scholarly journals Heterogeneity in sensitivity to cleavage by the restriction endonucleases ECORI and HindIII of circular kinetoplast DNA molecules of Crithidia acanthocephali.

1978 ◽  
Vol 79 (2) ◽  
pp. 329-341 ◽  
Author(s):  
D L Fouts ◽  
D R Wolstenholme ◽  
H W Boyer
Keyword(s):  
Restriction Endonucleases ◽  
Single Site ◽  
Contour Length ◽  
Kinetoplast Dna ◽  
Hindiii Site ◽  
Characteristic Structure ◽  
Sensitive Site ◽  
Partial Denaturation ◽  
Sequential Digestion ◽  
Topological Interlocking

Kinetoplast DNA (kDNA) of the protozoan Crithidia acanthocephali consists mainly of an association of approximately 27,000 covalently closed, 0.8-micron (1.58 X 10(6) daltons) circular molecules apparently held together in a particular structural configuration by topological interlocking. The sensitivities of circular kDNA molecules to the restriction endonucleases EcoRI and HindIII have been studied using agarose gel electrophoresis and electron microscopy. Digestion with EcoRI or HindIII of collections of single circular molecules obtained from sonicated kDNA associations resulted in a single cleavage of 9.3 and 12% of the molecules, respectively. Digestion of intact kDNA associations with EcoRI or HindIII resulted in cleavage of 9.2 and 10.4%, respectively, of the component circular molecules, but not in detectable disruption of the characteristic structure of the associations. Analysis of the products of sequential digestion of kDNA with the two enzymes indicated that approximately 8% of the circular molecules each contain a single site sensitive to EcoRI and a single site sensitive to HindIII; 1.5-3% contain only an EcoRI-sensitive site; 3-4% contain only a HindIII-sensitive site; and the remainder (approximately 86%) are insensitive to either enzyme. Further, data obtained from sequential digestion experiments and from studies of the partial denaturation products of the circular molecules digested with EcoRI or HindIII indicated that when they occur the EcoRI site and the HindIII site are each at a unique position in all molecules, 10-13% of the circular contour length apart. Similar digestion products were found for kDNAs from different cloned organisms, suggesting that the four different kinds of circular molecules, in regard to EcoRI and HindIII sensitivity, are found in similar proportions in the kDNA association of different organisms.

scholarly journals Isolation and characterization of kinetoplast DNA from bloodstream form of Trypanosoma brucei.

1978 ◽  
Vol 76 (2) ◽  
pp. 293-309 ◽  
Author(s):  
A H Fairlamb ◽  
P O Weislogel ◽  
J H Hoeijmakers ◽  
P Borst
Keyword(s):  
Electron Microscopy ◽  
Trypanosoma Brucei ◽  
Contour Length ◽  
Kinetoplast Dna ◽  
East African ◽  
S1 Nuclease ◽  
Isolation And Characterization ◽  
Trypanosoma Equiperdum ◽  
Double Networks

We have used restriction endonucleases PstI, EcoRI, HapII, HhaI, and S1 nuclease to demonstrate the presence of a large complex component, the maxi-circle, in addition to the major mini-circle component in kinetoplast DNA (kDNA) networks of Trypanosoma brucei (East African Trypanosomiasis Research Organization [EATRO] 427). Endonuclease PstI and S1 nuclease cut the maxi-circle at a single site, allowing its isolation in a linear form with a mol wt of 12.2 x 10(6), determined by electron microscopy. The other enzymes give multiple maxi-circle fragments, whose added mol wt is 12-13 x 10(6), determined by gel electrophoresis. The maxi-circle in another T. brucei isolate (EATRO 1125) yields similar fragments but appears to contain a deletion of about 0.7 x 10(6) daltons. Electron microscopy of kDNA shows the presence of DNA considerably longer than the mini-circle contour length (0.3 micron) either in the network or as loops extending from the edge. This long DNA never exceeds the maxi-circle length (6.3 microns) and is completely removed by digestion with endonuclease PstI. 5-10% of the networks are doublets with up to 40 loops of DNA clustered between the two halves of the mini-circle network and probably represent a division stage of the kDNA. Digestion with PstI selectively removes these loops without markedly altering the mini-circle network. We conclude that the long DNA in both single and double networks represents maxi-circles and that long tandemly repeated oligomers of mini-circles are (virtually) absent. kDNA from Trypanosoma equiperdum, a trypanosome species incapable of synthesizing a fully functional mitochondrion, contains single and double networks of dimensions similar to those from T. brucei but without any DNA longer than mini-circle contour length. We conclude that the maxi-circle of trypanosomes is the genetic equivalent of the mitochondrial DNA (mtDNA) of other organisms.

scholarly journals THE FORM AND STRUCTURE OF KINETOPLAST DNA OF CRITHIDIA

1972 ◽  
Vol 54 (2) ◽  
pp. 346-364 ◽  
Author(s):  
Hartmut C. Renger ◽  
David R. Wolstenholme
Keyword(s):  
Electron Microscopy ◽  
Cesium Chloride ◽  
Contour Length ◽  
Main Band ◽  
Thin Sections ◽  
Group A ◽  
Linear Molecules ◽  
Different Strains ◽  
Topological Interlocking

Cesium chloride centrifugation of each of the DNAs extracted from eight strains of Crithidia revealed a main band at ρ = 1.717 g/cm3 and a satellite band varying from ρ = 1.701 to 1.705 g/cm3 for the different strains By electron microscopy each DNA was shown to include circular molecules, 0.69–0.80 µ in mean contour length, and large, topologically two-dimensional masses of DNA in which the molecules appeared in the form of rosettes. DNA isolated from kinetoplast fractions of Crithidia acanthocephali was shown to consist of light satellite DNA and to be mainly in the form of large masses, 0.8 µ (mol wt = 1.54 x 106 daltons) circular molecules, and a few long, linear molecules. The results of experiments involving ultracentrifugation, heating, and quenching, sonication, and endodeoxyribonuclease digestion, combined with electron microscopy, are consistent with the following hypothesis. The large DNA masses are associations of 0.8 µ circles which are mainly covalently closed. The circles are held together in groups (the rosettes) of up to 46 by the topological interlocking of each circle with many other circles in the group. A group of circles is attached to an adjacent group by one or more circles, each interlocking with many circles of both groups. Each of the associations comprises, on the average, about 27,000 circles (total mol wt ≃ 41 x 109 daltons). A model is proposed for the in situ arrangement of the associations which takes into consideration their form and structure, and appearance in thin sections

scholarly journals The actions of restriction endonucleases on lampbrush chromosomes

1976 ◽  
Vol 21 (2) ◽  
pp. 303-313
Author(s):  
D.C. Gould ◽  
H.G. Callan ◽  
C.A. Thomas
Keyword(s):  
Tandem Repeats ◽  
Dnase I ◽  
Restriction Endonucleases ◽  
Notophthalmus Viridescens ◽  
Lampbrush Chromosomes ◽  
E Coli ◽  
Refractory Property ◽  
Sensitive Site ◽  
Chromosome Ii ◽  
Palindromic Sequences

Lampbrush chromosomes from oocytes of Notophthalmus viridescens were dispersed in media containing restriction endonucleases isolated from Haemophilus and E. coli. These endonucleases cleave duplex DNAs at specific palindromic sequences of nucleotides, and several sensitive sites occur per micron of DNA. The overwhelming majority of the lateral loops of lampbrush chromosomes are extensively fragmented by these endonucleases, but an occasional pair of loops is refractory. A notable example of loops showing this refractory property are the giant loops on chromosome II in the presence of Hae. These loops, whose DNA-containing axes are several hundred micra long, are sensitive to other nucleases such as EcoB, endonuclease I and pancreatic DNase I; their refractory behavior towards Hae therefore indicates that the sequence sensitive to this particular endonuclease is systematically absent. This anomalous property can be comprehended if it be assumed that the axial DNA of the giant loops consists of tandem repeats of a sequence which happens not to include the sensitive site.

Partial Denaturation Mapping of Phage T4 DNA at Low Temperature

1981 ◽  
Vol 36 (11-12) ◽  
pp. 980-987
Author(s):  
G. F. Grossi ◽  
M. F. Macchiato ◽  
G. Gialanella
Keyword(s):  
Electron Microscope ◽  
Low Temperature ◽  
Computer Program ◽  
Contour Length ◽  
High Concentration ◽  
Experimental Conditions ◽  
Endonucleolytic Cleavage ◽  
Phage T4 ◽  
Length Measurements ◽  
Partial Denaturation

Abstract The partial-denaturation map of T4 DNA is obtained by using benzyldodecyldimethyl ammonium chloride in the presence of a high concentration of formamide. In this way suitable conditions for preparation of electron microscope specimens and partial denaturation within a temperature range low enough to minimize the endonucleolytic cleavage, are realized. It is found that, under our experimental conditions, the denaturation increase depends mainly on the appearance of new denaturation sites rather than on the lengthening of DNA segments already denatured. Because of the DNA circular permutation it is necessary to align the measured maps to obtain the denaturation pattern. This is done through a computer program and informations on the distribution of the regions with highest (A-T) content along the genome are obtained. The results of contour length measurements of λ and T4 DNA’s are also reported.

scholarly journals Intramolecular heterogeneity of mitochondrial DNA of Drosophila melanogaster.

1977 ◽  
Vol 73 (2) ◽  
pp. 279-286 ◽  
Author(s):  
E S Goldring ◽  
W J Peacock
Keyword(s):  
Mitochondrial Dna ◽  
Drosophila Melanogaster ◽  
Base Composition ◽  
Gc Content ◽  
Contour Length ◽  
Regional Heterogeneity ◽  
Partial Denaturation

DNA from purified mitochondria of Drosophila melanogaster can be isolated as supercoiled molecules which when nicked have a contour length of 5.9 micron. Partial denaturation mapping shows regional heterogeneity of base composition with one early denaturing region, with a calculated GC content close to zero, extending over 20% of the genome. DNA isolated from unfertilized eggs shows nuclear and mitochondrial DNA in equal proportions; we found no evidence of other cytoplasmic species.

scholarly journals Replication of kinetoplast DNA of Crithidia acanthocephali. I. Density shift experiments using deuterium oxide.

1976 ◽  
Vol 70 (2) ◽  
pp. 406-418 ◽  
Author(s):  
J E Manning ◽  
D R Wolstenholme
Keyword(s):  
Nuclear Dna ◽  
Deuterium Oxide ◽  
Gradient Centrifugation ◽  
Unit Length ◽  
Buoyant Density ◽  
Cesium Chloride ◽  
Kinetoplast Dna ◽  
Open Circular ◽  
Linear Molecules ◽  
Topological Interlocking

The protozoan Crithidia acanthocephali contains, within a modified region of a mitochondrion, a mass of DNA known as kinetoplast DNA (kDNA). This DNA consists mainly of an association of approximately 27,000 covalently closed 0.8-mum circular molecules which are apparently held together in a definite ordered manner by topological interlocking. After culturing of C. acanthocephali cells for 25 generations in medium containing 75% deuterium oxide, both nuclear DNA (rhonative, nondeuterated=1.717 g/cm3) and kDNA (rhonative, nondeuterated=1.702 g/cm3) increased in buoyant density by 0.012 g/cm3. The replication of the two DNAs was studied by cesium chloride buoyant density analysis of DNAs from exponentially growing cells taken at 1.0, 1.4, 2.0, 3.0, and 4.0 cell doublings after transfer of cells from D2O-containing medium into medium containing only normal water. The results obtained from analysis of both native and denatured nuclear DNAs indicate that this DNA replicates semiconservatively. From an analysis of intact associations of kDNA, it appears that this DNA doubles once per generation and that the newly synthesized DNA does not segregate from parental DNA. Fractions of covalently closed single circular molecules and of open circular and unit length linear molecules were obtained from associations of kDNA by sonication, sucrose sedimentation, and cesium chloride-ethidium bromide equilibrium gradient centrifugation. Buoyant density profiles obtained from these fractions indicate that: (a) doubling of the kDNA results from the replication of each circular molecule rather than from repeated replication of a small fraction of the circular molecules; (b) replication of kDNA is semiconservative rather than conservative, but there is recombination between the circles at an undefined time during the cell cycle.

scholarly journals Characterization of the molecular components in kinetoplast-mitochondrial DNA of Trypanosoma equiperdum. Comparative study of the dyskinetoplastic and wild strains.

1979 ◽  
Vol 82 (1) ◽  
pp. 248-263 ◽  
Author(s):  
G F Riou ◽  
J M Saucier
Keyword(s):  
Mitochondrial Dna ◽  
Buoyant Density ◽  
Wild Strain ◽  
Restriction Endonucleases ◽  
Cleavage Sites ◽  
Kinetoplast Dna ◽  
Large Circle ◽  
Trypanosoma Equiperdum ◽  
Cellular Dna

The structure of the kinetoplast DNA of Trypanosoma equiperdum has been studied and compared to the structure of the circular mitochondrial DNA extracted from a dyskinetoplastic strain of T. equiperdum. In T. equiperdum wild type, the kinetoplast DNA constitutes approximately 6% of the total cellular DNA and is composed of approximately 3,000 supercoiled minicircles of 6.4 x 10(5) daltons and approximately 50 circular supercoiled molecules of 15.4 x 10(6) daltons topologically interlocked; The buoyant density in CsCl of the minicircles is 1.691 g/cm 3. The large circles have a buoyant density of 1.684 g/cm 3, are homogeneous in size and are selectively cleaved by several restriction endonucleases which do not cleave the minicircles. The cleavage sites of six different restriction endonucleases have been mapped on the large circle. The minicircles are cleaved by two other restriction endonucleases, and their cleavage sites have been mapped. The mitochondrial DNA extracted from the dyskinetoplastic strain of T. equiperdum represents 7% of the total DNA of the cell and is composed of supercoiled circles, heterogeneous in size, and topologically associated in catenated oligomers. Its buoyant density in CsCl is 1.688 g/cm 3. These molecules are not cleaved by any of the eight restriction endonucleases tested. The reassociation kinetics of in vitro labeled kDNA minicircles and large circles has been studied. The results indicate that the minicircles as well as the large circles are homogeneous in sequence and that the circular DNA of the dyskinetoplastic strain has no sequence in common with the kDNA of the wild strain.

Evaluation of the dark field method for large association of spread DNA

1978 ◽  
Vol 36 (2) ◽  
pp. 190-191
Author(s):  
Douglas C. Barker
Keyword(s):  
Electron Microscopy ◽  
Molecular Weight ◽  
Mitochondrial Dna ◽  
Extraction Method ◽  
Field Method ◽  
Dark Field ◽  
Kinetoplast Dna ◽  
Field Electron ◽  
Field Electron Microscopy ◽  
Dark Field Electron

A number of satisfactory methods are available for the electron microscopy of nicleic acids. These methods concentrated on fragments of nuclear, viral and mitochondrial DNA less than 50 megadaltons, on denaturation and heteroduplex mapping (Davies et al 1971) or on the interaction between proteins and DNA (Brack and Delain 1975). Less attention has been paid to the experimental criteria necessary for spreading and visualisation by dark field electron microscopy of large intact issociations of DNA. This communication will report on those criteria in relation to the ultrastructure of the (approx. 1 x 10-14g) DNA component of the kinetoplast from Trypanosomes. An extraction method has been developed to eliminate native endonucleases and nuclear contamination and to isolate the kinetoplast DNA (KDNA) as a compact network of high molecular weight. In collaboration with Dr. Ch. Brack (Basel [nstitute of Immunology), we studied the conditions necessary to prepare this KDNA Tor dark field electron microscopy using the microdrop spreading technique.

Studies of Circular DNA Using a New Electron Microscopic Technique

1973 ◽  
Vol 31 ◽  
pp. 596-597
Author(s):  
C. N. Gordon
Keyword(s):  
Electron Microscopy ◽  
Aqueous Salt Solution ◽  
Contour Length ◽  
Cleavage Surface ◽  
Mica Substrate ◽  
Electron Microscopic ◽  
Replica Technique ◽  
Circular Dna ◽  
Transmission Electron ◽  
Electron Microscopic Technique

Gordon and Kleinschmidt have described a new preparative technique for visualizing DNA by electron microscopy. This procedure, which is a modification of Hall's “mica substrate technique”, consists of the following steps: (a) K+ ions on the cleavage surface of native mica are exchanged for Al3+ ions by ion exchange. (b) The mica, with Al3+ in the exchange sites on the surface, is placed in a dilute aqueous salt solution of DNA for several minutes; during this period DNA becomes adsorbed on the surface. (c) The mica with adsorbed DNA is removed from the DNA solution, rinsed, dried and visualized for transmission electron microscopy by Hall's platinum pre-shadow replica technique.In previous studies of circular DNA by this technique, most of the molecules seen were either broken to linears or extensively tangled; in general, it was not possible to obtain suitably large samples of open extended molecules for contour length measurements.

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