Biomedical subjects

I N Roberts

Publications and source records attributed to I N Roberts.

28 records · Page 2Linked to original sources

PubMed1992

Heterologous gene expression in Aspergillus niger: a glucoamylase-porcine pancreatic prophospholipase A2 fusion protein is secreted and processed to yield mature enzyme.

The cDNA gene encoding porcine pancreatic prophospholipase A2 (proPLA2) was cloned into an Aspergillus niger expression vector downstream of the glucoamylase (glaA) gene promoter region. When this construct was transformed into A. niger, no detectable PLA2 was produced. Evidence was obtained showing that the PLA2 gene was transcribed and that PLA2 is extremely susceptible to both intracellular and extracellular proteases of A. niger, thus indicating that translation products would be rapidly degraded. By fusing the proPLA2-encoding sequence to the entire glaA gene, secreted yields of PLA2 up to 10 micrograms/ml were obtained from a transformed protease-deficient strain of A. niger. PLA2 was secreted in young cultures as a fusion protein, but in older cultures, it was processed from the glucoamylase carrier protein. Secreted PLA2 was shown to be enzymatically active and to have the correct N-terminal amino acid (aa) sequence, although another form of processed PLA2 was also produced. This form included two aa of the proregion from PLA2. The potential for improving yields of secreted heterologous proteins from A. niger still further is discussed.

Amino Acid Sequence↗

PubMed1992

CfT-I: an LTR-retrotransposon in Cladosporium fulvum, a fungal pathogen of tomato.

A retrotransposon from the fungal tomato pathogen Cladosporium fulvum (syn. Fulvia fulva) has been isolated and characterised. It is 6968 bp in length and bounded by identical long terminal repeats of 427 bp; 5 bp target-site duplications were found. Putative first- and second-strand primer binding sites were identified. Three long open reading frames (ORFs) are predicted from the sequence. The first has homology to retroviral gag genes. The second includes sequences homologous to protease, reverse transcriptase, RNAse H and integrase, in that order. Sequence comparisons of the predicted ORFs indicate that this element is closely related to the gypsy class of LTR retrotransposons. Races of the pathogen exhibit polymorphisms in their complement of at least 25 copies of the sequence. Virus-like particles which co-sediment with reverse transcriptase activity were observed in homogenates of the fungus. This is the first report of an LTR retrotransposon in a filamentous fungus.

Amino Acid Sequence↗

PubMed1992

Isolation and characterization of mutants of Aspergillus niger deficient in extracellular proteases.

In the present study, the extracellular protease activity in a strain of the filamentous fungus Aspergillus niger was investigated and mutant strains deficient in the production of extracellular proteases were isolated. The major protease, which is responsible for 80-85% of the total activity, is aspergillopepsin A, a protein of ca. 43 kDa, the activity of which is inhibited by pepstatin. In addition, a second protease, aspergillopepsin B, is produced, which is much less sensitive to inhibition by pepstatin. Several protease-deficient mutants were obtained by in vivo UV mutagenesis. In addition, a mutant lacking aspergillopepsin A was constructed by an in vitro gene replacement strategy. In this mutant, AB1.1, the entire coding region of the gene for aspergillopepsin A (pepA) is deleted. In three UV-induced mutants, aspergillopepsin A is also missing. One of these mutants, AB1.18, is mutated in the pepA gene, which is located on chromosome I. One of the other mutants, AB1.13, which has only 1-2% of the extracellular protease activity in the parent strain, is deficient in both aspergillopepsin A and aspergillopepsin B. The mutation involved, prt-13, has been localized to chromosome VI, and is probably a mutation in a regulatory gene. Another mutation involved in loss of protease function, prt-39, is located on chromosome VIII. Degradation of various heterologous proteins in culture media of the mutants is reduced but, even in strain AB1.13, not completely abolished.

Aspartic Acid Endopeptidases↗

PubMed1991

Heterologous protein production by filamentous fungi.

There are clearly many facets to successful production of heterologous proteins from filamentous fungi. The objectives are to exploit the natural ability of some species to secrete high levels of protein. The heterologous target proteins produced in a fungal host must be acceptable to the public and be economic to produce, i.e. the targets must be authentic (in structure and activity) and be produced in high yield to necessary levels of purity. The appearance of heterologous products from fungi on the market is testament to some success but, equally, there are considerable limitations in our ability to produce desired yields of many target proteins. We endorse the view of van den Hondel, Punt and van Gorcom (1991) that for the commercial production of heterologous proteins from filamentous fungi more information is required on transcriptional control, introns, mRNA stability and processing, translational efficiency, protein secretion, glycosylation and proteolysis. In addition, there is scope for yield improvement based on a better understanding of the physiology of growth/product secretion coupled to appropriate bioreactor operation. The authenticity of product is an aspect which will assume increasing importance, particularly for therapeutic proteins. The level at which the structures and functional activity of heterologous proteins are assessed will ultimately be determined by legislation. The analytical methods currently available are not always sufficient, for example, to reveal folded structures, and most proteins are not amenable to analysis by two-dimensional NMR. The authenticity of target heterologous proteins will also need to be assessed in relation to the glycosylation level and pattern. This is not easily done and explains the paucity of detailed information published to date on glycosylation of fungal proteins. Novel engineered proteins are already being produced from filamentous fungi where expression is an aid to investigation of structure-function relationships. Commercial production of such engineered proteins will require approval subject to a range of stringently applied tests and analyses. This imposes an even greater need to be able to specify and control, in a rational manner, the structures of recombinant proteins. The research needs for realization of improved yields are equally important in assuring authenticity of product. It is encouraging that progress is being made on all fronts, primarily with Aspergillus spp. and T. reesei, but also with other species, such as N. crassa.

Animals↗

PubMed1989

Expression of the Escherichia coli beta-glucuronidase gene in industrial and phytopathogenic filamentous fungi.

A chimaeric beta-glucuronidase (GUS) gene has been created by ligating the Aspergillus nidulans glyceraldehyde 3-phosphate dehydrogenase promoter to the coding sequence of the E. coli uidA gene. Co-transformation of this vector into A. nidulans, A. niger and the tomato pathogen Fulvia fulva (syn. Cladosporium fulvum (Cooke] resulted in the expression of beta-glucuronidase. GUS activity was detected by growth on agar media containing X-gluc and by enzyme assays of mycelial extracts. Expression of the gene in F. fulva transformants was also easily detectable during growth in plants and did not affect pathogenicity. These results form the basis for a versatile and sensitive reporter gene system for industrial and phytopathogenic filamentous fungi.

Aspergillus nidulans↗

PubMed1985

The cytoskeleton underlying side walls and cross walls in plants: molecules and macromolecular assemblies.

Plant cells organize their growth by reinforcing side walls during interphase (causing them to elongate) and by positioning and orienting the cross wall at cytokinesis. In the first part of this presentation we review progress made in identifying different cytoskeletal components that underlie side walls and that are involved in the deposition of the cross wall. During interphase, the cortical microtubule arrays co-distribute with an antigen recognized by a 'universal' monoclonal antibody to intermediate filaments. Using rhodaminyl-lysine-phalloidin no F-actin could be detected at the cortex but endoplasmic, axial cables were found. The cytokinetic apparatus--the phragmoplast--contains microtubules and we find that F-actin and the intermediate filament antigen also co-distribute with this array. We describe the three-dimensional arrangement of microtubules forming the interphase array in cells enlarging by both tip-growth and intercalary growth. In root hairs of higher plants and in apical cells of the filamentous stage of moss Physcomitrella patens, microtubules (MT) are detected at the apices and it is suggested from this that fragmentation of microtubules and absence of MTs from the tip are preparation artefacts. Using human serum from a scleroderma patient, possible microtubule nucleating sites are detected in meristematic cells; these segregate with the broad spindle poles and they surround the nucleus during early interphase--implying a peri-nuclear origin for the cortical MT array. The interphase microtubule array is described in terms of a dynamic helical model, which proposes: that the MT array is an integral complex; that microtubules form helices; that helices can change their pitch--the array converting to the various conformations.

Actins↗

PubMed1984

Pollen-stigma interactions in Brassica oleracea. I. Ultrastructure and physiology of the stigmatic papillar cells.

The osmotic potential (psi pi) of the stigmatic papillar cells of Brassica oleracea is -14.8 bars. In laboratory conditions each cell transpires water at rates within the range from 3 X 10(-5) to 5 X 10(-5) mm3h-1. A small increase in transpiration rate is detected following cross-(compatible) but not self-(incompatible)pollination. No significant changes in psi pi occur following pollinations of either compatibility. Electron microscopy reveals an active papillar cytoplasm apparently secreting proteins into the cell wall via small vesicles. The cuticle is discontinuous and freeze-fracture techniques indicate that channels transverse the cell wall, suggesting a possible pathway for the movement of protein molecules of high molecular weight from the cytoplasm to the stigma surface. Analysis of electron-microscopic autoradiographs of mature, self-incompatible papillae following pulse-chase experiments with L-[3H]leucine and treatment with cycloheximide shows that protein molecules secreted into the cell wall may return to the cytoplasm at a later stage. The significance of these results is discussed in terms of current models of the pollen-stigma interaction in Brassica.

Autoradiography↗

PubMed1984

Pollen-stigma interactions in Brassica oleracea. II. The fate of stigma surface proteins following pollination and their rôle in the self-incompatibility response.

Mature, self-incompatible stigmas exposed to cycloheximide for 2 h prior to pollination supported identical germination and growth of both cross and self pollen. Treatment of self-pollinated pistils with cycloheximide resulted in the germination of hitherto inactive pollen after some 2-4 h. Pollen germination and initial tube growth in an in vitro germination medium were not significantly affected by cycloheximide. A continuous synthesis of stigmatic proteins is therefore essential for the operation of the self-incompatibility (S.I.) system. However, light-microscope autoradiography of stigmas fed with L-[3H]leucine prior to pollination revealed no movement of stigmatic proteins into the pollen, independent of the compatibility of the pollen with respect to the stigma. Further, tunicamycin, when applied in the same way as cycloheximide, had no effect on the S.I. system. These results are discussed in terms of the proposed cycling of proteins in the papillar cell wall and the involvement of a stigmatic glycoprotein in the S.I. response.

Brassica↗

PubMed1980

Pollen-stigma interaction in Brassica oleracea: the role of stigmatic proteins in pollen grain adhesion.

The adhesion of pollen grains to the stigmas of Brassica oleracea was assayed after treatment of the stigmas wiuth protease and/or cycloheximide. Treatment with protease alone adversely affected pollen grain adhesion. However, the adhesive properties of the stigma recovered fully if the stigmas were not pollinated until 2 h after treatment. Immersion of the stigmas in cycloheximide after protease treatment prevented any recoveryt of the stigmas' adhesive properties. Cycloheximide treatment alone prevented pollen grain adhesion when pollination occurred later than 1--2 h after treatment but did not affect pollen grain adhesion if pollination occurred immediately after treatment. These results indicated not only that the surface-held proteins of the stigma are involved in pollen grain adhesion, but also that their turnover rate is rapid. Isoelectric focusing of extracts derived from stigmas after protease and cycloheximide treatment showed a marked decrease in staining intensity of 3 protein bands, one of which, a glycoprotein, is known to be present only when the self-incompativility system is fully functional. These observations suggest a specificity of adhesion between higher plant cells in the presence of the cell wall.

Brassica↗

PubMedDate not supplied

Expression of reverse transcriptase genes in Fulvia fulva.

Antibodies raised against intercellular fluid antigens isolated from diseased tomato leaves have revealed that the fungal pathogen Fulvia fulva expresses genes for a fungal reverse transcriptase (RNA-dependent DNA polymerase). This enzyme is required for the replication of retroviruses and retroviral-like transposable elements and could provide a mechanism for increasing the mutation rate of fungal pathogens, perhaps explaining their ability to evolve new races rapidly. We report here the DNA sequence of a 225-bp clone from a lambda gt11 genomic library of F. fulva. This clone, designated P5, exhibits a high degree of sequence homology with the reverse transcriptase (pol) gene of the Drosophila melanogaster copia-like retrotransposon 17.6. Southern blot analysis of genomic DNA of F. fulva showed that P5-related sequences are moderately reiterated with 30-100 copies, some of which exhibit restriction fragment length polymorphism in different races of the pathogen. Western blot analysis of extracts from F. fulva with antibodies raised to purified reverse transcriptase (from human immunodeficiency virus-1) revealed immunoreactive proteins. Reverse transcriptase previously has been detected in a variety of organisms including yeast, insects, protozoa, and mammals, but to our knowledge, this is the first report of its occurrence in filamentous fungi.

Amino Acid Sequence↗