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J Knowles

Publications and source records attributed to J Knowles.

At least 55 records · Page 3Linked to original sources
PubMed1987

Construction of cDNA libraries by blunt-end ligation: high-frequency cloning of long cDNAs from filamentous fungi.

A simplified cDNA synthesis and cloning method, suitable for efficient generation of cDNA libraries at frequencies up to 10(6) clones/micrograms mRNA, is described. Routine synthesis of transcripts of well over 4 kb is facilitated by the use of high-quality RNA template isolated from materials rich in RNases. Laborious cloning steps, like tailing or addition of linkers, can be omitted by the use of efficient blunt-end ligation to plasmid vectors, and rapid verification as well as characterization of the clones is possible by double-stranded plasmid sequencing. Using this method we have constructed several cDNA libraries of different filamentous fungi and show here the synthesis and cloning of cDNA copies larger than 1.8 kb corresponding to three Trichoderma reesei cellulases.

Bacteriophage lambda↗
PubMed1987

A versatile transformation system for the cellulolytic filamentous fungus Trichoderma reesei.

An efficient transformation system for the cellulolytic filamentous fungus Trichoderma reesei has been developed. Transformation was obtained with plasmid carrying the dominant selectable marker amdS or the argB gene of Aspergillus nidulans, which was found to complement the respective argB mutation of T. reesei. The transformation frequency can be up to 600 transformants per microgram of transforming DNA. The efficiency of co-transformation with unselected DNA was high (approx. 80%). The transforming DNA was found to be integrated at several different locations, often in multiple tandem copies in the T. reesei genome. In addition, the Escherichia coli beta-galactosidase was expressed in T. reesei in enzymatically active form from the A. nidulans gpd promoter.

Amidohydrolases↗
PubMed1987

Homologous domains in Trichoderma reesei cellulolytic enzymes: gene sequence and expression of cellobiohydrolase II.

Fungal cellobiohydrolases are unique enzymes capable of degrading highly ordered crystalline cellulose. We present here the isolation and complete sequence analysis of the chromosomal and cDNA copies of the structural gene (cbh2) coding for one of the major cellobiohydrolases (CBH II) of Trichoderma reesei. We also present data on expression of the cbh2 gene and show that the transcription start points of the cbh2 gene are heterogeneous and are located 32 to 52 bp downstream from a putative TATA box. The derived CBH II protein sequence is 471 amino acids long and the coding region is interrupted by three short introns. Most of the CBH II protein bears no apparent resemblance to CBH I and endoglucanase I. However, a short region of extensive homology is found in all Trichoderma cellulases characterized so far, suggesting that this region is important for cellulose hydrolysis. The implications of this information with regard to the evolution of fungal cellulase genes and the enzymology of cellulose hydrolysis are discussed.

Amino Acid Sequence↗
PubMed1986

Liquid chromatographic assay using electrochemical detection for the quantitation of indoramin in human plasma.

An automated high-performance liquid chromatographic method with electrochemical detection is described for the determination of indoramin, a new antihypertensive drug. The procedure involves a single extraction of the drug from alkaline plasma with chlorobutane. The chlorobutane extract is evaporated to dryness, reconstituted in methanol and injected into the chromatograph. Separation is achieved using a CN-bonded silica column and an isocratic elution with 0.01 M sodium phosphate in 50% acetonitrile. Detection is at 0.95 V applied potential on a glassy carbon electrode versus an Ag/AgCl electrode. Electrochemical methods that depend on a high oxidation potential suffer from rapid electrode passivation by significant amounts of impurities extracted from plasma. Therefore, the instrumentation includes a valve-switching unit to divert most of the impurities away from the electrode compartment; thus, maintaining sensitivity during the automated analysis of a large number of samples. An RSD of 5-15% was obtained in the concentration range of 0.5-100 ng/mL plasma. The limit of detection is 0.5 ng/mL. The method has been successfully used for the determination of indoramin in plasma samples from human subjects given a 50-mg oral dose of the drug.

Biological Availability↗
PubMed1986

Homology between cellulase genes of Trichoderma reesei: complete nucleotide sequence of the endoglucanase I gene.

The filamentous fungus Trichoderma reesei produces several endoglucanases (EG) and cellobiohydrolases (CBH) which are involved in cellulose hydrolysis in a complex synergistic manner. We have cloned and sequenced the gene and the full-length cDNA coding for the major endoglucanase EG-I, and compared this to the cbh1 gene sequence to clarify the relationship between the EG and CBH classes of cellulases. The deduced 437-amino acids (aa) long EG-I protein with a 22-aa long signal peptide is 45% identical in aa sequence with CBH-I. The best conserved region is found at the C terminus and shows about 70% homology. The data suggest that the two enzymes have arisen from a common ancestor by gene duplication. Despite this, the intron positions have not been conserved in these genes which both contain two short introns. The deduced EG-I sequence contains six putative N-glycosylation sites, and a putative O-glycosylated region is found near the C terminus, closely resembling a similar region at the C terminus of CBH-I. Comparison of the aa sequences suggests that the evolutionary divergence of EG-I from CBH-I has involved four separate 10-20 aa "deletions" from the ancestral protein.

Amino Acid Sequence↗
PubMed1986

The beta-glucanase gene from Bacillus amyloliquefaciens shows extensive homology with that of Bacillus subtilis.

The nucleotide sequence of a 1583-bp DNA fragment containing gene bg1A for endo-beta-1,3-1,4-glucanase (EC 3.2.1.73) of Bacillus amyloliquefaciens strain BE20/78, a high producer of secreted enzymes, has been determined. The gene bg1A comprises an open reading frame (ORF) of 717 bp (= 239 codons) starting with ATG at 469 up to the translation stop codon TAA at 1188. Upstream from the translation initiation codon ATG, the ribosome-binding sequence 5'-AAAAAAGGGGG-3' and two putative bglA promoters have been identified. A box of eleven AT out of twelve base pairs (bp) precedes the -35 region of promoter P1. Beyond the translation stop codon UAA, a sequence of 69 bp can be folded into a hook-like stem-loop structure which probably functions as a transcriptional terminator. The ORF region of the gene bglA reveals about 90% homology with another beta-glucanase gene, bglS of Bacillus subtilis C120 sequenced by Murphy et al. (1984). Three regions of frequent amino acid (aa) changes are indicated. However, the major difference between these is a set of deletions within the non-coding region separating the bglA gene from an unknown preceding ORF and by one deletion shortening the proposed signal peptide by three aa (Pro-Tyr-Leu-). The putative transcription terminator of gene bglA completely lacks homology with a B. subtilis bglS gene. The signification of deletions erasing the 'sacR-homology region' in B. amyloliquefaciens, which have been detected in proximity of the beta-glucanase gene of B. subtilis by Steinmetz and Aymerich (1986), is discussed.

Bacillus↗
PubMed1986

Computer software for the professional. Its evolution and current status.

The development of software during the past 30 years has been just as dramatic in many respects as that of hardware. Whereas future developments in hardware technology can be expected to provide smaller, cheaper, and more powerful computers as has been the case during the past 25 years, future developments in software technology can be expected to play the equally important role of expanding the user base for computers, thus making it economically feasible to exploit new technologies on the hardware side. Although today's computer software offerings are very broad indeed in terms of the variety of tasks they perform, one can reasonably expect that entirely new uses will be found for computers, through the development of new types of software, in the future. By exploiting the huge memory capacities of today's newer computers, future software will be much easier to use and will be capable of carrying out several tasks simultaneously. It will also be much more forgiving, adapting itself automatically to the work style (and probably also to the mistakes) of the user. By incorporating at least a part of what constitutes the stock of knowledge in particular applications, so-called "expert systems," one may expect future software, particularly in areas such as financial and statistical analysis, to enable the computer to work more as a partner or colleague than as an unintelligent (albeit very fast) tool. Perhaps to an even greater extent in the future than has been true to date, the evolution of the computer as a useful tool will depend on software, rather than hardware, innovation. The hardware advances necessary to place hearing, speaking, seeing, and even thinking computers on everyone's desktop, each of which would be able to provide ready access to truly mind-boggling quantities of information, is at hand. Only the software really remains to be developed.

Computers↗
PubMed1985

Construction and identification of a cDNA clone for human type II procollagen mRNA.

Double-stranded cDNA was constructed for poly(A)-containing RNA isolated from foetal human articular cartilage known to contain small amounts of pro alpha 1 (II) collagen mRNA. A 585 base pair PstI-EcoRI cDNA fragment was isolated and cloned into plasmid pBR322. A resulting recombinant plasmid pHCAR1 was shown to hybridize specifically to a 5.4 kilobase mRNA in cartilage but not in calvarial RNA. Definite identification of clone pHCAR1 was based on sequence analysis; marked homology with the corresponding chick gene and complete agreement with the human gene sequences available were observed.

Base Sequence↗
PubMed1985

Sequencing of the chicken non-erythroid spectrin cDNA reveals an internal repetitive structure homologous to the human erythrocyte spectrin.

Immunological screening of a chicken gizzard cDNA expression library was used to isolate two clones encoding a part of the non-erythroid spectrin-like protein. Clones were identified by immunoblotting of the polypeptides synthesized in Escherichia coli cells transformed with cDNA cloned in the pUC8 plasmid vector using polyclonal rabbit antibodies raised against bovine non-erythroid spectrin. The sequence of an approximately 1.5-kb cDNA insert of one clone was determined. Analysis of the predicted amino acid sequence reveals that, despite differences in immunological cross-reactivity and peptide maps, the chicken non-erythroid and the human erythrocyte spectrins are highly homologous proteins. Like the human erythrocyte spectrin, the chicken smooth muscle spectrin appears also to be constructed from repeated, homologous structures of 106 amino acid residues. This is probably a universal structure motif of spectrins.

Amino Acid Sequence↗
PubMed1984

Natural history of chronic mitral insufficiency: relation of peak systolic pressure/end-systolic volume ratio to morbidity and mortality.

The ratio of peak systolic pressure to end-systolic volume (PSP/ESV) is a measure of contractility that is relatively independent of loading conditions. To define the relation of this index to the natural history of chronic mitral insufficiency, follow-up studies were performed in 76 patients. All had isolated mitral insufficiency and were followed up for an average of 48 months. None underwent surgery. Cardiac volumes, ejection fraction and PSP/ESV ratio were calculated and Cox multiple regression analyses were performed to determine the relation of functional status, ejection fraction and PSP/ESV ratio to morbidity and mortality. Twenty-three patients died during follow-up; in 70% of those who died, the PSP/ESV ratio was reduced below the 20th percentile. However, as an independent predictor of mortality, this ratio was less sensitive (p greater than 0.05) than ejection fraction (p less than 0.01). Similarly, functional status change was predicted more accurately by ejection fraction (p less than 0.01) than by the PSP/ESV ratio (p greater than 0.05). Thus, although a decreased PSP/ESV ratio was associated with a higher mortality rate, other clinical and laboratory variables were superior to this index for determining morbidity and mortality in patients with isolated mitral insufficiency.

Adult↗
PubMed1983

Avian acute leukemia virus OK 10: analysis of its myc oncogene by molecular cloning.

Several DNAs representing the genome of the avian acute leukemia virus OK 10 were isolated by molecular cloning from a transformed quail cell line, 9C, which contained at least six OK 10 proviruses. Recombinant lambda phages harboring the OK 10 genome and additional flanking cellular DNA sequences were studied by restriction endonuclease mapping and hybridization to viral cDNA probes. Six of the clones represented complete proviruses with similar, if not identical, viral sequences integrated at different positions in the host DNA. The organization of the OK 10 genome was determined by electron-microscopic analysis of heteroduplexes formed between the cloned OK 10 DNA and DNAs representing the c-myc gene and the genomes of two other avian retroviruses, Rous-associated virus-1 and MC29. The results indicated that the OK 10 proviral DNA is about 7.5 kilobases in size with the following structure: 5'-LTR-gag-delta polmyc-delta env-LTR-3', where LTR indicates a long terminal repeat. The oncogene of OK 10, v-mycOK 10, forms a continuous DNA segment of around 1.7 kilobases between pol and env. It is similar in structure and length to the v-myc gene of MC29, as demonstrated by restriction endonuclease and heteroduplex analyses. Two of the OK 10 proviruses were tested in transfection experiments: both DNAs gave rise to virus with the transforming capacities of OK 10 when Rous-associated virus-1 was used to provide helper virus functions.

Animals↗
PubMed1982

The biology of SAA: identification of the inducer, in vitro synthesis, and heterogeneity demonstrated with monoclonal antibodies.

Continued studies of the macrophage-derived mediator of SAA synthesis (SAA Stimulating Factor) confirm our previous observations that SAASF copurified with leukocytic pyrogen (LP) and lymphocyte activating factor (LAF). Moreover, new data demonstrate three separate isoelectric points for human LP-LAF-SAASF each of which possess the three biological activities. During the purification of 15,000 MW LP from crude stimulated mononuclear cell supernatants, only those fractions with pyrogenic activity in rabbits caused augmented stimulation of lymphocytes (LAF) and induced SAA synthesis in mice. Purified human LP stimulated isolated mouse hepatocytes in vitro to synthesize SAA in a dose-responsive manner. Colchicine treatment of hepatocytes led to decreased secretion of SAA into the medium and to an intracellular accumulation of SAA. Messenger RNA was isolated from the livers of endotoxin-stimulated mice and translated in a wheat-germ cell-free system. A major product was identified at 13-14,000 MW. Immunoprecipitation with anti-mouse AA identified several bands on autoradiography of polyacrylamide gels. These larger SAA precursors may account for the previously noted heterogeneity of human SAA, comprising at least 6 SAA isomers, of similar molecular weight but different solubility and electrophoretic charge characteristics. Two monoclonal antibodies (IgM-K and IgG1-K) have been prepared using standard cell hybridization techniques. They are directed at the variable COOH terminal region of SAA since they detect differences between the 6 human SAAs but do not react with human, monkey, dog or mouse AA proteins, human AP, C-reactive protein, IgG nor albumin. These antibodies will be useful in examining the origin, structure and function of SAA.

Amyloid↗
PubMed1980

Genetic interactions in the control of mitochondrial function in Paramecium. II. Interactions between nuclear and mitochondrial genomes.

In an attempt to understand the genetic interactions between nuclear and mitochondrial genomes leading to mitochondrial biogenesis, different combinations of known nuclear and mitochondrial mutations have been constructed by microinjection. Eleven different tetrazolium resistant mutant strains, many clearly affecting mitochondrial function, were injected with mitochondria from four different erythromycin resistant mitochondrial mutants. Cases were found in which mutant mitochondria were unable to replicate in tetrazolium resistant mutants. The successful mitochondrial transfers were characterized for growth rate, temperature and cold sensitivity. Several selected combinations were characterised also for cytochrome spectra and cyanide resistance. Many different phenotypes were produced by the interaction of the different nuclear and mitochondrial mutations. These ranged from a positive interaction in which mutant mitochondria were selected by a nuclear mutant in preference to wild-type, through apparent absence of interaction, to negative interaction in which the mitochondrial-nuclear combination was temperature sensitive even though both 'parents' were thermoresistant. The possible molecular basis of these interactions is discussed.

Animals↗
PubMed1980

Comparison of results following three modalities of periodontal therapy related to tooth type and initial pocket depth.

Results following three modalities of periodontal therapy (subgingival curettage, modified Widman flap surgery, and pocket elimination or reduction surgery) in 78 patients over 8 years were compared for variations in pocket depth and clinical attachment level related to tooth types (maxillary molars, mandibular molars, maxillary biscupids, mandibular biscupids, maxillary anterior teeth, mandibular anterior teeth). The analysis was based on a classification of three severity groups according to initial crevice or pocket depth (Class I, 1-3 mm; Class II, 4-6 mm; and Class III, 7-12 mm) and with patient's means of measurements being the experimental units for the statistical analysis. Reduction in pocket depth and gain of clinical attachment for pockets 4 mm or deeper occurred following all three methods of treatment, and was well sustained over 8 years. No one modality of treatment was consistently superior to any of the other two with regards to sustained reduction of pocket depth and gain of clinical attachment. Surgical pocket elimination or reduction did not enhance the prognosis for maintenance of periodontal support in either moderate or advanced periodontal lesions anywhere in the mouth compared with more conservative modalities of treatment. In spite of prophylaxis and instruction in home care every 3 months, there was a slight progressive loss of attachment over time in areas of shallow crevices (1-3 mm).

Bicuspid↗
PubMed1979

Ultrastructure of intermediate lobe of the pituitary and melanocyte-stimulating hormone secretion in oestrogen-induced kidney tumours in male hamsters.

One group of male Syrian hamsters was killed after various periods of diethylstilboestrol (DES) treatment, ranging from 6 to 36 weeks. All developed renal tumours by 24 weeks. DES also induced hyperplastic and neoplastic changes in the pars intermedia of the pituitary with proliferation of MSH-secreting cells and elevated serum MSH concentration. Another group of male Syrian hamsters was given DES for 36 weeks followed by cessation of hormone treatment. The animals were killed at various intervals from 12 to 36 weeks after DES withdrawal. In these animals the DES-induced renal tumours underwent regression whilst the pituitary changes persisted until the termination of the experiment. The findings suggested that the hormone MSH may be implicated in renal carcinogenesis but plays no role in the maintenance of the established tumours.

Animals↗
PubMed1979

A reappraisal of the effects of ACTH on the response of the central nervous system to injury.

ACTH possesses powerful immuno-suppressive properties and also retards healing by inhibiting fibroblast proliferation and the deposition of collagen. In the present work this hormone was used to test both the 'barrier' and 'immune' hypotheses relating to the failure of CNS regeneration after injury, dosages being administered which had been found to stimulate maximal adrenal production of glucocorticoids in control animals. Although the scarring in the CNS and the systemic humoral and cellular immune responses were significantly depressed, no increase in CNS axon growth was noted, even when large doses of thyroxine were added. We conclude that both 'immune' and 'barrier' hypotheses should be rejected, and that the beneficial effects of thyroxine treatment reported by some workers are not substantiated.

Adrenocorticotropic Hormone↗