Evidence for chimeric sequences formed during random arbitrarily primed PCR

2003 ◽  
Vol 54 (3) ◽  
pp. 427-431 ◽  
Author(s):  
In Seop Chang ◽  
Jimmy D. Ballard ◽  
Lee R. Krumholz
Keyword(s):  
Chimeric Sequences ◽  
Arbitrarily Primed Pcr

scholarly journals FlrA, a σ54-Dependent Transcriptional Activator in Vibrio fischeri, Is Required for Motility and Symbiotic Light-Organ Colonization

2003 ◽  
Vol 185 (12) ◽  
pp. 3547-3557 ◽  
Author(s):  
Deborah S. Millikan ◽  
Edward G. Ruby
Keyword(s):  
Vibrio Fischeri ◽  
Transcriptional Activator ◽  
Pcr Analysis ◽  
Euprymna Scolopes ◽  
Wild Type ◽  
Flagellar Regulon ◽  
Colonization Factors ◽  
Recognition Motifs ◽  
Arbitrarily Primed Pcr ◽  
Organ Colonization

ABSTRACT Flagellum-mediated motility of Vibrio fischeri is an essential factor in the bacterium's ability to colonize its host, the Hawaiian squid Euprymna scolopes. To begin characterizing the nature of the flagellar regulon, we have cloned a gene, designated flrA, from V. fischeri that encodes a putative σ54-dependent transcriptional activator. Genetic arrangement of the flrA locus in V. fischeri is similar to motility master-regulator operons of Vibrio cholerae and Vibrio parahaemolyticus. In addition, examination of regulatory regions of a number of flagellar operons in V. fischeri revealed apparent σ54 recognition motifs, suggesting that the flagellar regulatory hierarchy is controlled by a similar mechanism to that described in V. cholerae. However, in contrast to its closest known relatives, flrA mutant strains of V. fischeri ES114 were completely abolished in swimming capability. Although flrA provided in trans restored motility to the flrA mutant, the complemented strain was unable to reach wild-type levels of symbiotic colonization in juvenile squid, suggesting a possible role for the proper expression of FlrA in regulating symbiotic colonization factors in addition to those required for motility. Comparative RNA arbitrarily primed PCR analysis of the flrA mutant and its wild-type parent revealed several differentially expressed transcripts. These results define a regulon that includes both flagellar structural genes and other genes apparently not involved in flagellum elaboration or function. Thus, the transcriptional activator FlrA plays an essential role in regulating motility, and apparently in modulating other symbiotic functions, in V. fischeri.

Clonal similarity of salivary and nasopharyngeal Fusobacterium nucleatum in infants with acute otitis media experience

2004 ◽  
Vol 53 (2) ◽  
pp. 161-165 ◽  
Author(s):  
Gunnsteinn Haraldsson ◽  
W. Peter Holbrook ◽  
Eija Könönen
Keyword(s):  
Oral Cavity ◽  
Otitis Media ◽  
Anaerobic Bacteria ◽  
Acute Otitis Media ◽  
Fusobacterium Nucleatum ◽  
Potential Source ◽  
History Of ◽  
Arbitrarily Primed Pcr ◽  
Inoculation Route ◽  
Media Experience

The environment of an infant's nasopharynx during acute otitis media (AOM) favours the growth of anaerobic bacteria, which can be recovered frequently during infection, but hardly at all if the infant is healthy. The aim of this investigation was to identify the potential source and inoculation route of anaerobes that were present in the nasopharynx. Eleven Fusobacterium nucleatum isolates that were collected through the nasal cavity from the nasopharynx of eight infants with a history of AOM, and 161 F. nucleatum isolates from the saliva of the same infants, were typed to the clonal level by using arbitrarily primed PCR (AP-PCR). In five of the eight infants examined, identical AP-PCR types were found among nasopharyngeal and salivary isolates. As anaerobes seem to be present only transiently in the nasopharynx and salivary contamination of the nasopharyngeal samples can be excluded, this observation indicates that the source of nasopharyngeal anaerobes is the oral cavity and that saliva is their transmission vehicle.

scholarly journals Fetal and Neonatal Exposure to the Endocrine Disruptor Methoxychlor Causes Epigenetic Alterations in Adult Ovarian Genes

Endocrinology ◽  
2009 ◽  
Vol 150 (10) ◽  
pp. 4681-4691 ◽  
Author(s):  
Aparna Mahakali Zama ◽  
Mehmet Uzumcu
Keyword(s):  
Dna Methylation ◽  
Dna Methyltransferase ◽  
Ovarian Function ◽  
Endocrine Disrupting Chemicals ◽  
Neonatal Exposure ◽  
Dna Hypermethylation ◽  
Promoter Regions ◽  
Altered Expression ◽  
Arbitrarily Primed Pcr ◽  
Methylation Patterns

Abstract Exposure to endocrine-disrupting chemicals during development could alter the epigenetic programming of the genome and result in adult-onset disease. Methoxychlor (MXC) and its metabolites possess estrogenic, antiestrogenic, and antiandrogenic activities. Previous studies showed that fetal/neonatal exposure to MXC caused adult ovarian dysfunction due to altered expression of key ovarian genes including estrogen receptor (ER)-β, which was down-regulated, whereas ERα was unaffected. The objective of the current study was to evaluate changes in global and gene-specific methylation patterns in adult ovaries associated with the observed defects. Rats were exposed to MXC (20 μg/kg·d or 100 mg/kg·d) between embryonic d 19 and postnatal d 7. We performed DNA methylation analysis of the known promoters of ERα and ERβ genes in postnatal d 50–60 ovaries using bisulfite sequencing and methylation-specific PCRs. Developmental exposure to MXC led to significant hypermethylation in the ERβ promoter regions (P < 0.05), whereas the ERα promoter was unaffected. We assessed global DNA methylation changes using methylation-sensitive arbitrarily primed PCR and identified 10 genes that were hypermethylated in ovaries from exposed rats. To determine whether the MXC-induced methylation changes were associated with increased DNA methyltransferase (DNMT) levels, we measured the expression levels of Dnmt3a, Dnmt3b, and Dnmt3l using semiquantitative RT-PCR. Whereas Dnmt3a and Dnmt3l were unchanged, Dnmt3b expression was stimulated in ovaries of the 100 mg/kg MXC group (P < 0.05), suggesting that increased DNMT3B may cause DNA hypermethylation in the ovary. Overall, these data suggest that transient exposure to MXC during fetal and neonatal development affects adult ovarian function via altered methylation patterns.

Arbitrarily Primed-PCR and Related DNA Methylation Methodologies

2004 ◽  
pp. 85-93
Author(s):  
Miguel Peinado ◽  
Jordi Frigola ◽  
Maria Paz ◽  
Manel Esteller
Keyword(s):  
Dna Methylation ◽  
Arbitrarily Primed Pcr

scholarly journals Dissemination of Two Methicillin-Resistant Staphylococcus aureus Clones Exhibiting Negative Staphylase Reactions in Intensive Care Units

1999 ◽  
Vol 37 (3) ◽  
pp. 504-509 ◽  
Author(s):  
Po-Ren Hsueh ◽  
Lee-Jene Teng ◽  
Pan-Chyr Yang ◽  
Hui-Ju Pan ◽  
Yu-Chi Chen ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Intensive Care ◽  
Intensive Care Units ◽  
Random Amplified Polymorphic Dna ◽  
Cellular Fatty Acid ◽  
University Hospital ◽  
Methicillin Resistant ◽  
Type Iv ◽  
Routine Identification ◽  
Arbitrarily Primed Pcr

From December 1997 to March 1998, 25 methicillin-resistantStaphylococcus aureus (MRSA) isolates exhibiting negative Staphylase (Oxoid Ltd., Basingstoke, England) reactions were identified from various clinical specimens from 13 patients in six intensive care units (ICUs) or in wards following a stay in an ICU at the National Taiwan University Hospital. The characteristics of these isolates have not been previously noted in other MRSA isolates from this hospital. Colonies of all these isolates were grown on Trypticase soy agar supplemented with 5% sheep blood and were nonhemolytic and unpigmented. Seven isolates, initially reported as Staphylococcus haemolyticus (5 isolates) and Staphylococcus epidermidis (2 isolates) by the routine identification scheme and with the Vitek GPI system (bioMerieux Vitek, Inc., Hazelwood, Mo.), were subsequently identified as S. aureus by positive tube coagulase tests, standard biochemical reactions, and characteristic cellular fatty acid chromatograms. The antibiotypes obtained by the E test, coagulase types, restriction fragment length polymorphism profiles of the staphylococcal coagulase gene, and random amplified polymorphic DNA patterns generated by arbitrarily primed PCR of the isolates disclosed that two major clones disseminated in the ICUs. Clone 1 (16 isolates) was resistant to clindamycin and was susceptible to trimethoprim-sulfamethoxazole (TMP-SMZ) and was coagulase type II. Clone 2 (eight isolates) was resistant to clindamycin and TMP-SMZ and was coagulase type IV. These two epidemic clones from ICUs are unique and underline the need for caution in identifying MRSA strains with colonial morphologies not of the typical type and with negative Staphylase reactions.

Comparison of Restriction Enzyme Analysis, Arbitrarily Primed PCR, and Protein Profile Analysis Typing for Epidemiologic Investigation of an Ongoing Clostridium difficileOutbreak

1998 ◽  
Vol 36 (10) ◽  
pp. 2957-2963 ◽  
Author(s):  
Mary Ellen Rafferty ◽  
Aldona L. Baltch ◽  
Raymond P. Smith ◽  
Lawrence H. Bopp ◽  
Carol Rheal ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
General Hospital ◽  
Restriction Enzyme ◽  
Profile Analysis ◽  
Protein Profile ◽  
Enzyme Analysis ◽  
Restriction Enzyme Analysis ◽  
Typing Methods ◽  
Arbitrarily Primed Pcr ◽  
Predominant Strain

During an outbreak of diarrhea in a general hospital in 1992, 166Clostridium difficile isolates from 102 patients were typed by restriction enzyme analysis (REA), arbitrarily primed PCR (AP-PCR), and protein profile analysis (PP) techniques. A total of 18 types and 5 subtypes were identified by REA, 32 types were identified by AP-PCR, and 9 types were identified by PP. Analysis of the data indicated the presence of a predominant strain among 76, 75, and 84% of the isolates by REA, AP-PCR, and PP, respectively. Subsequently, 45C. difficile isolates which had been collected in 1990 from 33 patients in the same hospital following a significant increase in the number of cases of diarrhea caused by C. difficile were studied by REA, AP-PCR, and PP typing techniques. Thirteen types and one subtype were identified by REA, 12 types were identified by AP-PCR, and 5 types were identified by PP. As with the isolates from 1992, a dominant strain was identified. This strain was represented by 53, 64, and 70% of the total number of isolates when the strains were typed by REA, AP-PCR, and PP, respectively. Every isolate (210 of 211) from both 1990 and 1992 that was available for typing was typeable by all three methods. Furthermore, the same dominant strain was identified in both 1990 and 1992 by each method. This study demonstrates that each of the three typing methods can be useful in epidemiologic investigations of C. difficileoutbreaks and that one strain can be dominant in an institution over a number of years.

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