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J K Knowles

Publications and source records attributed to J K Knowles.

At least 37 records · Page 2Linked to original sources
PubMed1990

Three-dimensional structure of cellobiohydrolase II from Trichoderma reesei.

The enzymatic degradation of cellulose is an important process, both ecologically and commercially. The three-dimensional structure of a cellulase, the enzymatic core of CBHII from the fungus Trichoderma reesei reveals an alpha-beta protein with a fold similar to but different from the widely occurring barrel topology first observed in triose phosphate isomerase. The active site of CBHII is located at the carboxyl-terminal end of a parallel beta barrel, in an enclosed tunnel through which the cellulose threads. Two aspartic acid residues, located in the center of the tunnel are the probable catalytic residues.

Amino Acid Sequence↗
PubMed1990

Structural homology among the peroxidase enzyme family revealed by hydrophobic cluster analysis.

A number of peroxidase amino acid sequences show limited homology to short regions comprising the known active site cleft of yeast cytochrome c peroxidase. Otherwise no clear homology is visible in linear alignments between this enzyme and other peroxidases. We have subjected eight peroxidase sequences to hydrophobic cluster analysis. Our results suggest that these peroxidases are evolutionary related and that they share many folding characteristics.

Amino Acid Sequence↗
PubMed1990

Random mutagenesis used to probe the structure and function of Bacillus stearothermophilus alpha-amylase.

Mutations that cover the sequence of Bacillus stearothermophilus alpha-amylase were produced by an efficient in vitro enzymatic random mutagenesis method and the mutant alpha-amylases were expressed in Escherichia coli, which also secreted the product. Ninety-eight mutants were identified by sequencing and their enzyme activities were classified into three classes: wild-type, reduced or null. A molecular model of the enzyme was constructed using the coordinates of Takaamylase A and a consensus alignment of mammalian, plant, and bacterial alpha-amylases. The location of mutant amino acids on the model indicate that mutations which destroy or decrease the catalytic activity are particularly clustered: (i) around the active site and along the substrate-binding groove and (ii) in the interface between the central alpha/beta barrel and the C-terminal domain. Exposed loops are typically tolerant towards mutations.

Amino Acid Sequence↗
PubMed1990

Molecular modelling study of antigen binding to oxazolone-specific antibodies: the Ox1 idiotypic IgG and its mature variant with increased affinity to 2-phenyloxazolone.

Structural models of the variable domains of the murine anti-2-phenyloxazolone IgG (Ox1 idiotype) and its somatic variant, which has higher affinity to the hapten 2-phenyloxazolone, were constructed by computer-aided model building using known structures of highly homologous immunoglobulins as templates. Molecular dynamics simulations were used to dock the hapten between the VL and VH domains. The hapten is predicted to bind to slightly different sites in the two models. Hypotheses concerning the role of a number of preferred mutations in anti-oxazolone variants are presented. These can be tested by mutagenesis and crystallography. In particular, the higher binding affinities of the different antibody variants are shown to correlate with better complementarity of electrostatics. The molecular dynamic simulations also suggest that two mobile tryptophans at the mouth of the pocket may play an important role in the binding of hapten.

Amino Acid Sequence↗
PubMed1989

A lignin peroxidase-encoding cDNA from the white-rot fungus Phlebia radiata: characterization and expression in Trichoderma reesei.

The nucleotide sequence of a cDNA coding for a lignin peroxidase (Lgp) of the white-rot fungus, Phlebia radiata, has been determined. By amino acid (aa) sequencing, it has been shown that the protein product of this gene is the LIII Lgp of Pb. radiata. The isolated gene and the putative aa sequence are about 60% homologous to published Lgp sequences from the fungus, Phanerochaete chrysosporium. The aa thought to be involved in the catalysis of LIII are revealed by comparison with the yeast cytochrome c peroxidase. The P. radiata Lgp-encoding gene (lgp3) was expressed in the fungus, Trichoderma reesei, under the cellobiohydrolase-encoding cbh1 gene promoter. Lgp3 mRNA was produced by the T. reesei transformants. No Lgp protein, however, could be detected.

Agaricales↗
PubMed1989

Treatment of severe anaemia in children in a rural hospital.

In a rural hospital in Malawi a protocol was developed for the treatment of severely anaemic children to improve the chances of survival of children with haemoglobin levels of 5 g/dl or less (referred to here as severe anaemia), and also to reduce the risks of transmission of human immunodeficiency virus (HIV) by blood transfusion in a unit where the routine screening of donated blood for HIV positivity is not available. Comparison of the results of applying the protocol (using digoxin, frusemide and selective delayed transfusion) with the results of using a widely accepted regimen of routine transfusion of all children with haemoglobin (Hb) of 5 g/dl or less suggests that stabilization of a severely anaemic child's cardiac state before transfusion improves the chances of survival. Many children with very low haemoglobin values do not then require transfusion, thus avoiding the dangers of transfusing blood that is potentially HIV positive. Those who do need transfusion withstand the procedure better.

Anemia↗
PubMed1988

Molecular analysis of a Phanerochaete chrysosporium lignin peroxidase gene.

The basidiomycete fungus Phanerochaete chrysosporium produces a number of extracellular peroxidases which appear to be important for lignin degradation. We present here the isolation and complete nucleotide (nt) sequence of a gene (lpo) coding for lignin peroxidase (LPO), the coding region of which is identical to a lpo cDNA sequence which had previously been described [M. Tien and C.-P.D. Tu, Nature 326 (1987) 520-523]. The deduced amino acid (aa) sequence corresponds to 372 aa residues and the coding region is interrupted by eight short introns that range in size from 50 to 62 nt. Southern blot experiments using the cloned lpo gene as hybridization probe revealed a complex restriction fragment pattern, indicating that there are a number of lpo-related nucleotide sequences present in P. chrysosporium DNA which cross-hybridize. We also present data on the in vivo expression of the lpo genes and show that they are regulated at the RNA level and that the structure of the transcripts as judged from S1 experiments is complex. These data are consistent with the idea that there are a number of related lpo genes in P. chrysosporium which constitute a gene family.

Base Sequence↗
PubMed1988

EGIII, a new endoglucanase from Trichoderma reesei: the characterization of both gene and enzyme.

A novel endoglucanase from Trichoderma reesei, EGIII, has been purified and its catalytic properties have been studied. The gene for that enzyme (egl3) and cDNA have been cloned and sequenced. The deduced EGIII protein shows clear sequence homology to a Schizophyllum commune enzyme (M. Yaguchi, personal communication), but is very different from the three other T. reesei cellulases with known structure. Nevertheless, all the four T. reesei cellulases share two common, adjacent sequence domains, which apparently can be removed by proteolysis. These homologous sequences reside at the N termini of EGIII and the cellobiohydrolase CBHII, but at the C termini of EGI and CBHI. Comparison of the fungal cellulase structures has led to re-evaluation of hypotheses concerning the localization of the active sites.

Amino Acid Sequence↗
PubMed1988

Efficient secretion of two fungal cellobiohydrolases by Saccharomyces cerevisiae.

Two different cellobiohydrolases, CBHI and CBHII, of the filamentous fungus Trichoderma reesei both hydrolyse highly crystalline cellulose. Cellulolytic strains of the yeast Saccharomyces cerevisiae were constructed by transferring cDNAs coding for these enzymes into yeast on an expression plasmid. These cellulolytic yeasts were able to secrete efficiently the large, heterologous proteins to the culture medium. The recombinant cellulases were observed to be heterogeneous in Mr due, at least partly, to variable N-glycosylation. Recombinant CBHII was able to bind to crystalline cellulose, although slightly less efficiently than the native enzyme. Both of the two recombinant cellulases were able to degrade amorphous cellulose. In a fermenter cultivation, around 100 micrograms/ml of CBHII was secreted into the yeast growth medium.

Cellulose↗
PubMed1988

A new method for random mutagenesis of complete genes: enzymatic generation of mutant libraries in vitro.

A new efficient in vitro mutagenesis method for the generation of complete random mutant libraries, containing all possible single base substitution mutations in a cloned gene is described. The method is based on controlled use of polymerases. Four populations of DNA molecules are first generated by primer elongation so that they terminate randomly, but always just before a known type of base (before A, C, G or T respectively). Each of the four populations is then mutagenized in a separate misincorporation reaction, where the correct base can now be omitted. The regeneration of wild-type sequences can thus be efficiently avoided. Also, the misincorporating nucleotide concentrations can be optimized to give the three possible single mutations in close to equal ratio. The mutagenesis can be precisely localized within a predetermined target region of any size, and vector sequences remain intact. We have mutagenized the DNA coding for the alpha-fragment of Escherichia coli beta-galactosidase, and identified 176 different base substitution mutations by sequencing. The present method gives mutant yields of 40-60%, when the mutants contain about one amino acid change per protein molecule. All types of base substitution mutations can be generated and deletions are rare. The efficiency of this method permits the use of relatively elaborate screening systems to isolate mutants of either structural genes or regulatory regions.

Amino Acid Sequence↗
PubMed1987

Expression of two Trichoderma reesei endoglucanases in the yeast Saccharomyces cerevisiae.

The cDNA copies of the two endo-beta-1,4-glucanase genes, egl1 and egl3, from the filamentous fungus Trichoderma reesei were expressed in yeast Saccharomyces cerevisiae under the control of the yeast phosphoglycerate kinase gene promoter. Active EGI and EGIII enzyme was produced and secreted by yeast into the growth medium. The recombinant EGI enzyme was larger and more heterogeneous in size than the native enzyme secreted by Trichoderma, due to differences in the extent of N-glycosylation between these two organisms. The morphology of the yeast cells producing EGI or EGIII was clearly different from control strain.

Blotting, Western↗
PubMed1987

Efficient secretion of Bacillus amyloliquefaciens alpha-amylase by [corrected] its own signal peptide from Saccharomyces cerevisiae host cells [corrected].

The expression and secretion of Bacillus amyloliquefaciens alpha-amylase was studied in yeast Saccharomyces cerevisiae. The Bacillus promoter was removed by BAL 31 digestion and three forms of the alpha-amylase gene were constructed: the Bacillus signal sequence was either complete (YEp alpha a1), partial (YEp alpha a2) or missing (YEp alpha a3). Secretion of alpha-amylase into the culture medium was obtained with the complete signal sequence only. The secreted alpha-amylase was glycosylated and its signal peptide was apparently processed. The glycosylated alpha-amylase remained active. The enzyme produced by the other constructions was not glycosylated and thus probably remained in the cytoplasm.

Amino Acid Sequence↗
PubMed1979

Absence of detectable mitochondrial recombination in Paramecium.

An extensive search for recombination between mitochondrial markers was carried out in Paramecium tetraurelia. Thirty-two combinations, altogether involving 24 different markers, were studied. The markers belonged to the three main categories of mitochondrial mutations presently available in this organism, (a) Spontaneous or UV-induced antibiotic resistance mutations, most probably affecting mitochondrial ribosomes, (b) nitrosoguanidine-induced antibiotic resistance markers displaying thermosensitivity or slow growth, enabling easy selection of possible wild-type recombinants, and (c) mitochondrial partial suppressors of a nuclear gene, probably corresponding to molecular alterations distinct from the preceding two categories. In addition, different genetic configurations were analyzed (i.e., mutant X mutant, double-mutant X wild-type, etc.).--None of the combinations yielded any evidence for the occurrence of recombined genomes despite the fact that: (1) all of them were studied on a large scale involving the screening of at least several thousand mitochondrial genomes (often several millions), (2) in many of them the detection level was sufficiently high to enable the isolation of spontaneous mutants in control cells, and (3) in several of them, reconstitution experiments carried out in parallel show that the conditions were fully adequate to detect recombinant genotypes. The results are in marked contrast with those obtained on the few other organisms in which mitochondrial recombination has been studied, particularly Saccharomyces cerevisiae, in which mitochondrial recombination is intense.--The most likely basis for the various manifestations of mitochondrial genetic autonomy in Paramecium, described in this as well as in previous publications, is that the chondriome of this organism is made up of thousands of structurally discrete, noninteracting units.

Animals↗
PubMed1977

Characterization of mitochondrial and cytoplasmic ribosomes from Paramecium aurelia.

The ribosomes extracted from the mitochondria of the ciliate, Paramecium aurelia, have been shown to sediment at 80S in sucrose gradients. The cytoplasmic ribosomes also sediment at 80S but can be distinguished from their mitochondrial counterparts by a number of criteria. Lowering of the Mg++ concentration, addition of EDTA, or high KCl concentrations results in the dissociation of the cytoplasmic ribosomes into 60S and 40S subunits, whereas the mitochondrial ribosomes dissociate into a single sedimentation class at 55S. Furthermore, the relative sensitivity of the two types of ribosome to dissociating conditions can be distinguished. Electron microscopy of negatively stained 80S particles from both sources has also shown that the two types can be differentiated. The cytoplasmic particles show dimensions of 270 X 220 A whereas the mitochondrial particles are larger (330 X 240 A). In addition, there are several distinctive morphological features. The incorporation of [14C]leucine into nascent polypeptides associated with both mitochondrial and cytoplasmic ribosomes has been shown: the incorporation into cytoplasmic 80S particles is resistant to erythromycin and chloramphenicol but sensitive to cycloheximide, whereas incorporation into the mitochondrial particles is sensitive to erythromycin and chloramphenicol but resistant to cycloheximide.

Animals↗
PubMed1976

Interspecies transfer of mitochondria in Paramecium aurelia.

Erythromycin-resistant mitochondria from species 1, 5 and 7 of P. aurelia were injected into erythromycin-sensitive paramecia of each of the same three species. Mitochondria from species 1 and 5 were successfully transferred to all three species, but species 7 mitochondria failed to develop in species 1 and 5. Minor differences were indicated in the frequency of successful transfers of species 1 mitochondria into species 1 and 5 cells. From studies on the transferability of mitochondria from "hybrid" cells, containing mitochondria from one species and nuclei from another, it was concluded that mitochondrial compatibility was mainly under control of the nuclear genome, with a possible minor control also by the mitochondrial genome.

Drug Resistance↗