A multilocus gene genealogy concordant with host preference indicates segregation of a new species, Magnaporthe oryzae, from M. grisea

Magnaporthe oryzae is described as a new species distinct from M. grisea. Gene trees were inferred for Magnaporthe species using portions of three genes: actin, beta-tubulin, and calmodulin. These gene trees were found to be concordant and distinguished two distinct clades within M. grisea. One clade is associated with the grass genus Digitaria and is therefore nomenclaturally tied to M. grisea. The other clade is associated with Oryza sativa and other cultivated grasses and is described as a new species, M. oryzae. While no morphological characters as yet distinguish them, M. oryzae is distinguished from M. grisea by several base substitutions in each of three loci as well as results from laboratory matings; M.oryzae and M. grisea are not interfertile. Given that M. oryzae is the scientifically correct name for isolates associated with rice blast and grey leaf spot, continued use of M. grisea for such isolates would require formal nomenclatural conservation.     

Time course of bioluminescent signal in orthotopic and heterotopic brain tumors in nude mice

In vivo bioluminescence imaging is becoming increasingly popular. Quantification of bioluminescence signals requires knowledge of the variability and reproducibility of this technique. The objective of this study was to analyze the time course of luminescent signal emitted from firefly luciferase-expressing tumors in two locations, following luciferin injection and at different times after tumor cell implantation. Knowledge of the kinetics of the bioluminescent signals is required for the reliable quantification and comparison of signal during longitudinal studies. The kinetics of bioluminescence was evaluated in orthotopic and heterotopic brain tumors in mice using a human brain tumor cell line constitutively expressing luciferase. Tumor cells were implanted in the brains and flanks of the animals, and whole-body images revealing tumor location were obtained. Tumor burden was monitored over time by the quantitation of photon emission. The magnitude of bioluminescence measured in vivo varied with time after the injection of luciferin, as well as with dose, which necessitated that the comparison of the quantitative results take into consideration the time after injection. Heterotopic and orthotopic tumors exhibited significantly different time courses; however, time after implantation as characterized by kinetic studies performed on days 4 and 14 after cell implantation revealed no significant differences in orthotopic tumors. Future quantitative longitudinal studies must take into account the differences in the kinetics of different models.     

Mitochondrial proteome: altered cytochrome c oxidase subunit levels in prostate cancer

Laser capture microdissection was combined with reverse phase protein lysate arrays to quantitatively analyze the ratios of mitochondrial encoded cytochrome c oxidase subunits to nuclear encoded cytochrome c oxidase subunits, and to correlate the ratios with malignant progression in human prostate tissue specimens. Cytochrome c oxidase subunits I-III comprise the catalytic core of the enzyme and are all synthesized from mitochondrial DNA. The remaining subunits (IV-VIII) are synthesized from cellular nuclear DNA. A significant (P < 0.001, 30/30 prostate cases) shift in the relative concentrations of nuclear encoded cytochrome c oxidase subunits IV, Vb, and VIc compared to mitochondrial encoded cytochrome c oxidase subunits I and II was noted during the progression of prostate cancer from normal epithelium through premalignant lesions to invasive carcinoma. Significantly, this shift was discovered to begin even in the premalignant stage. Reverse phase protein lysate array-based observations were corroborated with immunohistochemistry, and extended to a few human carcinomas in addition to prostate. This analysis points to a role for nuclear DNA encoded mitochondrial proteins in carcinogenesis; underscoring their potential as targets for therapy while highlighting the need for full characterization of the mitochondrial proteome.     

Clonality analysis after retroviral-mediated gene transfer to CD34+ cells from the cord blood of ADA-deficient SCID neonates

A clinical trial of retroviral-mediated transfer of the adenosine deaminase (ADA) gene into umbilical cord blood CD34(+) cells was started in 1993. ADA-containing peripheral blood mononuclear cells (PBMCs) have persisted in patients from this trial, with T lymphocytes showing the highest prevalence of gene marking. To gain a greater understanding of the nature and number of the transduced cells that were engrafted, we used linear amplification-mediated PCR (LAM-PCR) to identify clonal vector proviral integrants. In one patient, a single vector integrant was predominant in T lymphocytes at a stable level over most of the eight-year time span analyzed and was also detected in some myeloid samples. T-cell clones with the predominant integrant, isolated after eight years, showed multiple patterns of T-cell receptor (TCR) gene rearrangement, indicating that a single pre-thymic stem or progenitor cell served as the source of the majority of the gene-marked cells over an extended period of time. It is important to distinguish the stable pattern of monoclonal gene marking that we observed here from the progressive increase of a T-cell clone with monoclonal gene marking that results from leukemic transformation, as observed in two subjects in a clinical trial of gene therapy for X-linked severe combined immunodeficiency (SCID).     

C5 cytosine methylation at CpG sites enhances sequence selectivity of mitomycin C-DNA bonding

We have established that UvrABC nuclease is equally efficient in cutting mitomycin C (MC)-DNA monoadducts formed at different sequences and that the degree of UvrABC cutting represents the extent of drug-DNA bonding. Using this method we determined the effect of C5 cytosine methylation on the DNA monoalkylation by MC and the related analogues N-methyl-7-methoxyaziridinomitosene (MS-NMA) and 10-decarbamoylmitomycin C (DC-MC). We have found that C5 cytosine methylation at CpG sites greatly enhances MC and MS-NMA DNA adduct formation at those sites while reducing adduct formation at non-CpG sequences. In contrast, although DC-MC DNA bonding at CpG sites is greatly enhanced by CpG methylation, its bonding at non-CpG sequences is not appreciably affected. These cumulative results suggest that C5 cytosine methylation at CpG sites enhances sequence selectivity of drug-DNA bonding. We propose that the methylation pattern and status (hypo- or hypermethylation) of genomic DNA may determine the cells' susceptibility to MC and its analogues, and these effects may, in turn, play a crucial role in the antitumor activities of the drugs.     

Rho transcription factor: symmetry and binding of bicyclomycin

The antibiotic bicyclomycin inhibits rho-dependent termination processes by interfering with RNA translocation by preventing RNA binding at the translocation site or by uncoupling the translocation process from ATP hydrolysis. Previous studies have shown that bicyclomycin binds near the ATP hydrolysis pocket on rho. The hexameric structure of rho indicates that it is in a class of enzymes with strong sequence similarity to F(1)-ATP synthase. The bicyclomycin derivative 5a-formylbicyclomycin, an inhibitor comparable to bicyclomycin, was previously shown to form a stable imine with rho and when reduced to the amine with NaBH(4) to singly label five of the six rho subunits. Lysine-336 was identified by mass spectrometric analysis of trypsin-digested fragments as the site of 5a-formylbicyclomycin adduction. A model of rho was made by threading the rho sequence on the known crystal structure of the alpha and beta subunits of F(1)-ATP synthase. The model, along with information concerning the extent and site of 5a-formylbicyclomycin adduction, indicates an overall C6 symmetry for rho subunit organization. We propose that the sequence similarity between rho and F(1)-ATP synthase extends to a similar quaternary structure and an equivalent enzyme mechanism. The proposed mechanism of RNA translocation coupled with ATP hydrolysis changes the overall symmetry of rho from C6 to C6/C3.