scholarly journals Generation of a Stable Plasmid forIn VitroandIn VivoStudies of Staphylococcus Species

2016 ◽  
Vol 82 (23) ◽  
pp. 6859-6869 ◽  
Author(s):  
Christina N. Krute ◽  
Kelsey L. Krausz ◽  
Mary A. Markiewicz ◽  
Jason A. Joyner ◽  
Srijana Pokhrel ◽  
...  
Keyword(s):  
Shuttle Vector ◽  
Antibiotic Usage ◽  
Open Reading Frames ◽  
Antibiotic Selection ◽  
Content Type ◽  
Plasmid Maintenance ◽  
Genetic Tools ◽  
Reading Frames

ABSTRACTA major shortcoming to plasmid-based genetic tools is the necessity of using antibiotics to ensure plasmid maintenance. While selectable markers are very powerful, their use is not always practical, such as duringin vivomodels of bacterial infection. During previous studies, it was noted that the uncharacterized LAC-p01 plasmid inStaphylococcus aureusUSA300 isolates was stable in the absence of a known selection and therefore could serve as a platform for new genetic tools forStaphylococcusspecies. LAC-p01 was genetically manipulated into anEscherichia coli-S. aureusshuttle vector that remained stable for at least 100 generations without antibiotic selection. The double- and single-stranded (dsoandsso) origins were identified and found to be essential for plasmid replication and maintenance, respectively. In contrast, deletion analyses revealed that none of the four LAC-p01 predicted open reading frames were necessary for stability. Subsequent to this, the shuttle vector was used as a platform to generate two plasmids. The first plasmid, pKK22, contains all genes native to the plasmid for use inS. aureusUSA300 strains, while the second, pKK30, lacks the four predicted open reading frames for use in non-USA300 isolates. pKK30 was also determined to be stable inStaphylococcus epidermidis. Moreover, pKK22 was maintained for 7 days postinoculation during a murine model ofS. aureussystemic infection and successfully complemented anhlamutant in a dermonecrosis model. These plasmids that eliminate the need for antibiotics during bothin vitroandin vivoexperiments are powerful new tools for studies ofStaphylococcus.IMPORTANCEPlasmid stability has been problematic in bacterial studies, and historically antibiotics have been used to ensure plasmid maintenance. This has been a major limitation duringin vivostudies, where providing antibiotics for plasmid maintenance is difficult and has confounding effects. Here, we have utilized the naturally occurring plasmid LAC-p01 from anS. aureusUSA300 strain to construct stable plasmids that obviate antibiotic usage. These newly modified plasmids retain stability over a multitude of generationsin vitroandin vivowithout antibiotic selection. With these plasmids, studies requiring genetic complementation, protein expression, or genetic reporter systems would not only overcome the burden of antibiotic usage but also eliminate the side effects of these antibiotics. Thus, our plasmids can be used as a powerful genetic tool for studies ofStaphylococcusspecies.

scholarly journals Use of an Endogenous Plasmid Locus for Stable intransComplementation in Borrelia burgdorferi

2014 ◽  
Vol 81 (3) ◽  
pp. 1038-1046 ◽  
Author(s):  
Irene N. Kasumba ◽  
Aaron Bestor ◽  
Kit Tilly ◽  
Patricia A. Rosa
Keyword(s):  
Borrelia Burgdorferi ◽  
Shuttle Vector ◽  
Targeted Mutagenesis ◽  
Multiple Cloning Site ◽  
Stable Gene ◽  
Content Type ◽  
Shuttle Vectors ◽  
Stable Maintenance

ABSTRACTTargeted mutagenesis and complementation are important tools for studying genes of unknown function in the Lyme disease spirocheteBorrelia burgdorferi. A standard method of complementation is reintroduction of a wild-type copy of the targeted gene on a shuttle vector. However, shuttle vectors are present at higher copy numbers thanB. burgdorferiplasmids and are potentially unstable in the absence of selection, thereby complicating analyses in the mouse-tick infectious cycle.B. burgdorferihas over 20 plasmids, with some, such as linear plasmid 25 (lp25), carrying genes required by the spirochetein vivobut relatively unstable duringin vitrocultivation. We propose that complementation on an endogenous plasmid such as lp25 would overcome the copy number andin vivostability issues of shuttle vectors. In addition, insertion of a selectable marker on lp25 could ensure its stable maintenance by spirochetes in culture. Here, we describe the construction of a multipurpose allelic-exchange vector containing a multiple-cloning site and either of two selectable markers. This suicide vector directs insertion of the complementing gene into thebbe02locus, a site on lp25 that was previously shown to be nonessential during bothin vitroandin vivogrowth. We demonstrate the functional utility of this strategy by restoring infectivity to anospCmutant through complementation at this site on lp25 and stable maintenance of theospCgene throughout mouse infection. We conclude that this represents a convenient and widely applicable method for stable gene complementation inB. burgdorferi.

scholarly journals Genomic Sequence of Rhesus Cytomegalovirus 180.92: Insights into the Coding Potential of Rhesus Cytomegalovirus

2006 ◽  
Vol 80 (8) ◽  
pp. 4179-4182 ◽  
Author(s):  
Pierre Rivailler ◽  
Amitinder Kaur ◽  
R. Paul Johnson ◽  
Fred Wang
Keyword(s):  
Genomic Sequence ◽  
Open Reading Frames ◽  
Rhesus Cytomegalovirus ◽  
Human Cmv ◽  
Coding Capacity ◽  
Coding Potential ◽  
Reading Frames ◽  
Genetic Rearrangements

ABSTRACT A pathogenic isolate of rhesus cytomegalovirus (rhCMV 180.92) was cloned, sequenced, and annotated. Comparisons with the published rhCMV 68.1 genome revealed 8 open reading frames (ORFs) in isolate 180.92 that are absent in 68.1, 10 ORFs in 68.1 that are absent in 180.92, and 34 additional ORFs that were not previously annotated. Most of the differences appear to be due to genetic rearrangements in both isolates from a region that is frequently altered in human CMV (hCMV) during in vitro passage. These results indicate that the rhCMV ORF repertoire is larger than previously recognized. Like hCMV, understanding of the complete coding capacity of rhCMV is complicated by genomic instability and may require comparisons with additional isolates in vitro and in vivo.

E1−E4+ Adenoviral Gene Transfer Vectors Function as a “Pro-Life” Signal to Promote Survival of Primary Human Endothelial Cells

Blood ◽  
1999 ◽  
Vol 93 (9) ◽  
pp. 2936-2944 ◽  
Author(s):  
Ramachandran Ramalingam ◽  
Shahin Rafii ◽  
Stefan Worgall ◽  
Douglas E. Brough ◽  
Ronald G. Crystal
Keyword(s):  
Endothelial Cells ◽  
Open Reading Frames ◽  
Adenoviral Gene Transfer ◽  
Vector Genome ◽  
Transfer Vectors ◽  
Reading Frames ◽  
Human Endothelium ◽  
Pro Life

Abstract Although endothelial cells are quiescent and long-lived in vivo, when they are removed from blood vessels and cultured in vitro they die within days to weeks. In studies of the interaction of E1−E4+ replication–deficient adenovirus (Ad) vectors and human endothelium, the cells remained quiescent and were viable for prolonged periods. Evaluation of these cultures showed that E1−E4+ Ad vectors provide an “antiapoptotic” signal that, in association with an increase in the ratio of Bcl2 to Bax levels, induces the endothelial cells to enter a state of “suspended animation,” remaining viable for at least 30 days, even in the absence of serum and growth factors. Although the mechanisms initiating these events are unclear, the antiapoptoic signal requires the presence of E4 genes in the vector genome, suggesting that one or more E4 open reading frames of subgroup C Ad initiate a “pro-life” program that modifies cultured endothelial cells to survive for prolonged periods.

scholarly journals Characterization of Endogenous Retroviruses in Sheep

2003 ◽  
Vol 77 (20) ◽  
pp. 11268-11273 ◽  
Author(s):  
Nikolai Klymiuk ◽  
Mathias Müller ◽  
Gottfried Brem ◽  
Bernhard Aigner
Keyword(s):  
Endogenous Retrovirus ◽  
Ovis Aries ◽  
Endogenous Retroviruses ◽  
Open Reading Frames ◽  
In Vivo Experiments ◽  
Pregnant Sheep ◽  
Reading Frames

ABSTRACT Endogenous retrovirus (ERV) sequences have been found in all mammals. In vitro and in vivo experiments revealed ERV activation and cross-species infection in several species. Sheep (Ovis aries) are used for various biotechnological purposes; however, they have not yet been comprehensively screened for ERV sequences. Therefore, the aim of the study was to classify the ERV sequences in the ovine genome (OERV) by analyzing the retroviral pro-pol sequences. Three OERV β families and nine OERV γ families were revealed. Novel open reading frames (ORF) in the amplified proviral fragment were found in one OERV β family and two OERV γ families. Hybrid OERV produced by putative recombination events were not detected. Quantitative analysis of the OERV sequences in the ovine genome revealed no relevant variations in the endogenous retroviral loads of different breeds. Expression analysis of different tissues from fetal and pregnant sheep detected mRNA from both gammaretrovirus families, showing ORF fragments. Thus, the release of retroviruses from sheep cells cannot be excluded.

scholarly journals Depletion of the Trypanosome Pumilio Domain Protein PUF2 or of Some Other Essential Proteins Causes Transcriptome Changes Related to Coding Region Length

2014 ◽  
Vol 13 (5) ◽  
pp. 664-674 ◽  
Author(s):  
Bhaskar Anand Jha ◽  
Abeer Fadda ◽  
Clementine Merce ◽  
Elisha Mugo ◽  
Dorothea Droll ◽  
...  
Keyword(s):  
Rna Binding ◽  
Rna Binding Proteins ◽  
Mrna Translation ◽  
Open Reading Frames ◽  
Mrna Levels ◽  
Coding Region ◽  
Content Type ◽  
Puf Proteins ◽  
Reading Frames

ABSTRACT Pumilio domain RNA-binding proteins are known mainly as posttranscriptional repressors of gene expression that reduce mRNA translation and stability. Trypanosoma brucei has 11 PUF proteins. We show here that PUF2 is in the cytosol, with roughly the same number of molecules per cell as there are mRNAs. Although PUF2 exhibits a low level of in vivo RNA binding, it is not associated with polysomes. PUF2 also decreased reporter mRNA levels in a tethering assay, consistent with a repressive role. Depletion of PUF2 inhibited growth of bloodstream-form trypanosomes, causing selective loss of mRNAs with long open reading frames and increases in mRNAs with shorter open reading frames. Reexamination of published RNASeq data revealed the same trend in cells depleted of some other proteins. We speculate that these length effects could be caused by inhibition of the elongation phase of transcription or by an influence of translation status or polysomal conformation on mRNA decay.

scholarly journals An in vivo assay for the reverse transcriptase of human retrotransposon L1 in Saccharomyces cerevisiae.

1994 ◽  
Vol 14 (7) ◽  
pp. 4485-4492 ◽  
Author(s):  
B A Dombroski ◽  
Q Feng ◽  
S L Mathias ◽  
D M Sassaman ◽  
A F Scott ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Consensus Sequence ◽  
Unknown Origin ◽  
Open Reading Frames ◽  
Long Terminal Repeats ◽  
Base Pairs ◽  
In Vivo Assay ◽  
Reading Frames

L1 elements constitute a highly repetitive human DNA family (50,000 to 100,000 copies) lacking long terminal repeats and ending in a poly(A) tail. Some L1 elements are capable of retrotransposition in the human genome (Kazazian, H. H., Jr., C. Wong, H. Youssoufian, A. F. Scott, D. G. Phillips, and S.E. Antonarakis, Nature (London) 332:164-166, 1988). Although most are 5' truncated, a consensus sequence of complete L1 elements is 6 kb long and contains two open reading frames (ORFs) (Scott, A. F., B. J. Schmeckpeper, M. Abdelrazik, C. T. Comey, B. O'Hara, J. P. Rossiter, T. Cooley, P. Health, K. D. Smith, and L. Margolet, Genomics 1:113-125, 1987). The protein encoded by ORF2 has reverse transcriptase (RT) activity in vitro (Mathias, S. L., A. F. Scott, H. H. Kazazian, Jr., J. D. Boeke, and A. Gabriel, Science 254:1808-1810, 1991). Because L1 elements are so numerous, efficient methods for identifying active copies are required. We have developed a simple in vivo assay for the activity of L1 RT based on the system developed by Derr et al. (Derr, L. K., J. N. Strathern, and D. J. Garfinkel, Cell 67:355-364, 1991) for yeast HIS3 pseudogene formation. L1 ORF2 displays an in vivo RT activity similar to that of yeast Ty1 RT in this system and generates pseudogenes with unusual structures. Like the HIS3 pseudogenes whose formation depends on Ty1 RT, the HIS3 pseudogenes generated by L1 RT are joined to Ty1 sequences and often are part of complex arrays of Ty1 elements, multiple HIS3 pseudogenes, and hybrid Ty1/L1 elements. These pseudogenes differ from those previously described in that there are base pairs of unknown origin inserted at several of the junctions. In two of three HIS3 pseudogenes studied, the L1 RT appears to have jumped from the 5' end of a Ty1/L1 transcript to the poly(A) tract of the HIS3 RNA.

scholarly journals Production, Secretion, and Cell Surface Display of Recombinant Sporosarcina ureae S-Layer Fusion Proteins in Bacillus megaterium

2011 ◽  
Vol 78 (2) ◽  
pp. 560-567 ◽  
Author(s):  
Denise Knobloch ◽  
Kai Ostermann ◽  
Gerhard Rödel
Keyword(s):  
Cell Surface ◽  
Bacillus Megaterium ◽  
Fusion Proteins ◽  
Shuttle Vector ◽  
Surface Display ◽  
Fluorescent Labeling ◽  
Content Type ◽  
Sporosarcina Ureae

ABSTRACTMonomolecular crystalline bacterial cell surface layers (S-layers) have broad application potential in nanobiotechnology due to their ability to generate functional supramolecular structures. Here, we report thatBacillus megateriumis an excellent host organism for the heterologous expression and efficient secretion of hemagglutinin (HA) epitope-tagged versions of the S-layer protein SslA fromSporosarcina ureaeATCC 13881. Three chimeric proteins were constructed, comprising the precursor, C-terminally truncated, and N- and C-terminally truncated forms of the S-layer SslA protein tagged with the human influenza hemagglutinin epitope. For secretion of fusion proteins, the open reading frames were cloned into theEscherichia coli-Bacillus megateriumshuttle vector pHIS1525. After transformation of the respective plasmids intoBacillus megateriumprotoplasts, the recombinant genes were successfully expressed and the proteins were secreted into the growth medium. The isolated S-layer proteins are able to assemblein vitrointo highly ordered, crystalline, sheetlike structures with the fused HA tag accessible to antibody. We further show by fluorescent labeling that the secreted S-layer fusion proteins are also clustered on the cell envelope ofBacillus megaterium, indicating that the cell surface can servein vivoas a nucleation point for crystallization. Thus, this system can be used as a display system that allows the dense and periodic presentation of S-layer proteins or the fused tags.

scholarly journals Ancient Adversary – HERV-K (HML-2) in Cancer

2021 ◽  
Vol 11 ◽  
Author(s):  
Eoin Dervan ◽  
Dibyangana D. Bhattacharyya ◽  
Jake D. McAuliffe ◽  
Faizan H. Khan ◽  
Sharon A. Glynn
Keyword(s):  
Tumour Progression ◽  
Endogenous Retroviruses ◽  
Open Reading Frames ◽  
Human Endogenous Retroviruses ◽  
Viral Particles ◽  
Metastatic Capacity ◽  
Coding Capacity ◽  
Reading Frames

Human endogenous retroviruses (HERV), ancient integrations of exogenous viruses, make up 8% of our genome. Long thought of as mere vestigial genetic elements, evidence is now accumulating to suggest a potential functional role in numerous pathologies including neurodegenerative diseases, autoimmune disorders, and multiple cancers. The youngest member of this group of transposable elements is HERV-K (HML-2). Like the majority of HERV sequences, significant post-insertional mutations have disarmed HERV-K (HML-2), preventing it from producing infectious viral particles. However, some insertions have retained limited coding capacity, and complete open reading frames for all its constituent proteins can be found throughout the genome. For this reason HERV-K (HML-2) has garnered more attention than its peers. The tight epigenetic control thought to suppress expression in healthy tissue is lost during carcinogenesis. Upregulation of HERV-K (HML-2) derived mRNA and protein has been reported in a variety of solid and liquid tumour types, and while causality has yet to be established, progressively more data are emerging to suggest this phenomenon may contribute to tumour growth and metastatic capacity. Herein we discuss its potential utility as a diagnostic tool and therapeutic target in light of the current in vitro, in vivo and clinical evidence linking HERV-K (HML-2) to tumour progression.

scholarly journals New Shuttle Vectors for Real-Time Gene Expression Analysis in Multidrug-Resistant Acinetobacter Species: In Vitro and In Vivo Responses to Environmental Stressors

2019 ◽  
Vol 85 (18) ◽  
Author(s):  
Massimiliano Lucidi ◽  
Daniela Visaggio ◽  
Elisa Prencipe ◽  
Francesco Imperi ◽  
Giordano Rampioni ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Analysis ◽  
Gene Expression Analysis ◽  
Multidrug Resistant ◽  
Antibiotic Selection ◽  
Content Type ◽  
Shuttle Vectors ◽  
Reporter Systems

ABSTRACT The Acinetobacter genus includes species of opportunistic pathogens and harmless saprophytes. The type species, Acinetobacter baumannii, is a nosocomial pathogen renowned for being multidrug resistant (MDR). Despite the clinical relevance of infections caused by MDR A. baumannii and a few other Acinetobacter spp., the regulation of their pathogenicity remains elusive due to the scarcity of adequate genetic tools, including vectors for gene expression analysis. Here, we report the generation and testing of a series of Escherichia coli-Acinetobacter promoter-probe vectors suitable for gene expression analysis in Acinetobacter spp. These vectors, named pLPV1Z, pLPV2Z, and pLPV3Z, carry both gentamicin and zeocin resistance markers and contain lux, lacZ, and green fluorescent protein (GFP) reporter systems downstream of an extended polylinker, respectively. The presence of a toxin-antitoxin gene system and the high copy number allow pLPV plasmids to be stably maintained even without antibiotic selection. The pLPV plasmids can easily be introduced by electroporation into MDR A. baumannii belonging to the major international lineages as well as into species of the Acinetobacter calcoaceticus-A. baumannii complex. The pLPV vectors have successfully been employed to study the regulation of stress-responsive A. baumannii promoters, including the DNA damage-inducible uvrABC promoter, the ethanol-inducible adhP and yahK promoters, and the iron-repressible promoter of the acinetobactin siderophore biosynthesis gene basA. A lux-tagged A. baumannii ATCC 19606T strain, carrying the iron-responsive pLPV1Z::PbasA promoter fusion, allowed in vivo and ex vivo monitoring of the bacterial burden in the Galleria mellonella infection model. IMPORTANCE The short-term adaptive response to environmental cues greatly contributes to the ecological success of bacteria, and profound alterations in bacterial gene expression occur in response to physical, chemical, and nutritional stresses. Bacteria belonging to the Acinetobacter genus are ubiquitous inhabitants of soil and water though some species, such as Acinetobacter baumannii, are pathogenic and cause serious concern due to antibiotic resistance. Understanding A. baumannii pathobiology requires adequate genetic tools for gene expression analysis, and to this end we developed user-friendly shuttle vectors to probe the transcriptional responses to different environmental stresses. Vectors were constructed to overcome the problem of antibiotic selection in multidrug-resistant strains and were equipped with suitable reporter systems to facilitate signal detection. By means of these vectors, the transcriptional response of A. baumannii to DNA damage, ethanol exposure, and iron starvation was investigated both in vitro and in vivo, providing insights into A. baumannii adaptation during stress and infection.

Translational activation of GCN4 mRNA in a cell-free system is triggered by uncharged tRNAs

1990 ◽  
Vol 10 (8) ◽  
pp. 4375-4378
Author(s):  
G Krupitza ◽  
G Thireos
Keyword(s):  
Open Reading Frames ◽  
Yeast Cells ◽  
Free System ◽  
In Vitro System ◽  
A Cell ◽  
Translational Activation ◽  
Reading Frames ◽  
Small Open Reading Frames

Translation of GCN4 mRNA is activated when yeast cells are grown under conditions of amino acid limitation. In this study, we established the conditions through which translation of the GCN4 mRNA could be activated in a homologous in vitro system. This activation paralleled the in vivo situation: it required the small open reading frames located in the 5' untranslated region of the GCN4 mRNA, and it was coupled with reduced rates of 43S preinitiation complex formation. Translational derepression in vitro was triggered by uncharged tRNA molecules, demonstrating that deacylated tRNAs are more proximal signals for translational activation of the GCN4 mRNA.

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