scholarly journals Identification of a Cytolethal Distending Toxin Gene Locus and Features of a Virulence-Associated Region inActinobacillus actinomycetemcomitans

1999 ◽  
Vol 67 (3) ◽  
pp. 1227-1237 ◽  
Author(s):  
Marcia P. A. Mayer ◽  
Lina C. Bueno ◽  
Eric J. Hansen ◽  
Joseph M. DiRienzo
Keyword(s):  
Dna Sequences ◽  
Recombinant Strain ◽  
Integration Site ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Amino Acid Sequence Similarity ◽  
Toxin Gene ◽  
Virulence Plasmid ◽  
Cytolethal Distending Toxin ◽  
Oral Pathogen

ABSTRACT A genetic locus for a cytolethal distending toxin (CDT) was identified in a polymorphic region of the chromosome ofActinobacillus actinomycetemcomitans, a predominant oral pathogen. The locus was comprised of three open reading frames (ORFs) that had significant amino acid sequence similarity and more than 90% sequence identity to the cdtABC genes of some pathogenicEscherichia coli strains and Haemophilus ducreyi, respectively. Sonic extracts from recombinant E. coli, containing the A. actinomycetemcomitans ORFs, caused the distension and killing of Chinese hamster ovary cells characteristic of a CDT. Monoclonal antibodies made reactive with the CdtA, CdtB, and CdtC proteins of H. ducreyi recognized the corresponding gene products from the recombinant strain. CDT-like activities were no longer expressed by the recombinant strain when an ΩKan-2 interposon was inserted into the cdtA andcdtB genes. Expression of the CDT-like activities inA. actinomycetemcomitans was strain specific. Naturally occurring expression-negative strains had large deletions within the region of the cdt locus. The cdtABC genes were flanked by an ORF (virulence plasmid protein), a partial ORF (integrase), and DNA sequences (bacteriophage integration site) characteristic of virulence-associated regions. These results provide evidence for a functional CDT in a human oral pathogen.

scholarly journals Possible Involvement of Organic Anion Transporting Polypeptide 1c1 in the Photoperiodic Response of Gonads in Birds

Endocrinology ◽  
2006 ◽  
Vol 147 (3) ◽  
pp. 1067-1073 ◽  
Author(s):  
Nobuhiro Nakao ◽  
Tsuyoshi Takagi ◽  
Masayuki Iigo ◽  
Toshiro Tsukamoto ◽  
Shinobu Yasuo ◽  
...  
Keyword(s):  
Thyroid Hormones ◽  
Dna Sequences ◽  
Chinese Hamster Ovary Cells ◽  
Organic Anion ◽  
Chinese Hamster ◽  
Functional Expression ◽  
Photoperiodic Response ◽  
Target Cells ◽  
Iodothyronine Deiodinase ◽  
Ovary Cells

The photoperiodic response of the gonads requires T3, which is generated photoperiodically from T4 by type 2 iodothyronine deiodinase in the hypothalamus. Although thyroid hormones were long thought to traverse the plasma membrane by passive diffusion due to their lipophilic nature, it is now known that several organic anion transporting polypeptides (Oatp) transport thyroid hormones into target cells. In this study, we have used database searches to isolate DNA sequences encoding members of the chicken Oatp family and constructed a molecular phylogenetic tree. Comprehensive expression analyses using in situ hybridization revealed strong expression of cOatp1c1 and weak expression of cOatp1b1 in the ventro-lateral walls of basal tuberal hypothalamus, whereas expression of four genes (cOatp1a1, cOatp1b1, cOatp1c1, and cOatp3a2) was observed in the choroid plexus. Expression levels of all these genes in both regions were not different between short-day and long-day conditions. Functional expression of cOatp1c1 in Chinese hamster ovary cells revealed that cOatp1c1 is a highly specific transporter for T4 with an apparent Km of 6.8 nm and a Vmax of 1.50 pmol per milligram of protein per minute. These results suggest that cOatp1c1 could be involved in the thyroxine transport necessary for the avian photoperiodic response of the gonads.

Complementation of a methotrexate uptake defect in Chinese hamster ovary cells by DNA-mediated gene transfer

1989 ◽  
Vol 9 (4) ◽  
pp. 1754-1758
Author(s):  
T M Underhill ◽  
W F Flintoff
Keyword(s):  
Gene Transfer ◽  
Dna Sequences ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Wild Type ◽  
Ovary Cells ◽  
Ovary Cell Line ◽  
Human Specific

A methotrexate-resistant Chinese hamster ovary cell line deficient in methotrexate uptake has been complemented to methotrexate sensitivity by transfection with DNA isolated from either wild-type Chinese hamster ovary or human G2 cells. Primary and secondary transfectants regained the ability to take up methotrexate in a manner similar to that of wild-type cells, and in the case of those transfected with human DNA, to contain human-specific DNA sequences. The complementation by DNA-mediated gene transfer of this methotrexate-resistant phenotype provides a basis for the cloning of a gene involved in methotrexate uptake.

Structural alterations of the aprt locus induced by deoxyribonucleoside triphosphate pool imbalances in Chinese hamster ovary cells

1984 ◽  
Vol 4 (9) ◽  
pp. 1792-1799
Author(s):  
O Goncalves ◽  
E Drobetsky ◽  
M Meuth
Keyword(s):  
Dna Sequences ◽  
Chinese Hamster Ovary Cells ◽  
Mutant Cell ◽  
Chinese Hamster ◽  
Small Region ◽  
Base Pairs ◽  
Ovary Cells ◽  
Mutator Strain ◽  
Aprt Gene ◽  
Deoxyribonucleoside Triphosphate

Mutants induced at the adenine phosphoribosyl transferase (aprt) locus by dTTP or dCTP pool imbalances were examined for alterations in genomic DNA sequences. No observable changes were detected by Southern blot analysis of most mutant DNAs, suggesting induction of base pair alterations or other events below our level of detection (approximately 30 base pairs). However, in a few strains (11 from a total collection of 125 mutant cell strains), we were able to localize these events to restriction endonuclease recognition sequences when the mutations resulted in the loss or gain of a particular site. The distribution of lost or gained sites in aprt-deficient mutants induced by the two types of pool imbalances clearly varied, with those occurring in a mutator strain with increased dCTP clustering at one end of the aprt gene. Mutants induced by dTTP also revealed novel events: multiple restriction site modifications in a small region of the aprt gene in one mutant and a small (approximately 50 base pairs) insertion or duplication of DNA sequences. As in previous studies, very few deletion or insertion mutants were detected at the aprt locus. The significance of these findings in terms of the known biochemical and genetic consequences of these pool imbalances is discussed.

Inducible expression of amplified human beta interferon genes in CHO cells

1984 ◽  
Vol 4 (1) ◽  
pp. 166-172
Author(s):  
F McCormick ◽  
M Trahey ◽  
M Innis ◽  
B Dieckmann ◽  
G Ringold
Keyword(s):  
Dihydrofolate Reductase ◽  
Dna Sequences ◽  
Chinese Hamster Ovary Cells ◽  
Acquired Resistance ◽  
Chinese Hamster ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Cellular Growth ◽  
Beta Interferon ◽  
Beta Gene

Plasmid DNA containing the human beta-interferon (IFN-beta) gene and mouse dihydrofolate reductase cDNA was transfected into dihydrofolate reductase-negative Chinese hamster ovary cells. Dihydrofolate reductase-positive transformants were obtained, and cells containing amplified copies of mouse dihydrofolate reductase were selected by exposure to increasing methotrexate concentrations. These cells were found to express high levels of human IFN-beta after polyriboinosinic acid-polyribocytidylic acid superinduction or NDV infection; this was a result of coamplification of the IFN-beta gene. Levels of expression of 1 U/cell per day were achieved on superinduction, giving corresponding titers of up to 10(10) U/liter medium in culture supernatants. Constitutive production of IFN-beta rates of about 0.5% of superinduced rates was observed; cells producing these levels of IFN-beta had acquired resistance to cytotoxic antiviral effects of IFN-beta. Two forms of human IFN-beta were produced; a major glycosylated 23,000-dalton form and an unglycosylated 18,500-dalton form. The latter had greatly reduced antiviral activity. IFN-beta production was very sensitive to cellular growth rate; the highest levels were produced by density-arrested cultures. Regulation of IFN-beta production by polyriboinosinic acid-polyribocytidylic acid or by cell density effects required the presence of DNA sequences 5' to the IFN-beta-coding sequences; replacement of these sequences with the simian virus 40 early promoter resulted in uninducible, density-independent production of IFN-beta.

scholarly journals Structural alterations of the aprt locus induced by deoxyribonucleoside triphosphate pool imbalances in Chinese hamster ovary cells.

1984 ◽  
Vol 4 (9) ◽  
pp. 1792-1799 ◽  
Author(s):  
O Goncalves ◽  
E Drobetsky ◽  
M Meuth
Keyword(s):  
Dna Sequences ◽  
Chinese Hamster Ovary Cells ◽  
Mutant Cell ◽  
Chinese Hamster ◽  
Small Region ◽  
Base Pairs ◽  
Ovary Cells ◽  
Mutator Strain ◽  
Aprt Gene ◽  
Deoxyribonucleoside Triphosphate

Mutants induced at the adenine phosphoribosyl transferase (aprt) locus by dTTP or dCTP pool imbalances were examined for alterations in genomic DNA sequences. No observable changes were detected by Southern blot analysis of most mutant DNAs, suggesting induction of base pair alterations or other events below our level of detection (approximately 30 base pairs). However, in a few strains (11 from a total collection of 125 mutant cell strains), we were able to localize these events to restriction endonuclease recognition sequences when the mutations resulted in the loss or gain of a particular site. The distribution of lost or gained sites in aprt-deficient mutants induced by the two types of pool imbalances clearly varied, with those occurring in a mutator strain with increased dCTP clustering at one end of the aprt gene. Mutants induced by dTTP also revealed novel events: multiple restriction site modifications in a small region of the aprt gene in one mutant and a small (approximately 50 base pairs) insertion or duplication of DNA sequences. As in previous studies, very few deletion or insertion mutants were detected at the aprt locus. The significance of these findings in terms of the known biochemical and genetic consequences of these pool imbalances is discussed.

scholarly journals Demethylation enhances removal of pyrimidine dimers from the overall genome and from specific DNA sequences in Chinese hamster ovary cells.

1989 ◽  
Vol 9 (4) ◽  
pp. 1594-1603 ◽  
Author(s):  
L Ho ◽  
V A Bohr ◽  
P C Hanawalt
Keyword(s):  
Dna Repair ◽  
Dna Sequences ◽  
Chinese Hamster Ovary ◽  
Cytosine Methylation ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Dhfr Gene ◽  
Ovary Cells ◽  
Pyrimidine Dimers ◽  
Uv Dose

We have examined the effects of changes in cytosine methylation on DNA repair in UV-irradiated Chinese hamster ovary (CHO) cells. A hypomethylated derivative of the CHO K1B11 line, B11aza, was established by passaging B11 cells over several months in increasing concentrations of 5-azacytidine; greater than 60% demethylation was consistently demonstrated in these conditioned cells. Following a UV dose of 10 J/m2, the amount of repair replication performed within 24 h was approximately twofold higher in B11aza cells than in control B11 cells. Removal of T4 endonuclease V-sensitive sites (ESS) from specific restriction fragments within and around the dihydrofolate reductase (DHFR) gene was then examined in B11aza cells and compared with that in B11 cells. Although demethylation had little or no effect on repair in the 5' half of the DHFR gene, within a nontranscribed sequence immediately downstream from the gene, or within an extragenic region further downstream from the DHFR gene, significant increases in repair were observed at the 3' end of the DHFR gene and within an extragenic region upstream of the DHFR gene. However, the increases in DNA repair were not accompanied by any changes in overall cellular resistance to UV when colony-forming ability was assayed. We suggest that the level of DNA methylation may play an indirect role in the regulation of DNA repair, perhaps through an effect on chromatin structure or transcriptional activity.

scholarly journals Identification of Virulence-Associated Characteristics in Clinical Isolates of Yersinia enterocolitica Lacking Classical Virulence Markers

1998 ◽  
Vol 66 (3) ◽  
pp. 1113-1120 ◽  
Author(s):  
Travis Grant ◽  
Vicki Bennett-Wood ◽  
Roy M. Robins-Browne
Keyword(s):  
Yersinia Enterocolitica ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Electron Microscopic Examination ◽  
Clinical Isolates ◽  
Virulence Plasmid ◽  
Virulence Determinants ◽  
Electron Microscopic ◽  
Virulence Markers ◽  
Culture Cells

ABSTRACT Yersinia enterocolitica is an important enteric pathogen which has well-defined virulence determinants that allow the bacteria to become established in their hosts and overcome host defenses. A number of strains obtained from patients with diarrhea, however, lack these genes. Accordingly, the mechanisms by which they cause disease are uncertain. Most of these isolates belong to biotype 1A. Strains of this biotype are also frequently isolated from a variety of nonclinical sources, such as food, soil, water, and healthy animals, and there is evidence that some of these strains are avirulent. In this study we investigated 111 strains of Y. enterocoliticabiotype 1A, 79 from symptomatic humans and 32 from nonclinical sources, for virulence-associated characteristics. DNA hybridization studies showed that none of the strains carried sequences homologous with pYV, the ∼70-kb Yersinia virulence plasmid. Some strains hybridized with DNA probes for one of the following chromosomal virulence-associated genes: ail (7.2%), myfA(11.7%), ystA (0.9%), and ystB (85%). In addition, 33 strains (29.7%) produced an enterotoxin that was reactive in infant mice. However, the frequencies of these virulence-associated properties in clinical and nonclinical isolates were similar. Clinical isolates invaded HEp-2 cells and Chinese hamster ovary cells to a significantly greater extent than nonclinical strains (P ≤ 0.002). In addition, clinical strains colonized the intestinal tracts of perorally inoculated mice for significantly longer periods than nonclinical isolates (P ≤ 0.01). Light and electron microscopic examination of tissue culture cells incubated with invasive yersiniae revealed that the bacteria invaded selected cells in large numbers but spared others, suggesting that biotype-1A strains of Y. enterocolitica may invade cells by a novel mechanism. These results indicate that some clinical isolates of Y. enterocolitica which lack classical virulence markers may be able to cause disease via virulence mechanisms which differ from those previously characterized in enteropathogenic Yersiniaspecies.

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