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C P Novotny

Publications and source records attributed to C P Novotny.

31 records · Page 2Linked to original sources
PubMed1988

Nucleotide base sequence of the mitochondrial COIII gene of Schizophyllum commune.

The cytochrome oxidase subunit III gene (COIII) of the Badidiomycete S. commune has been identified, cloned, and sequenced. The gene contains no introns, is AT-rich (69%) and exhibits a high degree of similarity to sequences from Ascomycetes. While most mitochondrial genes use both TGA and TGG to specify tryptophan, the COIII gene of Schizophyllum uses TGG exclusively. Translation requires no deviation from the universal code.

Amino Acid Sequence↗
PubMed1987

The isolation of specific genes from the basidiomycete Schizophyllum commune.

We have developed a routine way to isolate genes directly from the basidiomycete fungus, Schizophyllum commune. Plasmid DNA from a genomic gene library was used to isolate five specific genes by complementation of Schizophyllum mutations via transformation. The mutant strains were deficient in the ability to synthesize either adenine (ade2 and ade5), uracil (ura1, encoding orotidine-5'-phosphate decarboxylase; OMPdecase), tryptophan (trp1, encoding indole-3-glycerol phosphate synthetase; IGPS) or para aminobenzoic acid (pab1). In each case, Southern analysis revealed that transformation to prototrophy was concomitant with the integration of vector sequence into the genome of the S. commune mutant. Total DNA from transformants was restricted, religated, and used to transform E. coli. Ampicillin resistant plasmids were recovered from E. coli and tested for their ability to transform the corresponding mutant of S. commune. Plasmids complementing the ade2, ade5, pab1, trp1, and ura1 mutations were recovered.

Basidiomycota↗
PubMed1986

Isolation of the DNA sequence coding indole-3-glycerol phosphate synthetase and phosphoribosylanthranilate isomerase of Schizophyllum commune.

A Schizophyllum gene library was made in plasmid pRK9. Plasmids from this library were tested for their ability to complement several auxotrophic mutations of Escherichia coli. The goal was to isolate a Schizophyllum auxotrophic gene that could be used to transform a corresponding Schizophyllum auxotrophic mutant to prototrophy. Complementation was observed only for E. coli trpC indole 3-glycerol phosphate synthetase (IGPS) and phosphoribosyl-anthranilate isomerase (PRAI) mutations. Plasmids with a Schizophyllum sequence coding for both IGPS and PRAI activities were recovered from E. coli transformants. Expression of the Schizophyllum gene (TRP1) in E. coli is probably dependent on the Serratia marcescens promoter of plasmid pRK9. The DNA sequence containing the Schizophyllum TRP1 gene was not obviously rearranged in cloning.

Aldose-Ketose Isomerases↗
PubMed1986

Transformation of the basidiomycete, Schizophyllum commune.

Protoplasts of a Schizophyllum commune tryptophan auxotroph (trp1), deficient in indole-3-glycerol phosphate synthetase (IGPS), were transformed to trp+ with plasmid DNA containing the Schizophyllum TRP1 sequence. Efficiencies up to 30 transformants per microgram of plasmid DNA were obtained. Southern blots reveal that the transforming DNA is integrated in chromosomal DNA. The trp+ phenotype of transformants is stable in meiosis and mitosis. Transformants possess IGPS activity comparable to wild-type cells.

Basidiomycota↗
PubMed1979

Defective F pili and other characteristics of Flac and Hfr Escherichia coli mutants resistant to bacteriophage R17.

Mutants resistant to the donor-specific bacteriophage R17 were isolated from Hfr and Flac-containing strains of Escherichia coli K-12. Thirty-five mutants were examined for the presence of F pili by electron microscopy. The pilus morphology was studied, as were the abilities of the cells to retract their pili and to synthesize new pili. Measurements were made of the efficiency of the conjugal deoxyribonucleic acid transfer and of M13 and R17 phage infection. All mutants had noticeable defects in pilus production, structure, or function. Mutants were found which produced unusually long pili, displayed wide variations in the number of pili per cell, and were deficient in pilus retraction and synthesis. Evidence is presented that there may be two pathways of pilus retraction.

Coliphages↗
PubMed1978

Effects of high temperature on Escherichia coli F pili.

The effects of high temperatures (46 to 50 degrees C) on the production of F pili by Escherichia coli were studied by electron microscopy. Attached F pili rapidly disappeared at 48 and 50 degrees C but not at 46 degrees C. Free pili were not denatured at these temperatures. The pili that disappeared from the cells at 50 degrees C did not appear as free pili in the culture supernatant fluid, indicating that the pili had retracted to the cell surface or into the cell. The adsorption of either R17 phage or F pili antibody to the sides of pili prevented retraction. The disappearance of pili was accompanied by a loss in the ability to adsorb R17 phage but not M13 phage, suggesting that the tip of a pilus remains exposed after retraction.

Antibodies, Bacterial↗
PubMed1976

Evidence for the involvement of ribonucleic acid in the production of F pili.

The effects of rifampin and streptolydigin, inhibitors of ribonucleic acid (RNA) synthesis, on the production of F pili by Escherichia coli were studied by electron microscopy. The inhibition of RNA synthesis reduces the number of new pili produced by depiliated cells, but does not affect their length or the number of pili present at the time of inhibition or the retraction of pili. We suggest that the rifampin-sensitive step may be linked to the establishement of a site for pili production. Evidence is provided that chloramphenicol inhibits retraction. We suggest that retraction requires some protein whose pool size is limited.

Aminoglycosides↗
PubMed1974

Retraction of F pili.

The disappearance of F pili on Escherichia coli cells in the presence of 10(-2) M NaCN was studied by electron microscopy and serum-blocking power. The pili which disappeared from the cell did not appear as free pili in the culture medium, suggesting that the pili had retracted into the cell. New pili were produced at a normal rate approximately 3 min after NaCN was removed. The adsorption of either F pili antibody or R17 bacteriophage to the sides of pili and temperatures below 24 C prevented retraction. The disappearance of pili was accompanied by a loss in the ability of cells to adsorb R17 phage and the type of F pili antibody that inhibits R17 phage infection and mating. The ability to adsorb M13 phage and the type of F pili antibody that inhibits M13 phage infection was not affected. This suggests that the tips of retracted pili are exposed.

Adsorption↗
PubMed1974

Effect of thymine-5-bromouracil substitution on F pili.

The effect of thymine-5-bromouracil substitution on the regeneration and length of F pili produced by an F(+)Lac(+)/Lac(-)Thy(-) strain of Escherichia coli was studied by electron microscopy. When 5-bromouracil (5BU) incorporation into deoxyribonucleic acid (DNA) was maximal, the modal length of the pilus doubled and the number of pili per cell was approximately 50% that of thymine-grown cells. The ability of 5BU-grown cells to form mating pairs and to be infected by ribonucleic acid (R17) and DNA (M13) male-specific phages was also reduced by approximately 50%. Loss of function was not due to loss of sex factor as 5BU cells retained a sex factor that was susceptible to curing by acridine orange. Elongation of pili on 5BU-grown cells was more sensitive to irradiation at 253.7 nm than on thymine-grown cells, suggesting that DNA is the sensitive target.

Acridines↗
PubMed1972

Effects of growth inhibitors and ultraviolet irradiation on F pili.

The effects of chloramphenicol, nalidixic acid, mitomycin C, NaCN, and ultraviolet irradiation at 253.7 nm on F pili production by Escherichia coli cells was studied by electron microscopy. The results show that cells contain pools of pili protein, and that assembly does not require synthesis of protein or deoxyribonucleic acid (DNA). NaCN (2 x 10(-2)m) prevents the reappearance of pili and causes existing pili to disappear quickly from the cell surface. This suggests that energy is used in the assembly of pili and to retain pili on the cell. Cells irradiated with high doses (10(4)ergs/mm(2)) of 253. 7 nm light produce fewer pili, and these are shorter than normal. Dose-response curves for number of pili per cell and length of pili resemble single hit kinetics, showing 37% survival at 10(4) ergs/mm(2) and 2 x 10(4) ergs/mm(2), respectively. This suggests that DNA is at the site where pili are produced, and that it may be involved in the assembly of pili.

Bacterial Proteins↗
PubMed1971

Some effects of temperature on the growth of F pili.

The effect of temperature on the production of F pili by an F(+) strain of Escherichia coli B/r was studied by electron microscopy and by a technique involving serum-blocking power. The latter method is based on the ability of F pili to adsorb F pili antibody which inhibits male-specific phage infection. The total amount of pili in a sample was estimated by serum-blocking power; the length of F pili and number per cell was determined by electron microscopy. Cell extracts prepared by sonic oscillation lacked serum-blocking power, suggesting that F pili are not present in the cytoplasm. The number of F pili per cell varied with the growth temperature, but the average length of F pili remained constant. Maximum number of pili per cell occurs between 37 and 42 C; below 37 C the number decreases, reaching zero at about 25 C. When cells are grown at 37 C, blended, and resuspended in fresh media at 25 C, they make F pili. These pili are probably assembled from a pool of subunits that were synthesized during growth at 37 C. The rates of assembly at 25 and 37 C, as judged by the rate of increase in length of F pili, are similar. When cells were grown at 25 C and shifted up to 37 C, there was a 30-min lag in pili production followed by a period of rapid outgrowth. When cells were shifted down from 37 to 20 C, outgrowth (assembly) of pili ceased, and approximately 50% of the attached pili were released in 2 min. No release was observed when cells were shifted to 0 C. This suggests that pili may be released from the cell by a mechanism that requires metabolic activity, but not the outgrowth of F pili.

Adsorption↗