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Biomedical subjects

M A Marra

Publications and source records attributed to M A Marra.

At least 19 recordsLinked to original sources
PubMed2006

Identification of genes targeted by the androgen and PKA signaling pathways in prostate cancer cells.

Progression of prostate cancer to androgen independence is suspected to involve the androgen and protein kinase A (PKA) signaling pathways. Here for the first time, the transcriptomes associated with each pathway and common transcriptional targets in response to stimulation of both pathways were identified in human prostate cancer cells using Affymetrix GeneChip technology (Human Genome U133 plus2). Statistically significant changes in the levels of 858 genes in response to androgen and 303 genes in response to activation of the PKA pathway were determined using GeneSpring software. Expression of a subset of these genes (22) that were transcriptional targets for the androgen and/or PKA pathways were validated by reverse transcriptase-polymerase chain reaction and Western blot analyses. Application of small interfering RNAs to the androgen receptor (AR) revealed that in addition to KLK3, levels of expression of KLK2 and SESN1 were regulated by AR activated by both the androgen and PKA signaling pathways. SESN1 was identified as a gene repressed by activated AR. These results provide a broad view of the effects of the androgen and PKA signaling pathways on the transcriptional program of prostate cancer cells and indicate that only a limited number of genes are targeted by cross-talk between AR and PKA pathways.

Androgens↗
PubMed2002

Assembly of fingerprint contigs: parallelized FPC.

SUMMARY: One of the more common uses of the program FingerPrint Contigs (FPC) is to assemble random restriction digest 'fingerprints' of overlapping genomic clones into contigs. To improve the rate of assembling contigs from large fingerprint databases we have adapted FPC so that it can be run in parallel on multiple processors and servers. The current version of 'parallelized FPC' has been used in our laboratory to assemble mammalian BAC fingerprint databases, each containing more than 300000 BAC fingerprints. AVAILABILITY: This parallelized version of FPC is available under the GNU GPL licence, and can be downloaded from ftp://ftp.bcgsc.bc.ca/pub/fpcd.

Algorithms↗
PubMed2001

Initial sequencing and analysis of the human genome.

The human genome holds an extraordinary trove of information about human development, physiology, medicine and evolution. Here we report the results of an international collaboration to produce and make freely available a draft sequence of the human genome. We also present an initial analysis of the data, describing some of the insights that can be gleaned from the sequence.

Animals↗
PubMed2001

Changes in gene expression associated with developmental arrest and longevity in Caenorhabditis elegans.

Gene expression in a developmentally arrested, long-lived dauer population of Caenorhabditis elegans was compared with a nondauer (mixed-stage) population by using serial analysis of gene expression (SAGE). Dauer (152,314) and nondauer (148,324) SAGE tags identified 11,130 of the predicted 19,100 C. elegans genes. Genes implicated previously in longevity were expressed abundantly in the dauer library, and new genes potentially important in dauer biology were discovered. Two thousand six hundred eighteen genes were detected only in the nondauer population, whereas 2016 genes were detected only in the dauer, showing that dauer larvae show a surprisingly complex gene expression profile. Evidence for differentially expressed gene transcript isoforms was obtained for 162 genes. H1 histones were differentially expressed, raising the possibility of alternative chromatin packaging. The most abundant tag from dauer larvae (20-fold more abundant than in the nondauer profile) corresponds to a new, unpredicted gene we have named tts-1 (transcribed telomere-like sequence), which may interact with telomeres or telomere-associated proteins. Abundant antisense mitochondrial transcripts (2% of all tags), suggest the existence of an antisense-mediated regulatory mechanism in C. elegans mitochondria. In addition to providing a robust tool for gene expression studies, the SAGE approach already has provided the advantage of new gene/transcript discovery in a metazoan.

Animals↗
PubMed2001

An oligonucleotide fingerprint normalized and expressed sequence tag characterized zebrafish cDNA library.

The zebrafish is a powerful system for understanding the vertebrate genome, allowing the combination of genetic, molecular, and embryological analysis. Expressed sequence tags (ESTs) provide a rapid means of identifying an organism's genes for further analysis, but any EST project is limited by the availability of suitable libraries. Such cDNA libraries must be of high quality and provide a high rate of gene discovery. However, commonly used normalization and subtraction procedures tend to select for shorter, truncated, and internally primed inserts, seriously affecting library quality. An alternative procedure is to use oligonucleotide fingerprinting (OFP) to precluster clones before EST sequencing, thereby reducing the re-sequencing of common transcripts. Here, we describe the use of OFP to normalize and subtract 75,000 clones from two cDNA libraries, to a minimal set of 25,102 clones. We generated 25,788 ESTs (11,380 3' and 14,408 5') from over 16,000 of these clones. Clustering of 10,654 high-quality 3' ESTs from this set identified 7232 clusters (likely genes), corresponding to a 68% gene diversity rate, comparable to what has been reported for the best normalized human cDNA libraries, and indicating that the complete set of 25,102 clones contains as many as 17,000 genes. Yet, the library quality remains high. The complete set of 25,102 clones is available for researchers as glycerol stocks, filters sets, and as individual EST clones. These resources have been used for radiation hybrid, genetic, and physical mapping of the zebrafish genome, as well as positional cloning and candidate gene identification, molecular marker, and microarray development.

Animals↗
PubMed2001

Theories and applications for sequencing randomly selected clones.

Theory is developed for the process of sequencing randomly selected large-insert clones. Genome size, library depth, clone size, and clone distribution are considered relevant properties and perfect overlap detection for contig assembly is assumed. Genome-specific and nonrandom effects are neglected. Order of magnitude analysis indicates library depth is of secondary importance compared to the other variables, especially as clone size diminishes. In such cases, the well-known Poisson coverage law is a good approximation. Parameters derived from these models are used to examine performance for the specific case of sequencing random human BAC clones. We compare coverage and redundancy rates for libraries possessing uniform and nonuniform clone distributions. Results are measured against data from map-based human-chromosome-2 sequencing. We conclude that the map-based approach outperforms random clone sequencing, except early in a project. However, simultaneous use of both strategies can be beneficial if a performance-based estimate for halting random clone sequencing is made. Results further show that the random approach yields maximum effectiveness using nonbiased rather than biased libraries.

Cloning, Molecular↗
PubMed2001

Left atrial volume: important risk marker of incident atrial fibrillation in 1655 older men and women.

OBJECTIVE: To evaluate the contribution of left atrial (LA) volume in predicting atrial fibrillation (AF). PATIENTS AND METHODS: In this retrospective cohort study, a random sample of 2200 adults was identified from all Olmsted County, Minnesota, residents who had undergone transthoracic echocardiographic assessment between 1990 and 1998 and were 65 years of age or older at the time of examination, were in sinus rhythm, and had no history of AF or other atrial arrhythmias, stroke, pacemaker, congenital heart disease, or valve surgery. The LA volume was measured off-line by using a biplane area-length method. Clinical characteristics and the outcome event of incident AF were determined by retrospective review of medical records. Echocardiographic data were retrieved from the laboratory database. From this cohort, 1655 patients in whom LA size data were available were followed from baseline echocardiogram until development of AF or death. The clinical and echocardiographic associations of AF, especially with respect to the role of LA volume in predicting AF, were determined. RESULTS: A total of 666 men and 989 women, mean +/- SD age of 75.2 +/- 7.3 years (range, 65-105 years), were followed for a mean +/- SD of 3.97 +/- 2.75 years (range, < 1.00-10.78 years); 189 (11.4%) developed AF. Cox model 5-year cumulative risks of AF by quartiles of LA volume were 3%, 12%, 15%, and 26%, respectively. With Cox proportional hazards multivariate models, logarithmic LA volume was an independent predictor of AF, incremental to clinical risk factors. After adjusting for age, sex, valvular heart disease, and hypertension, a 30% larger LA volume was associated with a 43% greater risk of AF, incremental to history of congestive heart failure (hazard ratio [HR], 1.887; 95% confidence interval [CI], 1.230-2.895; P = .004), myocardial infarction (HR, 1.751; 95% CI, 1.189-2.577; P = .004), and diabetes (HR, 1.734; 95% CI, 1.066-2.819; P = .03). Left atrial volume remained incremental to combined clinical risk factors and M-mode LA dimension for prediction of AF (P < .001). CONCLUSION: This study showed that a larger LA volume was associated with a higher risk of AF in older patients. The predictive value of LA volume was incremental to that of clinical risk profile and conventional M-mode LA dimension.

Aged↗
PubMed2000

Identifying potential tumor markers and antigens by database mining and rapid expression screening.

Genes expressed specifically in malignant tissue may have potential as therapeutic targets but have been difficult to locate for most cancers. The information hidden within certain public databases can reveal RNA transcripts specifically expressed in transformed tissue. To be useful, database information must be verified and a more complete pattern of tissue expression must be demonstrated. We tested database mining plus rapid screening by fluorescent-PCR expression comparison (F-PEC) as an approach to locate candidate brain tumor antigens. Cancer Genome Anatomy Project (CGAP) data was mined for genes highly expressed in glioblastoma multiforme. From 13 mined genes, seven showed potential as possible tumor markers or antigens as determined by further expression profiling. Now that large-scale expression information is readily available for many of the commonly occurring cancers, other candidate tumor markers or antigens could be located and evaluated with this approach.

Algorithms↗
PubMed1999

High-throughput plasmid DNA purification for 3 cents per sample.

To accommodate the increasingly rapid rates of DNA sequencing we have developed and implemented an inexpensive, expeditious method for the purification of double-stranded plasmid DNA clones. The robust nature, high throughput, low degree of technical difficulty and extremely low cost have made it the plasmid DNA preparation method of choice in both our expressed sequence tag (EST) and genome sequencing projects. Here we report the details of the method and describe its application in the generation of more than 700 000 ESTs at a rate exceeding 16 000 per week.

DNA, Bacterial↗
PubMed1999

A public database for gene expression in human cancers.

A public database, SAGEmap, was created as a component of the Cancer Genome Anatomy Project to provide a central location for depositing, retrieving, and analyzing human gene expression data. This database uses serial analysis of gene expression to quantify transcript levels in both malignant and normal human tissues. By accessing SAGEmap (http://www.ncbi.nlm.nih.gov/SAGE) the user can compare transcript populations between any of the posted libraries. As an initial demonstration of the database's utility, gene expression in human glioblastomas was compared with that of normal brain white matter. Of the 47,174 unique transcripts expressed in these two tissues, 471 (1.0%) were differentially expressed by more than 5-fold (P<0.001). Classification of these genes revealed functions consistent with the biological properties of glioblastomas, in particular: angiogenesis, transcription, and cell cycle related genes.

Brain↗
PubMed1999

Identification and characterization of a serine hydroxymethyltransferase isoform in Caenorhabditis briggsae.

In the nematode Caenorhabditis elegans, the maternal effect lethal gene mel-32 encodes a serine hydroxymethyltransferase isoform. Since interspecies DNA comparison is a valuable tool for identifying sequences that have been conserved because of their functional importance or role in regulating gene activity, mel-32(SHMT) genomic DNA from C. elegans was used to screen a genomic library from the closely related nematode Caenorhabditis briggsae. The C. briggsae genomic clone identified fully rescues the Mel-32 phenotype in C. elegans, indicating functional and regulatory conservation. Computer analysis reveals that CbMEL-32(SHMT) is 92% identical (97% similar) to CeMEL-32(SHMT) at the amino acid level over the entire length of the protein (484 amino acids), whereas the coding DNA is 82.5% identical (over 1455 nucleotides). Several highly conserved non-coding regions upstream and downstream of the mel-32(SHMT) gene reveal potential regulatory sites that may bind trans-acting protein factors.

Amino Acid Sequence↗
PubMed1999

Functional genomics in Caenorhabditis elegans: An approach involving comparisons of sequences from related nematodes.

Comparative genomic analysis was used to investigate the gene structure of the bli-4 locus from two related Caenorhabditis species, C. elegans and C. briggsae. In C. elegans, bli-4 is a complex gene encoding a member of the kex2/subtilisin-like family of proprotein convertases. Genomic sequence comparisons coupled with RT-PCR analysis identified five additional coding exons that had not been identified previously using standard recombinant DNA techniques. The C. briggsae gene was able to rescue both viable blistered and developmentally arrested mutants of C. elegans bli-4, demonstrating functional conservation. In addition, deletion analysis of conserved sequences outside of coding regions, combined with phenotypic rescue experiments, identified regulatory elements that alter the expression of the bli-4 gene. These results demonstrate the utility of genomic sequence comparisons of homologous genes in related species as an effective tool with which to dissect the functional information of complex genes.

Alternative Splicing↗
PubMed1999

The CMT2D locus: refined genetic position and construction of a bacterial clone-based physical map.

Charcot-Marie-Tooth (CMT) disease is a progressive neuropathy of the peripheral nervous system, typically characterized by muscle weakness of the distal limbs. CMT is noted for its genetic heterogeneity, with four distinct loci already identified for the axonal form of the disease (CMT2). In 1996, linkage analysis of a single large family revealed the presence of a CMT2 locus on chromosome 7p14 (designated CMT2D). Additional families have been linked subsequently to the same genomic region, including one with distal spinal muscular atrophy (dSMA) and one with mixed features of dSMA and CMT2; symptoms in both of these latter families closely resemble those seen in the original CMT2D family. There is thus a distinct possibility that CMT2 and dSMA encountered in these families reflect allelic heterogeneity at a single chromosome 7 locus. In the study reported here, we have performed more detailed linkage analysis of the original CMT2D family based on new knowledge of the physical locations of various genetic markers. The region containing the CMT2D gene, as defined by the original family, overlaps with those defined by at least two other families with CMT2 and/or dSMA symptoms. Both yeast artificial chromosome (YAC) and bacterial clone-based [bacterial artificial chromosome (BAC) and P1-derived artificial chromosome (PAC)] contig maps spanning approximately 3.4 Mb have been assembled across the combined CMT2D critical region, with the latter providing suitable clones for systematic sequencing of the interval. Preliminary analyses have already revealed at least 28 candidate genes and expressed-sequence tags (ESTs). The mapping information reported here in conjunction with the evolving sequence data should expedite the identification of the CMT2D/dSMA gene or genes.

Bacteriophage P1↗
PubMed1997

Representation of cloned genomic sequences in two sequencing vectors: correlation of DNA sequence and subclone distribution.

Representation of subcloned Caenorhabditis elegans and human DNA sequences in both M13 and pUC sequencing vectors was determined in the context of large scale genomic sequencing. In many cases, regions of subclone under-representation correlated with the occurrence of repeat sequences, and in some cases the under-representation was orientation specific. Factors which affected subclone representation included the nature and complexity of the repeat sequence, as well as the length of the repeat region. In some but not all cases, notable differences between the M13 and pUC subclone distributions existed. However, in all regions lacking one type of subclone (either M13 or pUC), an alternate subclone was identified in at least one orientation. This suggests that complementary use of M13 and pUC subclones would provide the most comprehensive subclone coverage of a given genomic sequence.

Animals↗
PubMed1997

High throughput fingerprint analysis of large-insert clones.

As part of the Human Genome Project, the Washington University Genome Sequencing Center has commenced systematic sequencing of human chromsome 7. To organize and supply the effort, we have undertaken the construction of sequence-ready physical maps for defined chromosomal intervals. Map construction is a serial process composed of three main activities. First, candidate STS-positive large-insert PAC and BAC clones are identified. Next, these candidate clones are subjected to fingerprint analysis. Finally, the fingerprint data are used to assemble sequence-ready maps. The fingerprinting method we have devised is key to the success of the overall approach. We present here the details of the method and show that the fingerprints are of sufficient quality to permit the construction of megabase-size contigs in defined regions of the human genome. We anticipate that the high throughput and precision characteristic of our fingerprinting method will make it of general utility.

Base Sequence↗