Biomedical subjects

J W Kronstad

Publications and source records attributed to J W Kronstad.

31 records · Page 2Linked to original sources

PubMed1994

Disruption of two genes for chitin synthase in the phytopathogenic fungus Ustilago maydis.

The phytopathogenic fungus Ustilago maydis exhibits a dimorphic transition in which non-pathogenic, yeast-like cells mate to form a pathogenic, filamentous dikaryon. Northern analysis indicated that two chitin synthase genes, chs1 and chs2, from U. maydis are expressed at similar levels in yeast-like cells and in cells undergoing the mating reaction leading to the filamentous cell type. A mutation was constructed in each of the chitin synthase genes by targeted gene disruption. Each mutant showed a reduction in the level of trypsin-activated enzyme activity, compared with a wild-type strain, but retained the wild-type morphology, the ability to mate and the ability to form the filamentous pathogenic cell type.

Chitin Synthase↗

PubMed1993

Construction of chimeric alleles with altered specificity at the b incompatibility locus of Ustilago maydis.

Multiallelic incompatibility systems found in many fungi and plants function to limit inbreeding by mediating self versus nonself recognition. The plant pathogenic fungus Ustilago maydis has a locus called b that governs incompatibility. Two multiallelic genes, bE and bW, are present at the b locus. Fusion of haploid strains carrying different alleles at bE and bW establishes an infectious dikaryon capable of pathogenesis on maize (Zea mays). Cells carrying a single type of b locus, whether haploid or dikaryotic, are nonpathogenic. To identify sequences within the bE gene that determine allelic specificity, targeted gene replacement was employed to produce a series of chimeras between the b1E and b2E alleles. Incompatibility tests with strains carrying the chimeric alleles identified a 30- to 48-amino acid region responsible for specificity. Suprisingly, the chimeras with recombination points within this region had a specificity different from both parent alleles. Overall, these results define an important domain in bE involved in self versus nonself recognition.

Alleles↗

PubMed1993

Conservation of the b mating-type gene complex among bipolar and tetrapolar smut fungi.

In the phytopathogenic fungus Ustilago hordei, one locus with two alternate alleles, MAT-1 and MAT-2, controls mating and the establishment of the infectious dikaryon (bipolar mating). In contrast, for U. maydis, these functions are associated with two different gene complexes, called a and b (tetrapolar mating); the a complex has two alternate specificities, and the b gene complex is multiallelic. We have found homologs for the b gene complex in U. hordei and have cloned one from each mating type using sequences from one bEast allele of U. maydis as a probe. Sequence analysis revealed two divergent open reading frames in each b complex, which we called bW (bWest) and bE (bEast) in analogy with the b gene complex of U. maydis. The predicted bW and bE gene products from the two different mating types showed approximately 75% identity when homologous polypeptides were compared. All of the characterized bW and bE gene products have variable amino-terminal regions, conserved carboxy-terminal regions, and similar homeodomain motifs. Sequence comparisons with the bW1 and bE1 genes of U. maydis showed conservation in organization and structure. Transformation of the U. hordei b gene complex into a U. hordei strain of opposite mating type showed that the b genes from the two mating types are functional alleles. The U. hordei b genes, when introduced into U. maydis, rendered the haploid transformants weakly pathogenic on maize. These results indicate that structurally and functionally conserved b genes are present in U. hordei.

Alleles↗

PubMed1991

Expression of the cryIB crystal protein gene of Bacillus thuringiensis.

The cryIB gene of Bacillus thuringiensis subsp. thuringiensis HD-2 codes for a Mr 139492 protein that is lethal to certain lepidopteran larvae. We used primer extension to map transcriptional initiation sites and found that cryIB was transcribed from two sites that are activated at different times during sporulation. The presumed promoter regions for the two start sites are very similar to the two promoters preceding the cryIA (a) gene, and the in vivo transcriptional start sites were found to be identical. Variable amounts of the full-length cryIB protein were detected by immunoblotting of extracts of recombinant cells of Escherichia coli; larger amounts were found when the TTG translational start codon was changed to ATG and when an htpR- strain of E. coli was used as the recipient for transformation. When expressed in E. coli, the cryIB protein was found to be toxic to the larvae of Artogeia rapae (LC50 of 58 ng/cm2) and exhibited little toxicity to the larvae of Manduca sexta (LC50 greater than 5000 ng/cm2).

Animals↗

PubMed1990

The b mating-type locus of Ustilago maydis contains variable and constant regions.

The b locus of the phytopathogenic fungus Ustilago maydis encodes a multiallelic recognition function that controls the ability of the fungus to form a dikaryon and complete the sexual stage of the life cycle. The b locus has at least 25 alleles and any combination of two different alleles, brought together by mating between haploid cells, allows the fungus to cause disease and undergo sexual development within the plant. An open reading frame of 410 amino acids has been shown to specify a polypeptide responsible for the activity of the b1 allele, and comparisons of the predicted amino acid sequences for 6 b alleles allowed identification of variable and constant regions within the coding region of the gene. Haploid strains carrying a null mutation at the b locus, created by gene disruption, are viable but fail to interact with formerly compatible strains to give an infectious dikaryon. Analysis of mutants carrying a null allele indicated that the products of different alleles of the b locus combine to form a new regulatory activity and that this activity directly or indirectly turns on the pathway leading to sexual development and pathogenesis.

Alleles↗

PubMed1989

Isolation of metabolic genes and demonstration of gene disruption in the phytopathogenic fungus Ustilago maydis.

A cDNA library was constructed in the yeast expression vector pYcDE8 using mRNA from the phytopathogenic fungus Ustilago maydis and cDNAs capable of complementing mutations in three yeast genes, URA3, LEU2 and TPI1, were identified. Nucleotide sequence analysis indicated that the cDNA clone, which complemented the yeast ura3 mutation, carries the pyr6 gene encoding orotidine-5'-phosphate decarboxylase. The genomic copy of the pyr6 gene was isolated by hybridization with the cDNA and used to complement a pyr- mutant of U. maydis. One-step gene disruption was demonstrated by transforming U. maydis with a copy of the pyr6 gene interrupted in the coding region by a selectable marker for resistance to hygromycin B.

Amino Acid Sequence↗

PubMed1989

Mutation in a heat-regulated hsp70 gene of Ustilago maydis.

Four genes (ums1, ums2, ums3 and ums4) representing an hsp70-related gene family were isolated from a genomic library of Ustilago maydis. All four genes are transcriptionally active during normal growth. Following a heat shock, the mRNA levels of ums1 and ums2 increase by approximately 5-fold, whereas the ums3 transcript becomes less abundant. The amount of ums4 mRNA remains relatively unchanged after heat treatment. The nucleotide sequence of the 5' non-coding and a portion of the ums2 coding region was determined. The sequence encoding the first 90 amino acids is 73% identical to corresponding regions of the Drosophila and yeast (SSA1) hsp70 genes. To investigate the effect of a mutation in ums2, a plasmid was constructed in which most of the transcriptional unit of ums2 was deleted and substituted with the Escherichia coli hygromycin B (hygB) phosphotransferase gene. Transcription of this gene is controlled by the ums2 promoter, allowing the expression of hygB resistance in Ustilago. The marker was introduced into diploid cells as a linear sequence with termini homologous to the 5' and 3' flanking regions of ums2. In approximately 50% of transformants examined, one of the two wild-type ums2 alleles had been replaced by the mutated sequence, demonstrating the feasibility of using one-step gene disruption to create heterozygous diploids in Ustilago. The ums2/ums2::hygBr heterozygote produced teliospores after injection into corn plants, but only cells carrying functional ums2 were found among their meiotic progeny. Therefore ums2::hygBr segregates as a recessive lethal, which strongly suggests that ums2 is essential for growth in Ustilago.

Amino Acid Sequence↗

PubMed1989

Isolation of two alleles of the b locus of Ustilago maydis.

The basidiomycete Ustilago maydis is the causative agent of the disease corn smut. To be pathogenic, strains of U. maydis must be heterozygous for a locus called "b," which appears to control both pathogenicity and sexual development. Two alleles of the b locus of U. maydis were isolated by complementation and hybridization. The clones have the specificities of b1 and b2 alleles as demonstrated by their effects on the colony morphology and pathogenicity of haploid and diploid strains upon transformation. For example, nonpathogenic haploid and diploid strains of U. maydis carrying b2 alleles became pathogenic when transformed with the cloned b1 allele. Furthermore, an a2 b2 haploid strain could be transformed to an a2 b1 genotype by gene replacement using a DNA fragment containing a b1 allele and a selectable marker. The isolation of b alleles represents an important step toward understanding the control of dicaryon formation, dicaryon maintenance, and pathogenicity in U. maydis.

Alleles↗

PubMed1987

A yeast operator overlaps an upstream activation site.

The product of the BAR1 gene of Saccharomyces cerevisiae is synthesized only in the a cell type and inactivates alpha-factor, the mating pheromone made by alpha cells. The MAT alpha 2 protein represses the transcription of a-cell-specific genes, including BAR1, in alpha and a/alpha diploid cells. Transcription of BAR1 in a cells in stimulated upon exposure to alpha-factor. Deletion analysis of the 5' noncoding region of the BAR1 gene revealed that the major upstream activation site (UAS) overlaps the 31 bp operator sequence required for MAT alpha 2 repression. This result has implications for the negative control of transcription in yeast. The deletion analysis also indicated that the sequence TGAAACA mediates alpha-factor stimulation.

Base Sequence↗

PubMed1986

Three classes of homologous Bacillus thuringiensis crystal-protein genes.

Four homologous genes encoding insecticidal crystal proteins from Bacillus thuringiensis have been cloned in Escherichia coli. Differences in lengths of HindIII restriction fragments containing the 5' ends of the genes allowed the identification of three classes termed the '4.5- and 5.3- and 6.6-kb-class genes'. A survey of 24 strains from subspecies kurstaki and thuringiensis revealed strains containing one, two, or three of these classes of crystal protein genes. The 4.5- and 6.6-kb-class genes encode polypeptides of Mr 133,500 and 133,330, respectively, while the two 5.3-kb-class genes encode a ca. 130-kDa polypeptide. The polypeptide composition of crystals from the B. thuringiensis strains agreed with the composition predicted from the content of the three classes of genes. The representative genes from each class were isolated from B. thuringiensis plasmids of different sizes. An analysis of plasmid DNA flanking three of these genes revealed a complex pattern of two different inverted repeat (IR) sequences, IR2150 and IR1750. Four variant forms of IR1750 were found. The IR elements were located near deletions and rearrangements adjacent to crystal protein genes and may account for the diversity of plasmids carrying crystal protein genes in other subspecies of B. thuringiensis.

Bacillus thuringiensis↗

PubMed1984

Inverted repeat sequences flank a Bacillus thuringiensis crystal protein gene.

Two sets of inverted repeat DNA sequences, IR2150 and IR1750, were discovered flanking the crystal protein gene on the 75-kilobase plasmid of Bacillus thuringiensis subsp. kurstaki HD73. A restriction map of ca. 40 kilobases around the crystal protein gene was constructed, and the positions of the copies of IR2150 and IR1750 were determined. Three copies of IR2150 were found flanking the crystal protein gene in an inverted orientation, and one partial and three intact copies of IR1750 were found in both inverted and direct orientations around the gene. Hybridization experiments with fragments from within IR2150 and IR1750 demonstrated the presence of multiple copies of these sequences on the chromosome of B. thuringiensis subsp. kurstaki HD73 and also revealed a strong correlation between the presence of these sequences and the presence of the crystal protein gene on plasmids from 14 strains of B. thuringiensis.

Bacillus thuringiensis↗

PubMed1983

Diversity of locations for Bacillus thuringiensis crystal protein genes.

The location of crystal protein genes in 22 crystalliferous Bacillus thuringiensis strains representing 14 subspecies was investigated by hybridization of an intragenic restriction fragment from a cloned crystal protein gene to whole plasmid preparations. Hybridization was found to a single plasmid in eight strains, to more than one plasmid in seven strains, and to one or both of two large, unresolved plasmids in two strains. The sizes of the hybridized plasmids ranged from 33 to over 150 megadaltons. In one additional subspecies, hybridization was only to linear DNA fragments, suggesting a chromosomal crystal protein gene, and for four other subspecies, not reported to be toxic to lepidopteran insects, no hybridization was found to either plasmids or to total cell DNA. Hybridization to restriction digests of plasmids and total cell DNA of several strains of subspecies thuringiensis and kurstaki revealed that all homology to the cloned crystal protein gene was plasmid associated and that several of these strains contained multiple regions of homology, implying the presence of multiple crystal protein genes.

Bacillus thuringiensis↗

PubMedDate not supplied

Identification and complementation of a mutation to constitutive filamentous growth in Ustilago maydis.

Pathogenicity of the corn smut fungus Ustilago maydis involves the formation of a filamentous, infectious dikaryon by fusion of compatible, yeastlike haploid cells. The mating-type loci, a and b, regulate cell fusion and establishment of the dikaryotic cell type, respectively. On solid medium, compatibility at the mating-type loci, in particular heterozygosity at the b locus, is manifested by the formation of aerial hyphae on colonies formed by mating cells. We have employed this "fuzzy" phenotype to identify haploid mutants that constitutively form hyphal filaments and forego cell division by budding. A total of 125 such mutants have been isolated; characterization of one mutant (termed rem1-1) revealed that it can participate in infection of the host plant, although it must be paired with a compatible, wild-type mating partner. That is, mutation to the mycelial phenotype is not sufficient to allow a haploid strain to be pathogenic by itself. A cosmid has been isolated that restores the ability of an rem1-1 mutant to grow with a budding phenotype. Localization of the complementing region on cosmid DNA allowed the construction of an additional mutation by gene disruption. Coinoculation of plants with two compatible strains, each carrying the disruption mutation, gave greatly reduced disease symptoms. The analysis of the rem1 gene should contribute to an understanding of dimorphic growth and pathogenesis in U. maydis.

Base Sequence↗