Sequence Assembly Validation by Restriction Digest Fingerprint Comparison

DNA sequence analysis depends on the accurate assembly of fragment reads for the determination of a consensus sequence. Genomic sequences frequently contain repeat elements that may confound the fragment assembly process, and errors in fragment assembly, and errors in fragment assembly may seriously impact the biological interpretation of the sequence data. Validating the fidelity of sequence assembly by experimental means is desirable. This report examines the use of restriction digest analysis as a method for testing the fidelity of sequence assembly. Restriction digest fingerprint matching is an established technology for high resolution physical map construction, but the requirements for assembly validation differ from those of fingerprint mapping. Fingerprint matching is a statistical process that is robust to the presence of errors in the data and independent of absolute fragment mass determination. Assembly validation depends on the recognition of a small number of discrepant fragments and is very sensitive to both false positive and false negative errors in the data. Assembly validation relies on the comparison of absolute masses derived from sequence with masses that are experimenally determined, making absolute accuracy as well as experimental precision important. As the size of a sequencing project increases, the difficulties in assembly validation by restriction fingerprinting befcome more severe. Simulation studies are used to demonstrate that large-scale errors in sequence assembly can escape detection in fingerprint pattern comparison. Alternative technologies for sequence assembly validation are discussed

Assembly and Analysis of Extended Human Genomic Contig Regions

The Human Genome Project (HGP) has led to the deposit of human genomic sequence in the form of sequenced clones into various databases such as the DNA Data Bank of Japan (DDBJ) (Tateno and Gojobori, 1997), the European Molecular Biology Laboratory (EMBL) Nucleotide Sequence Database (Stoesser, et. al., 1999), and GenBank (Benson, et. al., 1998). Many of these sequenced clones occur in regions where sequencing has taken place either within the same sequencing center or other centers throughout the world. The assembly of extended segments of genomic sequence by looking at overlapping end segments is desired and is currently availabel only in a limited sense from the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/genome/seq/) and Oak Ridge National Laboratories\u27 (ORNL) Genome Channel (http://compbio.ornl.gov/tools/channel/). We attempt to collate a definitive set of nonredundant extended segments of human genomic sequence by taking individual human entires in GenBank greater than 25 kilobases (kb) and extending them on either end. We address the several difficulties that arise when attempting to extend segments

Compositional Analysis of Homogeneous Regions in Human Genomic DNA

Due to increased production of human DNA sequence, it is now possible to explore and understand human genomic organization at the sequence level. In particular, we have studied one of the major organizational components of vertebrate genome organization previously described as isochores (Bernardi, 1993), which are compositionally homogeneous DNA segments based on G+C content. We have examined sequence data for the existence of compositionally differing regions and report that while compositionally homogeneous regions are present in the human genome, current isochore classification schemes are too brad for sequence-level data

Computational Detection of CpG Islands in DNA

Regions of DNA rich in CpG dinucleotides, also known as CpG islands, are often located upstream of the transcription start side in both tissue specific and housekeeping genes. Overall, CPG dinucleotides are observed at a density of 25% the expected level from base composition alone, partially due to 5-methylcytosine decay (Bird, 1993). Since CpG dinucleotides typically occur with low frequency, CpG islands can be distinguished statistically in the genome. Our method of detecting CpG islands involves a heuristic algorithm employing classic changepoint methods and log-likelihood statistics. A Java applet has been created to allow for user interaction and visualization of the segmentation resulting from the changepoint analysis. The model is tested using several sequences obtainable from GenBank (NCBI, 1997), including a 220 Kb fragment of human X chromosome from the filanin (FLM) gene to the glucose-6-phosphate dehydrogenase (G6PD) gene which has been experimentally studied (Rivella, et. al., 1995; E.Y. Chen, et. all., 1996). Preliminary results suggest a breakpoint segmentation that is consistent with observable manual analysis. About 56% of human genes have associated CpG rich islands (Antequera and Bird, 1993). By identifying the CpG islands, it is thought that regions of DNA coding for housekeeping or tissue-specific genes can be located (Antequera and Bird, 1993) even in the absence of transcriptional activity. Biological experiments searching for such genes can then be narrowed given the locations of the CpG islands

Sodium arsenite and hyperthermia modulate cisplatin-DNA damage responses and enhance platinum accumulation in murine metastatic ovarian cancer xenograft after hyperthermic intraperitoneal chemotherapy (HIPEC)

<p>Abstract</p> <p>Background</p> <p>Epithelial ovarian cancer (EOC) is the leading cause of gynecologic cancer death in the USA. Recurrence rates are high after front-line therapy and most patients eventually die from platinum (Pt) - resistant disease. Cisplatin resistance is associated with increased nucleotide excision repair (NER), decreased mismatch repair (MMR) and decreased platinum uptake. The objective of this study is to investigate how a novel combination of sodium arsenite (NaAsO₂) and hyperthermia (43°C) affect mechanisms of cisplatin resistance in ovarian cancer.</p> <p>Methods</p> <p>We established a murine model of metastatic EOC by intraperitoneal injection of A2780/CP70 human ovarian cancer cells into nude mice. We developed a murine hyperthermic intraperitoneal chemotherapy model to treat the mice. Mice with peritoneal metastasis were perfused for 1 h with 3 mg/kg cisplatin ± 26 mg/kg NaAsO₂at 37 or 43°C. Tumors and tissues were collected at 0 and 24 h after treatment.</p> <p>Results</p> <p>Western blot analysis of p53 and key NER proteins (ERCC1, XPC and XPA) and MMR protein (MSH2) suggested that cisplatin induced p53, XPC and XPA and suppressed MSH2 consistent with resistant phenotype. Hyperthermia suppressed cisplatin-induced XPC and prevented the induction of XPA by cisplatin, but it had no effect on Pt uptake or retention in tumors. NaAsO₂prevented XPC induction by cisplatin; it maintained higher levels of MSH2 in tumors and enhanced initial accumulation of Pt in tumors. Combined NaAsO₂and hyperthermia decreased cisplatin-induced XPC 24 h after perfusion, maintained higher levels of MSH2 in tumors and significantly increased initial accumulation of Pt in tumors. ERCC1 levels were generally low except for NaAsO₂co-treatment with cisplatin. Systemic Pt and arsenic accumulation for all treatment conditions were in the order: kidney > liver = spleen > heart > brain and liver > kidney = spleen > heart > brain respectively. Metal levels generally decreased in systemic tissues within 24 h after treatment.</p> <p>Conclusion</p> <p>NaAsO₂and/or hyperthermia have the potential to sensitize tumors to cisplatin by inhibiting NER, maintaining functional MMR and enhancing tumor platinum uptake.</p

Generalized Phase Space Representation of Operators

Introducing asymmetry into the Weyl representation of operators leads to a variety of phase space representations and new symbols. Specific generalizations of the Husimi and the Glauber-Sudarshan symbols are explicitly derivedComment: latex, 8 pages, expanded version accepted by J. Phys.

Use of a Natural Isotopic Signature in Otoliths to Evaluate Scale-Based Age Determination for American Shad

We used delta O-18 signatures in otoliths as a natural tag for hatch year to evaluate the scale-based age determination method used for adult American shad Alosa sapidissima in the York River, Virginia. Juveniles of the 2002 year-class exhibited high delta O-18 values in otolith cores that identified adult members of the cohort as they returned to spawn. Recruitment of the 2002 cohort was monitored for three consecutive years, identifying age-4, age-5, and age-6 individuals of the York River stock. The scale-based age determination method was not suitable for aging age-4, age-5, or age-6 American shad in the York River. On average, 50% of the individuals from the 2002 year-class were aged incorrectly using the scale-based method. These results suggest that the standard age determination method used for American shad is not applicable to the York River stock. Scientists and managers should use caution when applying scale-based age estimates to stock assessments for American shad in the York River and throughout their range, as the applicability of the scale-based method likely varies for each stock. This study highlights a promising new direction for otolith geochemistry to provide cohort-specific markers, and it identifies several factors that should be considered when applying the technique in the future

Slogging and Stumbling Toward Social Justice in a Private Elementary School: The Complicated Case of St. Malachy

This case study examines St. Malachy, an urban Catholic elementary school primarily serving children traditionally marginalized by race, class, linguistic heritage, and disability. As a private school, St. Malachy serves the public good by recruiting and retaining such traditionally marginalized students. As empirical studies involving Catholic schools frequently juxtapose them with public schools, the author presents this examination from a different tack. Neither vilifying nor glorifying Catholic schooling, this study critically examines the pursuit of social justice in this school context. Data gathered through a 1-year study show that formal and informal leaders in St. Malachy adapted their governance, aggressively sought community resources, and focused their professional development to build the capacity to serve their increasingly pluralistic student population. The analysis confirms the deepening realization that striving toward social justice is a messy, contradictory, and complicated pursuit, and that schools in both public and private sectors are allies in this pursuit

On the Importance of Engaging Students in Crafting Definitions

In this paper we describe an activity for engaging students in crafting definitions. We explore the strengths of this particular activity as well as the broader implications of engaging students in crafting definitions more generally