Expression of recombinant DNA introduced into Chlamydia trachomatis by electroporation

1994 ◽  
Vol 40 (7) ◽  
pp. 583-591 ◽  
Author(s):  
Jeffrey E. Tam ◽  
Carolyn H. Davis ◽  
Priscilla B. Wyrick
Keyword(s):  
Chlamydia Trachomatis ◽  
Plasmid Dna ◽  
Southern Hybridization ◽  
Recombinant Dna ◽  
Chloramphenicol Acetyltransferase ◽  
Developmentally Regulated ◽  
Reticulate Body ◽  
Body Development ◽  
Coli Plasmid

Electroporation was used to introduce DNA into the elementary bodies of the obligate parasitic bacterium Chlamydia trachomatis. The source of DNA for these experiments was the chimeric plasmid pPBW100, which was constructed from the well-characterized 7.5-kb plasmid of C. trachomatis and the Escherichia coli plasmid pBGS9. To select directly for C. trachomatis carrying pPBW100, an in-frame gene fusion between the chlamydial promoter P7248 and a promoterless chloramphenicol acetyltransferase (cat) cassette was incorporated into the plasmid. After infection of McCoy cells with electroporated elementary bodies containing pPBW100, the following were observed: (i) the plasmid DNA was detected inside the chloramphenicol-resistant chlamydial inclusions by in situ and Southern hybridization analyses; (ii) both physical and biochemical evidence showed that chloramphenicol acetyltransferase was synthesized by the electroporated C. trachomatis; (iii) expression of P7248::cat was developmentally regulated and occurred during the early stages of chlamydial reticulate body development; and (iv) although the expression from P7248::cat was mainly transient, there were rare instances where chloramphenicol-resistant C. trachomatis were observed after four passages.Key words: chlamydia, electroporation, chimeric plasmid, expression.

scholarly journals The effect of penicillin on Chlamydia trachomatis DNA replication

Microbiology ◽  
2006 ◽  
Vol 152 (9) ◽  
pp. 2573-2578 ◽  
Author(s):  
Paul R. Lambden ◽  
Mark A. Pickett ◽  
Ian N. Clarke
Keyword(s):  
Dna Replication ◽  
Chlamydia Trachomatis ◽  
Plasmid Dna ◽  
Host Cells ◽  
Developmental Cycle ◽  
Chromosomal Dna ◽  
Rapid Phase ◽  
Reticulate Body ◽  
Transmission Electron ◽  
Infected Cells

Chlamydia trachomatis L2 was used to infect BGMK cells at an m.o.i. of 1.0, and the developmental cycle was followed by transmission electron microscopy and quantitative PCR (QPCR) for both chromosomal and plasmid DNA. Samples were taken at sequential 6 h time points. Subsequent analysis by QPCR showed that there was an initial slow replication period (0–18 h), followed by a rapid phase (18–36 h) coinciding with exponential division when the DNA doubling time was 4.6 h. Chromosomal DNA was amplified 100–200-fold corresponding to 7–8 generations for the complete developmental cycle. Penicillin (10 and 100 units ml−1) was added to cultures at 20 h post-infection (p.i.). This blocked binary fission and also prevented reticulate body (RB) to elementary body transition. However, exposure to penicillin did not prevent chromosomal or plasmid DNA replication. After a short lag period, following the addition of penicillin, chlamydial chromosomal DNA replication resumed at the same rate as in control C. trachomatis-infected cells. C. trachomatis-infected host cells exposed to penicillin did not lyse, but instead harboured large, aberrant RBs in massive inclusions that completely filled the cell cytoplasm. In these RBs, the DNA continued to replicate well beyond the end of the normal developmental cycle. At 60 h p.i. each aberrant RB contained a minimum of 16 chromosomal copies.

mRNA localization by Electron microscopic in situ hybridization

1991 ◽  
Vol 49 ◽  
pp. 430-431
Author(s):  
J. A. Pollock ◽  
M. Martone ◽  
T. Deerinck ◽  
M. H. Ellisman
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Nucleic Acids ◽  
Recombinant Dna ◽  
Light And Electron Microscopy ◽  
Electron Microscopic ◽  
High Voltage Electron Microscopy ◽  
Functional Sites ◽  
Voltage Electron

Localization of specific proteins in cells by both light and electron microscopy has been facilitate by the availability of antibodies that recognize unique features of these proteins. High resolution localization studies conducted over the last 25 years have allowed biologists to study the synthesis, translocation and ultimate functional sites for many important classes of proteins. Recently, recombinant DNA techniques in molecular biology have allowed the production of specific probes for localization of nucleic acids by “in situ” hybridization. The availability of these probes potentially opens a new set of questions to experimental investigation regarding the subcellular distribution of specific DNA's and RNA's. Nucleic acids have a much lower “copy number” per cell than a typical protein, ranging from one copy to perhaps several thousand. Therefore, sensitive, high resolution techniques are required. There are several reasons why Intermediate Voltage Electron Microscopy (IVEM) and High Voltage Electron Microscopy (HVEM) are most useful for localization of nucleic acids in situ.

scholarly journals Retention of relict satellite DNA sequences in Anemone (Ranunculaceae)

2013 ◽  
Vol 72 (1) ◽  
pp. 1-133 ◽  
Author(s):  
Višnja Besendorfer ◽  
Jelena Mlinarec
Keyword(s):  
Dna Sequences ◽  
Southern Hybridization ◽  
Repeat Unit ◽  
Similar Species ◽  
Genomic Component ◽  
Library Hypothesis ◽  
Species Specific ◽  
Eukaryotic Organisms ◽  
Fish Signal

Abstract Satellite DNAis a genomic component present in virtually all eukaryotic organisms. The turnover of highly repetitive satellite DNAis an important element in genome organization and evolution in plants. Here we study the presence, physical distribution and abundance of the satellite DNAfamily AhTR1 in Anemone. Twenty-two Anemone accessions were analyzed by PCR to assess the presence of AhTR1, while fluorescence in situ hybridization and Southern hybridization were used to determine the abundance and genomic distribution of AhTR1. The AhTR1 repeat unit was PCR-amplified only in eight phylogenetically related European Anemone taxa of the Anemone section. FISH signal with AhTR1 probe was visible only in A. hortensis and A. pavonina, showing localization of AhTR1 in the regions of interstitial heterochromatin in both species. The absence of a FISH signal in the six other taxa as well as weak signal after Southern hybridization suggest that in these species AhTR1 family appears as relict sequences. Thus, the data presented here support the »library hypothesis« for AhTR1 satellite evolution in Anemone. Similar species-specific satellite DNAprofiles in A. hortensis and A. pavonina support the treatment of A. hortensis and A. pavonina as one species, i.e. A. hortensis s.l.

Integration of Bacteria Capture via Filtration and in Situ Lysis for Recovery of Plasmid DNA under Industry-Compatible Conditions

2007 ◽  
Vol 23 (4) ◽  
pp. 895-903 ◽  
Author(s):  
Kevin O'Mahony ◽  
Ruth Freitag ◽  
Frank Hilbrig ◽  
Ivo Schumacher ◽  
Patrick Müller
Keyword(s):  
Plasmid Dna

scholarly journals Effects ofMentha suaveolensEssential Oil onChlamydia trachomatis

2015 ◽  
Vol 2015 ◽  
pp. 1-7 ◽  
Author(s):  
Rosa Sessa ◽  
Marisa Di Pietro ◽  
Fiorenzo De Santis ◽  
Simone Filardo ◽  
Rino Ragno ◽  
...  
Keyword(s):  
Chlamydia Trachomatis ◽  
Substantial Reduction ◽  
Developmental Cycle ◽  
Elementary Body ◽  
Promising Candidate ◽  
Common Cause ◽  
Sexually Transmitted ◽  
Reticulate Body ◽  
Preventative Strategy

Chlamydia trachomatis, the most common cause of sexually transmitted bacterial infection worldwide, has a unique biphasic developmental cycle alternating between the infectious elementary body and the replicative reticulate body.C. trachomatisis responsible for severe reproductive complications including pelvic inflammatory disease, ectopic pregnancy, and obstructive infertility. The aim of our study was to evaluate whetherMentha suaveolensessential oil (EOMS) can be considered as a promising candidate for preventingC. trachomatisinfection. Specifically, we investigated thein vitroeffects of EOMS towardsC. trachomatisanalysing the different phases of chlamydial developmental cycle. Our results demonstrated that EOMS was effective towardsC. trachomatis, whereby it not only inactivated infectious elementary bodies but also inhibited chlamydial replication. Our study also revealed the effectiveness of EOMS, in combination with erythromycin, towardsC. trachomatiswith a substantial reduction in the minimum effect dose of antibiotic. In conclusion, EOMS treatment may represent a preventative strategy since it may reduceC. trachomatistransmission in the population and, thereby, reduce the number of new chlamydial infections and risk of developing of severe sequelae.

Identification and characterization of normal length nonfluorescent Y chromosomes: cytogenetic analysis, Southern hybridization and non-isotopic in situ hybridization

1990 ◽  
Vol 85 (6) ◽  
pp. 569-575 ◽  
Author(s):  
Frank Speleman ◽  
Bart Van der Auwera ◽  
Kathelijne Mangelschots ◽  
Miet Vercruyssen ◽  
Ton Raap ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Cytogenetic Analysis ◽  
Southern Hybridization ◽  
Normal Length ◽  
Y Chromosomes ◽  
Identification And Characterization

scholarly journals Chlamydial infection and disease in the koala

2005 ◽  
Vol 26 (2) ◽  
pp. 65 ◽  
Author(s):  
Peter Timms
Keyword(s):  
Chlamydia Trachomatis ◽  
Chlamydial Infection ◽  
Developmental Cycle ◽  
Elementary Body ◽  
Obligate Intracellular ◽  
Molecular Approaches ◽  
Reticulate Body ◽  
Wide Range ◽  
The Family ◽  
Serious Disease

Chlamydiae are obligate intracellular bacterial pathogens able to infect and cause serious disease in humans, birds and a remarkably wide range of warm and cold-blooded animals. The family Chlamydiaciae have traditionally been defined by their unique biphasic developmental cycle, involving the interconversion between an extracellular survival form, the elementary body and an intracellular replicative form, the reticulate body. However, as with many other bacteria, molecular approaches including 16SrRNA sequence are becoming the standard of choice. As a consequence, the chlamydiae are in a taxonomic state of flux. Prior to 1999, the family Chlamydiaceae consisted of one genus, Chlamydia, and four species, Chlamydia trachomatis, C. psittaci, C. pecorum and C. pneumoniae. In 1999, Everett et al proposed a reclassification of Chlamydia into two genera (Chlamydia and Chlamydophila) and nine species (Chlamydia trachomatis, C. suis, and C. muridarum and Chlamydophila psittaci, C. pneumoniae, C. felis, C. pecorum, C. abortus, and C. caviae). While some of these species are thought to be host specific (C. suis ? pigs, C. muridarum ? mice, C. felis ? cats, C. caviae ? guinea pigs) many are known to infect and cause disease in a wide range of hosts.

Detection of transcription and translation in situ with biotinylated molecular probes in cells transfected with recombinant DNA plasmids

1986 ◽  
Vol 156 (1) ◽  
pp. 17-24 ◽  
Author(s):  
G.H. Smith ◽  
P.J. Doherty ◽  
R.B. Stead ◽  
C.M. Gorman ◽  
D.E. Graham ◽  
...  
Keyword(s):  
Recombinant Dna ◽  
Molecular Probes ◽  
In Cells

Genetic transformation of Rhodopseudomonas sphaeroides by plasmid DNA

1982 ◽  
Vol 152 (1) ◽  
pp. 89-97
Author(s):  
C S Fornari ◽  
S Kaplan
Keyword(s):  
Plasmid Dna ◽  
Recombinant Dna ◽  
Effective Means ◽  
Viable Cell ◽  
Broad Host Range ◽  
Dna Uptake ◽  
Rhodopseudomonas Sphaeroides ◽  
Open Circular ◽  
Polyethylene Glycol 6000

A broad-host-range cloning vector, pUI81, was constructed in vitro from plasmids RSF1010 and pSL25 (a pBR322 derivative) and used to assay for transformation in Rhodopseudomonas sphaeroides. Washing cells with 500 mM Tris was an effective means of inducing competence for DNA uptake. Transformation frequencies as high as 10(-5) (transformants per viable cell) have been achieved by incubating Tris-treated cells with plasmid DNA, 100 mM CaCl2, and 20% polyethylene glycol 6000. Maximum frequencies were obtained when recipient cells were spread onto selective media after a 6.5-h outgrowth period in antibiotic-free medium. The structure (open circular versus closed, covalent circular), size, and concentration of plasmid DNA all significantly affected the transformation frequency. Four different plasmids, all small and suitable as cloning vectors, have been introduced by transformation into several different R. sphaeroides strains. Recombinant DNA carried on small, nonconjugative plasmids with broad host ranges can now be directly transferred to R. sphaeroides by this method.

Detection and identification of Leishmania parasites by in situ hybridization with total and recombinant DNA probes

1991 ◽  
Vol 73 (3) ◽  
pp. 345-353 ◽  
Author(s):  
G.J. Schoone ◽  
G.J.J.M. van Eys ◽  
G.S. Llgthart ◽  
F.E. Taub ◽  
J. Zaal ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Recombinant Dna ◽  
Dna Probes ◽  
Detection And Identification ◽  
Leishmania Parasites
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