Draft Macronuclear Genome Sequence of the Ruminal Ciliate Entodinium caudatum.

The transcriptionally active macronucleus of a ruminal ciliate, Entodinium caudatum MZG-1, was sequenced using the Illumina MiSeq and Oxford Nanopore MinION platforms. This is the first draft macronuclear genome sequence of a ruminal protozoon, and the genomic information will provide useful insight into the metabolism, physiology, and ecology of ruminal ciliates.

Restriction site associated DNA (RAD) for de novo sequencing and marker discovery in sugarcane borer, Diatraea saccharalis Fab. (Lepidoptera: Crambidae).

We present the development of a genomic library using RADseq (restriction site associated DNA sequencing) protocol for marker discovery that can be applied on evolutionary studies of the sugarcane borer Diatraea saccharalis, an important South American insect pest. A RADtag protocol combined with Illumina paired-end sequencing allowed de novo discovery of 12 811 SNPs and a high-quality assembly of 122.8M paired-end reads from six individuals, representing 40 Gb of sequencing data. Approximately 1.7 Mb of the sugarcane borer genome distributed over 5289 minicontigs were obtained upon assembly of second reads from first reads RADtag loci where at least one SNP was discovered and genotyped. Minicontig lengths ranged from 200 to 611 bp and were used for functional annotation and microsatellite discovery. These markers will be used in future studies to understand gene flow and adaptation to host plants and control tactics.

First Report of Leek yellow stripe virus in Garlic in Ohio.

Leek yellow stripe virus (LYSV), genus Potyvirus, family Potyviridae, infects a wide range of Allium species worldwide. LYSV is one of several viruses that chronically infect garlic, Allium sativum L. The garlic virus complex, which includes LYSV, Onion yellow dwarf virus, and Garlic common latent virus, is perpetuated by asexual propagation (4) and is transmitted to clean planting material by aphids (3). This virus complex can reduce garlic bulb weight by nearly three quarters (2), and LYSV-only infections can result in approximately a one-quarter reduction in bulb weight (2). Garlic is grown as a small-scale, specialty crop in Ohio. During late May and early June 2013, garlic plants with virus-like symptoms were collected from Medina, Holmes, and Wayne counties, Ohio. Plants exhibited chlorotic streaking, foliar dieback, dwarfing, small bulbs, and cylindrical bulbs that failed to differentiate into cloves. Incidence of affected plants in the fields was up to 5% and all fields had early season aphid infestations. Flexuous rods were observed in TEM micrographs of plant sap from symptomatic leaves. Five symptomatic plants and six asymptomatic plants (from fields with symptomatic plants) were evaluated for LYSV by DAS-ELISA (Agdia, Inc., Elkhart, IN). Reverse transcriptase (RT)-PCR with LYSV-specific primers LYSV-WA and LYSV-WAR (3) was performed with cDNA generated by the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA). Both foliar and bulb tissues were tested using both detection methods. Forty percent of symptomatic plants and 67% of asymptomatic plants tested positive for LYSV with both ELISA and RT-PCR. LYSV was detected in both foliar and bulb tissues, including both tissues from asymptomatic plants. Five PCR amplicons generated from both foliar and bulb tissue were sequenced and shown to share 96 to 98% maximum identity with an LYSV polyprotein gene accession in GenBank (AY842136). This provided additional support that the detected virus was LYSV. LYSV was initially difficult to detect in Ohio fields due to low disease incidence and subtle symptom development. Use of virus-tested garlic bulbs can improve yield for several years, even following viral reinfection by aphids, compared to growing garlic from chronically infected bulbs (1). However, many growers routinely save bulbs from year to year and lack access to or knowledge of virus-tested sources of garlic bulbs. Conducive conditions, chronic infections, or co-infections with other viruses enhance the severity of symptoms and yield loss (2). LYSV has previously been reported in garlic producing regions of the northwestern United States (3), and to our knowledge, this is the first report of LYSV in garlic in Ohio. References: (1) V. Conci et al. Plant Dis. 87:1411, 2003. (2) P. Lunello et al. Plant Dis. 91:153, 2008. (3) H. Pappu et al. Plant Health Progress 10, 2008. (4) L. Parrano et al. Phytopathol. Mediterr. 51:549, 2012.

Complete sequence and development of a full-length infectious clone of an Ohio isolate of Maize dwarf mosaic virus (MDMV).

Maize dwarf mosaic virus (MDMV) is an important and widespread aphid-transmitted virus of maize. It is a member of the genus Potyvirus in the family Potyviridae with a monopartite (+) ssRNA genome. Here we report the complete genome sequence and construction and testing of infectious clones of an Ohio isolate of MDMV. Full-length MDMV cDNA was cloned into the vector pSPORT. Full-length cDNA PCR-amplified from the vector constructs were used as template for in vitro transcription, and transcripts were inoculated to maize seeds by vascular puncture inoculation. Plants inoculated by this procedure showed symptoms typical of MDMV infection, and infection was confirmed by RT-PCR and mechanical transmission to new plants.

Binding patterns of human norovirus-like particles to buccal and intestinal tissues of gnotobiotic pigs in relation to A/H histo-blood group antigen expression.

Histo-blood group antigen (HBGA) phenotypes have been associated with susceptibility to human noroviruses (HuNoVs). Our aims were: (i) to determine the patterns of A/H HBGA expression in buccal and intestinal tissues of gnotobiotic (Gn) pigs; (ii) to determine if virus-like particles (VLPs) of HuNoV genogroup I (GI) and GII bind to A- or H-type tissues; (iii) to compare A/H expression and VLP binding patterns and confirm their binding specificities by blocking assays; (iv) to develop a hemagglutination inhibition test using buccal cells from live pigs to determine the Gn pig's A/H phenotype and to match viral strains with previously determined HuNoV VLP binding specificities; and (v) to determine the A/H phenotypes and compare these data to the infection outcomes of a previous study of 65 Gn pigs inoculated with HuNoV GII/4 strain HS66 and expressing A and/or H or neither antigen on their buccal and intestinal tissues (S. Cheetham, M. Souza, T. Meulia, S. Grimes, M. G. Han, and L. J. Saif, J. Virol. 80:10372-10381, 2006). We found that the HuNoV GI/GII VLPs of different clusters bound to tissues from four pigs tested (two A+ and two H+). The GI/1 and GII/4 VLPs bound extensively to duodenal and buccal tissues from either A+ or H+ pigs, but surprisingly, GII/1 and GII/3 VLPs bound minimally to the duodenum of an A+ pig. The VLP binding was partially inhibited by A-, H1-, or H2-specific monoclonal antibodies, but was completely blocked by porcine mucin. Comparing the A/H phenotypes of 65 HS66-inoculated Gn pigs from our previous study, we found that significantly more A+ and H(+) pigs (51%) than non-A+ and non-H+ pigs (12.5%) shed virus. From the 22 convalescent pigs, significantly more A+ or H+ pigs (66%) than non-A+ or H+ pigs (25%) seroconverted.

Identification and Host Relations of Turnip ringspot virus, A Novel Comovirus from Ohio.

Viruslike chlorotic ring spot symptoms and line patterns of unknown origin were observed on a greenhouse-grown turnip plant. The suspected virus was mechanically transmissible to plants in the Brassicaceae. Electron microscopic analysis revealed icosahedral particles approximately 28 nm in diameter. Reverse transcriptase-polymerase chain reaction (RT-PCR) analyses suggested that the pathogen is a comovirus, an observation that was confirmed by analysis of portions of the genomic sequence. This virus was provisionally named Turnip ringspot virus (TuRSV). Based on the RNA 1 sequence, TuRSV is most similar to Radish mosaic virus, another pathogen that infects members of the Brassicaceae. Arabidopsis thaliana is susceptible to TuRSV, and 12 out of the 23 ecotypes studied showed symptoms when inoculated with the virus. TuRSV induced a variety of responses on ecotypes from death to no infection. Some ecotypes showed one or two rounds of symptom display followed by recovery when inoculated with TuRSV. About half of the ecotypes (11/23) analyzed showed no symptoms when inoculated with TuRSV. Col-0 plants showed no symptoms, and infectious virus was not recovered from systemic leaves, although it could be detected by RT-PCR. Col-0 plants harboring mutations impairing the ethylene, jasmonic acid, or salicylic acid signaling pathways did not show symptoms when inoculated with TuRSV.

Maize fine streak virus, a New Leafhopper-Transmitted Rhabdovirus.

ABSTRACT A previously uncharacterized virus was isolated from fall-planted sweet corn (Zea mays L., Syngenta GSS 0966) leaves showing fine chlorotic streaks. Symptomatic plants were negative in enzyme-linked immunosorbent assay against many maize viruses, but reacted weakly with antisera to Sorghum stunt mosaic virus suggesting a distant relationship between the viruses. The virus was readily transmitted by vascular puncture inoculation (VPI), but not by leaf-rub inoculation. Symptoms on maize included dwarfing and fine chlorotic streaks along intermediate and small veins that developed 12 to 17 days post-VPI. The isolated virus was bacilliform (231 +/- 5 nm long and 71 +/- 2 nm wide), with a knobby surface, and obvious helical structure typical of rhabdovirus morphology. Nucleorhabdovirus virions were observed by transmission electron microscopy of infected maize leaf tissue sections. Proteins unique to infected plants were observed in extracts of infected leaves, and the isolated virion contained three proteins with molecular masses 82 +/- 2, 50 +/- 3, and 32 +/- 2 kDa. Preliminary sequence analysis indicated the virus had similarity to members of the family Rhabdoviridae. The virus was transmitted by Graminella nigrifrons under persistent conditions. The data indicate the virus, provisionally designated Maize fine streak virus, is a new species in the genus Nucleorhabdovirus.

Precise detection and tracing of Trichoderma hamatum 382 in compost-amended potting mixes by using molecular markers.

Randomly amplified polymorphic DNA (RAPD) analysis and the PCR assay were used in combination with dilution plating on a semiselective medium to detect and enumerate propagules of Trichoderma hamatum 382, a biocontrol agent utilized in compost-amended mixes. Distinct and reproducible fingerprints were obtained upon amplification of purified genomic DNA of T. hamatum 382 with the random primers OPE-16, OPH-19, and OPH-20. Three amplified DNA fragments of 0.35 (OPE-16(0.35)), 0.6 (OPH-19(0.6)), and 0.65 (OPH-20(0.65)) kb were diagnostic for T. hamatum 382, clearly distinguishing it from 53 isolates of four other Trichoderma spp. tested. Some isolates of T. hamatum shared these low-molecular-weight fragments with T. hamatum 382. However, RAPD analysis of isolates of T. hamatum with all three random primers used in consecutive PCR tests distinguished T. hamatum 382 from other isolates of T. hamatum. These three RAPD amplicons were cloned and sequenced, and pairs of oligonucleotide primers for each cloned fragment were designed. Use of the primers in the PCR assay resulted in the amplification of DNA fragments of the same size as the cloned RAPD fragments from genomic DNA of T. hamatum 382. A combination of dilution plating on a semiselective medium for Trichoderma spp. and PCR, with the RAPD primers OPH-19, OPE-16, and OPH-20 or the three sequence-characterized primers, was used successfully to verify the presence of T. hamatum 382 propagules in nine different soil, compost, and potting mix samples. All 23 Trichoderma isolates recovered on semiselective medium from commercial potting mixes fortified with T. hamatum 382 were identified as T. hamatum 382, whereas 274 Trichoderma isolates recovered from the other nine samples were negative in the PCR assay. Thus, this highly specific combination of techniques allowed detection and enumeration of propagules of T. hamatum 382 in fortified compost-amended potting mixes. Sequence-characterized amplified region markers also facilitated the development of a very simple procedure to amplify DNA of T. hamatum 382 directly from fortified compost-amended potting mixes.

Common mechanisms for the control of eukaryotic transcriptional elongation.

Regulation of transcriptional elongation is emerging as an important control mechanism for eukaryotic gene expression. In this essay, we review the basis of the current view of the regulation of elongation in the human c-myc gene and discuss similarities in elongation control among the c-myc, Drosophila hsp70 and the HIV-1 genes. Based upon these similarities, we propose a model for control of expression of these genes at the elongation phase of transcription. This model suggests that distinct promoter elements direct the assembly of RNA polymerase II transcription complexes which differ in their elongation efficiency.

Distinct properties of c-myc transcriptional elongation are revealed in Xenopus oocytes and mammalian cells and by template titration, 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), and promoter mutagenesis.

A block to c-myc transcription elongation has been observed in Xenopus oocytes and mammalian cells. Here, we show that the distribution of RNA polymerase II transcription complexes in the c-myc promoter proximal region in Xenopus oocytes is different from that observed previously in mammalian cells. Thus, there are major differences in the c-myc elongation block observed in the two systems. In addition, as first reported for a Xenopus tubulin gene (K. M. Middleton and G. T. Morgan, Mol. Cell. Biol. 10:727-735, 1990). c-myc template titration experiments reveal the existence of two classes of RNA polymerase II transcription complexes in oocytes: one (at low template concentration) that is capable of reading through downstream sites of premature termination, and another (high template concentration) that does not. We show that these classes of polymerases are distinct from those previously identified by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), which distinguishes transcription complexes on the basis of transcribed distance, rather than on the basis of differential elongation through sites of premature termination. We also show that mutations that affect the efficiency of initiation of transcription from the c-myc P2 promoter can influence premature termination by at least two mechanisms: TATA box mutations function by the titration effect (decrease in transcription initiation results in a relative decrease in premature termination), while an upstream activator (E2F) site functions by contributing to the assembly of polymerase complexes competent to traverse the downstream sites of premature termination.

The block to transcriptional elongation within the human c-myc gene is determined in the promoter-proximal region.

A conditional block to transcriptional elongation is an important mechanism for regulating c-myc gene expression. This elongation block within the first c-myc exon was defined originally in mammalian cells by nuclear run-on transcription analyses. Subsequent oocyte injection and in vitro transcription analyses suggested that sequences near the end of the first c-myc exon are sites of attenuation and/or premature termination. We report here that the mapping of single stranded DNA in vivo with potassium permanganate (KMnO4) and nuclear run-on transcription assays reveal that polymerase is paused near position +30 relative to the major c-myc transcription initiation site. Deletion of 350 bp, including the sites of 3'-end formation and intrinsic termination defined in oocyte injection and in vitro transcription assays does not affect-the pausing of polymerase in the promoter-proximal region. In addition, sequences upstream of +47 are sufficient to confer the promoter-proximal pausing of polymerases and to generate the polarity of transcription farther downstream. Thus, the promoter-proximal pausing of RNA polymerase II complexes accounts for the block to elongation within the c-myc gene in mammalian cells. We speculate that modification of polymerase complexes at the promoter-proximal pause site may determine whether polymerases can read through intrinsic sites of termination farther downstream.

Sequences in the human c-myc P2 promoter affect the elongation and premature termination of transcripts initiated from the upstream P1 promoter.

A conditional block to transcription elongation provides one mechanism for controlling the steady-state levels of c-myc RNA in mammalian cells. Although prematurely terminated c-myc RNAs are not detectable in mammalian cells, truncated c-myc RNAs with 3' ends that map near the end of the first exon are transcribed from human c-myc templates injected into Xenopus oocytes germinal vesicles. A series of linker scanner and deletion mutants within the c-myc P2 promoter was tested in the Xenopus oocyte injection assay to determine the potential contribution of promoter elements to the elongation or premature termination of c-myc transcription. Although this analysis failed to identify sequences in the P2 promoter that significantly affect the elongation or termination of P2-initiated transcripts, our results suggest that sequences within the P2 promoter contribute to the premature termination of transcripts initiated at the upstream P1 promoter. A subset of these sequences is essential for the efficient elongation of P1-initiated transcripts through intrinsic sites of termination at the end of exon 1. These sequences affect P1 elongation when they are downstream of the site of initiation, and we hypothesize that they may be analogous to a class of prokaryotic elements required for antitermination.

Tissue-specific factors and glucocorticoid receptors present in nuclear extracts bind next to each other in the promoter region of mouse mammary tumor virus.

Nuclear extracts from different mouse tissues have been used to study the interaction of factors with the steroid-inducible promoter of mouse mammary tumor virus. In addition to the glucocorticoid receptor that interacts with the distal region of the promoter, several tissue-specific proteins were found to bind to the 5' flanking region of the receptor-binding site and to the basal promoter region. The differences in the pattern of protection observed suggest that tissue-specific factors might co-operate with steroid receptors and result in a cell-type-dependent modulation of hormone action.