DOCSLIB.ORG

Transduction by Bacteriophage Ti * Henry Drexler

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Transduction by Bacteriophage Ti * Henry Drexler Proceedings of the National Academy of Sciences Vol. 66, No. 4, pp. 1083-1088, August 1970 Transduction by Bacteriophage Ti * Henry Drexler DEPARTMENT OF MICROBIOLOGY, BOWMAN GRAY SCHOOL OF MEDICINE, WAKE FOREST UNIVERSITY, WINSTON-SALEM, NORTH CAROLINA Communicated by Edward L. Tatum, May 25, 1970 Abstract. Amber mutants of the virulent coliphage T1 are able to transduce a wide variety of genetic characteristics from permissive to nonpermissive K strains of Escherichia coli. The virulent coliphage T1 is not known to be related to any temperate phage. Certain of the characteristics of T1 seem to be incompatible with its potential existence as a temperate phage; for example, the average latent period for T1 is only 13 min' and about 70% of its DNA is derived from the host.2 In order to demonstrate transduction by T1 it is necessray to provide condi- tions in which potential transductants are able to survive; this is accomplished by using amber mutants of T1 to transduce nonpermissive recipients. Experi- ments which show that T1 is a transducing phage are presented and have been designed chiefly to illustrate the following points: (1) Infection by T1 is able to cause heritable changes in recipients; (2) the genotype of the donor host is im- portant in determining what characteristics can be transferred to recipients; (3) the changes in the recipients are not caused by transformation; and (4) there is a similarity between the transducing activity and the plaque-forming ability of T1 with respect to serology, host range, and density. Materials and Methods. Bacterial strains: The abbreviations and symbols of Demerec et al.3 and Taylor and Trotter4 are used to describe all pertinent genotypes. The phenotype of each strain is given below, together with the symbols used to identify them, and are repeated at each point where their inclusion is useful for clarity. All strains used are members of the species Escherichia coli. KB-5 is a streptomycin-resistant (StrR) strain which is a permissive host (Su+) for all the amber mutants of T1 used here. KB-3 is able to ferment lactose (Lac+) and galac- tose (Gal+) and is independent of any requirement for arginine (Arg+), tryptophan (Trp+), serine (Ser+), threonine (Thr+), and biotin (Bio+). A tryptophan-dependent (Trp-) variant of KB-3 which requires tryptophan to satisfy its growth requirement was used in one experiment. CS1006 is a streptomycin-sensitive (StrS) strain which is Su+. A spontaneous StrR variant of CS100 was isolated in this laboratory. The strain W33507 is a nonpermissive host (Su-) for the amber mutants used here; it is StrS and is unable to ferment either galactose (Gal-) or lactose (Lac-). Mutants of W3350 which are de- pendent on either arginine (Arg-), tryptophan (Trp-), or serine and threonine (Ser-Thr-) were obtained by exposing W3350 to nitrosoguanidine. W3350/1 is a spontaneous, T1- resistant mutant isolated in this laboratory. Strain R9018 was derived from W3350 (X); it has a deletion from galE through XQ and also from XB through chlA including one or more genes controlling the biosynthesis of biotin; phenotypically R901 is Su-Gal-Bio-. Strains KB-3, CS100, W3350, and R901 were kindly provided by Drs. W. Michalke, J. R. Christensen, E. Six, and A. Campbell respectively. 1083 Downloaded by guest on September 29, 2021 10841084GENETICS: H. DREXLER PROC. N. A. S. Phage strains: T1 amber mutants (am) are from Michalke.' Efficiencies of plat- ing strains Tlamll and Tlam5amll on W3350 compared to KB-3 were 10-6 and less than 10-8 respectively. T1 plaques were less than 4 mm in diameter on the various selective media. The infected recipient cells were diluted to give less than 40 plaques per plate. Materials: L broth,9 L agar,9 saltless nutrient broth,10 synthetic T medium with glucose and agar (TMGA),"1 and complete eosin methylene blue agar (EMB)12 have been described elsewhere. T1 resuspension medium is a sterile solution of 1.21% tris(hydroxymethyl)aminomethane (Trizma Base, Sigma Chemical Corp.), 0.05% CaCl2, 0.06% MgSO4, and 0.15% gelatin. Streptomycin is an aqueous solution of streptomycin sulfate (Eli Lilly and Co.). Specific amino acids were used to supplement the T medium in 0.002% amounts. Biotin, when needed, was used at concentrations of 5 ,ug/ml. DNase was a solution of a crystalline preparation of Nutritional Biochem. Corp. CsCl solution contained 1.12 g/ml of CsCl dissolved in 1.21% Trizma base. Methods: In experiments, cells were grown to log phase in L broth and harvested at a titer of about 5 X 108/ml. Cells were centrifuged three times and resuspended each time in saltless nutrient broth and finally concentrated to about 1-2 X 109/ml. Cells and phage (multiplicity of infection of about 1) were incubated at 37°C for 7 min, chilled, and a representative sample was centrifuged in order to test the supernatant for un- adsorbed phage. The adsorption mixture was maintained in an ice bath during plating. In experiments where the transfer of the streptomycin-resistant phenotype was being selected, a sample of the adsorption mixture was plated by overlay on a L agar plates; these plates were incubated at 37°C for 3 hr and streptomycin solution was added via an additional overlay and incubation continued.'0 In experiments other than the strepto- mycin experiments, the adsorption mixture was spread on the surface of either EMB plates or TMGA plates. A volume of uninfected recipient cells equal in number to the number of cells exposed to T1 were plated as controls in all experiments. Anti-Ti serum wasl)repared by injecting wild type T1 which had been grown on Shi- gella dysenteriae into a rabbit; before use the complement was inactivated by incubation at 56°C for 30 min. Before use, all stocks of Ti were washed several times in Ti resuspension medium, re- frigerated overnight, and filtered. All stocks were routinely tested for sterility. With CsCl density gradient centrifugation, 2 ml of stock phage (about 1-3 X 1011 plaque-forming units/ml) were mixed with 3 ml of CsCl solution. The mixture was cen- trifuged for 18 hr in a model L, Beckman utracentrifuge in a SW50L rotor at 100,000 X g (36,000 rpm). Results: Transduction of various markers: Table 1 presents data from experiments in which Tlam phage grown on the Su+, Gal+, Arg+, Trp+, and StrR strain KB-3 were used to infect variants of W3350; all W3350 variants are Gal- and StrS; Arg- and Trp- isolates of W3350 were also used. In experiment 3 it can be observed that strain CS100 Su + StrR is also able to serve as a donor for Ti-mediated transduction. The results in Table 1 demonstrate the generalized nature of Tl transduction. Since the amber mutants of T1 which were used here (i.e., Tlamll and Tlam- 5aml 1) kill W3350 even though no phage are produced, transductants must arise from cells which obtained a transducing particle (containing donor DNA but little or no phage DNA") but no phage particle. The efficiency of transduction is given as the number of tranductants per milliliter less control values per singly infected cell as calculated using the multiplicity of infection, the number of cells, and the Poisson distribution. When KB-3Su+ is used as the donor, the number of transductants per singly infected cell varies from about 3 X 10- to 1 X 10-. Importance of the previous host: Tlam5amll was grown on CS100SutStrR Downloaded by guest on September 29, 2021 VOL. 66, 1970 GENETICS: H. DREXLER 1085 TABLE 1. Generalized tranduction by Ti; importance of previous host; host range of trans- ducing particles. * Colonies Transductants/ Phenotype Su + Su - with selected singly infected Expt. selected donor recipient phenotype cells 1 Gal + KB-3Gal + W335OGal- 157 5 X 10-7 Control 0 2 Arg+ KB-3Arg + W3350Arg- 211 3 X 10-7 Control 0 3 StrR CS10OStrR W335OStrS 2155 9 X 10-6 CSLOOStrS W335OStrS 35 7 X 10-8 Control 13 4 Trp + KB-3Trp+ W3350Trp- 129 1 X 10-6 KB-3Trp- W3350Trp- 21 7 X 10-8 Control 1 5 StrR KB-3StrR W335OStrS 248 1 X 10-6 Control 0 W335OStrS/1 0 Control 0 * Tlamll was used in Expts. 1, 2, and 5. Tlam5amll was used in Expts. 3 and 4. and CS100Su+StrS respectively. The third experiment in Table 1 shows that phage grown on CS100Su+StrR can transfer streptomycin resistance to W3350- Su-StrS but that phage grown on CS100Su+StrS cannot. Experiment 4 of Table 1 shows that if a tryptophan-dependent auxotroph (KB-3Su-Trp-), which requires tryptophan to satisfy its requirement, is used as the host for Tlam5amll, the phage is either not able to transfer tryptophan independence to a recipient with a similar requirement (W3350Su-Trp-) or it does so at a very low rate compared to phage grown on Trp+ donors. Since KB-3Trp- donors and W3350Trp- recipients do not necessarily represent iden- tical point mutations, a small amount of transduction may occur in this instance. Host range of transducing activity: Experiment 5 in Table 1 gives the results of a representative experiment in which attempts were made to transduce the Tl-resistant, Su-, StrS strain W3350/1 using Tlaml which had been grown on KB-3Su+StrR. It can be seen that while W335OSu-StrS could be transduced for StrR by the phage, no W3350/1 transductants were observed. Effect of specific anti-Ti serum: Antiserum was used in concentrations which, on the one hand, would inactivate 70-90% of the phage and which, on the other hand, would inactivate less than 10% of the phage after a 1:10 dilution of the antiserum.
Load more

Recommended publications
  • Mobile Genetic Elements in Streptococci
    Curr. Issues Mol. Biol. (2019) 32: 123-166. DOI: https://dx.doi.org/10.21775/cimb.032.123 Mobile Genetic Elements in Streptococci Miao Lu#, Tao Gong#, Anqi Zhang, Boyu Tang, Jiamin Chen, Zhong Zhang, Yuqing Li*, Xuedong Zhou* State Key Laboratory of Oral Diseases, National Clinical Research Center for Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, PR China. #Miao Lu and Tao Gong contributed equally to this work. *Address correspondence to: [email protected], [email protected] Abstract Streptococci are a group of Gram-positive bacteria belonging to the family Streptococcaceae, which are responsible of multiple diseases. Some of these species can cause invasive infection that may result in life-threatening illness. Moreover, antibiotic-resistant bacteria are considerably increasing, thus imposing a global consideration. One of the main causes of this resistance is the horizontal gene transfer (HGT), associated to gene transfer agents including transposons, integrons, plasmids and bacteriophages. These agents, which are called mobile genetic elements (MGEs), encode proteins able to mediate DNA movements. This review briefly describes MGEs in streptococci, focusing on their structure and properties related to HGT and antibiotic resistance. caister.com/cimb 123 Curr. Issues Mol. Biol. (2019) Vol. 32 Mobile Genetic Elements Lu et al Introduction Streptococci are a group of Gram-positive bacteria widely distributed across human and animals. Unlike the Staphylococcus species, streptococci are catalase negative and are subclassified into the three subspecies alpha, beta and gamma according to the partial, complete or absent hemolysis induced, respectively. The beta hemolytic streptococci species are further classified by the cell wall carbohydrate composition (Lancefield, 1933) and according to human diseases in Lancefield groups A, B, C and G.
    [Show full text]
  • Horizontal Gene Transfer
    Genetic Variation: The genetic substrate for natural selection Horizontal Gene Transfer Dr. Carol E. Lee, University of Wisconsin Copyright ©2020; Do not upload without permission What about organisms that do not have sexual reproduction? In prokaryotes: Horizontal gene transfer (HGT): Also termed Lateral Gene Transfer - the lateral transmission of genes between individual cells, either directly or indirectly. Could include transformation, transduction, and conjugation. This transfer of genes between organisms occurs in a manner distinct from the vertical transmission of genes from parent to offspring via sexual reproduction. These mechanisms not only generate new gene assortments, they also help move genes throughout populations and from species to species. HGT has been shown to be an important factor in the evolution of many organisms. From some basic background on prokaryotic genome architecture Smaller Population Size • Differences in genome architecture (noncoding, nonfunctional) (regulatory sequence) (transcribed sequence) General Principles • Most conserved feature of Prokaryotes is the operon • Gene Order: Prokaryotic gene order is not conserved (aside from order within the operon), whereas in Eukaryotes gene order tends to be conserved across taxa • Intron-exon genomic organization: The distinctive feature of eukaryotic genomes that sharply separates them from prokaryotic genomes is the presence of spliceosomal introns that interrupt protein-coding genes Small vs. Large Genomes 1. Compact, relatively small genomes of viruses, archaea, bacteria (typically, <10Mb), and many unicellular eukaryotes (typically, <20 Mb). In these genomes, protein-coding and RNA-coding sequences occupy most of the genomic sequence. 2. Expansive, large genomes of multicellular and some unicellular eukaryotes (typically, >100 Mb). In these genomes, the majority of the nucleotide sequence is non-coding.
    [Show full text]
  • Recombination in Bacteria
    Recombination in Bacteria 1. Conjugation DNA from a donor cell is transferred to a recipient cell through a conjugation tube (pili). 2. Transformation Uptake of naked DNA molecule from remains of one bacterium (donor cell) by another bacterium (recipient cell). 3. Transduction Bacterial genes are carried from a donor cell to a recipient cell by a bacteriophage. { Generalized { Specialized Conjugation Ability to conjugate located on the F-plasmid F+ Cells act as donors F- Cells act as recipients F+/F- Conjugation: { F Factor “replicates off” a single strand of DNA. { New strand goes through pili to recipient cell. { New strand is made double stranded. { If entire F-plasmid crosses, then recipient cell becomes F+, otherwise nothing happens Conjugation with Hfr Hfr cell (High Frequency Recombination) cells have F-plasmid integrated into the Chromosome. Integration into the Chromosome is unique for each F-plasmid strain. When F-plasmid material is replicated and sent across pili, Chromosomal material is included. (Figure 6.10 in Klug & Cummings) When chromosomal material is in recipient cell, recombination can occur: { Recombination is double stranded. { Donor genes are recombined into the recipient cell. { Corresponding genes from recipient cell are recombined out of the chromosome and reabsorbed by the cell. Interrupted Mating Mapping 1. Allow conjugation to start Genes closest to the origin of replication site (in the direction of replication) are moved through the pili first. 2. After a set time, interrupt conjugation Only those genes closest to the origin of replication site will conjugate. The long the time, the more that is able to conjugate. 3.
    [Show full text]
  • Microbial Genetics by Dr Preeti Bajpai
    Dr. Preeti Bajpai Genes: an overview ▪ A gene is the functional unit of heredity ▪ Each chromosome carry a linear array of multiple genes ▪ Each gene represents segment of DNA responsible for synthesis of RNA or protein product ▪ A gene is considered to be unit of genetic information that controls specific aspect of phenotype DNA Chromosome Gene Protein-1 Prokaryotic Courtesy: Team Shrub https://twitter.com/realscientists/status/927 cell 667237145767937 Genetic exchange within Prokaryotes The genetic exchange occurring in bacteria involve transfers of genes from one bacterium to another. The gene transfer in prokaryotic cells is thus unidirectional and the recombination events usually occur between a fragment of one chromosome (from a donor cell) and a complete chromosome (in a recipient cell) Mechanisms for genetic exchange Bacteria exchange genetic material through three different parasexual processes* namely transformation, conjugation and transduction. *Parasexual process involves recombination of genes from genetically distinct cells occurring without involvement of meiosis and fertilization Principles of Genetics-sixth edition Courtesy: Beatrice the Biologist.com by D. Peter Snustad & Michael J. Simmons (http://www.beatricebiologist.com/2014/08/bacterial-gifts/) Transformation: an introduction Transformation involves the uptake of free DNA molecules released from one bacterium (the donor cell) by another bacterium (the recipient cell). Frederick Griffith discovered transformation in Streptococcus pneumoniae (pneumococcus) in 1928. In his experiments, Griffith used two related strains of bacteria, known as R and S. The R bacteria (nonvirulent) formed colonies, or clumps of related bacteria, that Frederick Griffith 1877-1941 had a rough appearance (hence the abbreviation "R"). The S bacteria (virulent) formed colonies that were rounded and smooth (hence the abbreviation "S").
    [Show full text]
  • Comprehensive Analysis of Mobile Genetic Elements in the Gut Microbiome Reveals Phylum-Level Niche-Adaptive Gene Pools
    bioRxiv preprint doi: https://doi.org/10.1101/214213; this version posted December 22, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Comprehensive analysis of mobile genetic elements in the gut microbiome 2 reveals phylum-level niche-adaptive gene pools 3 Xiaofang Jiang1,2,†, Andrew Brantley Hall2,3,†, Ramnik J. Xavier1,2,3,4, and Eric Alm1,2,5,* 4 1 Center for Microbiome Informatics and Therapeutics, Massachusetts Institute of Technology, 5 Cambridge, MA 02139, USA 6 2 Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA 7 3 Center for Computational and Integrative Biology, Massachusetts General Hospital and Harvard 8 Medical School, Boston, MA 02114, USA 9 4 Gastrointestinal Unit and Center for the Study of Inflammatory Bowel Disease, Massachusetts General 10 Hospital and Harvard Medical School, Boston, MA 02114, USA 11 5 MIT Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 12 02142, USA 13 † Co-first Authors 14 * Corresponding Author bioRxiv preprint doi: https://doi.org/10.1101/214213; this version posted December 22, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 15 Abstract 16 Mobile genetic elements (MGEs) drive extensive horizontal transfer in the gut microbiome. This transfer 17 could benefit human health by conferring new metabolic capabilities to commensal microbes, or it could 18 threaten human health by spreading antibiotic resistance genes to pathogens. Despite their biological 19 importance and medical relevance, MGEs from the gut microbiome have not been systematically 20 characterized.
    [Show full text]
  • Molecular Model for the Transposition and Replication of Bacteriophage
    Proc. Natl. Acad. Sci. USA Vol. 76, No. 4, pp. 1933-1937, April 1979 Genetics Molecular model for the transposition and replication of bacteriophage Mu and other transposable elements (DNA insertion elements/nonhomologous recombination/site-specific recombination/replicon fusion/topoisomerases) JAMES A. SHAPIRO Department of Microbiology, The University of Chicago, Chicago, Illinois 60637 Communicated by Hewson Swift, December 11, 1978 ABSTRACT A series of molecular events will explain how B genetic elements can transpose from one DNA site to another, B y generate a short oligonucleotide duplication at both ends of the I % new insertion site, and replicate in the transposition process. I These events include the formation of recombinant molecules A a% /; C which have been postulated to be intermediates in the trans- position process. The model explains how the replication of bacteriophage Mu is obligatorily associated with movement to z x x new genetic sites. It postulates that all transposable elements replicate in the transposition process so that they remain at their z original site while moving to new sites. According to this model, the mechanism of transposition is very different from the in- V sertion and excision of bacteriophage X. y FIG. 1. Replicon fusion. The boxes with an arrow indicate copies Recent research on transposable elements in bacteria has pro- of a transposable element. The letters mark arbitrary regions of the vided important insights into the role of nonhomologous re- two replicons to indicate the relative positions of the elements in the combination in genetic rearrangements (1-4). These elements donor and cointegrate molecules. include small insertion sequences (IS elements), transposable resistance determinants (Tn elements), and bacteriophage Mu results in the duplication of a short oligonucleotide sequence (3).
    [Show full text]
  • Section 4. Guidance Document on Horizontal Gene Transfer Between Bacteria
    306 - PART 2. DOCUMENTS ON MICRO-ORGANISMS Section 4. Guidance document on horizontal gene transfer between bacteria 1. Introduction Horizontal gene transfer (HGT) 1 refers to the stable transfer of genetic material from one organism to another without reproduction. The significance of horizontal gene transfer was first recognised when evidence was found for ‘infectious heredity’ of multiple antibiotic resistance to pathogens (Watanabe, 1963). The assumed importance of HGT has changed several times (Doolittle et al., 2003) but there is general agreement now that HGT is a major, if not the dominant, force in bacterial evolution. Massive gene exchanges in completely sequenced genomes were discovered by deviant composition, anomalous phylogenetic distribution, great similarity of genes from distantly related species, and incongruent phylogenetic trees (Ochman et al., 2000; Koonin et al., 2001; Jain et al., 2002; Doolittle et al., 2003; Kurland et al., 2003; Philippe and Douady, 2003). There is also much evidence now for HGT by mobile genetic elements (MGEs) being an ongoing process that plays a primary role in the ecological adaptation of prokaryotes. Well documented is the example of the dissemination of antibiotic resistance genes by HGT that allowed bacterial populations to rapidly adapt to a strong selective pressure by agronomically and medically used antibiotics (Tschäpe, 1994; Witte, 1998; Mazel and Davies, 1999). MGEs shape bacterial genomes, promote intra-species variability and distribute genes between distantly related bacterial genera. Horizontal gene transfer (HGT) between bacteria is driven by three major processes: transformation (the uptake of free DNA), transduction (gene transfer mediated by bacteriophages) and conjugation (gene transfer by means of plasmids or conjugative and integrated elements).
    [Show full text]
  • Molecular Biology and Applied Genetics
    MOLECULAR BIOLOGY AND APPLIED GENETICS FOR Medical Laboratory Technology Students Upgraded Lecture Note Series Mohammed Awole Adem Jimma University MOLECULAR BIOLOGY AND APPLIED GENETICS For Medical Laboratory Technician Students Lecture Note Series Mohammed Awole Adem Upgraded - 2006 In collaboration with The Carter Center (EPHTI) and The Federal Democratic Republic of Ethiopia Ministry of Education and Ministry of Health Jimma University PREFACE The problem faced today in the learning and teaching of Applied Genetics and Molecular Biology for laboratory technologists in universities, colleges andhealth institutions primarily from the unavailability of textbooks that focus on the needs of Ethiopian students. This lecture note has been prepared with the primary aim of alleviating the problems encountered in the teaching of Medical Applied Genetics and Molecular Biology course and in minimizing discrepancies prevailing among the different teaching and training health institutions. It can also be used in teaching any introductory course on medical Applied Genetics and Molecular Biology and as a reference material. This lecture note is specifically designed for medical laboratory technologists, and includes only those areas of molecular cell biology and Applied Genetics relevant to degree-level understanding of modern laboratory technology. Since genetics is prerequisite course to molecular biology, the lecture note starts with Genetics i followed by Molecular Biology. It provides students with molecular background to enable them to understand and critically analyze recent advances in laboratory sciences. Finally, it contains a glossary, which summarizes important terminologies used in the text. Each chapter begins by specific learning objectives and at the end of each chapter review questions are also included.
    [Show full text]
  • The Art of Transfection (Poster / Pdf)
    TRANSDUCTION NON-VIRAL TRANSFECTION Transduction is the process of using vectors including retroviruses, lentiviruses, adenoviruses, PACKAGE DELIVERY: Chemical Chemical transfection adeno-associated viruses, or hybrids to deliver genetic payloads into cells. Generally, a plasmid transfection reagent containing mRNA carrying genes flanked by viral sequences is first transfected into a producer cell with other reagent virus-associated (packaging) plasmids. In the producer cells, virions form that contain the gene The Art of Transfection of interest. For safety, no plasmid used in the process contains all of the necessary sequences Inserting genetic material into mammalian and insect cells without killing them can be a challenge, CHEMICAL TRANSFECTION for virion formation, and only the plasmid carrying the gene of interest contains signals that but scientists have developed several ways to perform this intricate task. Transfection is the process of Functional proteins or allow it to be packaged into virions. Researchers then extract, purify, and use the virions from Complexation structural components released Chemical carriers represent the most straightforward and widespread tools for gene delivery the producer cells to insert DNA into other cells to stably or transiently express the DNA of introducing nucleic acids (plasmid DNA or messenger, short interfering, or micro RNA) into a cell. from cell or into cytoplasm experiments in mammalian cells. Chemical transfection experiments follow a simple workflow and interest. The transferred genetic material, which lacks viral genes, cannot generate new viruses. Researchers accomplish this with nonviral methods (chemical or physical transfection), or with viral provide high efficiency nucleic acid delivery for the most commonly used cells as well as many methods, commonly referred to as transduction.
    [Show full text]
  • Transducing Units (TU) Determination 4
    1. Bioinformatic and Gene expression 2. Viral based vectors 3. In vitro cell assays Transducing units (TU) determination 4. In vivo models Vectalys is an R&D company with a state-of-the-art technology platform for customized viral vector production. The company has developed a unique process enabling the production of high titer high purity lentiviral vectors for optimal knock-down or over expression in relevant models: primary and stem cells for target gene validation and specific tissue for animal models. Method of titration Titers of lentiviral vectors and wild type viruses critically depend on the method of titration used. The titer can be estimated by measuring the number of efficient viral particles or transducing units. In this case, the titer obtained only includes those particles which are able to transduce cells. > from cell binding to viral DNA integration, the titer corresponds to the number of integrated genomes and is quantified by qPCR. > from cell binding to protein expression, the titer corresponds to the number of transducing units and is quantified by FACS. This is a functional titer. Titers determined by qPCR are normalized with our standard GFP expressing vector titrated by FACS. So, all our qPCR or FACS titers are strictly comparable for any transgene. 1 2 3 4 5 Integrated qPCR genomes 6 1. Receptor recognition 2. Entry 7 3. Reverse transcription 4. Nuclear translocation Expression 5. Integration 8 FACS 6. Transcription of GFP 7. Splicing 8. Translation Both quantifications are performed after the transduction of cells and critically depend on cell and vector characteristics. > First, the cells must be readily permissive to all steps of viral transduction.
    [Show full text]
  • Genetic Exchange in Bacteria
    Systems Microbiology Monday Oct 16 - Ch 10 -Brock Genetic Exchange in Bacteria •• HomologousHomologous recombinationrecombination •• TransformationTransformation •• PlasmidsPlasmids andand conjugationconjugation •• TransposableTransposable elementselements •• TransductionTransduction (virus(virus mediatedmediated xchangexchange)) Gene exchange in bacteria • Transfer of DNA from one bacterium to another is a common means of gene dispersal. It has a big effect on bacterial evolution, and tremendous practical implications. For example, lateral transfer is responsible for the spread drug resistance determinants between bacterial species. • Three common mechanisms of lateral gene exchange : – Transformation (extracellular DNA uptake) – Conjugation (bacterial mating systems) – Transduction (viral mediated gene exchange) RecA mediated Homologous recombination Images removed due to copyright restrictions. See Figures 10-9 and 10-10 in Madigan, Michael, and John Martinko. Brock Biology of Microorganisms.11th ed. Upper Saddle River, NJ: Pearson Prentice Hall, 2006. ISBN: 0131443291. Gene exchange in bacteriaTransformation The Griffith Experiment S Injection Dead mouse; • Discovered by Griffith in yields S1 cells 1928 during the course of his Live "smooth" (encapsulated) studies of virulence in type 1 pneumococci (S1) Streptococcus pneumoniae. Dead S Live mouse Heat-killed S1 • S=smooth colony Live mouse morphotype Live "rough" (unencapsulated) pneumococci (R1 or R2) derived by subculture from S1 or S2, respectively • R=rough colony Dead mouse; yields S cells morphotype R1 + Dead S + 1 Live R1 Killed S1 R2 + Dead S + Dead mouse; yields S1 cells Live R2 Killed S2 Figure by MIT OCW. Gene exchange mechanisms in bacteria Transformation The Griffith Experiment Injection Dead mouse; S yields S cells Avery, MacLeod, and McCarthy (1944) 1 fractionation studies led to conclusion that Live "smooth" (encapsulated) type 1 pneumococci (S ) transformation principle is DNA.
    [Show full text]
  • Ability to Ferment Galactose) to Gal- Tmutants of Escherichia Coli K12 Has Been Described in a Previous Report (MORSE, LEDERBERG,And LEDERBERG1956)
    TRANSDUCTIONAL HETEROGENOTES IN ESCHERICHIA COLI' M. L. MORSE: ESTHER M. LEDERBERG, AND JOSHUA LEDERBERG Department of Genetics, University of Wisconsin, Madison Received April 12, 1956 HE transduction of the Gal+ factor (ability to ferment galactose) to Gal- Tmutants of Escherichia coli K12 has been described in a previous report (MORSE, LEDERBERG,and LEDERBERG1956). The galactose positive transduction clones were often found to be unstable and to throw off Gal- types about once per thousand divisions. We postulated that the transformed cells were heterogenotic (heterozygous for the transduced fragment), and that the instability was a result of segregation. This process has been studied in more detail with several non-allelic Gal- mutants. Since transduction genetics is a system analogous but not identical with sexual crossing, which also occurs in these strains, a distinctive terminology is a useful tool for integrating hypothesis and experiment. The following definitions are given for reference at this point. Their applications will be amplified in the experimental report. GLOSSARY AND SYMBOLS Genetic transduction-transfer of a genetic fragment from one cell to another. Exogenote-a chromosome fragment; usually relates to the donor in transduction. Endogenote-homologous part of the intact chromosome which corresponds to a given exogenote; usually relates to the recipient in transduction. Syngenote-(cf. synkaryon) a cell whose genetic complement includes an exo- genote (i.e. is hyperploid for a fragment). Heterogenote-(cf. heterozygote) a syngenote in which the exogenote and endo- genote differ in one or more markers. Homogenote-(cf. homozygote) a syngenote in which the exogenote and endo- genote carry the same marker.
    [Show full text]