scholarly journals Cloning and characterization of KNR4, a yeast gene involved in (1,3)-beta-glucan synthesis.

1994 ◽  
Vol 14 (2) ◽  
pp. 1017-1025 ◽  
Author(s):  
Z Hong ◽  
P Mann ◽  
N H Brown ◽  
L E Tran ◽  
K J Shaw ◽  
...  
Keyword(s):  
Cell Wall ◽  
Killer Toxin ◽  
Structural Defects ◽  
Beta Glucan ◽  
Calculated Molecular Mass ◽  
Beta Glucanase ◽  
Reading Frame ◽  
Glucan Synthesis ◽  
The Right

k9 killer toxin from Hansenula mrakii was used to select a number of resistant mutants from Saccharomyces cerevisiae. Preliminary biochemical and genetic studies showed that some of them acquired structural defects in the cell wall. One of these mutants, the knr4-1 mutant, displays a number of cell wall defects, including osmotic sensitivity; sensitivity to cercosporamide, a known antifungal agent; and resistance to Zymolyase, a (1,3)-beta-glucanase. We report here the isolation and analysis of the KNR4 gene. DNA sequence analysis revealed an uninterrupted open reading frame which contains five potential start codons. The longest coding template encodes a protein of 505 amino acids with a calculated molecular mass of 57,044 Da. A data base search revealed 100% identity with a nuclear protein, SMI1p. Disruption of the KNR4 locus does not result in cell death; however, it leads to reduced levels of both (1,3)-beta-glucan synthase activity and (1,3)-beta-glucan content in the cell wall. The gene was mapped to the right arm of chromosome VII.

Cloning and characterization of KNR4, a yeast gene involved in (1,3)-beta-glucan synthesis

1994 ◽  
Vol 14 (2) ◽  
pp. 1017-1025
Author(s):  
Z Hong ◽  
P Mann ◽  
N H Brown ◽  
L E Tran ◽  
K J Shaw ◽  
...  
Keyword(s):  
Cell Wall ◽  
Killer Toxin ◽  
Structural Defects ◽  
Beta Glucan ◽  
Calculated Molecular Mass ◽  
Beta Glucanase ◽  
Reading Frame ◽  
Glucan Synthesis ◽  
The Right

k9 killer toxin from Hansenula mrakii was used to select a number of resistant mutants from Saccharomyces cerevisiae. Preliminary biochemical and genetic studies showed that some of them acquired structural defects in the cell wall. One of these mutants, the knr4-1 mutant, displays a number of cell wall defects, including osmotic sensitivity; sensitivity to cercosporamide, a known antifungal agent; and resistance to Zymolyase, a (1,3)-beta-glucanase. We report here the isolation and analysis of the KNR4 gene. DNA sequence analysis revealed an uninterrupted open reading frame which contains five potential start codons. The longest coding template encodes a protein of 505 amino acids with a calculated molecular mass of 57,044 Da. A data base search revealed 100% identity with a nuclear protein, SMI1p. Disruption of the KNR4 locus does not result in cell death; however, it leads to reduced levels of both (1,3)-beta-glucan synthase activity and (1,3)-beta-glucan content in the cell wall. The gene was mapped to the right arm of chromosome VII.

scholarly journals Characterization of the yeast (1-->6)-beta-glucan biosynthetic components, Kre6p and Skn1p, and genetic interactions between the PKC1 pathway and extracellular matrix assembly.

1994 ◽  
Vol 127 (2) ◽  
pp. 567-579 ◽  
Author(s):  
T Roemer ◽  
G Paravicini ◽  
M A Payton ◽  
H Bussey
Keyword(s):  
Cell Wall ◽  
Beta Glucan ◽  
Homologous Proteins ◽  
Synthetic Lethal ◽  
Cell Wall Assembly ◽  
Cell Biol ◽  
Synthetic Lethal Interactions ◽  
Glucan Synthesis ◽  
Wall Assembly

A characterization of the S. cerevisiae KRE6 and SKN1 gene products extends previous genetic studies on their role in (1-->6)-beta-glucan biosynthesis (Roemer, T., and H. Bussey. 1991. Yeast beta-glucan synthesis: KRE6 encodes a predicted type II membrane protein required for glucan synthesis in vivo and for glucan synthase activity in vitro. Proc. Natl. Acad. Sci. USA. 88:11295-11299; Roemer, T., S. Delaney, and H. Bussey. 1993. SKN1 and KRE6 define a pair of functional homologs encoding putative membrane proteins involved in beta-glucan synthesis. Mol. Cell. Biol. 13:4039-4048). KRE6 and SKN1 are predicted to encode homologous proteins that participate in assembly of the cell wall polymer (1-->6)-beta-glucan. KRE6 and SKN1 encode phosphorylated integral-membrane glycoproteins, with Kre6p likely localized within a Golgi subcompartment. Deletion of both these genes is shown to result in a dramatic disorganization of cell wall ultrastructure. Consistent with their direct role in the assembly of this polymer, both Kre6p and Skn1p possess COOH-terminal domains with significant sequence similarity to two recently identified glucan-binding proteins. Deletion of the yeast protein kinase C homolog, PKC1, leads to a lysis defect (Levin, D. E., and E. Bartlett-Heubusch. 1992. Mutants in the S. cerevisiae PKC1 gene display a cell cycle-specific osmotic stability defect. J. Cell Biol. 116:1221-1229). Kre6p when even mildly overproduced, can suppress this pkc1 lysis defect. When mutated, several KRE pathway genes and members of the PKC1-mediated MAP kinase pathway have synthetic lethal interactions as double mutants. These suppression and synthetic lethal interactions, as well as reduced beta-glucan and mannan levels in the pkc1 null wall, support a role for the PKC1 pathway functioning in cell wall assembly. PKC1 potentially participates in cell wall assembly by regulating the synthesis of cell wall components, including (1-->6)-beta-glucan.

scholarly journals A mutational analysis of killer toxin resistance in Saccharomyces cerevisiae identifies new genes involved in cell wall (1-->6)-beta-glucan synthesis.

Genetics ◽  
1993 ◽  
Vol 133 (4) ◽  
pp. 837-849 ◽  
Author(s):  
J L Brown ◽  
Z Kossaczka ◽  
B Jiang ◽  
H Bussey
Keyword(s):  
Cell Wall ◽  
Mutational Analysis ◽  
Killer Toxin ◽  
Similar Proportion ◽  
Wild Type ◽  
Beta Glucan ◽  
Toxin Resistance ◽  
Recessive Mutations ◽  
New Genes ◽  
Glucan Synthesis

Abstract Recessive mutations leading to killer resistance identify the KRE9, KRE10 and KRE11 genes. Mutations in both the KRE9 and KRE11 genes lead to reduced levels of (1-->6)-beta-glucan in the yeast cell wall. The KRE11 gene encodes a putative 63-kD cytoplasmic protein, and disruption of the KRE11 locus leads to a 50% reduced level of cell wall (1-->6)-glucan. Structural analysis of the (1-->6)-beta-glucan remaining in a kre11 mutant indicates a polymer smaller in size than wild type, but containing a similar proportion of (1-->6)- and (1-->3)-linkages. Genetic interactions among cells harboring mutations at the KRE11, KRE6 and KRE1 loci indicate lethality of kre11 kre6 double mutants and that kre11 is epistatic to kre1, with both gene products required to produce the mature glucan polymer at wild-type levels. Analysis of these KRE genes should extend knowledge of the beta-glucan biosynthetic pathway, and of cell wall synthesis in yeast.

Preparation and characterization of yeast cell wall beta-glucan encapsulated humic acid nanoparticles as an enhanced aflatoxin B1 binder

2019 ◽  
Vol 203 ◽  
pp. 185-192 ◽  
Author(s):  
Zeinab Hamza ◽  
Maher El-Hashash ◽  
Soher Aly ◽  
Amal Hathout ◽  
Ernesto Soto ◽  
...  
Keyword(s):  
Cell Wall ◽  
Humic Acid ◽  
Yeast Cell ◽  
Aflatoxin B1 ◽  
Yeast Cell Wall ◽  
Beta Glucan

Mutational analysis of the functional domains of yeast K1 killer toxin

1991 ◽  
Vol 11 (1) ◽  
pp. 175-181
Author(s):  
H Zhu ◽  
H Bussey
Keyword(s):  
Cell Wall ◽  
Receptor Binding ◽  
Mutational Analysis ◽  
Killer Toxin ◽  
Alpha Subunit ◽  
Functional Domains ◽  
Beta Subunits ◽  
Channel Formation ◽  
Beta Glucan ◽  
Beta Toxin

To determine the functional domains of K1 killer toxin, we analyzed the phenotypes of a set of mutations throughout regions encoding the alpha- and beta-toxin subunits that allow secretion of mutant toxins. A range of techniques have been used to examine the ability of mutant toxins to bind to beta-glucan cell wall receptor and to form lethal ion channels. Our results indicate that both the alpha and beta subunits are involved in beta-glucan receptor binding. Defects in ion channel formation and toxin immunity are confined to the hydrophobic alpha subunit of the toxin.

scholarly journals Molecular Cloning and Characterization of cgs, theBrucella abortus Cyclic β(1-2) Glucan Synthetase Gene: Genetic Complementation of Rhizobium meliloti ndvB andAgrobacterium tumefaciens chvB Mutants

1998 ◽  
Vol 180 (17) ◽  
pp. 4392-4400 ◽  
Author(s):  
Nora Iñón de Iannino ◽  
Gabriel Briones ◽  
Marcelo Tolmasky ◽  
Rodolfo A. Ugalde
Keyword(s):  
Amino Acids ◽  
Membrane Protein ◽  
Rhizobium Meliloti ◽  
Nodule Development ◽  
Nucleotide Sequencing ◽  
Insertion Mutant ◽  
Reading Frame ◽  
Glucan Synthesis ◽  
Glucan Synthetase

ABSTRACT The animal pathogen Brucella abortus contains a gene,cgs, that complemented a Rhizobium melilotinodule development (ndvB) mutant and an Agrobacterium tumefaciens chromosomal virulence (chvB) mutant. The complemented strains recovered the synthesis of cyclic β(1-2) glucan, motility, virulence in A. tumefaciens, and nitrogen fixation in R. meliloti; all traits were strictly associated with the presence of an active cyclic β(1-2) glucan synthetase protein in the membranes. Nucleotide sequencing revealed the presence in B. abortus of an 8.49-kb open reading frame coding for a predicted membrane protein of 2,831 amino acids (316.2 kDa) and with 51% identity to R. meliloti NdvB. Four regions of the B. abortus protein spanning amino acids 520 to 800, 1025 to 1124, 1284 to 1526, and 2400 to 2660 displayed similarities of higher than 80% with R. meliloti NdvB. Tn3-HoHo1 mutagenesis showed that the C-terminal 825 amino acids of the Brucella protein, although highly conserved inRhizobium, are not necessary for cyclic β(1-2) glucan synthesis. Confirmation of the identity of this protein as B. abortus cyclic β(1-2) glucan synthetase was done by the construction of a B. abortus Tn3-HoHo1 insertion mutant that does not form cyclic β(1-2) glucan and lacks the 316.2-kDa membrane protein. The recovery of this mutant from the spleens of inoculated mice was decreased by 3 orders of magnitude compared with that of the parental strain; this result suggests that cyclic β(1-2) glucan may be a virulence factor inBrucella infection.

SKN1 and KRE6 define a pair of functional homologs encoding putative membrane proteins involved in beta-glucan synthesis

1993 ◽  
Vol 13 (7) ◽  
pp. 4039-4048
Author(s):  
T Roemer ◽  
S Delaney ◽  
H Bussey
Keyword(s):  
Genomic Library ◽  
Killer Toxin ◽  
Resistant Mutant ◽  
Null Alleles ◽  
Dependent Manner ◽  
Wild Type ◽  
Beta Glucan ◽  
Glucan Synthesis ◽  
Dose Dependent

KRE6 encodes a predicted type II membrane protein which, when disrupted, results in a slowly growing, killer toxin-resistant mutant possessing half the normal level of a structurally wild-type cell wall (1-->6)-beta-glucan (T. Roemer and H. Bussey, Proc. Natl. Acad. Sci. USA 88:11295-11299, 1991). The mutant phenotype and structure of the KRE6 gene product, Kre6p, suggest that it may be a beta-glucan synthase component, implying that (1-->6)-beta-glucan synthesis in Saccharomyces cerevisiae is functionally redundant. To examine this possibility, we screened a multicopy genomic library for suppression of both the slow-growth and killer resistance phenotypes of a kre6 mutant and identified SKN1, which encodes a protein sharing 66% overall identity to Kre6p. SKN1 suppresses kre6 null alleles in a dose-dependent manner, though disruption of the SKN1 locus has no effect on killer sensitivity, growth, or (1-->6)-beta-glucan levels. skn1 kre6 double disruptants, however, showed a dramatic reduction in both (1-->6)-beta-glucan levels and growth rate compared with either single disruptant. Moreover, the residual (1-->6)-beta-glucan polymer in skn1 kre6 double mutants is smaller in size and altered in structure. Since single disruptions of these genes lead to structurally wild-type (1-->6)-beta-glucan polymers, Kre6p and Skn1p appear to function independently, possibly in parallel, in (1-->6)-beta-glucan biosynthesis.

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