STIS Spectral Imagery of the OB Stars in NGC 604: Describing the Extraction Technique for a Crowded Stellar Field

We have developed a data reduction procedure to extract multiple spectra from a single two-dimensional Space Telescope Imaging Spectrograph (STIS) image of a crowded stellar field. This paper provides a description of our new technique, utilizing a STIS ultraviolet spectral image, acquired with the G140L grating and the 52 arcsec x 2 arcsec aperture, sampling a concentration of O and B stars in the central region of the NGC 604 starburst in M33. The software routines can disentangle and produce reliable ultraviolet spectra of stars with angular separations as small as 0.055 arcsec. Use of the extraction slit, based upon our model of the spectral cross dispersion profile, generates spectra with slightly higher resolution than the STScI standard processing. Our results clearly show that the spectral imaging capability of STIS represents a powerful tool for studying luminous stars in the star-forming regions of the Local Group.Comment: LaTeX, 23 pages total (including 11 figures and 1 table). To be published in June 2003 of The Astronomical Journal. Companion paper to "STIS Spectral Imagery of the OB Stars in NGC 604: The Most Luminous Stars" by Bruhweiler, Miskey, & Smith Neubi

Regulation of DNA transposition by CpG methylation and chromatin structure in human cells

BACKGROUND: The activity of transposable elements can be regulated by different means. DNA CpG methylation is known to decrease or inhibit transpositional activity of diverse transposons. However, very surprisingly, it was previously shown that CpG methylation of the Sleeping Beauty (SB) transposon significantly enhanced transposition in mouse embryonic stem cells. RESULTS: In order to investigate the unexpected response of SB transposition to CpG methylation, related transposons from the Tc1/mariner superfamily, that is, Tc1, Himar1, Hsmar1, Frog Prince (FP) and Minos were tested to see how transposition was affected by CpG methylation. A significant increase of >20-fold in transposition of SB, FP and Minos was seen, whereas Tc1, Himar1 and Hsmar1 showed no difference in transposition upon CpG-methylation. The terminal inverted repeats (TIRs) of the SB, FP and Minos elements share a common structure, in which each TIR contains two functionally important binding sites for the transposase (termed the IR/DR structure). The group of IR/DR elements showed increased excision after CpG methylation compared to untreated transposon donor plasmids. We found that de novo CpG methylation is not required for transposition. A mutated FP donor plasmid with depleted CpG sites in both TIRs was as efficient in transposition as the wild-type transposon, indicating that CpG sites inside the TIRs are not responsible for altered binding of the factors potentially modulating transposition. By using an in vivo one-hybrid DNA-binding assay in cultured human cells we found that CpG methylation had no appreciable effect on the affinity of SB transposase to its binding sites. However, chromatin immunoprecipitation indicated that CpG-methylated transposon donor plasmids are associated with a condensed chromatin structure characterized by trimethylated histone H3K9. Finally, DNA compaction by protamine was found to enhance SB transposition. CONCLUSIONS: We have shown that DNA CpG methylation upregulates transposition of IR/DR elements in the Tc1/mariner superfamily. CpG methylation provokes the formation of a tight chromatin structure at the transposon DNA, likely aiding the formation of a catalytically active complex by facilitating synapsis of sites bound by the transposase

Retargeting transposon insertions by the adeno-associated virus Rep protein

The Sleeping Beauty (SB), piggyBac (PB) and Tol2 transposons are promising instruments for genome engineering. Integration site profiling of SB, PB and Tol2 in human cells showed that PB and Tol2 insertions were enriched in genes, whereas SB insertions were randomly distributed. We aimed to introduce a bias into the target site selection properties of the transposon systems by taking advantage of the locus-specific integration system of adeno-associated virus (AAV). The AAV Rep protein binds to Rep recognition sequences (RRSs) in the human genome, and mediates viral integration into nearby sites. A series of fusion constructs consisting of the N-terminal DNA-binding domain of Rep and the transposases or the N57 domain of SB were generated. A plasmid-based transposition assay showed that Rep/SB yielded a 15-fold enrichment of transposition at a particular site near a targeted RRS. Genome-wide insertion site analysis indicated that an approach based on interactions between the SB transposase and Rep/N57 enriched transgene insertions at RRSs. We also provide evidence of biased insertion of the PB and Tol2 transposons. This study provides a comparative insight into target site selection properties of transposons, as well as proof-of-principle for targeted chromosomal transposition by composite protein-protein and protein-DNA interactions

12^{12}C/13^{13}C ratio in planetary nebulae from the IUE archives

We investigated the abundance ratio of $^{12}$C/$^{13}$C in planetary nebulae by examining emission lines arising from \ion{C}{3} 2s2p ^3P_{2,1,0} \to 2s^2 ^1S_0. Spectra were retrieved from the International Ultraviolet Explorer archives, and multiple spectra of the same object were coadded to achieve improved signal-to-noise. The $^{13}$C hyperfine structure line at 1909.6 \AA was detected in NGC 2440. The $^{12}$C/$^{13}$C ratio was found to be $\sim4.4\pm$1.2. In all other objects, we provide an upper limit for the flux of the 1910 \AA line. For 23 of these sources, a lower limit for the $^{12}$C/$^{13}$C ratio was established. The impact on our current understanding of stellar evolution is discussed. The resulting high signal-to-noise \ion{C}{3} spectrum helps constrain the atomic physics of the line formation process. Some objects have the measured 1907/1909 flux ratio outside the low-electron density theoretical limit for $^{12}$C. A mixture of $^{13}$C with $^{12}$C helps to close the gap somewhat. Nevertheless, some observed 1907/1909 flux ratios still appear too high to conform to the presently predicted limits. It is shown that this limit, as well as the 1910/1909 flux ratio, are predominantly influenced by using the standard partitioning among the collision strengths for the multiplet $^1S_0$--$^3P_J$ according to the statistical weights. A detailed calculation for the fine structure collision strengths between these individual levels would be valuable.Comment: ApJ accepted: 19 pages, 3 Figures, 2 Table

HST Observations and Photoionization Modeling of the LINER Galaxy NGC 1052

We present a study of available Hubble Space Telescope (HST) spectroscopic and imaging observations of the low ionization nuclear emission line region (LINER) galaxy NGC 1052. The WFPC2 imagery clearly differentiates extended nebular Halpha emission from that of the compact core. Faint Object Spectrograph (FOS) observations provide a full set of optical and UV data (1200-6800 Angstroms). These spectral data sample the innermost region (0."86 x 0."86 ~ 82pc x 82pc) and exclude the extended Halpha emission seen in the WFPC2 image. The derived emission line fluxes allow a detailed analysis of the physical conditions within the nucleus. The measured flux ratio for Halpha/Hbeta, F{Halpha}/F{Hbeta}=4.53, indicates substantial intrinsic reddening, E(B-V)=0.42, for the nuclear nebular emission. This is the first finding of a large extinction of the nuclear emission line fluxes in NGC 1052. If the central ionizing continuum is assumed to be attenuated by a comparable amount, then the emission line fluxes can be reproduced well by a simple photoionization model using a central power law continuum source with a spectral index of alpha = -1.2 as deduced from the observed flux distribution. A multi-density, dusty gas gives the best fit to the observed emission line spectrum. Our calculations show that the small contribution from a highly ionized gas observed in NGC 1052 can also be reproduced solely by photoionization modeling. The high gas covering factor determined from our model is consistent with the assumption that our line of sight to the central engine is obscured.Comment: 23 pages, 7 Postscript figures, 1 jpeg figure ; uses aaspp4.sty, 11pt to appear in The Astrophysical Journa

Far-Ultraviolet Surveys of Globular Clusters: Hunting for the Products of Stellar Collisions and Near Misses

Globular clusters are gravitationally bound stellar systems containing on the order of 100,000 stars. Due to the high stellar densities in the cores of these clusters, close encounters and even physical collisions between stars are inevitable. These dynamical interactions can produce exotic types of single and binary stars that are extremely rare in the galactic field, but which may be important to the dynamical evolution of their host clusters. A common feature of these dynamically-formed stellar populations is that many of their members are relatively hot, and thus bright in the far-ultraviolet (FUV) waveband. In this short review, I describe how space-based FUV observations are being used to find and study these populations.Comment: 15 pages, 6 figures; invited "Brief Review" for Modern Physics Letters

A single amino acid switch converts the sleeping beauty transposase into an efficient unidirectional excisionase with utility in stem cell reprogramming

The Sleeping Beauty (SB) transposon is an advanced tool for genetic engineering and a useful model to investigate cut-and-paste DNA transposition in vertebrate cells. Here, we identify novel SB transposase mutants that display efficient and canonical excision but practically unmeasurable genomic re-integration. Based on phylogenetic analyses, we establish compensating amino acid replacements that fully rescue the integration defect of these mutants, suggesting epistasis between these amino acid residues. We further show that the transposons excised by the exc(+)/int(-) transposase mutants form extrachromosomal circles that cannot undergo a further round of transposition, thereby representing dead-end products of the excision reaction. Finally, we demonstrate the utility of the exc(+)/int(-) transposase in cassette removal for the generation of reprogramming factor-free induced pluripotent stem cells. Lack of genomic integration and formation of transposon circles following excision is reminiscent of signal sequence removal during V(D)J recombination, and implies that cut-and-paste DNA transposition can be converted to a unidirectional process by a single amino acid change

A Helitron transposon reconstructed from bats reveals a novel mechanism of genome shuffling in eukaryotes

Helitron transposons capture and mobilize gene fragments in eukaryotes, but experimental evidence for their transposition is lacking in the absence of an isolated active element. Here we reconstruct Helraiser, an ancient element from the bat genome, and use this transposon as an experimental tool to unravel the mechanism of Helitron transposition. A hairpin close to the 3'-end of the transposon functions as a transposition terminator. However, the 3'-end can be bypassed by the transposase, resulting in transduction of flanking sequences to new genomic locations. Helraiser transposition generates covalently closed circular intermediates, suggestive of a replicative transposition mechanism, which provides a powerful means to disseminate captured transcriptional regulatory signals across the genome. Indeed, we document the generation of novel transcripts by Helitron promoter capture both experimentally and by transcriptome analysis in bats. Our results provide mechanistic insight into Helitron transposition, and its impact on diversification of gene function by genome shuffling

Molecular and functional characterization of BDNF-overexpressing human retinal pigment epithelial cells established by sleeping beauty transposon-mediated gene transfer

More and more patients suffer from multifactorial neurodegenerative diseases, such as age-related macular degeneration (AMD). However, their pathological mechanisms are still poorly understood, which complicates the development of effective therapies. To improve treatment of multifactorial diseases, cell-based gene therapy can be used to increase the expression of therapeutic factors. To date, there is no approved therapy for dry AMD, including late-stage geographic atrophy. We present a treatment option for dry AMD that transfers the brain-derived neurotrophic factor (BDNF) gene into retinal pigment epithelial (RPE) cells by electroporation using the plasmid-based Sleeping Beauty (SB) transposon system. ARPE-19 cells and primary human RPE cells were co-transfected with two plasmids encoding the (SB100X) transposase and the transposon carrying a BDNF transcription cassette. We demonstrated efficient expression and secretion of BDNF in both RPE cell types, which were further increased in ARPE-19 cell cultures exposed to hydrogen peroxide. BDNF-transfected cells exhibited lower apoptosis rates and stimulated neurite outgrowth in human SH-SY5Y cells. This study is an important step in the development of a cell-based BDNF gene therapy that could be applied as an advanced therapy medicinal product to treat dry AMD or other degenerative retinal diseases