William C. Nierman

Chromosomal assignment of 38 human brain expressed sequence tags (ESTs) by analyzing fluorescently labeled PCR products from hybrid cell panels

We have localized 38 human brain cDNA sequences to individual human chromosomes. PCR primers were designed from expressed sequence tags and tested for specific amplification from human genomic DNA. The sizes of amplification products from DNA of somatic cell hybrid mapping panels were determined electrophoretically using an automated fluorescence detection system. Chromosomal assignments were made by discordancy analysis.

The human thrombin receptor and proteinase activated receptor-2 genes are tightly linked on chromosome 5q13.

The thrombin receptor (TR) and proteinase activated receptor‐2 (PAR‐2) may represent the prototypes of an emerging family of cell‐surface receptors that effect cell activation events mediated by serine proteases generated during inflammatory, fibrinolytic or haemostatic‐regulated pathways. To further characterize the molecular genetics of these receptors, we have refined the genetic and physical mapping of both PAR‐2 and TR. Utilization of two distinct radiation hybrid mapping panels with different levels of resolution demonstrated that both genes are tightly linked to the microsatellite markers D5S424, D5S1977, D5S2529 and D5S2596 (in order of decreasing LOD scores, from 13.7 for D5S424 to 7.7 for D5S2596). Physical mapping using yeast artificial chromosomes (YACs) and inversion field gel electrophoresis demonstrated that they are maximally separate by 90 kb. If the association of TR and PAR‐2 genes resulted from a relatively recent gene duplication event from a common ancestral gene, these observations provide a general framework for the identification of gene transcripts representing alternative proteolytically activated receptors which may be clustered within this region of the human genome. These observations are especially relevant given recent evidence that murine and human platelets express alternative signalling mechanisms or receptors for thrombin.

Mapping the human corticotropin releasing hormone binding protein gene (CRHBP) to the long arm of chromosome 5 (5q11.2–q13.3)

Unexpected stimulation or stress activates the heat shock protein (hsp) system at the cellular level and the hypothalamic-pituitary-adrenal (HPA) axis at the level of the whole organism. At the molecular level, these two systems communicate through the functional interaction between hsp90 and glucocorticoid receptor (GR). The corticotropin releasing hormone (CRH) system regulates the mammalian stress response by coordinating the activity of the HPA axis. It consists of the 41-amino-acid-long principal hypothalamic secretagogue for pituitary adrenocorticotropic hormone (ACTH), CRH, its receptor (CRHR), and its binding protein (CRHBP). Because of its central role in the coordination of stress response and whole body homeostasis, the CRH system has been implicated in the pathogenesis of neuroendocrine and psychiatric disease. 19 refs., 1 fig.

259 Human Brain Expressed Sequence Tags (ESTs): Chromosome Localization, Subregional Assignment, and Sequence Analysis

Using PCR with automated product analysis, 259 human brain cDNA sequences have been assigned to individual human chromosomes, 99 with subregional localizations. Primers were designed from single-pass cDNA sequences (expressed sequence tags or ESTs) and tested for specific amplification from human genomic DNA. Primers were then used in PCR reactions with DNA from somatic cell hybrid mapping panels as templates, often with multiplexing. Amplification products were identified using automated fluorescence detection and chromosomal assignments were made by discordancy analysis.

Applications of Recombinant DNA Technology to the Diagnosis of Genetic Disease: Molecular Methods for Detecting the Genetic Basis of Diseases

Disease in humans can be caused by mutations that alter the DNA sequence of the coding or control regions of a gene. These mutations may result in the inappropriate expression or the nonexpression of a functional gene product, or in the production of a nonfunctional gene product. The mutations can be of various categories, ranging from point mutations or small insertions or deletions that are not cytologically detectable, to grosser abnormalities, including rearrangements and aneuploidy. The mutations can be in the germ line and heritable, and thus be studied by analysis of family members, or can arise somatically and be studied by comparing affected cells with nonmutated ones. Diseases have been identified that exemplify each of these genetic alterations. In addition, normally occurring somatic mutations, such as the rearrangements characteristic of immunoglobulin or T cell receptor genes, can be indicative of the differentiation and clonal status of various hematological malignancies. The diagnosis of these diseases, or the determination of carrier status for recessive heritable disorders, can thus be approached by analyzing DNA.