Integration of DNA into bacterial chromosomes from plasmids without a counter-selection marker.

Most bacteria can only be transformed with circular plasmids, so robust DNA integration methods for these rely upon selection of single-crossover clones followed by counter-selection of double-crossover clones. To overcome the limited availability of heterologous counter-selection markers, here we explore novel DNA integration strategies that do not employ them, and instead exploit (i) activation or inactivation of genes leading to a selectable phenotype, and (ii) asymmetrical regions of homology to control the order of recombination events. We focus here on the industrial biofuel-producing bacterium Clostridium acetobutylicum, which previously lacked robust integration tools, but the approach we have developed is broadly applicable. Large sequences can be delivered in a series of steps, as we demonstrate by inserting the chromosome of phage lambda (minus a region apparently unstable in Escherichia coli in our cloning context) into the chromosome of C. acetobutylicum in three steps. This work should open the way to reliable integration of DNA including large synthetic constructs in diverse microorganisms. © 2011 The Author(s)

On The Age Estimation of LBDS 53W091

The recent spectral analysis of LBDS 53W091 by Spinrad and his collaborators has suggested that this red galaxy at z=1.55 is at least 3.5 Gyr old. This imposes an important constraint on cosmology, suggesting that this galaxy formed at z > 6.5, assuming recent estimates of cosmological parameters. We have performed chi^2 tests to the continuum of this galaxy using its UV spectrum and photometric data (RJHK). We have used the updated Yi models that are based on the Yale tracks. We find it extremely difficult to reproduce such large age estimates, under the assumption of the most probable input parameters. Using the same configuration as in Spinrad et al. (solar abundance models), our analysis suggests an age of approximately 1.4 -- 1.8 Gyr. The discrepancy between Spinrad et al.'s age estimate (based on the 1997 Jimenez models) and ours originates from the large difference in the model integrated spectrum: the Jimenez models are much bluer than the Yi models and the Bruzual \& Charlot (BC) models. Preliminary tests favor the Yi and BC models. The updated age estimate of LBDS 53W091 would suggest that this galaxy formed approximately at z=2-3.Comment: LaTeX, 18 eps files Accepted for publication in ApJ (Feb 10, 2000, vol 530), uses emulateapj.st

Spores of Clostridium engineered for clinical efficacy and safety cause regression and cure of tumors in vivo.

Spores of some species of the strictly anaerobic bacteria Clostridium naturally target and partially lyse the hypoxic cores of tumors, which tend to be refractory to conventional therapies. The anti-tumor effect can be augmented by engineering strains to convert a non-toxic prodrug into a cytotoxic drug specifically at the tumor site by expressing a prodrug-converting enzyme (PCE). Safe doses of the favored prodrug CB1954 lead to peak concentrations of 6.3 μM in patient sera, but at these concentration(s) known nitroreductase (NTR) PCEs for this prodrug show low activity. Furthermore, efficacious and safe Clostridium strains that stably express a PCE have not been reported. Here we identify a novel nitroreductase from Neisseria meningitidis, NmeNTR, which is able to activate CB1954 at clinically-achievable serum concentrations. An NmeNTR expression cassette, which does not contain an antibiotic resistance marker, was stably localized to the chromosome of Clostridium sporogenes using a new integration method, and the strain was disabled for safety and containment by making it a uracil auxotroph. The efficacy of Clostridium-Directed Enzyme Prodrug Therapy (CDEPT) using this system was demonstrated in a mouse xenograft model of human colon carcinoma. Substantial tumor suppression was achieved, and several animals were cured. These encouraging data suggest that the novel enzyme and strain engineering approach represent a promising platform for the clinical development of CDEPT

Galaxy Morphology from NICMOS Parallel Imaging

We present high resolution NICMOS images of random fields obtained in parallel to other HST observations. We present galaxy number counts reaching H=24. The H-band galaxy counts show good agreement with the deepest I- and K-band counts obtained from ground-based data. We present the distribution of galaxies with morphological type to H<23. We find relatively fewer irregular galaxies compared to an I-band sample from the Hubble Deep Field, which we attribute to their blue color, rather than to morphological K-corrections. We conclude that the irregulars are intrinsically faint blue galaxies at z<1.Comment: 13 pages, including 4 figures. Accepted for publication in ApJ Letter

All-Sky spectrally matched UBVRI-ZY and u'g'r'i'z' magnitudes for stars in the Tycho2 catalog

We present fitted UBVRI-ZY and u'g'r'i'z' magnitudes, spectral types and distances for 2.4M stars, derived from synthetic photometry of a library spectrum that best matches the Tycho2 BtVt, NOMAD Rn and 2MASS JHK_{2/S} catalog magnitudes. We present similarly synthesized multi-filter magnitudes, types and distances for 4.8M stars with 2MASS and SDSS photometry to g<16 within the Sloan survey region, for Landolt and Sloan primary standards, and for Sloan Northern (PT) and Southern secondary standards. The synthetic magnitude zeropoints for BtVt, UBVRI, ZvYv, JHK_{2/S}, JHK_{MKO}, Stromgren uvby, Sloan u'g'r'i'z' and ugriz are calibrated on 20 calspec spectrophotometric standards. The UBVRI and ugriz zeropoints have dispersions of 1--3%, for standards covering a range of color from -0.3 < V-I < 4.6; those for other filters are in the range 2--5%. The spectrally matched fits to Tycho2 stars provide estimated 1-sigma errors per star of ~0.2, 0.15, 0.12, 0.10 and 0.08 mags respectively in either UBVRI or u'g'r'i'z'; those for at least 70% of the SDSS survey region to g<16 have estimated 1-sigma errors per star of ~0.2, 0.06, 0.04, 0.04, 0.05 in u'g'r'i'z' or UBVRI. The density of Tycho2 stars, averaging about 60 stars per square degree, provides sufficient stars to enable automatic flux calibrations for most digital images with fields of view of 0.5 degree or more. Using several such standards per field, automatic flux calibration can be achieved to a few percent in any filter, at any airmass, in most workable observing conditions, to facilitate inter-comparison of data from different sites, telescopes and instruments.Comment: 36 pages, 30 figures, 3 printed tables, several electronic tables, accepted PASP Dec 201

6-Deoxyhexoses froml-Rhamnose in the Search for Inducers of the Rhamnose Operon: Synergy of Chemistry and Biotechnology

In the search for alternative non‐metabolizable inducers in the l ‐rhamnose promoter system, the synthesis of fifteen 6‐deoxyhexoses from l ‐rhamnose demonstrates the value of synergy between biotechnology and chemistry. The readily available 2,3‐acetonide of rhamnonolactone allows inversion of configuration at C4 and/or C5 of rhamnose to give 6‐deoxy‐d ‐allose, 6‐deoxy‐d ‐gulose and 6‐deoxy‐l ‐talose. Highly crystalline 3,5‐benzylidene rhamnonolactone gives easy access to l ‐quinovose (6‐deoxy‐l ‐glucose), l ‐olivose and rhamnose analogue with C2 azido, amino and acetamido substituents. Electrophilic fluorination of rhamnal gives a mixture of 2‐deoxy‐2‐fluoro‐l ‐rhamnose and 2‐deoxy‐2‐fluoro‐l ‐quinovose. Biotechnology provides access to 6‐deoxy‐l ‐altrose and 1‐deoxy‐l ‐fructose