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PubMed2026

Evolutionary history and recombination in the mitochondrial carrier SLC25 superfamily analyzed by similarities in the exon and transmembrane α-helix sequences.

Mitochondrial carriers (MCs), which constitute a superfamily also called the solute carrier family 25 (SLC25), are characterized by conserved signature motif sequences and a six-transmembrane α-helical transporter domain. They transport a wide variety of substrates ranging from protons, inorganic ions, citric acid cycle intermediates, and amino acids to nucleotides and cofactors. The superfamily members can be divided into subfamilies, each with a distinct substrate specificity. In an attempt to understand how different subfamilies have evolved, we analyzed the protein sequences of the exons (with conserved boundaries) and the six transmembrane α-helices of MCs from highly diverged organisms. The results show that some MC subfamilies have all exons and transmembrane α-helices most similar to a closely related subfamily, which is consistent with a scenario of gene duplication and mutational divergence from a last common ancestor. However, several MC subfamilies appear to be mosaics of exons and transmembrane α-helices most similar to different and distant subfamilies, which in some cases could be explained by recombination between the superfamily genes during evolution. It seems that this latter mechanism could have played a role in the formation of new subfamilies with different substrate specificities by the combination of MC transporter domain segments that had been optimized previously for binding specific portions of the substrates. This study presents novel evolutionary relationships between MC subfamilies and may provide clues for how protein superfamilies have expanded and how to investigate their evolution.

Evolution, Molecular↗
PubMed2005

Genome-wide analysis of restriction-modification system in unicellular and filamentous cyanobacteria.

Cyanobacteria are an ancient group of gram-negative bacteria with strong genome size variation ranging from 1.6 to 9.1 Mb. Here, we first retrieved all the putative restriction-modification (RM) genes in the draft genome of Spirulina and then performed a range of comparative and bioinformatic analyses on RM genes from unicellular and filamentous cyanobacterial genomes. We have identified 6 gene clusters containing putative Type I RMs and 11 putative Type II RMs or the solitary methyltransferases (MTases). RT-PCR analysis reveals that 6 of 18 MTases are not expressed in Spirulina, whereas one hsdM gene, with a mutated cognate hsdS, was detected to be expressed. Our results indicate that the number of RM genes in filamentous cyanobacteria is significantly higher than in unicellular species, and this expansion of RM systems in filamentous cyanobacteria may be related to their wide range of ecological tolerance. Furthermore, a coevolutionary pattern is found between hsdM and hsdR, with a large number of site pairs positively or negatively correlated, indicating the functional importance of these pairing interactions between their tertiary structures. No evidence for positive selection is found for the majority of RMs, e.g., hsdM, hsdS, hsdR, and Type II restriction endonuclease gene families, while a group of MTases exhibit a remarkable signature of adaptive evolution. Sites and genes identified here to have been under positive selection would provide targets for further research on their structural and functional evaluations.

Bacterial Proteins↗
PubMed1996

[Antioncogene p53 and apoptosis response: new hypotheses on the molecular bases of tumor resistance to radiotherapy].

Recent experimental data indicate that in normal or neoplastic cells, DNA damage induced by ionizing radiation may work as a signal to trigger apoptosis, a characteristic suicide program inherent to multicellular organisms. The morphological signatures of apoptosis are cell shrinkage, nuclear condensation and genomic fragmentation down to the size of individual nucleosome units (apoptotic bodies). A number of genes have been identified as involved regulators of apoptosis. The tumor suppressor gene p53 has emerged as a main modulator and inducer of apoptosis. Cells containing wild-type p53 function undergo G1 arrest following DNA damage by ionizing radiations. DNA repair processes are, then, activated before the damaged genome can be replicated, with resultant genetic instability. If DNA repair fails, the wild p53 gene may trigger apoptosis and the cell with persisting DNA damage, dies. Cells with loss of functional p53 gene by either gene mutation or deletion appear unable to undergo radiation-induced apoptosis. This evidence may underlie the increased likelihood for p53 deficient cells to develop oncogenetic processes after irradiation exposure. According to p53-triggering apoptosis patterns, cancer cells with a normal p53 gene might be more sensitive to radiotherapy and less proliferating than tumor cells with p53 deficient function. If these data are confirmed, tumors harboring p53 gene mutation might be managed best with particularly aggressive or experimental treatment protocols. Conversely, a higher therapeutic index between malignant and normal cells might be obtained in tumors with fast activation of p53 dependent apoptotic response. Before any definitive conclusions are drawn, further knowledge of the different genes which are known to be involved in the regulation of apoptosis, is warranted.

Apoptosis↗
PubMed1999

The tomato I-box binding factor LeMYBI is a member of a novel class of myb-like proteins.

The RBCS3A gene of tomato belongs to a small gene family consisting of five members. Although the RBCS1, RBCS2 and RBCS3A promoters contain closely related cis regulatory sequences, the expression patterns of the genes are different. Whereas the RBCS1 and RBCS2 genes are expressed in both leaves and young fruit, the RBCS3A promoter is highly active in leaves, but not in young fruit. This lack of transcription could be due to a mutation in the RBCS3A promoter creating the so-called F-box, a protein binding site located between the activating cis elements, the I-box and G-box. In order to identify proteins that bind to the RBCS3A I-box/F-box region, the yeast one-hybrid system was used. One clone, LeMYBI was isolated which contains strong similarity to plant myb transcription factors. The encoded LeMYBI protein is at least 188 amino acids in length and contains two myb-like domains located at the amino terminus and close to the carboxy terminus, separated by a negatively charged domain. The protein contains a SHAQKYF amino acid signature motif in the second myb-like repeat, which is highly conserved in a number of recently identified plant myb-related genes, thus defining a new class of plant DNA-binding proteins. LeMYBI binds specifically to the I-box sequence of the RBCS1, RBCS2 and RBCS3A promoters, therefore representing the first cloned I-box binding factor. LeMYBI acts as a transcriptional activator in yeast and plants, and binds to the I-box with a DNA-binding domain located in the carboxyterminal domain.

Amino Acid Sequence↗
PubMed2001

The phosphatase C(X)5R motif is required for catalytic activity of the Saccharomyces cerevisiae Acr2p arsenate reductase.

Acr2p detoxifies arsenate by reduction to arsenite in Saccharomyces cerevisiae. This reductase has been shown to require glutathione and glutaredoxin, suggesting that thiol chemistry might be involved in the reaction mechanism. Acr2p has a HC(X)(5)R motif, the signature sequence of the phosphate binding loop of the dual-specific and protein-tyrosine phosphatase family. In Acr2p these are residues His-75, Cys-76, and Arg-82, respectively. Acr2p has another sequence, (118)HCR, that is absent in phosphatases. Acr2p also has a third cysteine residue at position 106. Each of these cysteine residues was changed individually to serine residues, whereas the histidine and arginine residues were altered to alanines. Cells of Escherichia coli heterologously expressing the majority of the mutant ACR2 genes retained wild type resistance to arsenate, and the purified altered Acr2p proteins exhibited normal enzymatic properties. In contrast, cells expressing either the C76S or R82A mutations lost resistance to arsenate, and the purified proteins were inactive. These results suggest that Acr2p utilizes a phosphatase-like Cys(X)(5)Arg motif as the catalytic center to reduce arsenate to arsenite.

Adenosine Triphosphatases↗
PubMed2005

Cross-talk between cAMP and calcium signalling in Aspergillus niger.

Very little is known about cross-talk between cAMP and calcium signalling in filamentous fungi. The aim of this study was to analyse the influence of cAMP and protein kinase A (PKA)-dependent phosphorylation on calcium signalling in Aspergillus niger. For this purpose, cytosolic free calcium ([Ca2+]c) was measured in living hyphae expressing codon-optimized aequorin. The calcium signature following mechanical perturbation was analysed after applying dibutryl-cAMP or IBMX which increased intracellular cAMP, or H7 which inhibited phosphorylation by PKA. Calcium signatures were also measured in mutant strains in which phosphorylation by PKA was increased or lacking. The results indicated that calcium channels were activated by cAMP-mediated, PKA-dependent phosphorylation. Further evidence for cross-talk between cAMP and calcium signalling came from the analysis of a mutant in which the catalytic subunit of PKA was under the control of an inducible promoter. The consequence of PKA induction was a transient increase in [Ca2+]c which correlated with a polar-apolar transition in hyphal morphology. A transient increase in [Ca2+]c was not observed in this mutant when the morphological shift was in the opposite direction. The [Ca2+]c signatures in response to mechanical perturbation by polarized and unpolarized cells were markedly different indicating that these two cell types possessed different calcium signalling capabilities. These results were consistent with PKA-dependent phosphorylation increasing [Ca2+]c to induce a polar to apolar shift in hyphal morphology.

Aspergillus niger↗
PubMed2003

Arginyl-tRNA synthetase with signature sequence KMSK from Bacillus stearothermophilus.

ArgRS (arginyl-tRNA synthetase) belongs to the class I aaRSs (aminoacyl-tRNA synthetases), though the majority of ArgRS species lack the canonical KMSK sequence characteristic of class I aaRSs. A DNA fragment of the ArgRS gene from Bacillus stearothermophilus was amplified using primers designed according to the conserved regions of known ArgRSs. Through analysis of the amplified DNA sequence and known tRNA(Arg)s with a published genomic sequence of B. stearothermophilus, the gene encoding ArgRS ( argS ') was amplified by PCR and the gene encoding tRNA(Arg) (ACG) was synthesized. ArgRS contained 557 amino acid residues including the canonical KMKS sequence. Recombinant ArgRS and tRNA(Arg) (ACG) were expressed in Escherichia coli. ArgRS purified by nickel-affinity chromatography had no ATPase activity. The kinetics of ArgRS and cross-recognition between ArgRSs and tRNA(Arg)s from B. stearothermophilus and E. coli were studied. The activities of B. stearothermophilus ArgRS mutated at Lys(382) and Lys(385) of the KMSK sequence and at Gly(136) upstream of the HIGH loop were determined. From the mutation results, we concluded that there was mutual compensation of Lys(385) and Gly(136) for the amino acid-activation activity of B. stearothermophilus ArgRS.

Adenosine Triphosphatases↗
PubMed2003

Requirement of E6AP and the features of human papillomavirus E6 necessary to support degradation of p53.

E6 oncoproteins from human papillomavirus type 16 (16E6) and Bovine Papillomavirus type 1 (BE6) bind to leucine rich peptides (called charged leucine, LXXLL, or signature peptides) found on target cellular proteins. BE6 and 16E6 both bind the product of the UBE3A gene called E6AP on a charged leucine peptide, LQELL. E6AP is an E3 ubiquitin ligase that together with 16E6 interacts with p53 to target p53 degradation. Although both BE6 and 16E6 bind the LQELL peptide of E6AP, only 16E6 acts as an adapter to then bring p53 to E6AP. In order to determine how E6 proteins function as adapters, 16E6, p53, and E6AP were expressed in yeast, and were shown to form a tri-molecular complex. 16E6 mutants were selected that retained interactions with E6AP yet were defective for interaction with p53. Such 16E6 mutations were typically within the amino-terminus of 16E6. Through the use of E6AP null cells, transfected E6AP was shown to be necessary and sufficient for the degradation of p53 in the presence of 16E6. However, the interaction of 16E6 with E6AP was complex. While BE6 interacts only with the LQELL motif of E6AP, an intact LQELL motif is not necessary either for interaction of 16E6 with E6AP or for p53 degradation. In addition, 16E6 mutants that fail to bind the LQELL motif of E6AP can support p53 degradation. These results indicate that 16E6 may have multiple modes of interaction with E6AP and that assembly of p53 containing complexes for targeted degradation by E6AP may occur in more than one way. These results have implications for potential targeting of the interaction of 16E6 and E6AP in the therapy of HPV-induced cancer.

Amino Acid Sequence↗
PubMed2003

Open-state disulfide crosslinking between Mycobacterium tuberculosis mechanosensitive channel subunits.

The mechanosensitive channel of large conductance from Mycobacterium tuberculosis (Tb-MscL) was subjected to cysteine-scanning mutagenesis at several residues in the M1 region. The V15C channel displayed disulfide crosslinking in air, but not in the presence of 100 mM beta-mercaptoethanol. In single-channel experiments, the V15C channel was more sensitive to tension than was wild-type Tb-MscL. In air, Tb-MscL V15C occasionally displayed signature-events: at constant tension, there was first a sojourn in the highest conductance open state, then a series of transitions to substates. During a signature-event, these transitions do not appear to be reversible. Some sojourns in the lower conductance states lasted for > or =100 s. These signature-events were abolished by 100 mM beta-mercaptoethanol and did not occur in a cysteineless gain-of-function mutant, suggesting that the signature-events represent disulfide crosslinking between channel subunits. We conclude that the crosslinking occurs during an open state during asymmetric sojourns that bring the alpha-carbons of adjacent 15C side chains within 3.6-6.8 A. Such asymmetric structures must be considered in models of TB-MscL gating.

Binding Sites↗
PubMed2004

The transcriptome of prematurely aging yeast cells is similar to that of telomerase-deficient cells.

To help define the pathologies associated with yeast cells as they age, we analyzed the transcriptome of young and old cells isolated by elutriation, which allows isolation of biochemical quantities of old cells much further advanced in their life span than old cells prepared by the biotin-streptavidin method. Both 18-generation-old wild-type yeast and 8-generation-old cells from a prematurely aging mutant (dna2-1), with a defect in DNA replication, were evaluated. Genes involved in gluconeogenesis, the glyoxylate cycle, lipid metabolism, and glycogen production are induced in old cells, signifying a shift toward energy storage. We observed a much more extensive generalized stress response known as the environmental stress response (ESR), than observed previously in biotin-streptavidin-isolated cells, perhaps because the elutriated cells were further advanced in their life span. In addition, there was induction of DNA repair genes that fall in the so-called DNA damage "signature" set. In the dna2-1 mutant, energy production genes were also induced. The response in the dna2-1 strain is similar to the telomerase delete response, genes whose expression changes during cellular senescence in telomerase-deficient cells. We propose that these results suggest, albeit indirectly, that old cells are responding to genome instability.

Cell Cycle↗
PubMed1995

The case for heterogeneity in the etiology of schizophrenia.

To confirm etiological heterogeneity, it is required that schizophrenic patients may be separated into at least two classes having different known etiologies and, perhaps, different pathophysiological signatures. In contrast, the homogeneity hypothesis asserts that there is a single necessary and sufficient cause or configuration of causes of schizophrenia. Because the link between phenotypic heterogeneity and etiological heterogeneity is tenuous, attempts to use purely phenotypic data to infer etiologic heterogeneity must be viewed cautiously. We examined three candidate causes for schizophrenia: genes, obstetric complications and viral infection. Cytogenetic studies show that some rare cases of schizophrenia are due to gross abnormalities of chromosomes. As for the large majority of schizophrenic patients, the candidate cause data most certainly reject the most parsimonious version of the hypothesis of etiological homogeneity: that all schizophrenia is caused by exactly the same pattern of genetic mutations, birth related complications and exposure to the same viral infections. We conclude that the heterogeneity debate should consider the possibility of rewording the question: 'Heterogeneity: yes or no?' to 'Heterogeneity: how much?'

Chromosome Aberrations↗
PubMed2006

Genetic attributes of cerebrospinal fluid-derived HIV-1 env.

HIV-1 often invades the CNS during primary infection, eventually resulting in neurological disorders in up to 50% of untreated patients. The CNS is a distinct viral reservoir, differing from peripheral tissues in immunological surveillance, target cell characteristics and antiretroviral penetration. Neurotropic HIV-1 likely develops distinct genotypic characteristics in response to this unique selective environment. We sought to catalogue the genetic features of CNS-derived HIV-1 by analysing 456 clonal RNA sequences of the C2-V3 env subregion generated from CSF and plasma of 18 chronically infected individuals. Neuropsychological performance of all subjects was evaluated and summarized as a global deficit score. A battery of phylogenetic, statistical and machine learning tools was applied to these data to identify genetic features associated with HIV-1 neurotropism and neurovirulence. Eleven of 18 individuals exhibited significant viral compartmentalization between blood and CSF (P < 0.01, Slatkin-Maddison test). A CSF-specific genetic signature was identified, comprising positions 9, 13 and 19 of the V3 loop. The residue at position 5 of the V3 loop was highly correlated with neurocognitive deficit (P < 0.0025, Fisher's exact test). Antibody-mediated HIV-1 neutralizing activity was significantly reduced in CSF with respect to autologous blood plasma (P < 0.042, Student's t-test). Accordingly, CSF-derived sequences exhibited constrained diversity and contained fewer glycosylated and positively selected sites. Our results suggest that there are several genetic features that distinguish CSF- and plasma-derived HIV-1 populations, probably reflecting altered cellular entry requirements and decreased immune pressure in the CNS. Furthermore, neurological impairment may be influenced by mutations within the viral V3 loop sequence.

Amino Acid Sequence↗
PubMed2001

Description of staphylococcus serine protease (ssp) operon in Staphylococcus aureus and nonpolar inactivation of sspA-encoded serine protease.

Signature tagged mutagenesis has recently revealed that the Ssp serine protease (V8 protease) contributes to in vivo growth and survival of Staphylococcus aureus in different infection models, and our previous work indicated that Ssp could play a role in controlling microbial adhesion. In this study, we describe an operon structure within the ssp locus of S. aureus RN6390. The ssp gene encoding V8 protease is designated as sspA, and is followed by sspB, which encodes a 40.6-kDa cysteine protease, and sspC, which encodes a 12.9-kDa protein of unknown function. S. aureus SP6391 is an isogenic derivative of RN6390, in which specific loss of SspA function was achieved through a nonpolar allelic replacement mutation. In addition to losing SspA, the culture supernatant of SP6391 showed a loss of 22- to 23-kDa proteins and the appearance of a 40-kDa protein corresponding to SspB. Although the 40-kDa SspB protein could degrade denatured collagen, our data establish that this is a precursor form which is normally processed by SspA to form a mature cysteine protease. Culture supernatant of SP6391 also showed a new 42-kDa glucosaminidase and enhanced glucosaminidase activity in the 29 to 32 kDa range. Although nonpolar inactivation of sspA exerted a pleiotropic effect, S. aureus SP6391 exhibited enhanced virulence in a tissue abscess infection model relative to RN6390. Therefore, we conclude that SspA is required for maturation of SspB and plays a role in controlling autolytic activity but does not by itself exert a significant contribution to the development of tissue abscess infections.

Adhesins, Bacterial↗
PubMed2002

Hypocretin increases impulse flow in the septohippocampal GABAergic pathway: implications for arousal via a mechanism of hippocampal disinhibition.

Hypocretins (Hcrts), or orexins, are a recently described set of hypothalamic peptides that have been implicated in feeding, neuroendocrine regulation, sleep-wakefulness, and disorders of sleep, such as narcolepsy. Hcrt-containing neurons, which are located exclusively in the lateral hypothalamic area, provide a dense innervation to the medial septum/diagonal band of Broca (MSDB), a sleep-associated brain region that has been suggested to show intense axonal degeneration in canine narcoleptics. The MSDB, via its cholinergic and GABAergic projections to the hippocampus, controls the hippocampal theta rhythm and associated learning and memory functions that occur during exploratory behavior and rapid eye movement sleep. Neurons of the MSDB express the Hcrt receptor 2, which is mutated in canine narcoleptics, but lack the Hcrt receptor 1 mRNA. In the present study, we investigated the electrophysiological effects of Hcrt2 on MSDB neurons from rat brain slices. We report that Hcrt2 produces a reversible, reproducible, concentration-dependent and direct postsynaptic excitation of GABA-type neurons of the MSDB with an EC50 of 207 nm. This effect is sodium dependent but not potassium or chloride dependent and is attenuated by blockers of the Na+-Ca+ exchanger. Hcrt2 also increases impulse-dependent release of GABA within the MSDB. Using recordings from retrogradely labeled septohippocampal neurons, we found that Hcrt2-excited MSDB neurons project to the hippocampus and have a GABAergic physiological signature. Double-immunolabeling studies confirmed the presence of Hcrt receptor-2 immunoreactivity in septohippocampal GABAergic neurons, as well as the presence of Hcrt fibers adjacent to these neurons. Based on these results, we speculate that Hcrt2-induced activation of septohippocampal GABAergic neurons will, by engaging disinhibitory mechanisms in the hippocampus, promote generation of the hippocampal theta rhythm and associated behaviors.

Animals↗
PubMed2004

Two perfectly conserved arginine residues are required for substrate binding in a high-affinity nitrate transporter.

This study represents the first attempt to investigate the molecular mechanisms by which nitrate, an anion of significant ecological, agricultural, and medical importance, is transported into cells by high-affinity nitrate transporters. Two charged residues, R87 and R368, located within hydrophobic transmembrane domains 2 and 8, respectively, are conserved in all 52 high-affinity nitrate transporters sequenced thus far. Site-directed replacements of either of R87 or R368 residues by lysine were found to be tolerated, but such residue changes increased the K(m) for nitrate influx from micromolar to millimolar values. Seven other amino acid substitutions of R87 or R368 all led to loss of function and lack of growth on nitrate. No evidence was obtained of R87 or R368 forming a salt-bridge with conserved acidic residues. Remarkably, the phenotype of loss-of-function mutant R87T was found to be alleviated by an alteration to lysine of N459, present in the second copy of the nitrate signature (transmembrane domain 11), suggesting a structural or functional interplay between residues R87 and N459 in the three-dimensional NrtA protein structure. Failure of the potential reciprocal second site suppressor N168K (in the first nitrate signature copy of transmembrane domain 5) to revert R368T was observed. Taken with recent structural studies of other major facilitator superfamily proteins, the results suggest that R87 and R368 are involved in substrate binding and probably located in a region of the protein close to N459.

Anion Transport Proteins↗
PubMed2007

Oncogenic KRAS provides a uniquely powerful and variable oncogenic contribution among RAS family members in the colonic epithelium.

Activating mutations of the RAS family of small GTPases are among the most common genetic events in human tumorigenesis. Constitutive activation of the three canonical family members, KRAS, NRAS, and HRAS segregate strongly by tissue type. Of these, KRAS mutations predominate in human tumors, including those arising from the colon and lung. We sought to compare the oncogenic contributions of different RAS isoforms in a comparable genetic setting and to explore downstream molecular changes that may explain the apparent differential oncogenic effects of the various RAS family members. We utilized colorectal cancer cell lines characterized by oncogenic KRAS in parallel with isogenically derived lines in which the mutant allele has been disrupted. We additionally attempted to reconstitute the isogenic derivatives with oncogenic forms of other RAS family members and analyze them in parallel. Pairwise analysis of HCT 116 and DLD-1 cell lines as well as their isogenic derivatives reveals distinct K-RAS(G13D) signatures despite the genetic similarities of these cell lines. In DLD-1, for example, oncogenic K-RAS enhances the motility of these cells by downregulation of Rap1 activity, yet is not associated with increased ERK1/2 phosphorylation. In HCT 116, however, ERK1/2 phosphorylation is elevated relative to the isogenic derivative, but Rap1 activity is unchanged. K-RAS is uniquely oncogenic in the colonic epithelium, though the molecular aspects of its oncogenic contribution are not necessarily conserved across cell lines. We therefore conclude that the oncogenic contribution of K-RAS is a function of its multifaceted functionality and is highly context-dependent.

Cell Line, Tumor↗
PubMed1995

Establishment and characterization of a cell line (CUMC-3) derived from a human squamous carcinoma of the uterine cervix.

A new cell line, CUMC-3, has been derived from an invasive nonkeratinizing squamous cell carcinoma of the uterine cervix in a 32-year-old patient. It has been maintained in long-term culture for 59 months, and passaged over 310 times. Monolayer-cultured cells were polygonal in shape, showing a pavement-like arrangement and a tendency to pile up without contact inhibition. The epithelial nature of the cultured CUMC-3 cells was also confirmed by transmission electron microscopy which demonstrated the presence of desmosomes and tonofilaments. The cells were highly tumorigenic in nude mice and the transplanted tumors were poorly differentiated squamous carcinoma which closely resembled the original tumor. Cultured cells obtained from the CUMC-3-derived nude mouse tumor, CUMC-3N, also were studied for its characterization. Repeated chromosome analysis revealed a stable clone with the modal chromosome number of 78. The metaphase of this cell line had multiple structural aberrations of chromosomes 1, 3, 8, 10, 11, 20, and X and showed several markers of unknown origin. The results of isozyme analyses were distinct from the HeLa cell line. The identical genetic signature was demonstrated both in CUMC-3 and in CUMC-3N cells. Cultured CUMC-3 cells produced human chorionic gonadotropin beta-subunit and tumor antigen of squamous cell carcinoma (TA-4). Cytosol estrogen receptors were found in this cell line but progesterone receptors were not measured. HLA typing of CUMC-3 cells indicated the presence of DR4, DR8, DQw3, and DQw6. The result of oncogene analysis using Southern blotting technique revealed no amplification of oncogene c-myc. Analysis of the DNA samples extracted from the CUMC-3 cells showed the presence of human papillomavirus type 16 DNA. Using the single-strand conformation polymorphism technique, we have screened CUMC-3 cells for p53 mutation in exons 4 to 9. No mobility shift was observed in this cell line. This cell line may be useful in studying the in vitro and in vivo properties of human cervical carcinoma.

Adult↗
PubMed2005

Mechanism of nonhomologous end-joining in mycobacteria: a low-fidelity repair system driven by Ku, ligase D and ligase C.

DNA double-strand breaks (DSBs) can be repaired either via homologous recombination (HR) or nonhomologous end-joining (NHEJ). Both pathways are operative in eukaryotes, but bacteria had been thought to rely on HR alone. Here we provide direct evidence that mycobacteria have a robust NHEJ pathway that requires Ku and a specialized polyfunctional ATP-dependent DNA ligase (LigD). NHEJ of blunt-end and complementary 5'-overhang DSBs is highly mutagenic ( approximately 50% error rate). Analysis of the recombination junctions ensuing from individual NHEJ events highlighted the participation of several DNA end-remodeling activities, including template-dependent fill-in of 5' overhangs, nontemplated addition of single nucleotides at blunt ends, and nucleolytic resection. LigD itself has the template-dependent and template-independent polymerase functions in vitro that compose the molecular signatures of NHEJ in vivo. Another ATP-dependent DNA ligase (LigC) provides a backup mechanism for LigD-independent error-prone repair of blunt-end DSBs. We speculate that NHEJ allows mycobacteria to evade genotoxic host defense.

Antigens, Nuclear↗