Search PubMed⌕ Search

Biomedical subjects

P Borst

Publications and source records attributed to P Borst.

At least 289 records · Page 16Linked to original sources
PubMed1977

Particle-bound enzymes in the bloodstream form of Trypanosoma brucei.

We have screened the bloodstream form of Trypanosoma brucei for the presence of enzymes that could serve as markers for the microbodies and the highly repressed mitochondrion of this organism. None of seven known microbody enzymes were detected at all, but glycerol-3-phosphate oxidase, ATPase, isocitrate dehydrogenase, acid phosphatase and part of the hyperoxide dismutase and malate dehydrogenase activities were found to be particle-bound after fractionation of homogenates by differential centrifugation. Part of the ATPase activity was sensitive to oligomycin, an inhibitor of oxidative phosphorylation. This oligomycin-sensitive activity can serve as a specific marker for the mitochondria. More than 80% of the NAD+-linked glycerol-3-phosphate dehydrogenase in T. brucei was found to be particulate and latent. The enzyme could be activated by Triton X-100, by the combined action of sonication and salt, but not by salt alone, and partially by freezing and thawing. We conclude that the NAD+-linked glycerol-3-phosphate dehydrogenase is located inside an organelle.

Acid Phosphatase↗
PubMed1977

On the mechanism of oligonucleotide-primed RNA synthesis. I. Model studies with deoxyhomopolymer templates and Escherichia coli RNA polymerase.

We have studied the effect of temperature, primer chain length and primer concentration on the oligonucleotide-primed transcription by Escherichia coli RNA polymerase. Our experiments with the homopolymer model systems poly(dT) .oligo(A)n, poly(dA) .oligo(U)n and poly(dA) .oligo(dT)n lead to three main conclusions. First, de novo chain initiation on single-stranded templates is preferentially suppressed at higher temperatures. Second, stable annealing of template and primer is neither a prerequisite nor does it stimulate the primer-dependent transcription. Third, formation of the ternary enzyme-template-primer complex is a rate-limiting step in the oligonucleotide-primed RNA synthesis. The maximal rate of primer-stimulated RNA synthesis, moreover, is strongly dependent on the nature of the primer and decreases in the order oligo(A)-primed poly(A) synthesis greater than oligo(U)-primed poly(U) synthesis greater than oligo(dT)-primed poly(U) synthesis. We attribute this to differences in the rate at which the first nucleotide is added to the primer. Raising temperature and primer concentration renders transcription in the model systems almost completely primer-dependent. This can be useful in a transcription approach to DNA sequence analysis.

DNA-Directed RNA Polymerases↗
PubMed1977

On the mechanism of oligonucleotide-primed RNA synthesis. II. Synthesis of specific primer-initiated RNA copies suitable for DNA sequence analysis.

The effect of temperature and primer concentration on oligonucleotide-primed transcription has been studied using the separated strands of a well-defined natural DNA as template. Results were similar to those obtained in the homopolymer-directed model systems. At high temperature and excess primer concentration mainly primer-initiated RNA copies are synthesized. Omission of one ribonucleoside triphosphate also makes the termination specific. The unique RNA fragments thus obtained have been used to determine the perfectly-repeated sequence of 68 base pairs in this DNA.

Base Sequence↗
PubMed1976

The structure of kinetoplast DNA. 1. The mini-circles of Crithidia lucilae are heterogeneous in base sequence.

We have analysed limit digests of mini-circles from kinetoplast DNA of Crithidia luciliae by gel electrophoresis. Endonucleases HapII and AluI cut the circles into at least 37 and 21 fragments, respectively, and leave no circles intact. In both cases the added molecular weights of the fragments, estimated from mobility in gels, exceeds 18 X 10(6), i.e. more than 12 times the molecular weight of the mini-circle DNA. Endonucleases HindII + III, EcoRI and HpaI cut only part of the circles. These results show that the mini-circles are heterogeneous in base sequence. Different sequence classes are present in different amounts. DNA-DNA renaturation analysis of mini-circle DNA yields a complexity of about 3 X 10(6), i.e. twice the molecular weight on one mini-circle. The delta tm of native and renatured duplexes is about 1 degree C, showing that the sequence heterogeneity is a micro-heterogeneity. Electron microscopy, gel electrophoresis and sedimentation analysis show that the circles that are not cut by endonucleases HindII + III remain catenated in very large associations. These associations lack the 'rosette' structures and the long edge loops characteristic of intact kinetoplast DNA. This suggests that the mini-circle classes cut by endonucleases HindII + III are present throughout the network and that the maxi-circle component of the network (see accompanying paper) is not essential to hold the network together. Prolonged electrophoresis on 1.5% or 2% agarose gels resolves the open mini-circles into three and linearized mini-circles into four bands, present in different amounts. We conclude that the mini-circles are also heterogeneous in size, the difference in size between the two extreme size classes being 4% of the contour length. Digestion with endonuclease HapII shows that at least three out of these four bands differ in sequence. Possible mechanisms that could account for the micro-heterogeneity in sequence of mini-circles are discussed.

Animals↗
PubMed1976

The structure of kinetoplast DNA. 2. Characterization of a novel component of high complexity present in the kinetoplast DNA network of Crithidia luciliae.

1. Degradation of highly purified kinetoplast DNA (kDNA) networks with restriction endonucleases yields "extra" bands in agarose gels that are absent from digests of mini-circles. Each of the five endonucleases tested, i.e. AluI, HapII, EcoRI, Hsu and HindII + III, yields a unique set of "extra" bands. The "extra" bands consist of linear DNA; they are not mini-circle oligomers and their added molecular weight, calculated from mobility in gels, are around 2 X 10(7). Double digests with two restriction endonucleases yield a new set of "extra" bands, showing that the "extra" bands obtained with different enzymes are all derived from the same complex component of kDNA. In digests of 32P-labelled kDNA an average of 2.3% of the radioactivity is recovered in the "extra" bands. 2. Treatment of kDNA networks with the single-strand-specific S1 nuclease of Aspergillus oryzae preferentially releases a linear DNA with a molecular weight of 26 X 10(6), calculated from mobility in gels. We present evidence that the 'extra' bands obtained with restriction endonucleases are derived from this component. 3. DNA-DNA renaturation analysis of fragmented kDNA shows the presence of a minor complex component with a complexity of about 3 X 10(7), making up less than 10% of the total kDNA. 4. From these results we conclude that 3--5% of the kDNA consists of a homogeneous class of maxi-circles catenated in the mini-circle network. The molecular weight of these maxi-circles is about 26 X 10(6) and they contain a unique, non-repetitive, non-mini-circle nucleotide sequence. This component is a prime candidate for the true mitochondrial DNA of trypanosomes.

Animals↗
PubMed1976

The binding of poly(rA) and poly(rU) to denatured DNA. I. Model studies with homopolymers.

We have compared the properties of the poly(rA).oligo(dT) complex with those of the poly(rU).oligo(dA)n complex. Three main differences were found. First, poly(rA) and oligo(dT)n do not form a complex in concentrations of CsCl exceeding 2 M because the poly(rA) is insoluble in high salt. If the complex is made in low salt, it is destabilized if the CsCl concentration is raised. Complexes between poly(rU) and oligo(dA)n, on the other hand, can be formed in CsCl concentrations up to 6.6 M. Second, complexes between poly(rA) and oligo(dT)n are more rapidly destabilized with decreasing chain length than complexes between poly(rU) and oligo(dA)n. Third, the density of the complex between poly(rA) and poly(dT) in CsCl is slightly lower than that of poly(dT), whereas the density of the complex between poly(rU) and poly(dA) in CsCl is at least 300 g/cm3 higher than that of poly(dA). These results explain why denatured natural DNAs that bind poly(rU) in a CsCl gradient usually do not bind poly(rA).

Binding Sites↗
PubMed1976

The binding of poly (rA) and poly (rU) to denatured DNA. II. Studies with natural DNAs.

We have studied the interaction of poly(rA) and poly(rU) with natural DNAs containing (dA.dT)n sequences. The results indicate that hybridization of poly(rA) to denatured DNA can be used to estimate the size and frequency of large (dA.dT)n tracts, whereas hybridization with poly(rU) does not give reliable information on these points. In 6.6 M CsCl, poly(rU) can form stable complexes with denatured DNA containing short (dA)n tracts (n less than or equal to 6), whereas binding of poly(rA) to denatured DNA under these conditions requires much larger (dT)n tracts (estimated n greater than 13). Moreover, binding of poly(rA) requires pre-hybridization in low salt, because free poly(rA) precipitates in 6.6 M CsCl.

Base Sequence↗
PubMed1976

The presence of (dA.dT)20-25 tracts in the DNA of primitive eukaryotes.

Previous work by Jacobson et al. (1) has shown that the number and distribution of (dA.dT)25 tracts in the nuclear DNA of the slime mold Dictyostelium discoideum reflects the number and the distribution of transcriptional units. To investigate whether this is a general phenomenon we compared the nuclear DNAs of other primitive eukaryotes with respect to their content of large (dA.dT)n tracts via the thermal stability of their hybrids with poly(rA). The results of our analysis indicate that all nuclear DNAs tested have (dA.dT)20-25 tracts, but the frequency of such tracts varies from one per 5.4 X 10(6) daltons in Dictyostelium nuclear DNA to one per 2.8 X 10(8) daltons in Crithidia luciliae nuclear DNA. We conclude that the presence of (dA.dT)20-25 tracts is not an obligatory characteristic of the transcriptional unit in primitive eukaryotes. Chromatography of native DNAs on poly(rU) Sephadex columns shows that the large (dA.dT)n tracts occurring in the genomes of both primitive and higher eukaryotes are widely distributed throught these genomes.

Animals↗
PubMed1975

The organization of genes in yeast mitochondrial DNA II. The physical map of EcoRI and HindII + III fragments.

1. We have isolated large fragments of the mtDNA of the yeast Saccharomyces carlsbergensis and digested these with restriction endonucleases. The digestion products were separated by electrophoresis in agarose gels. 2. Endonucleases EcoRI, HindII + III, HpaI, HindIII and HapII yield 9, 11, 6, 0 and greater than 80 fragments, respectively. 3. By analysis of partial digestion products and by redigesting the fragments obtained with one endonuclease with a second, we have established the order of all EcoRI and HindII + III fragments. The map is circular and its contour length is 22.1 +/- 0.35 mum, in good agreement with earlier estimates of the size of yeast mtDNA, using electron microscopy and renaturation kinetics. 4. A comparison of the fragmentation pattern of mtDNAs from S. carlsbergensis and various strains of Saccharomyces cerevisiae with endonuclease HindII + III suggests that the overall gene order is similar.

DNA Restriction Enzymes↗
PubMed1975

Sequence heterogeneity of the mini-circles of kinetoplast DNA of Crithidia luciliae and evidence for the presence of a component more complex than mini-circle DNA in the kinetoplast network.

Exhaustive digestion of the 0.76 mum mini-circles of the kinetoplast DNA from Crithidia luciliae with endonuclease HapII yields at least 37 fragments with an added molecular weight of at least 24-10(6), i.e. about 16 times that of the mini-circle. The DNA isolated from cloned cells yields the same digestion pattern. Endonuclease EcoRI cuts only part of the mini-circles in each network. This proves that mini-circles are not homogeneous in sequence. Digestion of total kinetoplast DNA with HapII yields, in addition to the mini-circle fragments, 7 fragments with an added molecular weight of 16-10(6). We conclude that these are derived from a minor component of the network, with a higher sequence complexity than the mini-circles.

Animals↗