Search

Mutants of class I enolpyruvylshikimate 3-phosphate synthase (EPSPS) with resistance to glyphosate were produced in a previous study using the staggered extension process with aroA genes from S. typhimurium and E. coli. Two of these mutants shared a common amino acid substitution, T42M, near the hinge region between the large globular domains of EPSPS. Using site-directed mutagenisis, we produced the T42M mutants without the other amino acid changes of the original mutants. The T42M substitution alone produced enzymes with a 9- to 25-fold decreased K(m)[PEP] and a 21- to 26-fold increased K(i)[glyphosate] compared to the wild-type enzymes. These results provide more testimony for the powerful approach for protein engineering by the combination of directed evolution and rational design.

Ustilago maydis secretes ferrichrome-type siderophores, ferric-ion-binding compounds, in response to iron starvation. TA2701, a non-enterobactin-producing, non-ferrichrome-utilizing mutant of Salmonella typhimurium LT-2, was employed as a biological indicator in a novel screening method to isolate three N-methyl-N'-nitro-N-nitrosoguanidine-induced U. maydis mutants defective in the regulation of ferrichrome-type siderophore biosynthesis. These mutants displayed a constitutive phenotype; they produced siderophores in the presence of iron concentrations that would typically repress siderophore synthesis in wild-type strains. A 4.8-kb fragment of U. maydis genomic DNA capable of restoring normal regulation of siderophore biosynthesis in the constitutive mutants was identified. This segment of DNA contains an intronless open reading frame that specifies a protein of 950 amino acids containing two finger motifs similar to those found in the erythroid transcription factor GATA-1. Disruption of this open reading frame in a wild-type strain gave rise to cells that produced siderophores constitutively. Genetic studies indicated that the disruption mutation was allelic to the chemically induced mutations, confirming that the structural gene for a regulator rather than a suppressor gene had been cloned. Northern (RNA) analysis of the gene revealed a 4.2-kb transcript that is expressed constitutively at low levels in wild-type cells. The data support the hypothesis that this gene, which we designate urbs1 (Ustilago regulator of biosynthesis of siderophores), acts directly or indirectly to repress biosynthesis of siderophores in U. maydis.

Drosomycin(Drs) encoding an inducible 44-residue antifungal peptide is clustered with six additional genes,Dro1, Dro2, Dro3, Dro4, Dro5,andDro6, forming a multigene family on the 3L chromosome arm inDrosophila melanogaster. To get further insight into the regulation of each member of the drosomycin gene family, here we investigated gene expression patterns of this family by either microbe-free injury or microbial challenges using real time RT-PCR. The results indicated that among the sevendrosomycingenes,Drs, Dro2, Dro3, Dro4,andDro5showed constitutive expressions. Three out of five,Dro2, Dro3,andDro5, were able to be upregulated by simple injury. Interestingly,Drsis an only gene strongly upregulated whenDrosophilawas infected with microbes. In contrast to these five genes,Dro1andDro6were not transcribed at all in either noninfected or infected flies. Furthermore, by5′rapid amplification of cDNA ends, two transcription start sites were identified inDrsandDro2, and one inDro3, Dro4,andDro5. In addition, NF-κB binding sites were found in promoter regions ofDrs, Dro2, Dro3,andDro5, indicating the importance of NF-κB binding sites for the inducibility ofdrosomycingenes. Based on the analyses of flanking sequences of each gene inD. melanogasterand phylogenetic relationship ofdrosomycinsinD. melanogasterspecies-group, we concluded that gene duplications were involved in the formation of the drosomycin gene family. The possible evolutionary fates ofdrosomycingenes were discussed according to the combining analysis of gene expression pattern, gene structure, and functional divergence of these genes.

Extracellular polysaccharide (EPS) has long been regarded as one of the most important factors involved in wilting of plants by Pseudomonas solanacearum. By means of transposon Tn5 mutagenesis, we have isolated a class of mutants that have an afluidal colony morphology but retain the ability to cause severe wilting and death of tobacco plants. One such mutant, KD700, was studied in detail. By marker exchange mutagenesis, the altered colony morphology was shown to be the result of a single Tn5 insertion in a 14.3-kilobase EcoRI fragment. This defect could be corrected by introducing a homologous clone from a cosmid library of the wild-type, parental strain K60. The Tn5-containing fragment was introduced into other P. solanacearum wild-type strains by marker exchange, and these altered strains had the same afluidal phenotype as KD700. N-Acetylgalactosamine (GalNac), the major constituent of EPS of all wild-type strains of P. solanacearum, was not detected by gas chromatography-mass spectrometry analysis of vascular fluids from wilting plants infected by KD700. In contrast, GalNac was readily detected in similar fluids of plants infected by K60. Polysaccharides extracted from culture filtrates of KD700 contained approximately one-fifth of the GalNac present in polysaccharides from K60. No differences in growth rates in culture or in planta between the mutant and the parental strains were observed. Since strains that are deficient in EPS production can remain highly virulent to tobacco, we conclude that EPS, or at least its GalNac-containing component, may not be required for disease development by P. solanacearum.

The most economically significant Chinese cotton cultivar (Gossypium hirsutum L. cv. Zhongmian 35) was transformed via Agrobacterium tumefaciens-mediated DNA transfer. The aroA-M1 gene that confers resistance to the glyphosate was fused with a chloroplast-transit peptide of Arabidopsis thaliana 5-enolpyruvyl-3-phosphoshikimate synthase (ASP) and expressed in cotton plants under the control of a CaMV35S promoter. Transgenic plants were directly selected on medium containing glyphosate. Thirty-four independent transgenic lines were obtained after selection, giving a maximal 1.9% transformation frequency. The integration and expression of the aroA-M1 gene in T(0) plants and T(1) progeny were confirmed using DNA hybridization, Western blot and PCR techniques. An increased resistance of T(0) and T(1 )transgenic plants towards glyphosate was also observed.

Glyphosate or Roundup is the most extensively used herbicide for broad-spectrum control of weeds. Glyphosate inhibits 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), a key enzyme in the aromatic amino acid biosynthetic pathway in microorganisms and plants. Applying the staggered extension process, we randomly mutated and recombined the aroA genes of Salmonella typhimurium and Escherichia coli to obtain four variants that exhibit significantly enhanced tolerance to glyphosate. All four mutants are chimeras of the two parental genes and, in addition, three of them carry one or more de novo point mutations. None of the amino acid substitutions in the mutants was in a position previously known to be important for catalysis or substrate binding. Kinetic analysis of EPSPS activity from these mutants indicated that the tolerance was attributed to a 2-10-fold increased specific activity, 0.4-8-fold reduced affinity to glyphosate, and 2.5-19-fold decreased K(m) for phosphoenolpyruvate. Such mutants will be instrumental for the structural and function study of the enzyme and for the generation of transgenic crops resistant to the herbicide.

We are isolating and studying genes required for pathogenicity of Ustilago maydis, the causative agent of corn smut disease (Christensen, 1963). This phytopathogenic basidiomycete offers an attractive system in which to gain a molecular understanding of host-parasite interactions. The organism grows as a haploid yeast on defined laboratory media, mutants are readily generated by UV or chemical mutagenesis, stable diploids can be constructed for mitotic recombination and complementation analysis, and the fungus is amenable to Mendelian genetic analysis (Holliday, 1974). These attributes are not found in combination in any other phytopathogenic fungus. Although this pathogen is no longer a production constraint in North America, where resistant hybrid corn is grown, it continues to be a problem in third world countries where susceptible varieties are still cultivated. Moreover, we hope that an understanding of mechanisms of pathogenesis in this host-parasite interaction will have application to more economically important and difficult to study fungal diseases such as the bunts and rusts. We have initiated a molecular genetic analysis of two gene systems thought to control pathogenic growth of U. maydis in maize. These include genes that program sexual development and genes involved in the high affinity, siderophore-mediated iron uptake system of the fungus. In order to conduct these analyses, one of our first goals has been to develop tools which enable us to clone, study, and transfer genes in U. maydis.

In this study, we describe the use of complementation, gene replacement, and saturation mutagenesis to identify a new gene cluster that controls pathogenicity and HR in these particular strains of P. solanacearum. The results also indicate that expression of the two transcriptional units in this region may be increased in the presence of plant cells

The comparison of the nucleotide sequences of closely linked duplicated genes of higher eukaryotes has been important in the identification of molecular events that shape the evolution of mammalian genes, most notably recombinational events such as unequal crossovers and gene conversions. Toward this goal we have been comparing the nucleotide sequences of the paired gamma 1- and gamma 2-fetal globin genes from species of catarrhine primates. Previous comparisons document that, within each great ape species as in humans, the paired gamma-genes have been involved in gene conversion events. We now extend our analysis to the catarrhine superfamily Cercopithecoidea by obtaining the nucleotide sequence of the paired gamma 1- and gamma 2-genes of rhesus monkey (Macaca mulatta). The rhesus gamma 1- and gamma 2-genes diverge less from each other than from human, chimpanzee, gorilla, and orangutan gamma 1- or gamma 2-genes. This finding indicates that a species-specific gene conversion occurred between rhesus gamma 1- and gamma 2-genes. This gamma-gene conversion (labeled C14 in our series) involved at least 1898 base pairs, extending across the complete transcriptional region of the rhesus gamma-genes. C14 could have resulted from a single large conversion or several short conversion events which may have involved the (TG)n repetitive sequence element. Parsimony analysis of the enlarged body of gamma-gene sequence data also strengthens the evidence for the 14 previously suggested gamma-gene conversion events: labeled C2, C3, and C4 in Homo; C5, C6, and C7 in Pan; C8, C9, and C10 in Gorilla; C11, C12, C13 in Pongo; C1 in the stem to Homininae (the subfamily of Homo, Pan, and Gorilla) and CO in the stem of Hominidae (the family of Pongo and Homininae).