Isolation of proteins associated with kinetoplast DNA networks in vivo.

Xu, Chanxing; Ray, Dan S.

doi:10.1073/pnas.90.5.1786

Cited by 56 publications

(45 citation statements)

References 21 publications

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“…1. As reported previously (35), the amino acid sequence determined by amino-terminal sequence analysis of the purified proteins begins at the 10th residue of the predicted amino acid sequence for each protein, indicating the presence of a 9-aminoacid cleavable presequence on these proteins. (In the report of the amino-terminal sequence of p18, the nature of residue 11 of the mature p18 protein was uncertain, with lysine and aspartic acid being equally likely at that position.…”

Section: Resultsmentioning

confidence: 99%

“…We have developed methods recently for identifying and characterizing proteins that may play a role in organizing and condensing the kDNA network into the compact disc structure observed in vivo (35). Proteins bound to kDNA are covalently cross-linked to the DNA by treatment of the cells with formaldehyde and isolated subsequently from detergent lysates on the basis of the sedimentation properties of the cross-linked kDNA-protein complexes.…”

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confidence: 99%

“…A C. fasciculata cDNA library in the lambda Max1 phage constructed from C. fasciculata poly(A) ϩ RNA was purchased from Clontech Laboratories, Palo Alto, Calif. This library was screened with probes derived from partial cDNA clones of genes encoding proteins p16, p17, and p18 obtained previously (35). Positive clones having the largest inserts were converted to plasmid clones by in vivo excision and conversion into plasmid pYEUra by helper phage infection as recommended by the manufacturer.…”

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confidence: 99%

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Nucleus-Encoded Histone H1-Like Proteins Are Associated with Kinetoplast DNA in the Trypanosomatid Crithidia fasciculata

Hines

Engel

et al. 1996

Molecular and Cellular Biology

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Kinetoplast DNA (kDNA), the mitochondrial DNA of trypanosomatids, consists of thousands of minicircles and 20 to 30 maxicircles catenated into a single large network and exists in the cell as a highly organized compact disc structure. To investigate the role of kinetoplast-associated proteins in organizing and condensing kDNA networks into this disc structure, we have cloned three genes encoding kinetoplast-associated proteins. The KAP2, KAP3, and KAP4 genes encode proteins p18, p17, and p16, respectively. These proteins are small basic proteins rich in lysine and alanine residues and contain 9-amino-acid cleavable presequences. Proteins p17 and p18 are closely related to each other, with 48% identical residues and carboxyl tails containing almost exclusively lysine, alanine, and serine or threonine residues. These proteins have been expressed as Met-His 6 -tagged recombinant proteins and purified by metal chelate chromatography. Each of the recombinant proteins is capable of compacting kDNA networks in vitro and was shown to bind preferentially to a specific fragment of minicircle DNA. Expression of each of these proteins in an Escherichia coli mutant lacking the HU protein rescued a defect in chromosome condensation and segregation in the mutant cells and restored a near-normal morphological appearance. Proteins p16, p17, and p18 have been localized within the cell by immunofluorescence methods and appear to be present throughout the kDNA. Electron-microscopic immunolocalization of p16 shows that p16 is present both within the kDNA disc and in the mitochondrial matrix at opposite edges of the kDNA disc. Our results suggest that nucleus-encoded H1-like proteins may be involved in the organization and segregation of kDNA networks in trypanosomatids.The mitochondrial DNA of kinetoplastid protozoa consists of about 5,000 minicircles and 20 to 30 maxicircles. These circular DNAs are held together by catenation into a single two-dimensional sheet of DNA referred to as a kinetoplast DNA (kDNA) network. The minicircles exist within the network as relaxed covalently closed circles, with each minicircle linked on average to three other minicircles (3). Each cell has only one such network, which in purified form or in cell lysates has a diameter similar in size (8 to 10 m) to that of the whole cell. In vivo, the kDNA network exists as a highly condensed disc about 1 m in diameter and approximately 0.4 m thick (25; also see below). The kDNA disc is physically associated with the basal body of the cell and is oriented with the axis of the disc parallel to that of the flagellum. Electron micrographs of sections through the kDNA disc also show DNA fibers oriented parallel to the axis of the disc. For recent reviews of the structure and replication of kinetoplast DNA, see references 7 and 24.We have developed methods recently for identifying and characterizing proteins that may play a role in organizing and condensing the kDNA network into the compact disc structure observed in vivo (35). Proteins bound to kDNA are covalently...

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Section: Resultsmentioning

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Nucleus-Encoded Histone H1-Like Proteins Are Associated with Kinetoplast DNA in the Trypanosomatid Crithidia fasciculata

Hines

Engel

et al. 1996

Molecular and Cellular Biology

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“…The N-terminal sequence, ASXNAT SPXVXLDAIV, proved identical to the sequence of the GDH gene 41 amino acids downstream of the putative initiating methionine. The presequence may correspond to a mitochondrial signal sequence which is cleaved after the protein enters the mitochondrion (7,58).…”

Section: Identification Of a 110-kda Grna-binding Proteinmentioning

confidence: 99%

Mitochondrial Glutamate Dehydrogenase from Leishmania tarentolae Is a Guide RNA-Binding Protein

Bringaud

Stripecke

Frech

et al. 1997

Molecular and Cellular Biology

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To identify specific proteins interacting with guide RNAs (gRNAs) in mitochondrial ribonucleoprotein complexes from Leishmania tarentolae, fractionated and unfractionated mitochondrial extracts were subjected to UV cross-linking with added labeled gRNA and also with [␣-32 P]UTP-labeled endogenous RNA. An abundant 110-kDa protein (p110) localized in the T-V complex, which sediments in glycerol gradients at the leading edge of the 10S terminal uridylyltransferase peak, was found to interact with both types of labeled RNAs. The p110 protein was gel isolated and subjected to microsequence analysis, and the gene was cloned. The sequence revealed significant similarity with mitochondrial glutamate dehydrogenases. A polyclonal antiserum was raised against a recombinant fragment of the p110 gene and was used to demonstrate a stable and specific gRNA-binding activity by coimmunoprecipitation and competitive gel shift analyses. Complex formation was strongly inhibited by competition with poly(U) or by deletion or substitution of the gRNA 3 oligo(U) tail. Also, addition of a 3 oligo(U) tail to an unrelated transcript was sufficient for p110 binding. Both the gRNA-binding activity of the p110 protein and in vitro gRNA-independent and gRNA-dependent uridine insertion activities in the mitochondrial extract were inhibited by high concentrations of dinucleotides.RNA editing in trypanosomatid mitochondria involves the insertion and deletion of uridine (U) residues at multiple sites mainly within the coding regions of transcripts of more than half of the structural genes in the maxicircle genome (2, 24, 53, 57). The sequence information required for editing is provided by guide RNAs (gRNAs), which are small RNAs (40 to 70 nucleotides) that are complementary (if G:U and, rarely, C:A pairs are allowed) to edited blocks of the mature RNA (3, 42). The gRNAs also possess 3Ј-terminal nonencoded oligo(U) tails (4) that are created by the action of a mitochondrial terminal uridylyltransferase (TUTase) (1).Several models for the mechanism of the U insertion-deletion type of RNA editing have been proposed (3,5,12). Recent results from partial in vitro systems with both Trypanosoma brucei (28,48,49) and Leishmania tarentolae (10,15,20,21) suggest that the original enzyme cascade model is essentially correct. This model postulated an initial cleavage of the preedited mRNA at the first editing site, followed by an addition of U residues to the 3Ј end of the mRNA 5Ј fragment. It was initially proposed that the number of U's added was directly determined by base pairing with the guide A or G nucleotides in the cognate gRNA (3), but recent evidence suggests that multiple U's are first added to the 5Ј fragment and those terminal U's not base paired to the gRNA are then deleted by a 3Ј exonuclease (10,18,28,48). The final step is a ligation of the 5Ј fragment and the 3Ј fragment. Guide RNAdependent U deletions would involve the removal of all of the added U's and the encoded U's due to the lack of guiding nucleotides in the gRNA at that site. Rel...

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“…In bacteria (mitochondrial ancestors), basic proteins such as HU (Heat Unstable) and IHF (Integration Host Factor) have been identified as major DNA-binding proteins and are termed "bacterial histones" (Drlica and Rouviere-Yaniv 1987). In a primitive eukaryote trypanosome, 4 types of basic proteins that are similar to histone H1 have been identified as the major DNA-binding proteins in a kinetoplast (mt-nucleoid containing region of a trypanosome) and have been named kinetoplastassociated proteins (KAPs), (Xu and Ray 1993). Mitochondrial DNA-binding proteins in yeast and their animal homologues, termed autonomously replicating sequence binding factor 2 (Abf2p) (Miyakawa andSando 1987, Diffley andStillman 1991) and mitochondrial transcription factor A (TFAM) (Kang et al 2007), respectively, belong to the high mobility group (HMG) protein family, and are thought to be involved in the packaging of mtDNA in yeast and humans (Brewer et al 2003, Alam et al 2003.…”

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Organization of Mitochondrial-Nucleoids in BY-2 Cultured Tobacco Cells

et al. 2009

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SummaryThe molecular organization of mitochondrial-nucleoids (mt-nucleoids) isolated from BY-2 cultured tobacco (Nicotiana tabacum L.) cells was analyzed. Fluorescence microscopy using 4Ј,6-diamidino-2-phenylindole (DAPI) revealed that isolated mt-nucleoids retained the compact structural organization as that observed in vivo. Treatments of mt-nucleoids with DNase I, proteinase K and 2 M NaCl resulted in the disappearance, partial swelling, or complete dispersion of their DAPI-fluorescence, respectively, whereas treatment with RNase A did not affect their compact appearance. These results suggest that the compact structure of mt-nucleoids is maintained by electrostatic interaction between DNA and proteins. When mt-nucleoids that were treated with micrococcal nuclease (MNase) were analyzed by agarose gel electrophoresis, a ladder-like electrophoretic pattern of DNA, whose minimum fragment size was about 75 bp, was observed. This result suggests the presence of nucleosome-like, repetitive structures in the mt-nucleoids. Treatment of mt-nucleoids with increasing concentrations of NaCl gradually solubilized various proteins, including those with DNA-binding activities, which was accompanied by gradual dispersion of the mt-nucleoid structure. This result suggests that a variety of DNA-binding proteins with differing degrees of binding strength are present in the mt-nucleoids, and are involved in the compact organization of DNA-protein complexes at various levels. Detailed analyses of mt-nucleoids by Southwestern blotting, SDS-DNA PAGE, and DNA-cellulose affinity chromatography revealed the presence of many different DNA-binding proteins in the 2 M NaCl-soluble fraction of mt-nucleoids, which are expected to be involved in the compact organization of mt-nucleoids at various levels.

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Isolation of proteins associated with kinetoplast DNA networks in vivo.

Cited by 56 publications

References 21 publications

Nucleus-Encoded Histone H1-Like Proteins Are Associated with Kinetoplast DNA in the Trypanosomatid Crithidia fasciculata

Nucleus-Encoded Histone H1-Like Proteins Are Associated with Kinetoplast DNA in the Trypanosomatid Crithidia fasciculata

Mitochondrial Glutamate Dehydrogenase from Leishmania tarentolae Is a Guide RNA-Binding Protein

Organization of Mitochondrial-Nucleoids in BY-2 Cultured Tobacco Cells

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