We report here the complete genome sequences of 44 phages infecting Arthrobacter sp. strain ATCC 21022. These phages have double-stranded DNA genomes with sizes ranging from 15,680 to 70,707 bp and G+C contents from 45.1% to 68.5%. All three tail types (belonging to the families Siphoviridae, Myoviridae, and Podoviridae) are represented.

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.S. infecting a single Arthrobacter sp. strain. These phages include representatives of all three virion morphologies, and Jasmine is the first sequenced podovirus of an actinobacterial host. The phages also span considerable sequence diversity, and can be grouped into 10 clusters according to their nucleotide diversity, and two singletons each with no close relatives. However, the clusters/singletons appear to be genomically well separated from each other, and relatively few genes are shared between clusters. Genome size varies from among the smallest of siphoviral phages (15,319 bp) to over 70 kbp, and G+C contents range from 45-68%, compared to 63.4% for the host genome. Although temperate phages are common among other actinobacterial hosts, these Arthrobacter phages are primarily lytic, and only the singleton Galaxy is likely temperate.

The goal of this project was to develop a "normalized" extraction procedure to be used in conjunction with previously validated 3μL fast PCR reactions (42-51min utilizing KAPA2G™ Fast Multiplex PCR Kit) and alternative capillary electrophoresis (24-28min injection using POP-6™ Polymer and a 22cm array). This was the final phase of a workflow overhaul for the database unit at Cellmark Forensics to achieve a substantial reduction in processing time for forensic DNA database samples without incurring significant added costs and/or the need for new instrumentation, while still generating high quality STR profiles. Extraction normalization aimed to consistently yield a small range of DNA concentrations, thereby eliminating the need for sample quantification and dilution. This was specifically achieved using the ChargeSwitch® Forensic DNA Purification Kit and a reduction in extraction bead quantity, thereby forcing an increase in bead binding efficiency. Following development of this extraction procedure, an evaluation ensued to assess the combination of normalized extraction, 3μL fast PCR (with PowerPlex 16 HS, Identifiler Plus and Identifiler primer sets), and alternative CE detection - further referred to as new "first pass" procedures. These modifications resulted in a 37% reduction in processing time and were evaluated via an in depth validation, from which nearly 2000 STR profiles were generated, of which 554 profiles from 77 swab donors and 210 profiles from 35 buccal collector donors specifically arose from the new first pass procedures. This validation demonstrates the robustness of these processes for buccal swabs and Buccal DNA Collectors™ using the three primer sets evaluated and their ability to generate high quality STR profiles with 95-99% and 88-91% pass rates, respectively.

The goal of this project was to reduce capillary electrophoresis detection time on a 3130xl Genetic Analyzer for amplification product obtained from 4-dye and 5-dye STR amplification kits while still generating high quality STR profiles. This was accomplished by utilizing a more viscous polymer (POP-6™) and a shorter array (22 cm) than that which are typically used (POP-4(®) polymer and a 36 cm array) for human identification purposes. Spatial calibration and detection run modules were modified in response to the use of this polymer/array combination and to reduce detection time. Alternative detection resulted in 24-28 min run times, as compared to ∼45 min using traditional POP-4(®)/36 cm detection methods. POP-6™/22 cm detection run modules were validated for use with 4-dye Promega STR kits (e.g., PowerPlex(®) 16 and PowerPlex(®) 16HS) and 5-dye Life Technologies kits (e.g., Identifiler(®) and Identifiler(®) Plus). Three hundred ninety-five samples, controls and allelic ladders were used for the validation studies, which consisted of a comparison of alternative POP-6™/22 cm detection to traditional POP-4(®)/36 cm (including reproducibility/concordance of allele calls, resolution, ILS sizing quality, peak height and pass rates), a sizing study (precision and accuracy) and a sensitivity study to obtain a usable range of injection times. Compared to traditional POP-4(®)/36 cm detection, alternative detection resulted in 100% reproducible and concordant alleles, the ability to achieve one base resolution, slightly reduced ILS sizing quality, slightly reduced peak height and statistically similar pass rates (α=0.05). It should be noted that alternative detection offered improved resolution over that of traditional for amplicons less than ∼200 b, but had reduced resolution for products greater than ∼200 b. Additionally, alternative detection yielded acceptable precision and accuracy of sizing using Life Technologies criteria (<0.15 standard deviation of allele sizing and ±0.5b sizing differences for the same allele) and usable injection parameters of 2 kV 4-15s (compared to 3 kV 10s for traditional). The run modules developed and validated for 4-dye and 5-dye STR kits using POP-6™ polymer on a 22 cm array offer a tremendous reduction in detection time (∼40%) while still generating high quality STR profiles.

Amela and Verse are two Streptomyces phages isolated by enrichment on Streptomyces venezuelae (ATCC 10712) from two different soil samples. Amela has a genome length of 49,452, with 75 genes. Verse has a genome length of 49,483, with 75 genes. Both belong to the BD3 subcluster of Actinobacteriophage.

Paper describes the Phage Hunters Advancing Genomics and Evolutionary Science (PHAGES) program from the Howard Hughes Medical Institute’s Science Education Alliance as implemented at the University of North Texas.

Five newly isolated mycobacteriophages--Angelica, CrimD, Adephagia, Anaya, and Pixie--have similar genomic architectures to mycobacteriophage TM4, a previously characterized phage that is widely used in mycobacterial genetics. The nucleotide sequence similarities warrant grouping these into Cluster K, with subdivision into three subclusters: K1, K2, and K3. Although the overall genome architectures of these phages are similar, TM4 appears to have lost at least two segments of its genome, a central region containing the integration apparatus, and a segment at the right end. This suggests that TM4 is a recent derivative of a temperate parent, resolving a long-standing conundrum about its biology, in that it was reportedly recovered from a lysogenic strain of Mycobacterium avium, but it is not capable of forming lysogens in any mycobacterial host. Like TM4, all of the Cluster K phages infect both fast- and slow-growing mycobacteria, and all of them--with the exception of TM4--form stable lysogens in both Mycobacterium smegmatis and Mycobacterium tuberculosis; immunity assays show that all five of these phages share the same immune specificity. TM4 infects these lysogens suggesting that it was either derived from a heteroimmune temperate parent or that it has acquired a virulent phenotype. We have also characterized a widely-used conditionally replicating derivative of TM4 and identified mutations conferring the temperature-sensitive phenotype. All of the Cluster K phages contain a series of well conserved 13 bp repeats associated with the translation initiation sites of a subset of the genes; approximately one half of these contain an additional sequence feature composed of imperfectly conserved 17 bp inverted repeats separated by a variable spacer. The K1 phages integrate into the host tmRNA and the Cluster K phages represent potential new tools for the genetics of M. tuberculosis and related species.

This study constitutes a first approach to evaluate the use of genetic information and relatedness estimators in the investigation of questions related to mating system and parentage in ex situ and in situ populations of the Roseate Spoonbill. We assessed the parentage assignments in 17 supposed families from US captive populations and investigated the genetic relationships among 67 nestlings sampled within 28 nests in Brazilian natural breeding colonies. Estimations of genetic relatedness values, hypothesis testing methods, simulations and maximum likelihood approaches were performed on data from four microsatellite loci. Parentage was confirmed in 61.5% of the registered parent-offspring relationships at zoo parks. Inconsistencies in assignments were investigated and the likely parents were identified for most of the hatchlings. Matings among relatives, not previously noticed based on behavioral observations, were identified by the use of genetic analyses. In natural populations, 33% of the sampled dyads were confidently classified as full-sibs. Above 25% of the analyzed dyads were unrelated, indicating that more than one parent-pair may have been responsible for the progeny. Our results demonstrate that genetic information can augment the precision in parentage assignment in captive Roseate Spoonbill populations, and this approach can contribute to their management and conservation. Results obtained using three different methodologies are concordant and point to the existence of a mating system other than monogamy for this species in the wild. The approaches implemented in this study can be applied to other waterbird species in which capture of adults is difficult.

This article documents the addition of 238 microsatellite marker loci and 72 pairs of Single Nucleotide Polymorphism (SNP) sequencing primers to the Molecular Ecology Resources Database. Loci were developed for the following species: Adelges tsugae, Artemisia tridentata, Astroides calycularis, Azorella selago, Botryllus schlosseri, Botrylloides violaceus, Cardiocrinum cordatum var. glehnii, Campylopterus curvipennis, Colocasia esculenta, Cynomys ludovicianus, Cynomys leucurus, Cynomys gunnisoni, Epinephelus coioides, Eunicella singularis, Gammarus pulex, Homoeosoma nebulella, Hyla squirella, Lateolabrax japonicus, Mastomys erythroleucus, Pararge aegeria, Pardosa sierra, Phoenicopterus ruber ruber and Silene latifolia. These loci were cross-tested on the following species: Adelges abietis, Adelges cooleyi, Adelges piceae, Pineus pini, Pineus strobi, Tubastrea micrantha, three other Tubastrea species, Botrylloides fuscus, Botrylloides simodensis, Campylopterus hemileucurus, Campylopterus rufus, Campylopterus largipennis, Campylopterus villaviscensio, Phaethornis longuemareus, Florisuga mellivora, Lampornis amethystinus, Amazilia cyanocephala, Archilochus colubris, Epinephelus lanceolatus, Epinephelus fuscoguttatus, Symbiodinium temperate-A clade, Gammarus fossarum, Gammarus roeselii, Dikerogammarus villosus and Limnomysis benedeni. This article also documents the addition of 72 sequencing primer pairs and 52 allele specific primers for Neophocaena phocaenoides.

Six microsatellite loci were isolated and characterized to assess genetic diversity and determine parentage in three captive roseate spoonbill (Ajaia ajaja) populations. Analysis of 61 individuals from three zoological parks and one wild population at five polymorphic loci revealed an average of six alleles per locus and expected heterozygosities from 59% to 81% (average 70%). Since spoonbills do not exhibit obvious sexual dimorphism, Aaju4, which exhibited ZW-specific alleles, was exceptionally useful for sex identification. These loci will be valuable tools for investigating genetic diversity and documenting patterns of parentage in captive roseate spoonbill populations.

Ethanol has recently been reported to potentiate the accumulation of poly(ADP-ribose) in SV40 transformed mouse 3T3 cells following hyperthermia or N-methyl-N′-nitro-N-nitroso-guanidine (MNNG) treatment [1,2]. We became interested in understanding the mechanism by which ethanol potentiates polymer accumulation and how polymer synthesis might be affected by cellular redox metabolism in ethanol-treated cells and tissues. Figure 1 shows the effect of ethanol on the time course of poly(ADP-ribose) synthesis in nucleotide permeable cells in the presence and absence of very low doses of DNA damage. Polymer synthesis in undamaged control cells was essentially uneffected by ethanol up to 5%. This was in marked contrast to lightly damaged cells in which polymer synthesis was stimulated 3.5-fold by 1% ethanol. Higher ethanol concentrations also stimulated polymer synthesis, but were less effective than 1%. Control experiments (data not shown) indicated that this was not due to inactivation of DNAse I or altered polymer turnover under these conditions. In order to determine exactly how ethanol stimulates polymer synthesis, we conducted a series of kinetic experiments with partially purified enzyme and defined DNAs. We used restriction endonucleases to generate DNA fragments of pBR322 with flush ends and 5′-phosphates (Hae III fragments), flush ended-fragments with 5′-hydroxyls (alkaline phosphatase treated-Hae III fragments), and fragments with protruding 5′-phosphates (Hpa II fragments). Flush-ended fragments have been shown to be more effective stimulators of synthetase activity than fragments with protruding ends [5].

Poly(ADP-ribose) polymerase is a chromatin-associated enzyme which, in the presence of fragmented DNA, assembles branched homopolymers from the ADP-ribose moiety of NAD (reviewed in [1]). Fragmented DNA is an essential activator of the polymerase and is not modified by the reaction. This report presents data related to the kinetic mechanism of DNA activation of the poly(ADP-ribose) polymerase.