Examination of Campylobacter jejuni Putative Adhesins Leads to the Identification of a New Protein, Designated FlpA, Required for Chicken Colonization

ABSTRACT Campylobacter jejuni colonization of chickens is presumably dependent upon multiple surface-exposed proteins termed adhesins. Putative C. jejuni adhesins include CadF, CapA, JlpA, major outer membrane protein, PEB1, Cj1279c, and Cj1349c. We examined the genetic relatedness of 97 C. jejuni isolates recovered from human, poultry, bovine, porcine, ovine, and canine sources by multilocus sequence typing (MLST) and examined their profile of putative adhesin-encoding genes by dot blot hybridization. To assess the individual contribution of each protein in bacterium-host cell adherence, the C. jejuni genes encoding the putative adhesins were disrupted by insertional mutagenesis. The phenotype of each mutant was judged by performing in vitro cell adherence assays with chicken LMH hepatocellular carcinoma epithelial cells and in vivo colonization assays with broiler chicks. MLST analysis indicated that the C. jejuni isolates utilized in this study were genetically diverse. Dot blot hybridization revealed that the C. jejuni genes encoding the putative adhesins, with the exception of capA, were conserved among the isolates. The C. jejuni CadF, CapA, Cj1279c, and Cj1349c proteins were found to play a significant role in the bacterium's in vitro adherence to chicken epithelial cells, while CadF, PEB1, and Cj1279c were determined to play a significant role in the bacterium's in vivo colonization of broiler chicks. Collectively, the data indicate that Cj1279c is a novel adhesin. Because Cj1279c harbors fibronectin type III domains, we designated the protein FlpA, for fibronectin-like protein A.

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Detection of herpes simplex virus and adenovirus DNA by dot blot hybridization using in vitro synthesized RNA transcripts

Journal of Virological Methods ◽

10.1016/0166-0934(86)90054-6 ◽

1986 ◽

Vol 13 (4) ◽

pp. 291-299 ◽

Cited By ~ 9

Author(s):

Volker Schuster ◽

Bertfried Matz ◽

Helga Wiegand ◽

Brigitte Traub ◽

Dieter Neumann-Haefelin

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Blot Hybridization ◽

Dot Blot ◽

Rna Transcripts ◽

Dot Blot Hybridization ◽

Simplex Virus

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Attenuation of late simian virus 40 mRNA synthesis is enhanced by the agnoprotein and is temporally regulated in isolated nuclear systems.

Molecular and Cellular Biology ◽

10.1128/mcb.5.6.1327 ◽

1985 ◽

Vol 5 (6) ◽

pp. 1327-1334 ◽

Cited By ~ 17

Author(s):

N Hay ◽

Y Aloni

Keyword(s):

Gel Electrophoresis ◽

Blot Hybridization ◽

Simian Virus 40 ◽

Simian Virus ◽

Wild Type Virus ◽

Insertion Mutant ◽

Dot Blot ◽

Nuclear Systems

Studies were performed to verify the physiological significance of attenuation in the life cycle of simian virus 40 and the role of agnoprotein in this process. For these purposes, nuclei were isolated at various times after infection and incubated in vitro in the presence of [alpha-32P]UTP under the standard conditions which lead to attenuation. Attenuation was evident by the production of a 94-nucleotide attenuator RNA, revealed by gel electrophoresis. In parallel, the synthesis of agnoprotein was studied at various times after infection by labeling the cells for 3 h with [14C]arginine, lysing them, and analyzing the labeled proteins by gel electrophoresis. Both attenuation and the synthesis of agnoprotein were predominant towards the end of the infectious cycle. At earlier times, there was almost no attenuation and no synthesis of agnoprotein. Moreover, there was almost no attenuation even at the latest times after infection in nuclei isolated from cells infected with simian virus 40 deletion mutants that do not synthesize agnoprotein. Finally, analysis by dot blot hybridization showed higher amounts of cytoplasmic viral RNA in cells infected with an agnoprotein gene insertion mutant, delta 79, that does not produce agnoprotein, compared with cells infected with wild-type virus. The present studies indicate that attenuation is temporally regulated and suggest that agnoprotein enhances attenuation in isolated nuclei and that may also enhance it in vivo.

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A study of the messenger RNA encoding pyruvate kinase of Neurospora crassa

Bioscience Reports ◽

10.1007/bf01115007 ◽

1986 ◽

Vol 6 (2) ◽

pp. 201-208

Author(s):

M. Devchand ◽

M. Kapoor

Keyword(s):

Neurospora Crassa ◽

Pyruvate Kinase ◽

Messenger Rna ◽

Blot Hybridization ◽

Dot Blot ◽

Kinase Gene ◽

S1 Nuclease ◽

Coding Regions

In Neurospora crassa, there is a single pyruvate kinase (PK) consisting of four identical subunits of ∼60k daltons. Northern and dot blot hybridization studies, using most of the yeast pyruvate kinase gene as a probe, suggest the presence of two distinct mRNA species for pyruvate kinase, separable on the basis of the length of their polyadenylated tails, by oligo(dT)cellulose chromatography. These messages are present in polysomes, immuno-precipitated by anti-PK antibodies, indicating probable translation in vivo. Fractions containing both messages were translated in vitro in the heterologous systems as well as in a homologous N. crassa lysate, the newly-synthesized PK being detected by immunoadsorption. Protection studies using S1-nuclease suggest no major structural differences in the 5′-untranslated and most of the coding regions of the two messages.

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Transcription of the mouse ribosomal spacer region

Molecular and Cellular Biology ◽

10.1128/mcb.4.5.822-828.1984 ◽

1984 ◽

Vol 4 (5) ◽

pp. 822-828

Author(s):

K M Wood ◽

L H Bowman ◽

E A Thompson

Keyword(s):

Rna Polymerase ◽

Dna Sequences ◽

Blot Hybridization ◽

Rna Polymerase I ◽

Dot Blot ◽

Intact Cells ◽

Nuclease Mapping ◽

Polymerase I

This paper describes experiments designed to test the hypothesis that DNA sequences upstream from the mouse rRNA promoter are transcribed in vivo or in vitro. Plasmid pB28 contains a SalI restriction fragment that extends from -169 to -1,894 base pairs, with respect to the origin of transcription of pre-rRNA. Labeled RNA synthesized in intact cells does not hybridize to this region. Neither S1 nuclease mapping nor RNA dot blot hybridization revealed the presence of sequences complementary to this region. Transcriptional studies carried out in vitro indicated that this region is not transcribed under conditions that are optimal for utilization of the authentic rRNA promoter. Moreover, this region does not appear to form stable transcription complexes with RNA polymerase I transcription components. These data indicate that the mouse rDNA repeating unit differs from those of Xenopus spp. and Drosophila melanogaster in that reduplicated RNA polymerase I promoters are not found in the mouse rDNA spacer region.

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Detection of genes encoding aminoglycoside-modifying enzymes in staphylococci by polymerase chain reaction and dot blot hybridization

International Journal of Antimicrobial Agents ◽

10.1016/s0924-8579(99)00124-7 ◽

2000 ◽

Vol 13 (4) ◽

pp. 273-279 ◽

Cited By ~ 30

Author(s):

E.E Udo ◽

A.A Dashti

Keyword(s):

Polymerase Chain Reaction ◽

Blot Hybridization ◽

Dot Blot ◽

Chain Reaction ◽

Dot Blot Hybridization ◽

Genes Encoding ◽

Polymerase Chain

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Transcription of the mouse ribosomal spacer region.

Molecular and Cellular Biology ◽

10.1128/mcb.4.5.822 ◽

1984 ◽

Vol 4 (5) ◽

pp. 822-828 ◽

Cited By ~ 9

Author(s):

K M Wood ◽

L H Bowman ◽

E A Thompson

Keyword(s):

Rna Polymerase ◽

Dna Sequences ◽

Blot Hybridization ◽

Rna Polymerase I ◽

Dot Blot ◽

Intact Cells ◽

Nuclease Mapping ◽

Polymerase I

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RT-PCR and Dot Blot hybridization methods for a universal detection of tospoviruses

Fitopatologia Brasileira ◽

10.1590/s0100-41582001000200009 ◽

2001 ◽

Vol 26 (2) ◽

pp. 170-175 ◽

Cited By ~ 21

Author(s):

MARCELO EIRAS ◽

RENATO O. RESENDE ◽

ALEXANDRE A. MISSIAGGIA ◽

ANTÔNIO C. DE ÁVILA

Keyword(s):

Blot Hybridization ◽

Impatiens Necrotic Spot Virus ◽

Virus Species ◽

Rt Pcr ◽

Dot Blot ◽

Spot Virus ◽

Dot Blot Hybridization ◽

Tospovirus Species ◽

Universal Detection

Transcriptase reverse - polymerase chain reaction (RT-PCR) and dot blot hybridization with digoxigenin-labeled probes were applied for the universal detection of Tospovirus species. The virus species tested were Tomato spotted wilt virus, Tomato chlorotic spot virus, Groundnut ringspot virus, Chrysanthemum stem necrosis virus, Impatiens necrotic spot virus, Zucchini lethal chlorosis virus, Iris yellow spot virus. Primers for PCR amplification were designed to match conserved regions of the tospovirus genome. RT-PCR using distinct primer combinations was unable to simultaneously amplify all tospovirus species and consistently failed to detect ZLCV and IYSV in total RNA extracts. However, all tospovirus species were detected by RT-PCR when viral RNA was used as template. RNA-specific PCR products were used as probes for dot hybridization. This assay with a M probe (directed to the G1/G2 gene) detected at low stringency conditions all Tospovirus species, except IYSV. At low stringency conditions, the L non-radioactive probe detected the seven Tospovirus species in a single assay. This method for broad spectrum detection can be potentially employed in quarantine services for indexing in vitro germplasm.

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Expression of larval jelly antimicrobial peptide defensin1 in Apis mellifera colonies

Biologia ◽

10.2478/s11756-011-0153-8 ◽

2012 ◽

Vol 67 (1) ◽

Cited By ~ 14

Author(s):

Jaroslav Klaudiny ◽

Katarína Bachanová ◽

Lenka Kohútová ◽

Mária Dzúrová ◽

Ján Kopernický ◽

...

Keyword(s):

Apis Mellifera ◽

Antimicrobial Peptide ◽

Blot Hybridization ◽

Mrna Levels ◽

Dot Blot ◽

Recombinant Peptide ◽

Defense Function ◽

Made In

AbstractHoneybee brood food, larval jelly (LJ) contains antimicrobial peptide defensin1 that is able to inhibit in vitro growth of the pathogen causing American foulbrood (AFB). This fact suggests that LJ defensin1 could participate in defense of colonies against AFB. We assume that the potential defense function of defensin1 in vivo might depend on its amount in LJs. Therefore, we investigated the expression of defensin1 in colonies. The expression was examined on protein and mRNA levels in colonies of several Apis mellifera carnica lines collected in 3 apiaries (1 infected with AFB) with the aim to identify factors influencing the expression. Levels of defensin1 were determined in royal and worker jellies by a developed immunoblot procedure employing antibodies generated against the recombinant peptide. Defensin1 mRNA levels in nurse heads were explored by dot blot hybridization using transcript of two MRJP genes for normalization. Analyzed LJs contained various amounts of defensin1 (0.159–0.524 μg/mg jelly). Higher variations in defensin1 levels were observed among LJ samples collected from different colonies than among those collected within single colony. Colonies producing LJs with elevated defensin1 levels occurred among various honeybee lines. Levels of defensin1 mRNA varied in heads of nurses and the variations correlated with defensin1 peptide levels in LJs only in some colonies. Obtained data demonstrate that defensin1 is constitutively expressed into LJs in colonies and indicate that its levels in jellies are determined by genetic factors regulating transcription and/or translation/posttranslation processes in nurses. AFB infection, larval age and type of LJ do not seem to affect the levels of the peptide in LJs. Findings made in this work suggest that it should be possible to breed novel honeybee lines expressing higher amounts of defensin1 into LJs.

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Transposon Tn5281 is the main distributor of the aminoglycoside modifying enzyme gene among isolates of Enterococcus faecalis in Tehran hospitals

Canadian Journal of Microbiology ◽

10.1139/w08-073 ◽

2008 ◽

Vol 54 (10) ◽

pp. 887-890 ◽

Cited By ~ 6

Author(s):

Mohammad Mehdi Feizabadi ◽

Leila Shokrzadeh ◽

Sara Sayady ◽

Soroor Asadi

Keyword(s):

Enterococcus Faecalis ◽

High Frequency ◽

Blot Hybridization ◽

Dot Blot ◽

Enzyme Gene ◽

Long Pcr ◽

Dot Blot Hybridization ◽

Genes Encoding ◽

N 93 ◽

Pcr Targeting

Infections with high levels of gentamicin-resistant (HLGR) isolates of Enterococcus faecalis are common in Tehran hospitals. Genes encoding such resistance are transmissible by conjugation at high frequency. The purpose of this study was to determine the existence of Tn5281 and its flanking aminoglycoside modifying enzyme gene aac(6′)-aph(2″) among 102 HLGR isolates of E. faecalis cultured from patients at three hospitals in Tehran, Iran. These isolates were detected by disks containing 120 μg of gentamicin and made 65% of all E. faecalis during the study period. DNA was extracted from HLGR isolates and subjected to PCR assays targeting aac(6′)-aph(2″) and conjugative transposon Tn5281. The amplified aac(6′)-aph(2″) gene was labeled with digoxigenin and probed with Tn5281 amplicons in dot blot hybridization assays. The aac(6′)-aph(2″) gene was detected in 91%–92% (n = 93) of the HLGR isolates. All isolates containing aac(6′)-aph(2″) were positive in long-PCR targeting Tn5281 and the probe hybridized with Tn5281 amplicons. The number of HLGR isolates of E. faecalis has increased considerably in Tehran hospitals. Tn5281 is the main cause of transmission of aac(6′)-aph(2″) to different isolates of E. faecalis in the hospitals studied.

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