Biomedical subjects

Stephen A Krawetz

Publications and source records attributed to Stephen A Krawetz.

11 recordsLinked to original sources

PubMed2003

Onto-Tools, the toolkit of the modern biologist: Onto-Express, Onto-Compare, Onto-Design and Onto-Translate.

Onto-Tools is a set of four seamlessly integrated databases: Onto-Express, Onto-Compare, Onto-Design and Onto-Translate. Onto-Express is able to automatically translate lists of genes found to be differentially regulated in a given condition into functional profiles characterizing the impact of the condition studied upon various biological processes and pathways. OE constructs functional profiles (using Gene Ontology terms) for the following categories: biochemical function, biological process, cellular role, cellular component, molecular function and chromosome location. Statistical significance values are calculated for each category. Once the initial exploratory analysis identified a number of relevant biological processes, specific mechanisms of interactions can be hypothesized for the conditions studied. Currently, many commercial arrays are available for the investigation of specific mechanisms. Each such array is characterized by a biological bias determined by the extent to which the genes present on the array represent specific pathways. Onto-Compare is a tool that allows efficient comparisons of any sets of commercial or custom arrays. Using Onto-Compare, a researcher can determine quickly which array, or set of arrays, covers best the hypotheses studied. In many situations, no commercial arrays are available for specific biological mechanisms. Onto-Design is a tool that allows the user to select genes that represent given functional categories. Onto-Translate allows the user to translate easily lists of accession numbers, UniGene clusters and Affymetrix probes into one another. All tools above are seamlessly integrated. The Onto-Tools are available online at http://vortex.cs.wayne.edu/Projects.html.

Databases, Nucleic Acid↗

PubMed2003

Nuclear matrix association of the human beta-globin locus utilizing a novel approach to quantitative real-time PCR.

The human beta-globin locus is home to five genes that are regulated in a tissue-specific and developmental stage-specific manner. While the exact mode of expression remains somewhat enigmatic, a significant effort has been focused at the locus control region (LCR). The LCR is marked by five DNase I-hypersensitive sites (HS) approximately 15 kb upstream of the epsilon-globin gene. Nuclear matrix-associated regions (MARs) organize chromatin into functional domains and at least one of the HS appears bound to the nuclear matrix. We have employed an in vivo based PCR MAR assay to investigate the role of MAR-mediated regulation of the beta-globin locus. This was facilitated with a novel reaction efficiency based quantitative real-time PCR analysis software tool, Target Analysis Quantification. Using a log-linear regression strategy, discordances were eliminated. This allowed us to reliably estimate the relative amount of initial template associated with the nuclear matrix at 15 unique regions spanning the beta-globin locus in both non-expressing and expressing cell lines. A dynamic association dependent on expression status was revealed both at the LCR/5'HS region and within the second intron of the beta-globin gene. These results provide the first evidence that nuclear matrix association dynamically mediates the looping of the beta-globin locus to achieve transcriptional control.

Cell Line↗

PubMed2003

The structural organization of sperm chromatin.

The packaging of the male haploid genome within the differentiating spermatid nucleus is facilitated by small basic nuclear proteins called protamines. Although the majority of the DNA in human sperm chromatin is bound by these proteins, a small percentage retains a nucleosomal-like component. These histone-enriched regions may possess enhanced nuclease sensitivity and have been postulated to designate certain genes involved in early embryogenesis. We have shown previously that the chromatin domain containing the two human protamines PRM1 and PRM2 and the transition protein TNP2 forms a DNase I-sensitive conformation in pachytene spermatocytes, a requisite event prior to the haploid expression of its members in round spermatids (Kramer, J. A, McCarrey, J., Djakiew, D., and Krawetz, S. A. (1998) Development 125, 4749-4755). Interestingly, this configuration persists in mature spermatozoa subsequent to the transcriptional silencing of the locus. It was therefore postulated that the retained, enhanced DNase I-sensitive conformation of the PRM1-->PRM2-->TNP2 domain in human sperm may be preferentially histone-enriched. To address this tenet, we examined the chromatin structure of the human PRM1--> PRM2--> TNP2 domain using a PCR-based assay. The results show that this retained, enhanced DNase I sensitive domain reflects an enrichment of histones at discrete regions across the locus. In addition, a similar examination of other genes and repetitive sequences suggests the non-random distribution of histones and protamines within the sperm nucleus. A discussion of these results and their functional significance is presented.

Blotting, Southern↗

PubMed2003

Global functional profiling of gene expression.

The typical result of a microarray experiment is a list of tens or hundreds of genes found to be differentially regulated in the condition under study. Independent of the methods used to select these genes, the common task faced by any researcher is to translate these lists of genes into a better understanding of the biological phenomena involved. Currently, this is done through a tedious combination of searches through the literature and a number of public databases. We developed Onto-Express (OE) as a novel tool able to automatically translate such lists of differentially regulated genes into functional profiles characterizing the impact of the condition studied. OE constructs functional profiles (using Gene Ontology terms) for the following categories: biochemical function, biological process, cellular role, cellular component, molecular function, and chromosome location. Statistical significance values are calculated for each category. We demonstrate the validity and the utility of this comprehensive global analysis of gene function by analyzing two breast cancer datasets from two separate laboratories. OE was able to identify correctly all biological processes postulated by the original authors, as well as discover novel relevant mechanisms.

Breast Neoplasms↗

PubMed2003

Multitasking with molecular dynamics Typhoon: quantifying nucleic acids and autoradiographs.

With increased sensitivity and specificity, fluorescent assays are rapidly becoming the method of choice for nucleic acid quantification. The utility of the Typhoon scanner has now been extended to accurately measure low levels of DNA and RNA (5 ng ml(-1)) with PicoGreen and RiboGreen dyes. In addition, with a few simple modifications, autoradiographic film images can be scanned and quantified with the Typhoon series of scanners.

Autoradiography↗

PubMed2002

Spermatozoal RNA profiles of normal fertile men.

BACKGROUND: Findings from several studies support the conclusion that spermatozoa contain a complex repertoire of mRNAs. Even though these mRNAs are thought to provide an insight into past events of spermatogenesis, their complexity and function have yet to be established. Our aim was to determine whether we could use spermatozoal mRNAs to generate a genetic fingerprint of normal fertile men. METHODS: We used a suite of microarrays containing 27016 unique expressed sequence tags (ESTs) to investigate cDNAs from a pool of 19 testes, cDNAs from a pool of nine individual ejaculate spermatozoal mRNAs, and cDNAs constructed from spermatozoal mRNAs from a single ejaculate. We also used ontological data mining to determine the function of the genes identified in each EST profile. FINDINGS: The cDNAs from the testes, pooled ejaculate, and single ejaculate hybridised to 7157, 3281, and 2780 ESTs, respectively. The testicular population contained all of the ESTs identified by the cDNAs from the pooled and individual ejaculate. The pooled ejaculate population contained all but four ESTs identified from the individual ejaculate. A subset of the spermatozoal mRNAs was associated with embryo development. INTERPRETATION: The microarray data from testes and spermatozoa (pooled and individual) were concordant, supporting the view that a spermatozoal mRNA fingerprint can be obtained from normal fertile men. Thus, profiling can be used to monitor past events-ie, gene expression of spermatogenesis. Moreover, the data suggest that, in addition to delivering the paternal genome, spermatozoa provide the zygote with a unique suite of paternal mRNAs. Ejaculate spermatozoa can now be used as a non-invasive proxy for investigations of testis-specific infertility.

Adolescent↗

PubMed2002

Somatic mutations of the lysyl oxidase gene on chromosome 5q23.1 in colorectal tumors.

Lysyl oxidase (LOX), a copper-dependent amine oxidase, has been implicated in tumor suppression and cell growth regulation. The chromosomal locus of LOX, 5q23, is affected by loss of heterozygosity (LOH) in colon cancer, suggesting that the LOX gene could be affected by LOH and consequently, loss or reduction of LOX function contribute to the tumorigenic process. Identification of microsatellite markers within the LOX locus has allowed us to map the LOX gene within the 5q23.1 region. Analysis of this locus and flanking loci in matched tumor and blood DNA samples from a panel of colorectal cancer patients, demonstrated that 38% (16/42) of informative samples were affected by LOH or allelic imbalance. Furthermore, 75% (6/8) of these tumor samples were shown to have significantly reduced LOX mRNA levels. Similar reduction in LOX levels were detected in a panel of matched normal colon and colon tumor samples. Tumor samples demonstrating LOH by RFLP, were subject to mutational analysis, including RT-PCR, exonic deletion detection by PCR, cDNA and genomic DNA sequencing, and were found to have a spectrum of alterations and mutations affecting the LOX gene. These results confirm that loss or reduction of LOX function during tumor development is a direct consequence of somatic mutations and is associated with colon tumor pathogenesis.

Adenocarcinoma↗

PubMed2002

X-chromosome inactivation during spermatogenesis is regulated by an Xist/Tsix-independent mechanism in the mouse.

Transcriptional inactivation of the single X chromosome occurs in spermatogenic cells during male meiosis in mammals and has been shown to be coincident with expression of the Xist gene in spermatogonia and spermatocytes in mice. However, male mice carrying an ablated Xist gene show normal fertility. Here we examined expression from the Xist locus during spermatogenesis in wild-type mice and detected sense (Xist), but not antisense (Tsix) transcripts. In addition, we examined expression and chromatin conformation of X-linked structural genes in meiotic and postmeiotic spermatogenic cells from wild-type and Xist(-) mice and found no differences associated with the absence of a functional Xist gene. These results, along with the formation of a morphologically normal XY body in primary spermatocytes in Xist(-) mice, indicate that a functional Xist gene is not required for X-chromosome inactivation during spermatogenesis and that this process is therefore regulated by a different mechanism than that which regulates X-chromosome inactivation in female embryonic cells.

Animals↗

PubMed2002

Profiling gene expression using onto-express.

Gene expression profiles obtained through microarray or data mining analyses often exist as vast data strings. To interpret the biology of these genetic profiles, investigators must analyze this data in the context of other information such as the biological, biochemical, or molecular function of the translated proteins. This is particularly challenging for a human analyst because large quantities of less than relevant data often bury such information. To address this need we implemented an automated routine, called Onto-Express (http://vortex.cs.wayne.edu:8080), to systematically translate genetic fingerprints into functional profiles. Using strings of accession or cluster identification numbers, Onto-Express searches the public databases and returns tables that correlate expression profiles with the cytogenetic locations, biochemical and molecular functions, biological processes, cellular components, and cellular roles of the translated proteins. The profiles created by Onto-Express fundamentally increase the value of gene expression analyses by facilitating the translation of quantitative value sets to records that contain biological implications.

Gene Expression Profiling↗

PubMed2002

Identification, molecular characterization, and tissue expression of OVCOV1.

Key to the maternal-embryonic dialogue during early implantation are the extra embryonic trophoblast cells that play several critical roles including invasion of the maternal decidua. Cytotrophoblast proliferation and differentiation along an invasive pathway is regulated by a positive gradient of oxygen tension. We have employed transcript profiling to characterize the extra-embryonic tissues during early human implantation. We probed a cDNA library isolated from the ectoplacental cone region of stage 7.5 days post coitus mouse embryos with cDNA from hypoxic cultured human trophoblast cells. This identified the pattern of expression of a series of previously unknown genes that correlate to day 20 of human embryonic development. One EST was selected for further analysis since it is identical to the 1588-bp CGI-15 sequence assembled in silico. The human gene is organized into 10 exons 2063 bp in length. The open reading frame contains 1272 bp predicting protein composed of 365 amino acids. This gene is expressed in many tissues including those found in the reproductive tract. Its expression is regulated by oxygen tension and is unaffected by estradiol or progesterone treatment.

Animals↗

PubMed2002

A bioinformatic strategy to rapidly characterize cDNA libraries.

MOTIVATION: Complementary DNA libraries can define the genetic constituents of specific cells and/or tissues. Their sequencing will illuminate the transcriptome but it is a monumental task requiring considerable resources. RESULTS: We have employed a computational search in conjunction with a microarray-based strategy to alleviate the impediments of deriving a consensus of records that describe testis gene expression. This strategy identified 5681 unique testes-expressed genes of which 3265 were previously portrayed in the UniGene database. Interestingly, a total of 2416 novel testes-expressed genes were identified. This clearly demonstrates that microarrays can be used to rapidly discover a large number of new transcripts.

Adolescent↗