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Cosmid-Based System for Transient Expression and Absolute Off-to-On Transcriptional Control of Escherichia coli Genes. John E. Cronan, 2003.Cosmids are plasmids that contain the phage  sequences (cos) required for packaging of the phage DNA into the virion . Induction of a
prophage in an Escherichia coli strain carrying a cosmid results in lysates containing phage particles that are filled with cosmid DNA . However, the lysates also contain a large excess of infectious phage particles which complicate use of the packaged cosmids . I report that cosmids packaged by induction of a strain carrying a prophage with an altered cos region results in lysates containing very high levels (>1010/ml) of particles that contain cosmid DNA together with very few infectious phage particles . These lysates can be used to transduce cosmid DNA into all of the cells of a growing culture with minimal physiological disturbance . When the cosmid carries a conditionally active origin of replication, transductional introduction of the cosmid under nonreplicative conditions provides a system of transient expression . Transient expression has been used to make a recA strain temporarily recombination proficient and to temporarily introduce a site-specific recombinase . Transductional introduction of a cosmid also allows absolute off-to-on transcriptional control of nonessential genes . Two examples are given showing that when a strain carrying a null mutation in the gene of interest is transduced with a packaged cosmid carrying a functional copy of that gene, the expression of the gene rapidly goes from absolutely off to high-level expression . Additional possible uses of in vivo-packaged cosmids are proposed .
Formation of Pseudo-Terminal Restriction Fragments, a PCR-Related Bias Affecting Terminal Restriction Fragment Length Polymorphism Analysis of Microbial Community Structure. Markus Egert, 2003.Terminal restriction fragment length polymorphism (T-RFLP) analysis of PCR-amplified genes is a widely used fingerprinting technique in molecular microbial ecology . In this study, we show that besides expected terminal restriction fragments (T-RFs), additional secondary T-RFs occur in T-RFLP analysis of amplicons from cloned 16S rRNA genes at high frequency . A total of 50% of 109 bacterial and 78% of 68 archaeal clones from the guts of cetoniid beetle larvae, using MspI and AluI as restriction enzymes, respectively, were affected by the presence of these additional T-RFs . These peaks were called "pseudo-T-RFs" since they can be detected as terminal fluorescently labeled fragments in T-RFLP analysis but do not represent the primary terminal restriction site as indicated by sequence data analysis . Pseudo-T-RFs were also identified in T-RFLP profiles of pure culture and environmental DNA extracts . Digestion of amplicons with the single-strand-specific mung bean nuclease prior to T-RFLP analysis completely eliminated pseudo-T-RFs . This clearly indicates that single-stranded amplicons are the reason for the formation of pseudo-T-RFs, most probably because single-stranded restriction sites cannot be cleaved by restriction enzymes . The strong dependence of pseudo-T-RF formation on the number of cycles used in PCR indicates that (partly) single-stranded amplicons can be formed during amplification of 16S rRNA genes . In a model, we explain how transiently formed secondary structures of single-stranded amplicons may render single-stranded amplicons accessible to restriction enzymes . The occurrence of pseudo-T-RFs has consequences for the interpretation of T-RFLP profiles from environmental samples, since pseudo-T-RFs may lead to an overestimation of microbial diversity . Therefore, it is advisable to establish 16S rRNA gene sequence clone libraries in parallel with T-RFLP analysis from the same sample and to check clones for their in vitro digestion T-RF pattern to facilitate the detection of pseudo-T-RFs . Similarity of Tetracycline Resistance Genes Isolated from Fish Farm Bacteria to Those from Clinical Isolates. Manabu Furushita, 2003.Tetracycline-resistant (Tetr) bacteria were isolated from fishes collected at three different fish farms in the southern part of Japan in August and September 2000 . Of the 66 Tetr gram-negative strains, 29 were identified as carrying tetB only . Four carried tetY, and another four carried tetD . Three strains carried tetC, two strains carried tetB and tetY, and one strain carried tetC and tetG . Sequence analyses indicated the identity in Tetr genes between the fish farm bacteria and clinical bacteria: 99.3 to 99.9% for tetB, 98.2 to 100% for tetC, 99.7 to 100% for tetD, 92.0 to 96.2% for tetG, and 97.1 to 100% for tetY . Eleven of the Tetr strains transferred Tetr genes by conjugation to Escherichia coli HB-101 . All transconjugants were resistant to tetracycline, oxycycline, doxycycline, and minocycline . The donors included strains of Photobacterium, Vibrio, Pseudomonas, Alteromonas, Citrobacter, and Salmonella spp., and they transferred tetB, tetY, or tetD to the recipients . Because NaCl enhanced their growth, these Tetr strains, except for the Pseudomonas, Citrobacter, and Salmonella strains, were recognized as marine bacteria . Our results suggest that tet genes from fish farm bacteria have the same origins as those from clinical strains .
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