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Biomedical subjects

P Broda

Publications and source records attributed to P Broda.

At least 19 recordsLinked to original sources

The construction of Streptomyces cyaneus genomic libraries in Escherichia coli is dependent upon the use of Mcr-deficient strains.

Streptomyces cyaneus genomic DNA ligated into either lambda phage or plasmid vectors was very inefficiently cloned into standard Escherichia coli host strains. However, the same material could be efficiently cloned using Mcr-deficient E. coli strains. These results suggest that the S. cyaneus genome contains 5-methylcytosine residues, some of which occur within the recognition sequences of the E. coli Mcr restriction system.

Cloning, Molecular

Expression of a single lignin peroxidase-encoding gene in Phanerochaete chrysosporium strain ME446.

A previously described linked set of lignin peroxidase-encoding genes (Lpo) from Phanerochaete chrysosporium (P.c.) ME446 is not expressed under standard growth conditions for ligninolytic activity. However, a single unlinked Lpo gene, not previously described in P.c. strain ME446, is expressed. The transcription start points of this gene are mapped and the gene is assigned to a genetic linkage group by the use of restriction-site polymorphism segregation analysis. No transcripts from Lpo-related genes, including that normally expressed in ME446, could be detected within RNA extracted from three nonligninolytic mutant strains, but a hyper-ligninolytic strain showed an increased level of Lpo expression. This increase is due to expression of additional Lpo genes, rather than to an increased level of transcription from the normally expressed sequence.

Base Sequence

An integrated approach to studying regulation of production of the antibiotic methylenomycin by Streptomyces coelicolor A3(2).

A physiological and molecular biological study was made of the control of methylenomycin biosynthesis by Streptomyces coelicolor A3(2). A simple and reliable assay for this antibiotic was developed. Conditions that permit the synthesis of methylenomycin by S. coelicolor cultures grown in defined medium were elucidated: a readily assimilated carbon and nitrogen source is required. Under these conditions methylenomycin is produced late in the growth phase, at the time of transition from exponential to linear growth. Provided that the phosphate concentration in the medium is kept high, there is synthesis of methylenomycin but not of the other secondary metabolites that this strain can produce. These conditions were used to study the transcription of the methylenomycin gene cluster during the transition from primary to secondary metabolism. The biosynthetic genes of at least one of the mmy transcription units appear to be transcribed before the mmr resistance determinant. The possibility that methylenomycin induces the transcription of mmr is discussed.

Acetates

Nucleotide sequence of a gene from Phanerochaete chrysosporium that shows homology to the facA gene of Aspergillus nidulans.

Heterologous hybridisation was used to isolate a genomic DNA sequence from Phanerochaete chrysosporium using the facA (acetyl CoA synthetase) gene from Aspergillus nidulans as a probe. The cloned sequence hybridises to a 2.2 kb transcript in poly(A)+ RNA prepared from mycelium grown on acetate as the sole carbon source. Comparison of the DNA sequence obtained with those of the A. nidulans facA and N. crassa acu5 genes reveals an ORF that appears to be interrupted by five typical fungal introns. Two possible candidates for the translation initiation codon were observed. Homology with the facA and acu5 genes is revealed after the second ATG codon.

Acetate-CoA Ligase

The trpC gene of Phanerochaete chrysosporium is unique in containing an intron but nevertheless maintains the order of functional domains seen in other fungi.

The Phanerochaete chryososporium trpC gene has been isolated by complementation of an Escherichia coli trpC mutant. The full extent of the fungal gene, determined by sequence analysis, was found to be 2414bp. This includes a single intron of 50bp, the presence of which was confirmed by RNA-primed polymerase chain reaction analysis. This features makes the P. chrysosporium gene unique when compared to equivalent genes from other filamentous fungi. The P. chrysosporium trpC gene encodes a single protein containing three enzyme activities involved in tryptophan biosynthesis arranged in the order: NH2-GAT-IGPS-PRAI-COOH. This order is conserved in all filamentous fungi so far examined and, indeed, is the gene order within the E. coli trp operon.

Amino Acid Sequence

RFLP-based genetic map of Phanerochaete chrysosporium ME446: lignin peroxidase genes occur in clusters.

We describe the construction of an RFLP-based genetic map of the white rot fungus Phanerochaete chrysosporium ME446. The map is deduced from the allele distributions of 38 RFLP markers in a test set of 53 meiotically derived haploid recombinants of this strain. The map includes a cellulase gene, a matingtype locus and a family of lignin peroxidase (and related) genes that are arranged in two unlinked clusters.

Alleles

Genetic factors influencing lignin peroxidase activity in Phanerochaete chrysosporium ME446.

Haploid recombinant progeny of Phanerochaete chrysosporium ME446, genome compositions of which had been defined by RFLP-mapping, vary in their idiophasic behaviour. This allowed us to formulate a model of the sequence of idiophasic activities. One component of this variation, the amount of lignin peroxidase activity, is independent of the allele distributions of the lignin peroxidase gene clusters, but correlates with the allele distribution of another locus. This locus appears to control the spread of the lignin peroxidase-active state within the idiophasic mycelial mat and may be the mating-type locus. The successful determination of linkage relied on analysis of chromosome intervals rather than linkage to single markers; this approach should be generally useful for analysing quantitative characters by RFLP mapping.

Alleles

The identification, molecular cloning and characterisation of a gene from Phanerochaete chrysosporium that shows strong homology to the exo-cellobiohydrolase I gene from Trichoderma reesei.

We have identified a genomic DNA fragment from the lignin-degrading fungus Phanerochaete chrysosporium (P.c.) that hybridizes to a DNA probe encoding part of the exo-cellobiohydrolase I (CBHI) gene of Trichoderma reesei (T.r.). This fragment has been subcloned and its nucleotide sequence determined. We demonstrate that it could encode a 516 residue protein that shows strong homology with the known protein sequence of CBHI from T.r. Comparison of the two nucleotide sequences identifies two regions within the P.c. sequence that are not represented in T.r. Further inspection of these regions reveals sequences closely related to the conserved elements of filamentous fungal introns. We conclude that the P.c. genomic sequence contains the same number of introns (2) as found in T.r. but that these are located at different relative positions within the two genes. The transcript from the P.c. sequence is induced in the presence of cellulose but not by glucose and we therefore conclude that this sequence represents the first cellulase gene to have been described from this organism.

Amino Acid Sequence

Multiple ligninase-related genes from Phanerochaete chrysosporium.

We have investigated four distinct sequences in genomic DNA of Phanerochaete chrysosporium that show strong homology to a lignin peroxidase cDNA sequence [Tien and Tu: Nature 236 (1987) 520-523, and 328 (1987) 742]. The complete nucleotide sequence of one of these genes is presented here. Comparison of the sequenced gene, LIG1, with the published cDNA sequence reveals the presence of eight short intervening sequences. The open reading frame encodes a polypeptide of 372 amino acids (aa), which includes a 28-aa residue hydrophobic leader sequence. The predicted Mr of the mature protein product is 37,000. Partial sequencing of the other three genes reveals substantial nucleotide and amino acid homology.

Agaricales

Phenotypic classes of phenoloxidase-negative mutants of the lignin-degrading fungus Phanerochaete chrysosporium.

This paper reports the isolation of phenoloxidase-negative mutants of the white-rot fungus Phanerochaete chrysosporium and the results of a survey of idiophasic functions among these mutants. The mutant strains were isolated from a medium containing o-anisidine after gamma irradiation of wild-type spores and fell into four classes, divided by the manner in which they mineralized 14C-lignin wheat lignocellulose. Examples are strain LMT7, which degraded lignin at a rate similar to that of the wild type; strain LMT26, in which degradation was enhanced; strain LMT16, whose degradation rate was apparently unaffected, although the onset of lignin attack was delayed compared with that in the wild type; and strain LMT24, which was unable to evolve significant amounts of 14CO2 from the radiolabeled substrate. The mutants were not necessarily defective in other functions associated with idiophasic activities (intracellular cyclic AMP levels, sporulation, extracellular glucan production, veratryl alcohol synthesis). We conclude that phenoloxidase activity as detected by the o-anisidine plate test is not necessary for lignin degradation. In addition, mutations resulting in the loss of lignin-degrading ability were not necessarily pleiotropic with other idiophasic functions.

Catechol Oxidase

Molecular comparisons of plasmids isolated from colicinogenic strains of Escherichia coli.

The plasmid content of 14 colicinogenic strains of Escherichia coli has been examined. Four strains contained miniplasmids (1.2-2.0 kb). Small plasmids (4-7 kb) were detected in all the strains specifying group A colicins (colicins A, E1, E2, E3 and K; group I plasmids). Larger plasmids (55-130 kb) were detected in seven out of nine strains specifying group B colicins (colicins B, H, Ia, Ib, M, V and S1; group II plasmids). DNA-DNA hybridization with group II plasmids showed a wide variation in the degree of DNA sequence homology among its members. In contrast little (if any) DNA sequence homology was detected with the group I plasmids when the same group II plasmid DNAs were used as hybridization probes. The results of DNA-DNA hybridization studies with the two small group II plasmids (pcolG-CA46 and pcolD-CA23) suggested that these plasmids are equivalent to deleted forms of larger group II plasmids. Our hybridization data thus support the division of colicin plasmids into the two groups (I and II) that have been previously defined from genetic and physiological studies.

Bacteriocin Plasmids

Characterization by molecular cloning of insertion mutants in TOL catabolic functions.

A physical and genetic map of the Tol catabolic region of pWWO (TOL) was obtained by restriction endonuclease analysis of several DNA insertion mutants (xylA, xylA xylS, xylS, and xylR) of R plasmid--TOL derivatives. In two cases, the inserted DNA was shown from restriction, DNA hybridization, or heteroduplex analysis of cloned Hind III fragments to originate from within pWWO fragment Hind III-E. The effect of these DNA insertions on Tol catabolic activity and on structural alterations to the TOL plasmid is discussed.

Chromosome Mapping

Molecular relationships between pseudomonas INC P-9 degradative plasmids TOL, NAH, and SAL.

We have examined the extent to which the degradative plasmids SAL, NAH, and TOL of the Inc P-9 incompatibility group share common DNA sequences. The homology we observe using 32P-labeled SAL and NAH DNA probes can be assigned to six regions of the TOL (pWWO) restriction endonuclease cleavage map. At least three of these regions are probably related to transfer and replication functions, whereas a fourth region is related to the common metacleavage pathway. Restriction endonuclease maps of the SAL and NAH plasmids are derived and the relationships between these plasmids discussed.

Base Sequence

Identification of chromosomally integrated TOL DNA in cured derivatives of Pseudomonas putida PAW1.

Some plasmid-free Tol- strains derived from Pseudomonas putida PAW1 (which carries the TOL plasmid pWW0) have a segment of TOL DNA located chromosomally. Of three independently isolated strains, PAW86 had an integrated TOL segment of 16 kilobases and PAW85 had two copies of this segment in different chromosomal locations, whereas the chromosomal DNA of PAW82 showed no homology with the TOL plasmid. In cultures of the parental strain, it appears that a 56-kilobase TOL DNA segment is located chromosomally in some cells.

Base Composition