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S A Krawetz

Publications and source records attributed to S A Krawetz.

At least 19 recordsLinked to original sources
PubMed2005

LARALink: a web application for cytogenetic linkage analysis.

Genomic and expression data have increased dramatically over the last several years. This is primarily due to the completion of the human genome project as well as an upsurge in the use of various high-throughput technologies. Recent attempts to correlate genomic and expression data have stimulated the scientific community to determine how this data can be used within a clinical setting (P Khatri et al., Genomics 2002: 79: 266; LJ van't Veer et al., Nature 2002: 415: 530). LARALink (Loci Analysis for Rearrangements Link) is a database-driven web application that utilizes several public datasets to analyze clinical cytogenetic data to identify candidate genes. LARALink allows UniGene clusters or single-nucleotide polymorphisms (SNPs) to be queried for multiple patients by cytoband, chromosome marker, or base pair. The results can be further refined with the use of an anatomical site, developmental stage, pathology, or cell-type expression filter. Once a set of UniGene clusters (expressed genes) has been identified either for a single patient or for a shared region among multiple patients, the expression-distribution profile, expressed sequence tags (ESTs), or online mendelian inheritance in man (OMIM) entries are displayed. The utility of this tool is shown by its application to both research and clinical medicine. LARALink is a public resource available at: http://www.laralink.bioinformatics.wayne.edu:8080/unigene.

Alzheimer Disease↗
PubMed2003

From DNA structure to gene expression: mediators of nuclear compartmentalization and dynamics.

Eukaryotic genomes are functionally compartmentalized into chromatin domains by their attachment to a supporting structure that has traditionally been termed the nuclear matrix. Present evidence indicates the dynamics of this entity, which requires particular properties of the elements that mediate this kind of interaction. Above all, this is enabled by the so-called 'mass binding phenomenon' by which scaffold/matrix-attachment regions (S/MARs) reversibly associate with ubiquitous factors. Recent investigations and novel techniques have shown that these contacts can be altered by modulators as well as by specific interactions with the components of enhancers and locus control regions.

Chromatin↗
PubMed2003

Analysis of marker or complex chromosomal rearrangements present in pre- and post-natal karyotypes utilizing a combination of G-banding, spectral karyotyping and fluorescence in situ hybridization.

The significance of complex chromosomal rearrangements presents a diagnostic dilemma. In the past, the use of G-banding coupled with fluorescence in situ hybridization (FISH) has been the standard approach. The recent development of spectral karyotyping (SKY) and multicolor FISH (M-FISH) has resulted in an increased accuracy of identification of marker or other complex chromosomal rearrangements. However, owing to the additional cost and time associated with SKY or M-FISH, and the restricted availability of such imaging facilities in many centers, it is not feasible to perform these procedures routinely on every sample. In addition, the identification of an aberration by SKY or M-FISH will often require confirmation by FISH. A practical approach is needed to take advantage of the complementary strengths of each method. In our center we utilize an algorithm that dictates the use of routine G-banding for the initial preliminary evaluation of a patient, followed by SKY characterization if marker chromosomes or complex translocations are detected by the G-banding analysis. According to this algorithm, FISH is used to verify the results once the origin of the abnormal chromosome has been determined by SKY. To demonstrate the effectiveness of this algorithm, we have analyzed both amniocyte and lymphocyte slides, using a combination of G-banding, SKY, and FISH. Our results confirm that an algorithm which selectively uses SKY or M-FISH will provide an efficient and improved method for pre- and post-natal chromosomal analysis.

Algorithms↗
PubMed2002

Type II gonadotropin-releasing hormone receptor transcripts in human sperm.

GnRH regulates reproduction via the well-characterized mammalian pituitary GnRH receptor (type I). In addition, two homologous genes for a second form of the GnRH receptor (type II) are present in the human genome, one on chromosome 14 and the second on chromosome 1. The chromosome 14 gene is ubiquitously transcribed at high levels in the antisense orientation but lacks exon 1, required to encode a full-length receptor. In comparison, the chromosome 1 gene contains all three exons. The issue of whether this gene is transcribed in any human tissue(s), and whether these transcripts encode a functional receptor protein, remains unresolved. We have directly addressed this by screening a panel of human RNAs by hybridization and RT-PCR. These analyses showed that, unlike the chromosome 14 gene, chromosome 1 gene expression is limited and of low abundance. Exon 1-containing transcripts were detected by in situ hybridization in mature sperm and in human postmeiotic testicular cells. Further sequence analysis revealed that although all the potential coding segments were present, the human transcripts, like the gene, contain a stop codon within the coding region and a frame-shift relative to other mammalian GnRH receptors. Although this suggests that the human gene may be a transcribed pseudogene, a functional type II GnRH receptor cDNA has recently been cloned from monkeys. Given the well-established role of GnRH in spermatogenesis and reported evidence of type II GnRH receptor immunoreactivity in human tissues, it is possible that the chromosome 1 gene is functional.

Amino Acid Sequence↗
PubMed2001

Development of a 950-gene DNA array for examining gene expression patterns in mouse testis.

BACKGROUND: Over the past five years, interest in and use of DNA array technology has increased dramatically, and there has been a surge in demand for different types of arrays. Although manufacturers offer a number of pre-made arrays, these are generally of utilitarian design and often cannot accommodate the specific requirements of focused research, such as a particular set of genes from a particular tissue. We found that suppliers did not provide an array to suit our particular interest in testicular toxicology, and therefore elected to design and produce our own. RESULTS: We describe the procedures used by members of the US Environmental Protection Agency MicroArray Consortium (EPAMAC) to produce a mouse testis expression array on both filter and glass-slide formats. The approaches used in the selection and assembly of a pertinent, nonredundant list of testis-expressed genes are detailed. Hybridization of the filter arrays with normal and bromochloroacetic acid-treated mouse testicular RNAs demonstrated that all the selected genes on the array were expressed in mouse testes. CONCLUSION: We have assembled two lists of mouse (950) and human (960) genes expressed in the mouse and/or human adult testis, essentially all of which are available as sequence-verified clones from public sources. Of these, 764 are homologous and will therefore enable close comparison of gene expression between murine models and human clinical testicular samples.

Adult↗
PubMed2001

Whole-body gene expression by data mining.

To date, a comprehensive survey of the expression of lysyl oxidase (LOX), lysyl oxidase-like 1 (LOXL1), and lysyl oxidase-like 2 (LOXL2) has yet to be performed. The use of in vitro strategies to accomplish this task would prove daunting as it is both time-consuming and costly. We present a new in silico data mining strategy that directly addresses these limitations. Sequences corresponding to the 3' untranslated regions of LOX, LOXL1, and LOXL2 were individually queried against the human expressed sequence tag database (dbEST). In this manner, the entire tissue repertoire available in the dbEST was surveyed. This provided an estimate of the levels of mRNA transcripts in a variety of adult and fetal tissues. We have also employed this strategy to determine the pattern of expression and levels of a newly discovered gene, CGI-15. The veracity of this technique has been independently assessed by semiquantitative PCR analysis. The application of this technology is bounded only by the ever-growing information available in the GenBank, UniGene, and human EST databases. The utility of our data mining strategy to establish relative transcript levels in numerous tissues is presented.

3' Untranslated Regions↗
PubMed2001

Sperm nuclear matrix association of the PRM1-->PRM2-->TNP2 domain is independent of Alu methylation.

Genes or multigenic chromosomal regions are organized by the nuclear matrix into a series of functionally discrete genic domains. Biophysical analysis of the human chromosome 16p13.13 region has shown that the PRM1-->PRM2-->TNP2 protamine containing multigenic locus is bounded by two sperm nuclear matrix attachment regions (MAR). This domain exists in a transcriptionally readied or potentiated (i.e. open) chromatin state when associated with the nuclear matrix. The MAR-bounded PRM1-->PRM2-->TNP2 locus is nestled in an Alu repetitive element dense region. Fluorescence in-situ hybridization, analysis of sperm nuclear matrix/halo preparations showed that the PRM1-->PRM2-->TNP2 domain specifically localizes to the sperm nuclear matrix. This raised the question of whether nuclear matrix association and gene expression in this locus is mediated by Alu methylation. The methylation status of the various Alu elements contained within the human PRM1-->PRM2-->TNP2 locus was therefore assayed. The seven Alu elements tested, including those associated with the matrix attachment regions within the PRM1-->PRM2-->TNP2 locus, were fully methylated in sperm DNA. Conversely, these same Alu repeats were hypomethylated within the erythroleukaemic cell line, K562, which does not express any of the genes from this domain. This study shows that Alu methylation status is independent of attachment of PRM1-->PRM2-->TNP2 locus to the nuclear matrix and that Alu methylation does not play a leading role in the regulation of this domain.

Alu Elements↗
PubMed2001

Re-defining the chromatin loop domain.

It is commonly accepted that the loop domain represents the basic structural unit of eukaryotic chromatin associated with DNA replication, gene expression and higher order packaging. However, molecular-cytological information defining the loop domain is lacking. There are gaps in our knowledge of the loop structure and how it regulates gene expression. The combination of new data/reagents from the Human Genome Project plus the use of novel molecular cytological technology will provide answers. Here we briefly review the status of chromatin loop research and pose questions that need to be addressed. New experimental systems are also presented to target some long-standing issues regarding the structure and function of the chromatin loop domain and its relationship with the nuclear matrix. This new knowledge will have a profound impact for modern genetics and molecular medicine.

Animals↗
PubMed2001

The combination of SKY and specific loci detection with FISH or immunostaining.

Spectral karyotyping (SKY) represents an effective tool to detect individual chromosomes and analyze major karyotype abnormalities within an entire genome. We have tested the feasibility of combining SKY and FISH/protein detection in order to combine SKY's unique abilities with specific loci detection. Our experimental results demonstrate that various combined protocols involving SKY, FISH and immunostaining work well when proper procedures are used. This combined approach allows the tracking of key genes or targeted chromosome regions while monitoring changes throughout the whole genome. It is particularly useful when simultaneously monitoring the behavior of both protein complexes and DNA loci within the genome. The details of this methodology are described and systematically tested in this communication.

Animals↗
PubMed2001

Design and implementation of an introductory course for computer applications in molecular genetics. A case study.

Formal training in computational biology was initiated at Wayne State University in 1990 to meet the needs of the faculty. This was still at a time when the molecular databases and analysis tools could be housed in what is now equivalent to a modern but dated desktop computer. In 1995 the course was expanded to include graduate students to provide these senior students with a foundation in computational biology. This course has armed our students with a requisite set of basic skills that are necessary for a successful career in molecular genetics. It is now an integral component of the graduate program of the Center for Molecular Medicine and Genetics and our experiences in course delivery have been detailed (BioInformatics Methods and Protocols, S. Misener and S. A. Krawetz, eds., Humana Press, Totowa, NJ, 2000.). The course was expanded to a campus-wide unlimited enrollment program for the summer of 2000 to address the needs of our student body. In this review we present our experience with delivering a multidisciplinary campus-wide computational biology course to a new and widely diverse student body.

Computational Biology↗
PubMed2001

Recovering filter-based microarray data for pathways analysis using a multipoint alignment strategy.

The use of commercial microarrays is rapidly becoming the method of choice for profiling gene expression and assessing various disease states. Research Genetics has provided a series of biological and software tools to the research community for these analyses. The fidelity of data analysis using these tools is dependent on a series of well-defined reference control points in the array. During the course of our investigations, it became apparent that in some instances the reference control points that are required for analysis became lost in background noise. This effectively halted the analysis and the recovery of any information contained within that experiment. To recover this data and to increase analytical veracity, the simple strategy of superimposing a template of reference control points onto the experimental array was developed. The utility of this tool is established in this communication.

Filtration↗
PubMed2001

Characterization of the region encompassing the human lysyl oxidase locus.

A 46,823 bp region of human chromosome 5q23.1 encompassing the seven-exon lysyl oxidase gene was characterized at the primary sequence level. Approximately 17.4% of this region is comprised of repetitive elements. The gene colocalizes with microsatellite marker D5S467. It is flanked by two candidate nuclear matrix association regions (MARs). The 5' MAR centered at position 12,500 is of the AT-rich and curved DNA class. This is followed by a large CpG island containing fifty-seven putative regulatory elements which extend from just upstream of exon 1 to intron 2. The larger 3' MAR, spans position 35,050-39,750 and is characterized by a TG-rich kinked structure that also contains a topoisomerase II binding site. Based on these results model of the transcriptional regulation of the lysy/oxidase gene is presented.

Base Sequence↗
PubMed2000

Human spermatogenesis as a model to examine gene potentiation.

The first tier of control over the expression of genic domains utilizes chromatin structure. Before the onset of transcription, the chromatin domain that encompasses the gene(s) must assume an open conformation. This renders large segments of the genome available to the tissue-specific and ubiquitous trans-factors necessary for proper expression of the genes present. This process has been termed potentiation. It is a necessary obligate, but alone it is not sufficient for gene expression. Spermatogenesis, the development of a viable fertile male gamete, provides a unique model to begin to address the underlying mechanism(s) governing differentiation and tissue-specific gene expression. Male gametogenesis is typified by the activation of numerous genes whose products have novel functions, as well as testis-specific forms of constitutively expressed somatic genes. We have shown that mouse spermatogenesis represents a selective potentiative process (Kramer et al., 1998: Development 125:4749-4655), but little is known about its human counterpart. To fill this void we have examined the potentiative state of several spermatid-expressed genes during the latter stages of human spermatogenesis. We have shown that spermatidexpressed genes are potentiated by the pachytene stage of differentiation. Furthermore, we establish that a chromatin domain functions as a discrete structural unit during differentiation. Interestingly, some of these open structures are maintained in the mature spermatozoon.

Acrosin↗
PubMed2000

Characterizing a human lysyl oxidase chromosomal domain.

The expression of each locus in our genome is regulated by a gene-potentiative mechanism, whereby the gene first assumes the necessary structural conformation to enable transcription. This serves as the corner-stone for the three-tiered regulatory mechanism of potentiation, i.e., the opening of a chromatin domain, initiation of transcription, and transcript elongation. Although this is now generally accepted as the pathway that mediates gene expression, it has never been shown directly to control the expression of any heart-related gene. Lysyl oxidase enzymatically crosslinks members of the extracellular matrix, including elastin and collagen. Formation of these structures is essential to development and tissue repair. This system has enabled us to begin to address the underlying mechanism governing the selection of connective tissue genes for expression. However, before one can dissect this mechanism, it is necessary to define and characterize the locus, i.e., the corresponding genic domain. Our progress toward creating the resources necessary to unravel this mechanism is summarized in this review.

Chromosomes, Human, Pair 5↗
PubMed2000

Mammalian spermatozoal mRNAs: tools for the functional analysis of male gametes.

Mammalian spermatogenesis reflects a dynamic partnership between somatic genes which display altered expression patterns within the testes and germ cell specific genes expressed solely during male gametogenesis. Among the latter include the haploid-specific transition proteins and protamines which facilitate the molecular restructuring of the chromatin during the terminal differentiation of the spermatid nucleus. Previously, we have shown that the human protamines PRM1, PRM2 and transition protein TNP2 transcripts persist in mature spermatozoa subsequent to their functional role during spermiogenesis. We now demonstrate the conserved presence of these same spermatozoal mRNAs in mouse. While the basis for the persistence of these haploid-specific transcripts remains poorly defined, the results presented firmly establish that the presence of mRNA in mammalian spermatozoa is a general phenomenon. The potential use of these transcripts as a tool for investigating molecular pathologies is discussed.

Animals↗
PubMed1999

A complex population of RNAs exists in human ejaculate spermatozoa: implications for understanding molecular aspects of spermiogenesis.

The presence of mRNAs in human ejaculate spermatozoa is well established, yet little is known of the representation or function of these transcripts. To address these issues, the complexity of spermatozoal RNA was examined. As expected, testis-expressed mRNAs were detected by RT-PCR in mature human spermatozoa. Interestingly, when a testis cDNA library was probed with total spermatozoal RNA, less than 2% of plaques gave a strong hybridization signal, suggesting a rather unique sperm-derived population. To further define the sequence distribution, 18 strongly hybridizing clones were selected at random for end-sequence analysis. Twelve matched unique sequences in the EST, STS and NR databases, whereas five showed no similarity to any of the sequences in the databases. In addition, one clone belonged to the SINE repetitive element family. As demonstrated by sequencing randomly primed cloned inserts, short (SINE/MER) or long (LINE/ORF2) interspersed repeat-like sequences are also contained as part of the spermatozoal RNA fraction. It is now evident that human spermatozoa contain a rich repertoire of both known and unknown protein-encoding and non-coding RNAs. This provides a unique opportunity to identify and investigate the many genes responsible for the structure and function/dysfunction of the male gamete using spermatozoal RNA as the template.

Amino Acid Sequence↗