Isolation and Characterization of Phages That Bypass the Requirement for RNA-Mediated Antitermination.

Introduction: The Y75N mutation in the zinc-binding domain of the β' subunit of RNA polymerase blocks the RNA-based mechanism of transcription antitermination utilized by bacteriophage HK022.

Materials And Methods: Mutant phages that overcome the block imposed by the Y75N mutation are described. These phages, designated "" (overcomes ), carry mutations that create new promoters.

Structural basis of transcriptional regulation by a nascent RNA element, HK022 putRNA.

Transcription, in which RNA polymerases (RNAPs) produce RNA from DNA, is the first step of gene expression. As such, it is highly regulated either by trans-elements like protein factors and/or by cis-elements like specific sequences on the DNA. Lambdoid phage HK022 contains a cis-element, put, which suppresses pausing and termination during transcription of the early phage genes.

Intrathymic differentiation of natural antibody-producing plasma cells in human neonates.

The thymus is a central lymphoid organ primarily responsible for the development of T cells. A small proportion of B cells, however, also reside in the thymus to assist negative selection of self-reactive T cells. Here we show that the thymus of human neonates contains a consistent contingent of CD138 plasma cells, producing all classes and subclasses of immunoglobulins with the exception of IgD.

Arthroscopic removal of a solitary osteochondroma interfering with the podotrochlear apparatus in a foal.

Objective: To report the diagnostics, surgical treatment, and outcome of a juvenile foal with solitary osteochondroma (SO) interfering with the podotrochlear apparatus.

Study Design: Case report ANIMAL: One 36-day-old Arabian colt.

Methods: Clinical, radiographic, ultrasonographic, computed tomographic, and histopathologic examinations were required to characterize and treat an SO located at the palmar aspect of the diaphysis of the second phalanx of the left forelimb.

Whole genome sequencing identifies an allele responsible for clear vs. turbid plaque morphology in a Mycobacteriophage.

Background: Whole genome sequencing promises to revolutionize our ability to link genotypic and phenotypic variation in a wide range of model and non-model species.

Results: Here we describe the isolation and characterization of a novel mycobacteriophage named BGlluviae that grows on Mycobacterium smegmatis mc155. BGlluviae normally produces turbid plaques but a spontaneous clear plaque was also recovered.

Fc Receptor-Like 6 (FCRL6) Discloses Progenitor B Cell Heterogeneity That Correlates With Pre-BCR Dependent and Independent Pathways of Natural Antibody Selection.

B-1a cells produce "natural" antibodies (Abs) to neutralize pathogens and clear neo self-antigens, but the fundamental selection mechanisms that shape their polyreactive repertoires are poorly understood. Here, we identified a B cell progenitor subset defined by Fc receptor-like 6 (FCRL6) expression, harboring innate-like defense, migration, and differentiation properties conducive for natural Ab generation. Compared to FCRL6 pro B cells, the repressed mitotic, DNA damage repair, and signaling activity of FCRL6 progenitors, yielded V repertoires with biased distal segment accessibility, constrained diversity, and hydrophobic and charged CDR-H3 sequences.

Sequence Analysis of a Hybrid HK022/λ Bacteriophage and the Precise Identification of the λ and λ Deletion Endpoints.

Bacteriophage O276 is a laboratory-generated hybrid that carries the immunity region of bacteriophage HK022 and all remaining genes from phage λ. Its construction was instrumental in the discovery of RNA-mediated antitermination, an intriguing alternative to the protein-mediated mechanism of transcription antitermination found in most lambdoid phages.

Tales of diversity: Genomic and morphological characteristics of forty-six Arthrobacter phages.

The vast bacteriophage population harbors an immense reservoir of genetic information. Almost 2000 phage genomes have been sequenced from phages infecting hosts in the phylum Actinobacteria, and analysis of these genomes reveals substantial diversity, pervasive mosaicism, and novel mechanisms for phage replication and lysogeny. Here, we describe the isolation and genomic characterization of 46 phages from environmental samples at various geographic locations in the U.

Comparative genomics of Cluster O mycobacteriophages.

Mycobacteriophages--viruses of mycobacterial hosts--are genetically diverse but morphologically are all classified in the Caudovirales with double-stranded DNA and tails. We describe here a group of five closely related mycobacteriophages--Corndog, Catdawg, Dylan, Firecracker, and YungJamal--designated as Cluster O with long flexible tails but with unusual prolate capsids. Proteomic analysis of phage Corndog particles, Catdawg particles, and Corndog-infected cells confirms expression of half of the predicted gene products and indicates a non-canonical mechanism for translation of the Corndog tape measure protein.

A broadly implementable research course in phage discovery and genomics for first-year undergraduate students.

Unlabelled: Engaging large numbers of undergraduates in authentic scientific discovery is desirable but difficult to achieve. We have developed a general model in which faculty and teaching assistants from diverse academic institutions are trained to teach a research course for first-year undergraduate students focused on bacteriophage discovery and genomics. The course is situated within a broader scientific context aimed at understanding viral diversity, such that faculty and students are collaborators with established researchers in the field.

Inhibition of a transcriptional pause by RNA anchoring to RNA polymerase.

We describe a mechanism by which nascent RNA inhibits transcriptional pausing. PutL RNA of bacteriophage HK022 suppresses transcription termination at downstream terminators and pausing within a nearby U-rich sequence. In vitro transcription and footprinting assays reveal that this pausing results from backtracking of RNA polymerase and that binding of nascent putL RNA to polymerase limits backtracking by restricting re-entry of the transcript into the RNA exit channel.

Characterization of Salmonella bacteriophages isolated from swine lagoon effluent.

Four Salmonella bacteriophages that had been originally isolated from swine manure lagoons were characterized and compared to each other and to well-known Salmonella phages P22 and Felix 01. Host ranges of the lagoon phages were similar to each other in spot tests on reference strains of Salmonella, but differed slightly from each other on a panel of Salmonella lagoon strains. In single-step growth at 35 degrees C the lagoon phages had latent periods of 15 to 20 min and burst sizes from 100 to 230.

Protection of antiterminator RNA by the transcript elongation complex.

Nascent transcripts encoded by the putL and putR sites of phage HK022 bind the transcript elongation complex and suppress termination at downstream transcription terminators. We report here that the chemical stability of putL RNA is considerably greater than that of the typical Escherichia coli message because the elongation complex protects this RNA from degradation. When binding to the elongation complex was prevented by mutation of either putL or RNA polymerase, RNA stability decreased more than 50-fold.

A conserved zinc binding domain in the largest subunit of DNA-dependent RNA polymerase modulates intrinsic transcription termination and antitermination but does not stabilize the elongation complex.

An evolutionarily conserved zinc-binding motif is found close to the amino terminus of the largest subunits of DNA-dependent RNA polymerases from bacteria, archaea, and eukaryotes. In bacterial RNA polymerase, this motif, the zinc binding domain, has been implicated in protein-DNA interactions that stabilize the transcription elongation complex and that occur downstream of the catalytic center. Here, we show that this view is incorrect, and instead, the zinc binding domain interacts with product RNA located upstream of the catalytic center and the RNA-DNA hybrid, a view consistent with structural studies of the elongation complex.

Suppression of factor-dependent transcription termination by antiterminator RNA.

Nascent transcripts of the phage HK022 put sites modify the transcription elongation complex so that it terminates less efficiently at intrinsic transcription terminators and accelerates through pause sites. We show here that the modification also suppresses termination in vivo at two factor-dependent terminators, one that depends on the bacterial Rho protein and a second that depends on the HK022-encoded Nun protein. Suppression was efficient when the termination factors were present at physiological levels, but an increase in the intracellular concentration of Nun increased termination both in the presence and absence of put.

Sequence-specific interaction of nascent antiterminator RNA with the zinc-finger motif of Escherichia coli RNA polymerase.

The N-terminal Zn-finger motif of the beta' subunit of RNA polymerase contains two pairs of invariant cysteines flanking a moderately well-conserved segment of 13 amino acids that is rich in basic residues. Previous work showed that replacement of certain Zn-finger residues prevented transcription antitermination in response to phage HK022 put sites. Nascent put RNA binds to and modifies transcribing polymerase, so that it becomes resistant to termination.