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

Xu, Chanxing; Hines, Jane C.; Engel, Michele L.; Russell, David G.; Ray, Dan S.

doi:10.1128/mcb.16.2.564

Cited by 78 publications

(94 citation statements)

References 34 publications

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“…Because of the importance of this result, we questioned whether there might be an undiscovered splice acceptor site 5Ј of the octamer sequence located distal to the single identified splice acceptor. The known splice acceptor site was identified in several independent cDNA clones earlier by RT-PCR (26). Similar experiments have shown that the KAP3-GFP chimeric RNA is also spliced at this site in several independent isolates (data not shown).…”

Section: Resultssupporting

confidence: 65%

“…In the present work we have examined the sequence requirements for cycling of the C. fasciculata KAP3 gene, a gene encoding a histone H1-like protein associated with kinetoplast DNA (8,26). A plasmid construct containing the KAP3 gene with 5Ј and 3Ј flanking sequences was used to insert the green fluorescent protein (GFP) gene in frame within KAP3 for use as a sequence tag to permit easy discrimination of the expressed transcript from that of the chromosomal KAP3 mRNA during the cell cycle.…”

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confidence: 99%

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Sequence Elements in both the Intergenic Space and the 3′ Untranslated Region of the Crithidia fasciculata KAP3 Gene Are Required for Cell Cycle Regulation of KAP3 mRNA

Avliyakulov

Hines

2003

Eukaryot Cell

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mRNA levels of several Crithidia fasciculata genes involved in DNA metabolism have previously been found to cycle as cells progress through the cell cycle. Octamer consensus sequences in the 5 untranslated regions (5 UTRs) of these transcripts were shown to be required for cycling of these mRNAs. The KAP3 gene encodes a kinetoplast histone H1-like DNA binding protein, and its mRNA levels cycle in parallel with those of the kinetoplast DNA topoisomerase (TOP2), dihydrofolate reductase-thymidylate synthase (DHFR-TS), and the large subunit of the nuclear single-stranded DNA binding protein (RPA1). KAP3 mRNA contains two octamer consensus sequences in its 3 UTR but none in its 5 UTR. Mutation of these octamer sequences was not sufficient to prevent cycling of a sequence-tagged KAP3 mRNA expressed from a plasmid. Mutation of an octamer sequence contained on the precursor transcript but not on the mRNA, in addition to mutation of the two octamer sequences in the 3 UTR, was necessary to abolish cycling of the mRNA. The requirement for a sequence not present on the mature mRNA indicates that regulation of the mRNA levels by the octamer sequences occurs at or prior to splicing of the transcript. Incompletely processed RNAs containing octamer sequences were also found to accumulate during the cell cycle when the mRNA levels were lowest. These RNA species hybridize to both the KAP3 coding sequence and that of the downstream drug resistance gene, indicating a lack of processing within the intergenic region separating these genes. We propose a cell cycledependent interference in transcript processing mediated by octamer consensus sequences as a mechanism contributing to the cycling of such transcripts.Gene expression in trypanosomes and other kinetoplastid protozoa differs significantly from that in most other eukaryotes. These unicellular parasites are among the earliest diverging organisms containing a mitochondrion, and many of their biological features are unique (21). One unusual feature of these organisms is the arrangement of genes along their chromosomes. The sequence of the Leishmania major chromosome I has revealed a 1.6-kb region lacking open reading frames (16). Predicted open reading frames to the left of this region are all oriented toward the left telomere, and those to the right are all oriented toward the right telomere. Also, in contrast to genes in most other eukaryotes, genes in kinetoplastids lack introns, with only a single exception (14). Transcription of genes on the Leishmania chromosome I appears to be bidirectional from within the 1.6-kb region. Similar studies of other chromosomes indicate that both promoters and terminators are present in the regions in which the direction of transcription changes (P. Myler, cited in reference 3).Constitutive transcription of protein-coding genes generates polycistronic transcripts, which are processed to produce mature monocistronic mRNAs (reviewed in reference 3). Maturation of the polycistronic transcripts involves cleavage within intergenic spaces, trans-spli...

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Section: Resultssupporting

confidence: 65%

mentioning

confidence: 99%

Sequence Elements in both the Intergenic Space and the 3′ Untranslated Region of the Crithidia fasciculata KAP3 Gene Are Required for Cell Cycle Regulation of KAP3 mRNA

Avliyakulov

Hines

2003

Eukaryot Cell

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“…The C. fasciculata ligase gene is clearly distinct from the C. fasciculata nuclear DNA ligase I described (36) and also is unrelated to the mitochondrial DNA ligases (LIG III) of higher eukaryotes. However, the predicted N-terminal sequence of the kinetoplast ligase is consistent with the 9-aa cleavable sequences of several other kinetoplast-targeted proteins in C. fasciculata (37). Interestingly, the C. fasciculata nuclear LIG I gene is highly similar to LIG I genes of higher eukaryotes.…”

Section: Resultssupporting

confidence: 64%

Mitochondrial DNA ligase in Crithidia fasciculata

Sinha

Hines

Downey

2004

Proc. Natl. Acad. Sci. U.S.A.

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Kinetoplast DNA (kDNA), the form of mitochondrial DNA in trypanosomatids, consists of thousands of interlocked circular DNAs organized into a compact disk structure. A type II DNA topoisomerase, a DNA polymerase ␤, and a structure-specific endonuclease have been localized to antipodal sites flanking the kDNA disk along with nascent DNA minicircles. We have cloned a gene (LIG k) encoding a mitochondrial DNA ligase in the trypanosomatid Crithidia fasciculata, and we show that an epitope-tagged form of the ligase colocalizes with the other replication proteins at the antipodal sites and also at the two faces of the kDNA disk. DNA LIG k becomes adenylated in reactions with ATP, and the adenylate moiety is removed by incubation with pyrophosphate or nicked DNA. The ligase interacts physically with the ␤ polymerase and is proposed to be involved in the repair of gaps in the newly synthesized minicircles. In yeast and mammals, a single gene encodes both nuclear and mitochondrial forms of DNA ligase. The LIG K protein sequence has low similarity to mitochondrial DNA ligases in other eukaryotes and is distinct from the C. fasciculata nuclear DNA ligase (LIG I).T he mitochondrial DNA of trypanosomatid protozoa (kinetoplast DNA, or kDNA) consists of two forms of topologically interlocked circular DNAs, minicircles, and maxicircles (for recent reviews of kDNA structure and replication, see refs. 1-3). The kDNA of the trypanosomatid Crithidia fasciculata contains 5,000 minicircles of 2.5 kb each and 20-30 maxicircles of 37 kb each. The maxicircles encode genes for mitochondrial proteins and ribosomal RNAs. Minicircles encode small guide RNAs that serve as templates for RNA editing of maxicircle transcripts (4). In vivo, the kDNA exists as a condensed disk structure 1 m in diameter and Ϸ0.4 m thick. Each minicircle in the kDNA disk is linked to three other minicircles on average and is relaxed, unlike most other circular DNAs in nature, which are negatively supercoiled (5). Electron microscopic observation of sections through the kDNA disk shows fibers aligned parallel to the axis of the disk. This observation together with the thickness of the disk corresponding to approximately one half of the minicircle circumference (6) suggests that individual minicircles may have a topology like that of a rubber band stretched taut from opposite sides of the circle (7). Maxicircles appear to be present throughout the kDNA and exist as a catenated network within the overall network (8). The kDNA isolated from nonreplicating C. fasciculata is a planar elliptical network of interlocked circles with dimensions of Ϸ10 by 15 m.The kDNA disk is always located at the base of the flagellum, with the flagellum perpendicular to the face of the disk. Recent studies (9) have shown that in trypanosomes the kDNA disk is connected physically to the flagellar basal bodies and is segregated by them at cell division. The attachment was found to involve three components that penetrate the inner and outer mitochondrial membranes to link the kDNA physically ...

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“…H1299 cells (human lung large cell carcinoma) were purchased from ATCC. Anti-PIAS1 antibody was produced by immunizing New Zealand White rabbits with GST-PIAS1 (residues 47-180) as described previously (12). Other antibodies used were antip21 antibody (Upstate Biotechnology), anti-p53 DO-1 (Santa Cruz Biotechnology), anti-Flag (M2), and anti-actin antibodies (Sigma).…”

Section: Methodsmentioning

confidence: 99%

Activation of p53 by Protein Inhibitor of Activated Stat1 (PIAS1)

Megidish¹,

Xu²,

Xu³

2002

Journal of Biological Chemistry

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The tumor suppressor protein p53 functions as a transcriptional factor that activates genes controlling cell cycle arrest and apoptosis. Here, we report that protein inhibitor of activated Stat1 (PIAS1) interacts with the tetramerization and C-terminal regulatory domains of p53 in yeast two-hybrid analyses. Endogenous PIAS1 is also associated with endogenous p53 in mammalian cells. Ectopic expression of PIAS1 activates p53-mediated expression in mouse embryonic fibroblast cells (p53 ؊/؊ ) as well as a variety of other cell lines. Furthermore, ectopic expression of PIAS1 induces p53-mediated expression of cyclin-dependent kinase inhibitor p21 and G 1 arrest of the cell cycle in H1299 cells. In addition, a PIAS1 mutant without the RING-finger domain required for sumoylation could still activate p53-mediated gene expression, indicating that activation of p53 by PIAS1 does not require the RING-finger domain. Taken together, our results suggest that PIAS1 is a novel activator of p53.The tumor suppressor protein p53 is normally very short-lived and remains at very low levels through proteasomal degradation in unstressed mammalian cells. In response to damaged DNA, nucleotide depletion, hypoxia, oncogenes, and other genotoxic stresses, p53 accumulates dramatically in nucleus and functions as a transcriptional activator (1-4). Activation of p53 leads to cell cycle arrest or apoptosis by inducing a number of genes including cyclin-dependent kinase inhibitor p21 and apoptotic genes such as Bax. Although the exact mechanisms by which some of these stress signals are transduced to p53 are not known, signaling to p53 is thought to be mediated by upstream p53 regulators in at least three distinguishable pathways (5). The first pathway transduces signals of DNA damage induced by ionizing radiation, which activates ATM, Chk2, and subsequently p53. The second pathway is mediated by p14 ARF -MDM2 interaction (6), which is triggered by oncogenic signals such as Ras and Myc. The third pathway is turned on by a wide array of chemotherapeutic drugs, UV light, and protein kinase inhibitors, which requires kinases ATR and casein kinase II. There are additional proteins that can activate p53, but the significance of some of these interactions remains to be elucidated (1).The key negative regulator of the p53 network is MDM2. Transcription of MDM2 is itself activated by p53. However, MDM2 protein can both inhibit the transcriptional activity of p53 and target p53 for ubiquitin-mediated protein degradation (7-9), forming a negative regulatory loop for p53 function. Interference of the p53 and MDM2 interaction appears to be a main convergent point for many of the stress signals, resulting in stabilization and activation of p53 (10).The discovery of p63 and p73, the p53 family members, added a new level of complexity to understanding the p53 network (11). Both p63 and p73 produce multiple transcripts, which could either activate or inhibit p53-mediated gene expression. In contrast to p53, p63 and p73 do not interact with most viral proteins...

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

Cited by 78 publications

References 34 publications

Sequence Elements in both the Intergenic Space and the 3′ Untranslated Region of the Crithidia fasciculata KAP3 Gene Are Required for Cell Cycle Regulation of KAP3 mRNA

Sequence Elements in both the Intergenic Space and the 3′ Untranslated Region of the Crithidia fasciculata KAP3 Gene Are Required for Cell Cycle Regulation of KAP3 mRNA

Mitochondrial DNA ligase in Crithidia fasciculata

Activation of p53 by Protein Inhibitor of Activated Stat1 (PIAS1)

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