A first genome assembly of the barley fungal pathogen <Emphasis Type="Italic">Pyrenophora teres</Emphasis> f. <Emphasis Type="Italic">teres</Emphasis>

Background  

Pyrenophora teres f. teres is a necrotrophic fungal pathogen and the cause of one of barley's most important diseases, net form of net blotch. Here we report the first genome assembly for this species based solely on short Solexa sequencing reads of isolate 0-1. The assembly was validated by comparison to BAC sequences, ESTs, orthologous genes and by PCR, and complemented by cytogenetic karyotyping and the first genome-wide genetic map for P. teres f. teres.     

UV irradiation inhibits initiation of DNA replication from oriC in Escherichia coli

Summary Irradiation of Escherichia coli with UV light causes a transient inhibition of DNA replication. This effect is generally thought to be accounted for by blockage of the elongation of DNA replication by UV-induced lesions in the DNA (a cis effect). However, by introducing an unirradiated E. coli origin (oriC)-dependent replicon into UV-irradiated cells, we have been able to show that the environment of a UV-irradiated cell inhibits initiation of replication from oriC on a dimer-free replicon. We therefore conclude that UV-irradiation of E. coli leads to a trans-acting inhibition of initiation of replication. The inhibition is transient and does not appear to be an SOS function.     

Application of singular value decomposition to the analysis of time-resolved macromolecular x-ray data

Singular value decomposition (SVD) is a technique commonly used in the analysis of spectroscopic data that both acts as a noise filter and reduces the dimensionality of subsequent least-squares fits. To establish the applicability of SVD to crystallographic data, we applied SVD to calculated difference Fourier maps simulating those to be obtained in a time-resolved crystallographic study of photoactive yellow protein. The atomic structures of one dark state and three intermediates were used in qualitatively different kinetic mechanisms to generate time-dependent difference maps at specific time points. Random noise of varying levels in the difference structure factor amplitudes, different extents of reaction initiation, and different numbers of time points were all employed to simulate a range of realistic experimental conditions. Our results show that SVD allows for an unbiased differentiation between signal and noise; a small subset of singular values and vectors represents the signal well, reducing the random noise in the data. Due to this, phase information of the difference structure factors can be obtained. After identifying and fitting a kinetic mechanism, the time-independent structures of the intermediates could be recovered. This demonstrates that SVD will be a powerful tool in the analysis of experimental time-resolved crystallographic data.     

Cell Budding from Normal Appearing Epithelia: A Predictor of Colorectal Cancer Metastasis?

Background: Colorectal carcinogenesis is believed to be a multi-stage process that originates with a localized adenoma, which linearly progresses to an intra-mucosal carcinoma, to an invasive lesion, and finally to metastatic cancer. This progression model is supported by tissue culture and animal model studies, but it is difficult to reconcile with several well-established observations, principally among these are that up to 25% of early stage (Stage I/II), node-negative colorectal cancer (CRC) develop distant metastasis, and that circulating CRC cells are undetectable in peripheral blood samples of up to 50% of patients with confirmed metastasis, but more than 30% of patients with no detectable metastasis exhibit such cells. The mechanism responsible for this diverse behavior is unknown, and there are no effective means to identify patients with pending, or who are at high risk for, developing metastatic CRC.Novel findings: Our previous studies of human breast and prostate cancer have shown that cancer invasion arises from the convergence of a tissue injury, the innate immune response to that injury, and the presence of tumor stem cells within tumor capsules at the site of the injury. Focal degeneration of a capsule due to age or disease attracts lymphocyte infiltration that degrades the degenerating capsules resulting in the formation of a focal disruption in the capsule, which selectively favors proliferating or “budding” of the underlying tumor stem cells. Our recent studies suggest that lymphocyte infiltration also triggers metastasis by disrupting the intercellular junctions and surface adhesion molecules within the proliferating cell buds causing their dissociation. Then, lymphocytes and tumor cells are conjoined through membrane fusion to form tumor-lymphocyte chimeras (TLCs) that allows the tumor stem cell to avail itself of the lymphocyte''s natural ability to migrate and breach cell barriers in order to intravasate and to travel to distant organs. Our most recent studies of human CRC have detected nearly identical focal capsule disruptions, lymphocyte infiltration, budding cells, and the formation of TLCs. Our studies have further shown that age- and type-matched node-positive and -negative CRC have a significantly different morphological and immunohistochemical profile and that the majority of lymphatic ducts with disseminated cells are located within the mucosa adjacent to morphologically normal appearing epithelial structures that express a stem cell-related marker.New hypothesis: Based on these findings and the growth patterns of budding cells revealed by double immunohistochemistry, we further hypothesize that metastatic spread is an early event of carcinogenesis and that budding cells overlying focal capsule disruptions represent invasion- and metastasis-initiating cells that follow one of four pathways to progress: (1) to undergo extensive in situ proliferation leading to the formation of tumor nests that subsequently invade the submucosa, (2) to migrate with associated lymphocytes functioning as “seeds” to grow in new sites, (3) to migrate and intravasate into pre-existing vascular structures by forming TLCs, or (4) to intravasate into vascular structures that are generated by the budding cells themselves. We also propose that only node-positive cases harbor stem cells with the potential for multi-lineage differentiation and unique surface markers that permit intravasation.     

Crystallization and preliminary x-ray characterization of bovine growth hormone. Purification of bovine prolactin and growth hormone

A new purification scheme for both prolactin and growth hormone from bovine pituitaries has been developed which avoids the use of potentially damaging solution conditions. Both hormones were greater than 95% pure as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and had specific activities similar to or greater than standard samples of the same hormone as judged by several bioassays. Small single crystals of bovine growth hormone were obtained by vapor diffusion techniques. Examination of these crystals by x-ray diffraction, using the Cornell High Energy Synchrotron Source, showed that they were well ordered, and exhibited diffraction to 2.8-A resolution on still photographs. Precession and oscillation photographs showed that they belonged to the orthorhombic space group P2(1)2(1)2(1) (or P2(1)2(1)2) with unit cell dimensions a = 219 A, b = 51.9 A, c = 68.9 A. The density of the crystals was 1.19 +/- 0.02 g/ml from which the presence of eight 45,000-dalton dimers/unit cell was deduced. The protein content of the crystals was shown by isoelectric focusing to be identical to that of purified growth hormone in solution. These crystals appear suitable for use in the x-ray structure determination of bovine growth hormone to at least 3.2-A resolution.