A highly bent fragment of Crithidia fasciculata kinetoplast DNA.

Kinetoplast DNA, the mitochondrial DNA of trypanosomatids, is composed of several thousand minicircles and a few dozen maxicircles, all of which are topologically interlocked in a giant network. We have studied the replication of maxicircle DNA, using electron microscopy to analyze replication intermediates from both Crithidia fasciculata and Trypanosoma brucei. Replication intermediates were stabilized against branch migration by introducing DNA interstrand cross-links in vivo with 4,5',8-trimethylpsoralen and UV radiation. Electron microscopy of individual maxicircles resulting from a topoisomerase II decatenation of kinetoplast DNA networks revealed intact maxicircle theta structures. Analysis of maxicircle DNA linearized by restriction enzyme cleavage revealed branched replication intermediates derived from theta structures. Measurements of the linearized branched molecules in both parasites indicate that replication initiates in the variable region (a noncoding segment characterized by repetitive sequences) and proceeds unidirectionally, clockwise on the standard map.

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A trypanosomal CCHC-type zinc finger protein which binds the conserved universal sequence of kinetoplast DNA minicircles: isolation and analysis of the complete cDNA from Crithidia fasciculata.

Molecular and Cellular Biology ◽

10.1128/mcb.13.12.7766 ◽

1993 ◽

Vol 13 (12) ◽

pp. 7766-7773 ◽

Cited By ~ 26

Author(s):

H Abeliovich ◽

Y Tzfati ◽

J Shlomai

Keyword(s):

Amino Acid ◽

Nucleotide Sequence ◽

Dna Binding ◽

Zinc Finger ◽

Binding Protein ◽

Kinetoplast Dna ◽

Single Stranded Dna ◽

Crithidia Fasciculata ◽

Heavy Strand ◽

Minicircle Sequence

Replication of the kinetoplast DNA minicircle light strand initiates at a highly conserved 12-nucleotide sequence, termed the universal minicircle sequence. A Crithidia fasciculata single-stranded DNA-binding protein interacts specifically with the guanine-rich heavy strand of this origin-associated sequence (Y. Tzfati, H. Abeliovich, I. Kapeller, and J. Shlomai, Proc. Natl. Acad. Sci. USA 89:6891-6895, 1992). Using the universal minicircle sequence heavy-strand probe to screen a C. fasciculata cDNA expression library, we have isolated two overlapping cDNA clones encoding the trypanosomatid universal minicircle sequence-binding protein. The complete cDNA sequence defines an open reading frame encoding a 116-amino-acid polypeptide chain consisting of five repetitions of a CCHC zinc finger motif. A significant similarity is found between this universal minicircle sequence-binding protein and two other single-stranded DNA-binding proteins identified in humans and in Leishmania major. All three proteins bind specifically to single-stranded guanine-rich DNA ligands. Partial amino acid sequence of the endogenous protein, purified to homogeneity from C. fasciculata, was identical to that deduced from the cDNA nucleotide sequence. DNA-binding characteristics of the cDNA-encoded fusion protein expressed in bacteria were identical to those of the endogenous C. fasciculata protein. Hybridization analyses reveal that the gene encoding the minicircle origin-binding protein is nuclear and may occur in the C. fasciculata chromosome as a cluster of several structural genes.

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Dyskinetoplasty in two species of trypanosomatids

Journal of Cell Science ◽

10.1242/jcs.35.1.185 ◽

1979 ◽

Vol 35 (1) ◽

pp. 185-202

Author(s):

S.L. Hajduk

Keyword(s):

Fluorescence Microscopy ◽

Kinetoplast Dna ◽

Feulgen Staining ◽

Crithidia Fasciculata ◽

Caesium Chloride ◽

Electron And Fluorescence Microscopy ◽

Staining Techniques ◽

Trypanosoma Equiperdum ◽

In Cells

Dyskinetoplastic cells from both Crithidia fasciculata and Trypanosoma equiperdum lack detectable kinetoplast DNA (kDNA) by conventional staining techniques. Two dyskinetoplastic strains of T. equiperdum, either acriflavine-induced or spontaneously occurring, show normal amounts of kDNA (p = 1.692 g/cm3) in analytical caesium chloride, ultracentrifugation. Electron and fluorescence microscopy of the dyskinetoplastic strains of T. equiperdum suggest that the kDNA network is fragmented and dispersed throughout the mitochondrion. The fragmentation and dispersion of the kDNA, rather than a reduction in the amount of kDNA, is the cause of the lack of kinetoplast staining in the dyskinetoplastic strains of T. equiperdum. Acriflavine-treated cultures of C. fasciculata show a decrease in the amount of kDNA (p = 1.703 g/cm3) corresponding to the percentage of dyskinetoplastic cells in the cultures. Electron and fluorescence microscopy of acriflavine-treated cultures of C. fasciculata show the loss of the kDNA network in cells which lack Giemsa and Feulgen staining, confirming the hypothesis that the kDNA is lost in dyskinetoplastic trypanosomatids from insects. Possible modes of acriflavine action are considered and a proposed mechanism for acriflavine action in trypanosomes from mammals is presented.

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Replication of DNA minicircles in kinetoplasts isolated from Crithidia fasciculata: structure of nascent minicircles

Molecular and Cellular Biology ◽

10.1128/mcb.9.1.169-176.1989 ◽

1989 ◽

Vol 9 (1) ◽

pp. 169-176

Author(s):

C Sheline ◽

T Melendy ◽

D S Ray

Keyword(s):

Topoisomerase Ii ◽

Short Pulse ◽

Kinetoplast Dna ◽

Crithidia Fasciculata ◽

Open Circular ◽

Leading Strand ◽

Polymerase I ◽

Open Circular Form ◽

Lagging Strand ◽

Migration Component

We have previously described an isolated kinetoplast system from Crithidia fasciculata capable of ATP-dependent replication of kinetoplast DNA minicircles (L. Birkenmeyer and D.S. Ray, J. Biol. Chem. 261: 2362-2368, 1986). We present here the identification of two new minicircle species observed in short pulse-labeling experiments in this system. The earliest labeled minicircle species (component A) contains both nascent H and L strands and is heterogeneous in sedimentation and electrophoretic migration. Component A has characteristics consistent with a Cairns-type structure in which the L strand is the leading strand and the H strand is the lagging strand. The other new species (component B) has a nascent 2.5-kilobase linear L strand with a single discontinuity that mapped to either of two alternative origins located 180 degrees apart on the minicircle map. Component B could be repaired to a covalently closed form by Escherichia coli polymerase I and T4 ligase but not by T4 polymerase and T4 ligase. Even though component B has a single gap in one strand, it had an electrophoretic mobility on an agarose gel (minus ethidium bromide) similar to that of a supercoiled circle with three supertwists. Treatment of component B with topoisomerase II converted it to a form that comigrated with a nicked open circular form (replicative form II). These results indicate that component B is a knotted topoisomer of a kinetoplast DNA minicircle with a single gap in the L strand.

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A trypanosomal CCHC-type zinc finger protein which binds the conserved universal sequence of kinetoplast DNA minicircles: isolation and analysis of the complete cDNA from Crithidia fasciculata

Molecular and Cellular Biology ◽

10.1128/mcb.13.12.7766-7773.1993 ◽

1993 ◽

Vol 13 (12) ◽

pp. 7766-7773

Author(s):

H Abeliovich ◽

Y Tzfati ◽

J Shlomai

Keyword(s):

Amino Acid ◽

Nucleotide Sequence ◽

Dna Binding ◽

Zinc Finger ◽

Binding Protein ◽

Kinetoplast Dna ◽

Single Stranded Dna ◽

Crithidia Fasciculata ◽

Heavy Strand ◽

Minicircle Sequence

Replication of the kinetoplast DNA minicircle light strand initiates at a highly conserved 12-nucleotide sequence, termed the universal minicircle sequence. A Crithidia fasciculata single-stranded DNA-binding protein interacts specifically with the guanine-rich heavy strand of this origin-associated sequence (Y. Tzfati, H. Abeliovich, I. Kapeller, and J. Shlomai, Proc. Natl. Acad. Sci. USA 89:6891-6895, 1992). Using the universal minicircle sequence heavy-strand probe to screen a C. fasciculata cDNA expression library, we have isolated two overlapping cDNA clones encoding the trypanosomatid universal minicircle sequence-binding protein. The complete cDNA sequence defines an open reading frame encoding a 116-amino-acid polypeptide chain consisting of five repetitions of a CCHC zinc finger motif. A significant similarity is found between this universal minicircle sequence-binding protein and two other single-stranded DNA-binding proteins identified in humans and in Leishmania major. All three proteins bind specifically to single-stranded guanine-rich DNA ligands. Partial amino acid sequence of the endogenous protein, purified to homogeneity from C. fasciculata, was identical to that deduced from the cDNA nucleotide sequence. DNA-binding characteristics of the cDNA-encoded fusion protein expressed in bacteria were identical to those of the endogenous C. fasciculata protein. Hybridization analyses reveal that the gene encoding the minicircle origin-binding protein is nuclear and may occur in the C. fasciculata chromosome as a cluster of several structural genes.

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Kinetoplast DNA in the insect trypanosomes Crithidia luciliae and Crithidia fasciculata

Plasmid ◽

10.1016/0147-619x(82)90001-4 ◽

1982 ◽

Vol 7 (3) ◽

pp. 199-209 ◽

Cited By ~ 14

Author(s):

J.H.J. Hoeijmakers ◽

B. Schoutsen ◽

P. Borst

Keyword(s):

Kinetoplast Dna ◽

Crithidia Fasciculata ◽

Crithidia Luciliae

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Kinetoplast DNA replication: mechanistic differences between Trypanosoma brucei and Crithidia fasciculata.

The Journal of Cell Biology ◽

10.1083/jcb.126.3.631 ◽

1994 ◽

Vol 126 (3) ◽

pp. 631-639 ◽

Cited By ~ 32

Author(s):

M L Ferguson ◽

A F Torri ◽

D Pérez-Morga ◽

D C Ward ◽

P T Englund

Keyword(s):

Electron Microscopy ◽

Mitochondrial Dna ◽

Dna Replication ◽

Trypanosoma Brucei ◽

Protein Complexes ◽

Kinetoplast Dna ◽

Relative Movement ◽

Crithidia Fasciculata ◽

Trypanosomatid Parasites

Kinetoplast DNA, the mitochondrial DNA of trypanosomatid parasites, is a network containing several thousand minicircles and a few dozen maxicircles. We compared kinetoplast DNA replication in Trypanosoma brucei and Crithidia fasciculata using fluorescence in situ hybridization and electron microscopy of isolated networks. One difference is in the location of maxicircles in situ. In C. fasciculata, maxicircles are concentrated in discrete foci embedded in the kinetoplast disk; during replication the foci increase in number but remain scattered throughout the disk. In contrast, T. brucei maxicircles generally fill the entire disk. Unlike those in C. fasciculata, T. brucei maxicircles become highly concentrated in the central region of the kinetoplast after replication; then during segregation they redistribute throughout the daughter kinetoplasts. T. brucei and C. fasciculata also differ in the pattern of attachment of newly synthesized minicircles to the network. In C. fasciculata it was known that minicircles are attached at two antipodal sites but subsequently are found uniformly distributed around the network periphery, possibly due to a relative movement of the kinetoplast disk and two protein complexes responsible for minicircle synthesis and attachment. In T. brucei, minicircles appear to be attached at two antipodal sites but then remain concentrated in these two regions. Therefore, the relative movement of the kinetoplast and the two protein complexes may not occur in T. brucei.

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