Variables affecting the stability of multiple cloning sites and hairpin structures in retroviral vectors

Retroviruses are RNA viruses that replicate through a double-stranded DNA intermediate. The viral enzyme reverse transcriptase copies the retroviral genomic RNA into this DNA intermediate through the process of reverse transcription. Many variables can affect the fidelity of reverse transcriptase during reverse transcription, including specific sequences within the retroviral genome. ^ Previous studies have observed that multiple cloning sites (MCS) and sequences predicted to form stable hairpin structures are hotspots for deletion during retroviral replication. The studies described in this dissertation were performed to elucidate the variables that affect the stability of MCS and hairpin structures in retroviral vectors. Two series of retroviral vectors were constructed and characterized in these studies. ^ Spleen necrosis virus-based vectors were constructed containing separate MCS insertions of varying length, orientation, and symmetry. The only MCS that was a hotspot for deletion formed a stable hairpin structure. Upon more detailed study, the MCS previously reported as a hotspot for deletion was found to contain a tandem linker insertion that formed a hairpin structure. Murine leukemia virus-based vectors were constructed containing separate sequence insertions of either inverted repeat symmetry (122IR) that could form a hairpin structure, or little symmetry (122c) that would form a less stable structure. These insertions were made into either the neomycin resistance marker ( neo) or the hygromycin resistance marker (hyg) of the vector. 122c was stable in both neo and hyg, while 122IR was preferentially deleted in neo and was remarkably unstable in hyg. ^ These results suggest that MCS are hotspots for deletion in retroviral vectors if they can form hairpin structures, and that hairpin structures can be highly unstable at certain locations in retroviral vectors. This information may contribute to improved design of retroviral vectors for such uses as human gene therapy, and will contribute to a greater understanding of the basic science of retroviral reverse transcription. ^

An analysis of two methods for the classification of stage of readiness to obtain repeat mammography screening

Background and purpose: Breast cancer continues to be a health problem for women, representing 28 percent of all female cancers and remaining one of the leading causes of death for women. Breast cancer incidence rates become substantial before the age of 50. After menopause, breast cancer incidence rates continue to increase with age creating a long-lasting source of concern (Harris et al., 1992). Mammography, a technique for the detection of breast tumors in their nonpalpable stage when they are most curable, has taken on considerable importance as a public health measure. The lifetime risk of breast cancer is approximately 1 in 9 and occurs over many decades. Recommendations are that screening be periodic in order to detect cancer at early stages. These recommendations, largely, are not followed. Not only are most women not getting regular mammograms, but this circumstance is particularly the case among older women where regular mammography has been proven to reduce mortality by approximately 30 percent. The purpose of this project was to increase our understanding of factors that are associated with stage of readiness to obtain subsequent mammograms. A secondary purpose of this research was to suggest further conceptual considerations toward the extension of the Transtheoretical Model (TTM) of behavior change to repeat screening mammography. ^ Methods. A sample (n = 1,222) of women 50 years and older in a large multi-specialty clinic in Houston, Texas was surveyed by mail questionnaire regarding their previous screening experience and stage of readiness to obtain repeat screening. A computerized database, maintained on all women who undergo mammography at the clinic, was used to identify women who are eligible for the project. The major statistical technique employed to select the significant variables and to examine the man and interaction effects of independent variables on dependent variables was polychotomous stepwise, logistic regression. A prediction model for each stage of readiness definition was estimated. The expected probabilities for stage of readiness were calculated to assess the magnitude and direction of significant predictors. ^ Results. Analysis showed that both ways of defining stage of readiness for obtaining a screening mammogram were associated with specific constructs, including decisional balance and processes of the change. ^ Conclusions. The results of the present study demonstrate that the TTM appears to translate to repeat mammography screening. Findings in the current study also support finding of previous studies that suggest that stage of readiness is associated with respondent decisional balance and the processes of change. ^

Deterrence, Incapacitation, and Repeat Offenders

This paper develops an economic model of criminal enforcement that combines the goals of deterrence and incapacitation. Potential offenders commit an initial criminal act if the present value of net private gains is positive. A fraction of these offenders become habitual and commit further crimes immediately upon release from their initial prison term (if any). The optimal punishment scheme in this setting generally involves a finite prison term for first-time offenders (based on the goal of deterrence), and an infinite (life) sentence for repeat offenders (based on the goal of incapacitation).

Test, Edit, Repeat: Steps to Improve Your Web Site.

Presents ways to improve library Web sites. Description of the Hampshire College library Web site and how it is an integral part of the college's reference service; Usability analysis of Web sites; How to apply what librarians learn from usability analysis; Importance of testing; How to measure success. INSETS: Test Questions; Usability Guidelines.

A Simple Theory of Increasing Penalties for Repeat Offenders

A feature of many penal codes is that punishments are more severe for repeat offenders, yet economic models have had a hard time providing a theoretical justification for this practice. This paper offers an explanation based on the wage penalty suffered by individuals convicted of crime. While this penalty probably deters some first-timers from committing crimes, it actually hampers deterrence of repeat offenders because of their diminished employments opportunities. We show that in this setting, an escalating penalty scheme is optimal and time consistent.

DNA incisions stimulate genetic instability of triplet repeat sequences related to human hereditary neurological diseases

The molecular mechanisms responsible for the expansion and deletion of trinucleotide repeat sequences (TRS) are the focus of our studies. Several hereditary neurological diseases including Huntington's disease, myotonic dystrophy, and fragile X syndrome are associated with the instability of TRS. Using the well defined and controllable model system of Escherichia coli, the influences of three types of DNA incisions on genetic instability of CTG•CAG repeats were studied: DNA double-strand breaks (DSB), single-strand nicks, and single-strand gaps. The DNA incisions were generated in pUC19 derivatives by in vitro cleavage with restriction endonucleases. The cleaved DNA was then transformed into E. coli parental and mutant strains. Double-strand breaks induced deletions throughout the TRS region in an orientation dependent manner relative to the origin of replication. The extent of instability was enhanced by the repeat length and sequence (CTG•CAG vs. CGG•CCG). Mutations in recA and recBC increased deletions, mutations in recF stabilized the TRS, whereas mutations in ruvA had no effect. DSB were repaired by intramolecular recombination, versus an intermolecular gene conversion or crossover mechanism. 30 nt gaps formed a distinct 30 nt deletion product, whereas single strand nicks and gaps of 15 nts did not induce expansions or deletions. Formation of this deletion product required the CTG•CAG repeats to be present in the single-stranded region and was stimulated by E. coli DNA ligase, but was not dependent upon the RecFOR pathway. Models are presented to explain the DSB induced instabilities and formation of the 30 nucleotide deletion product. In addition to the in vitro creation of DSBs, several attempts to generate this incision in vivo with the use of EcoR I restriction modification systems were conducted. ^

The in vivo and in vitro formation of sticky DNA and the GAA.TTC trinucleotide repeat associated with Friedreich's ataxia

Friedreich's ataxia is caused by the expansion of the GAA•TTC trinucleotide repeat sequence located in intron 1 of the frataxin gene. The long GAA•TTC repeats are known to form several non-B DNA structures including hairpins, triplexes, parallel DNA and sticky DNA. Therefore it is believed that alternative DNA structures play a role in the loss of mRNA transcript and functional frataxin protein in FRDA patients. We wanted to further elucidate the characteristics for formation and stability of sticky DNA by evaluating the structure in a plasmid based system in vitro and in vivo in Escherichia coli. The negative supercoil density of plasmids harboring different lengths of GAA•TTC repeats, as well as either one or two repeat tracts were studied in E. coli to determine if plasmids containing two long tracts (≥60 repeats) in a direct repeat orientation would have a different topological effect in vivo compared to plasmids that harbored only one GAA•TTC tract or two tracts of < 60 repeats. The experiments revealed that, in fact, sticky DNA forming plasmids had a lower average negative supercoil density (-σ) compared to all other control plasmids used that had the potential to form other non-B DNA structures such as triplexes or Z-DNA. Also, the requirements for in vitro dissociation and reconstitution of the DNA•DNA associated region of sticky DNA were evaluated. Results conclude that the two repeat tracts associate in the presence of negative supercoiling and MgCl 2 or MnCl2 in a time and concentration-dependent manner. Interaction of the repeat sequences was not observed in the absence of negative supercoiling and/or MgCl2 or in the presence of other monovalent or divalent cations, indicating that supercoiling and quite specific cations are needed for the association of sticky DNA. These are the first experiments studying a more specific role of supercoiling and cation influence on this DNA conformation. To support our model of the topological effects of sticky DNA in plasmids, changes in sticky DNA band migration was measured with reference to the linear DNA after treatment with increasing concentrations of ethidium bromide (EtBr). The presence of independent negative supercoil domains was confirmed by this method and found to be segregated by the DNA-DNA associated region. Sequence-specific polyamide molecules were used to test the effect of binding of the ligands to the GAA•TTC repeats on the inhibition of sticky DNA. The destabilization of the sticky DNA conformation in vitro through this binding of the polyamides demonstrated the first conceptual therapeutic approach for the treatment of FRDA at the DNA molecular level. ^ Thus, examining the properties of sticky DNA formed by these long repeat tracts is important in the elucidation of the possible role of sticky DNA in Friedreich's ataxia. ^

Gene clustering of the small leucine -rich repeat gene family

The small leucine-rich repeat proteoglycans (or SLRPs) are a group of extracellular proteins (ECM) that belong to the leucine-rich repeat (LRR) superfamily of proteins. The LRR is a protein folding motif composed of 20–30 amino acids with leucines in conserved positions. LRR-containing proteins are present in a broad spectrum of organisms and possess diverse cellular functions and localization. In mammals, the SLRPs are abundant in connective tissues, such as bones, cartilage, tendons, skin, and blood vessels. We have discovered a new member of the class I small leucine rich repeat proteoglycan (SLRP) family which is distinct from the other class I SLRPs since it possesses a unique stretch of aspartate residues at its N-terminus. For this reason, we called the molecule asporin. The deduced amino acid sequence is about 50% identical (and 70% similar) to decorin and biglycan. However, asporin does not contain a serine/glycine dipeptide sequence required for the assembly of O-linked glycosaminoglycans and is probably not a proteoglycan. The tissue expression of asporin partially overlaps with the expression of decorin and biglycan. During mouse embryonic development, asporin mRNA expression was detected primarily in the skeleton and other specialized connective tissues; very little asporin message was detected in the major parenchymal organs. The mouse asporin gene structure is similar to that of biglycan and decorin with 8 exons. The asporin gene is localized to human chromosome 9q22-9g21.3 where asporin is part of a SLRP gene cluster that includes ECM2, osteoadherin, and osteoglycin. This gene cluster of four LRR-encoding genes is embedded in a 238 kilobase intron of another novel gene named Tes9orf that is expressed primarily in the testes of the adult mouse. The SLRP genes are not present in Drosophila or C. elegans , but reside in three separate gene clusters in the puffer fish, mice and humans. Targeted disruption of individual mouse SLRP genes display minor connective tissue defects such as skin fragility, tendon laxity, minor growth plate defects, and mild osteoporosis. However, double and triple knockouts of SLRP genes exacerbate these phenotypes. Both the double epiphycan/biglycan and the triple PRELP/fibromodulin/biglycan knockout mice exhibit premature osteoarthritis. ^

Adam vetus tandem laetus novum promat canticum : Adán de San Víctor y la novedad del canto victorino en la lírica mariana medieval

La poesía latina del maestro Adán de San Víctor (ca.11121192), oriundo de Bretaña y canónigo agustino de la abadía parisina de SaintVictor, representa, en su ámbito y en su época, una novedad trascendente dentro de la lírica litúrgica y particularmente de la lírica mariana. La ductilidad rítmica y retórica del subgénero de la sequentia lograda en sus piezas hace de este autor un paradigma de la creación poética litúrgica en la Europa de los siglos XII y XIII. En este sentido, tanto la mención a su obra como la inclusión de sus sequentiae (junto con otras del mismo período y "estilo" gótico) en diversos manuscritos hispánicos, desde las Cantigas de Santa María hasta el ms. Alcobacense 149, permiten considerar la relevante influencia de esta refinada lírica del siglo XII en la confección de poemas y compilaciones marianas del siglo siguiente en la Península Ibérica.

Adam vetus tandem laetus novum promat canticum : Adán de San Víctor y la novedad del canto victorino en la lírica mariana medieval

La poesía latina del maestro Adán de San Víctor (ca.11121192), oriundo de Bretaña y canónigo agustino de la abadía parisina de SaintVictor, representa, en su ámbito y en su época, una novedad trascendente dentro de la lírica litúrgica y particularmente de la lírica mariana. La ductilidad rítmica y retórica del subgénero de la sequentia lograda en sus piezas hace de este autor un paradigma de la creación poética litúrgica en la Europa de los siglos XII y XIII. En este sentido, tanto la mención a su obra como la inclusión de sus sequentiae (junto con otras del mismo período y "estilo" gótico) en diversos manuscritos hispánicos, desde las Cantigas de Santa María hasta el ms. Alcobacense 149, permiten considerar la relevante influencia de esta refinada lírica del siglo XII en la confección de poemas y compilaciones marianas del siglo siguiente en la Península Ibérica.

Adam vetus tandem laetus novum promat canticum : Adán de San Víctor y la novedad del canto victorino en la lírica mariana medieval

La poesía latina del maestro Adán de San Víctor (ca.11121192), oriundo de Bretaña y canónigo agustino de la abadía parisina de SaintVictor, representa, en su ámbito y en su época, una novedad trascendente dentro de la lírica litúrgica y particularmente de la lírica mariana. La ductilidad rítmica y retórica del subgénero de la sequentia lograda en sus piezas hace de este autor un paradigma de la creación poética litúrgica en la Europa de los siglos XII y XIII. En este sentido, tanto la mención a su obra como la inclusión de sus sequentiae (junto con otras del mismo período y "estilo" gótico) en diversos manuscritos hispánicos, desde las Cantigas de Santa María hasta el ms. Alcobacense 149, permiten considerar la relevante influencia de esta refinada lírica del siglo XII en la confección de poemas y compilaciones marianas del siglo siguiente en la Península Ibérica.

TanDEM-X ice thickness dataset for Wilkins Ice Shelf, Antarctic Peninsula, with link to GeoTIFF

The dataset shows the ice thickness over Wilkins Ice Shelf, Antarctic Peninsula derived from TanDEM-X Interferometry. The data has been acquired between June and August 2012. The TanDEM-X heights have been linked to CryoSAT-2 heights (V. Helm) from the respective time stamp. Elevations have been transformed from WGS84 ellipsoidal heights to the EGM2008 geoid. The ice shelf thickness was estimated assuming hydrostatic equilibrium and a mean ice density of 915 kg/m³.

Association between simple sequence repeat-rich chromosome regions and intergenomic translocation breakpoints in natural populations of allopolyploid wild wheats

Aegilops biuncialis y Aegilops geniculata son dos especies silvestres alotetraploides, con genomios UM, que constituyen un importante reservorio de genes de interés para la mejora del trigo. En este estudio se ha analizado la distribución cromosómica de diferentes secuencias de tipo microsatélites (?single sequence repeat?, SSR) y su relación con las translocaciones intergenómicas U/M, frecuentes en accesiones de ambas especies. En la mayoría de los cromosomas U y en algunos M, se ha localizado una única señal pericéntromérica de la secuencia (ACG)n, mientras que la secuencia (GAA)n aparece como grandes ?clusters? de localización pericentromérica y, en ocasiones, intersticial. En las 5 accesiones portadoras de translocaciones U/M analizadas, se ha comprobado una asociación estadísticamente significativa entre el punto de rotura-reunión de la reordenación y regiones cromosómicas ricas en secuencias SSR.

Preceptorium Nider, hoc est, opus preclarissimu[m] ... Iohannis Nider ... in expositionem preceptorum decalogi : diligentissime nunc tandem cum originalibus collatum et recognitum ac multis locis tersum et emendatum.

Hojas imp. por ambas caras