and mapped to an 8.5-cM region on chromosome 12. A high-resolution genetic map was developed using these ten CAPS markers and a segregating population consisting of 3202 individuals. A BAC contig containing four BAC clones was constructed, and Spl1 was localized to a 423-kb region. Seven spl1 mutants were obtained from the IR64 deletion mutant collection, and molecular analysis using these mutants delimited the Spl1 gene to a 70-kb interval, covered by two BAC clones. These results provide the basis for cloning this gene, which is involved in cell death and disease resistance in rice.Communicated by R. HagemannThe first two authors contributed equally to the work     

Construction of squalene-accumulatingSaccharomyces cerevisiae mutants by gene disruption through homologous recombination

Saccharomyces cerevisiae synthesizes ergosterol via squalene, but squalene is hardly detected in aerobically grown cells. To obtain a stable squalene-accumulating yeast strain, we attempted to disrupt a gene required in the conversion of squalene to ergosterol, by homologous recombination with a short piece of the gene fragment conjugated with an integration plasmid vector carrying theLEU2 gene. Two mutants that required ergosterol at least for fast growth were isolated. In an aerobic cultivation and with ergosterol supplementation, the two mutants accumulated squalene up to 5 mg/g dry cells. Southern hybridization analysis indicated that both mutants had acquired the vector DNA integrated in the same gene, or nearby genes, on chromosome 12.     

Escherichia coli cad operon functions as a supplier of carbon dioxide

We examined the gene expression of the Escherichia coli cad operon, which consisted of the genes cadB and cadA (lysine decarboxylase), using cells possessing cadB–lacZ fusion gene. The cad operon was expressed when O₂ was limited, and the expression was optimal at pH6.3. The β-galactosidase activity was lowered by the addition of sodium carbonate to the medium. The expression of the cad operon was reduced in cells containing the plasmid-encoding ornithine decarboxylase, which produced carbon dioxide, indicating that the gene expression of the cad operon was regulated by carbon dioxide (or its derivatives). It is known that the Krebs cycle is a major pathway for producing carbon dioxide, and that its activity is repressed when O₂ is limited. Thus, our present results suggested that the physiological role of the cad operon is to supply carbon dioxide when its internal level is lowered under O₂-limiting conditions at a low pH.     

Elements of theS-gene complex VI. Mutations of the self-incompatibility gene,pseudo-compatibility and origin of new self-incompatibility alleles

Spontaneously occurring mutations of theS gene, involving both theS _I and theS _FI classes of alleles, were studied inNicotiana alata. The results showed that while almost all of the irradiation-induced mutants of theS gene requiredS-bearing duplication for their survival, usually in the form of a free fragment, most of the spontaneous mutants in the same species, surprisingly, did not have such a requirement. This difference has been attributed to the greater depth of mutations produced in response to the ionizing radiations, which necessitated complementation for the survival of the mutants. There is a possibility from the data that theS _FI class of alleles may have even less need for the duplication than the S_I class of alleles. Both pollen- and stylar-part mutations of theS gene were obtained, but the majority of the mutations were partial, producing less than half the normal complement of seeds per pod in the mutants. Complementation was observed in the style between a -part mutant alleleS _infF11 ^sup and a normal alleleS _F10, which was the other allele in the parental plant that produced the mutant. No complementation occurred with another normal unrelated alleleS ₂. This observation was similar to that previously recorded in the study of induced mutants inN. alata.In a cross where the two alleles of the pollen parent were both compatible the allele which was also a mutant had an advantage over the other, normal, allele. This suggests that in maize, where the occurrence of mutant forms of theS gene has been demonstrated, the preferential fertilization of ovules by pollen containing the B-chromosomes may be due to the presence of a mutant form of theS gene on the B-chromosome.Besides clear-cutS-gene mutants, there were others, showing mostly irregular, slight compatibility, which did not appear to be directly related to theS-gene mutation. In some of the progeny of certain of these mutants, partial or complete lack of the specificity of one or bothS alleles in the style was observed; in certain others all progeny were normal. This pseudo-compatibility is attributed to cytoplasmic mutations affecting the products of theS gene; however, the possibility of an effect of chance polygenic modifier combinations is not ruled out.Recent literature on theS-gene structure, mutational specificity ofS alleles, and genetic control of pseudo-compatibility is reviewed. The time ofS-gene action is discussed in relation to the mechanism of the generation of new self-incompatibility alleles.     

Effect of the local anesthetics phenethyl alcohol and procaine on hns mutants of the acid-induced biodegradative arginine (adi) and lysine (cad) decarboxylases of Escherichia coli

The environmentally responsive biodegradative arginine (adi) and lysine (cad) decarboxylases are maximally induced when Escherichia coli is cultured under acidic, anaerobic conditions in rich medium. Previously, transposon mutagenesis led to the identification of hns (encoding H-NS, a histone-like DNA binding protein) as being a trans-acting regulatory factor of both systems. The hns mutants show derepressed expression of adi or cad (i.e., their expression is increased). The effects of the local anesthetics phenethyl alcohol (PEA) and procaine (both environmental perturbants) were investigated with lacZ operon fusions to either adi or cad and their respective hns mutants. These results indicate that wild-type fusion strains are insensitive to either PEA or procaine, but that hns mutants show decreased -galactosidase synthesis in the presence of one or both of the local anesthetics. This is the first report of the effect of local anesthetics on hns mutants in this or any other environmentally responsive system.     

The mapping of phytochrome genes and photomorphogenic mutants of tomato

The map positions of five previously described phytochrome genes have been determined in tomato (Lycopersicon esculentum Mill.) The position of the yg-2 gene on chromosome 12 has been confirmed and the classical map revised. The position of the phytochrome A (phy A)-deficient fri mutants has been refined by revising the classical map of chromosome 10. The position of the PhyA gene is indistinguishable from that of the fri locus. The putative phyB1-deficient tri mutants were mapped by classical and RFLP analysis to chromosome 1. The PhyB1 gene, as predicted, was located at the same position. Several mutants with the high pigment (hp) phenotype, which exaggerates phytochrome responses, have been reported. Allelism tests confirmed that the hp-2 mutant is not allelic to other previously described hp (proposed here to be called hp-1) mutants and a second stronger hp-2 allele (hp-2 ^j ) was identified. The hp-2 gene was mapped to the classical, as well as the RFLP, map of chromosome 1. Received: 24 May 1996 / Accepted: 14 June 1996     

Pleiotropic mutants of Aspergillus nidulans altered in carbon metabolism.

Summary Mutants altered in carbon catabolite regulation have been isolated by selecting for mutants of theareA217 strain capable of using acetamide as the sole nitrogen source in the presence of sucrose. In addition tocreA mutants described previously by Arst and Cove, strains with mutations in two new genes,creB andcreC, have been found. ThecreB andcreC mutants grow poorly on some sole carbon sources and have low levels of some enzymes of carbon catabolism e.g. -galactosidase and D-quinate dehydrogenase. ThecreB andcreC mutants are hypersitive to fluoroacetate, fluoroacetamide and allyl alcohol in the presence of glucose or sucrose but not glycerol; and the enzymes, acetamidase, and alcohol dehydrogenase, are less sensitive to carbon catabolite repression than the wild-type strain. Extracellular protease and -glucosidase enzyme activities are elevated increB andcreC mutants, while L-proline and L-glutamate uptake capacities are lower in both the presence and absence of glucose. Interactions betweencreA, B and C mutations have been investigated in double mutants, and the dominance properties ofcreB andcreC mutants determined. The results indicate that thecreB andcreC genes may have a regulatory role in the control of carbon catabolism.     

ADrosophila homologue of theSchizosaccharomyces pombe act2 gene

Diverse proteins that are 35% to 55% identical to actins have been discovered recently in yeasts, nematodes, and vertebrates. In order to study these proteins systematically and relate their functions to those of conventional actins, we are isolating the corresponding genes from the genetically tractable eukaryote,Drosophila melanogaster. Here we report the isolation and partial characterization of aDrosophila homologue of theSchizosaccharomyces pombe act2 gene. Degenerate oligonucleotide primers specifying peptides that are highly conserved within the actin protein superfamily were used in conjunction with polymerase chain reaction (PCR) to amplify a portion of theDrosophila gene that we have namedactr66B. The corresponding full-length cDNA sequence encodes a protein of 418 residues that is 65% identical to the product of theS. pombe act2 gene, 80% identical to the bovineact2 homologue, but only 48% identical to the principalDrosophila cytoplasmic actin encoded by theAct5C actin gene. Alignment of the yeast, bovine, andDrosophila actin-related proteins shows that they have four peptide insertions, relative to conventional actins, three of which are well placed to modify actin polymerization and one that is likely to perturb the binding of myosin. Locations of two of the fiveactr66B introns are conserved betweenDrosophila and yeast genes, further attesting that they evolved from a common ancestor and are likely to encode proteins having similar functions. We demonstrate that theDrosophila gene is located on the left arm of chromosome 3, within subdivision 66B. Finally, we show by RNA blot-hybridization that the gene is expressed at low levels, relative to conventional nonmuscle actin, in all developmental stages. From these and other observations we infer that the actr66B protein is a minor component of all cells, perhaps serving to modify the polymerization, structure, and dynamic behavior of actin filaments. Our work was supported by grants from the NIH and the Muscular Dystrophy Association to E.A.F. Sequences described herein have been filed in the GenBank Database under Accession Number X71789.     

A ribosomal RNA gene ofEscherichia coli (rrnD) on λ daroE specialized transducing phages

Summary Lambda-transducing phages carrying segments of theEscherichia coli chromosome in thearoE-trkA region have been isolated and shown by hybridization to carry an rRNA gene (rrnD). The most likely gene order istrkA aroE rrnD. TheEcoRI andSmaI endonuclease cutting pattern of therrnD gene is identical with the one ofrrnB, but differented fromrrnC.