2006 expressed-sequence tags derived from human chromosome 7-enriched cDNA libraries.

Abstract

The establishment and mapping of gene-specific DNA sequences greatly complement the ongoing efforts to map and sequence all human chromosomes. To facilitate our studies of human chromosome 7, we have generated and analyzed 2006 expressed-sequence tags (ESTs) derived from a collection of direct selection cDNA libraries that are highly enriched for human chromosome 7 gene sequences. Similarity searches indicate that approximately two-thirds of the ESTs are not represented by sequences in the public databases, including those in dbEST. In addition, a large fraction (68%) of the ESTs do not have redundant or overlapping sequences within our collection. Human DNA-specific sequence-tagged sites (STSs) have been developed from 190 of the ESTs. Remarkably, 180 (96%) of these STSs map to chromosome 7, demonstrating the robustness of chromosome enrichment in constructing the direct selection cDNA libraries. Thus far, 140 of these EST-specific STSs have been assigned unequivocally to YAC contigs that are distributed across the chromosome. Together, these studies provide > 2000 ESTs highly enriched for chromosome 7 gene sequences, 180 new chromosome 7 STSs corresponding to ESTs, and a definitive demonstration of the ability to enrich for chromosome-specific cDNAs by direct selection. Furthermore, the libraries, sequence data, and mapping information will contribute to the construction of a chromosome 7 transcript map.

Gene Expression Profile of Human Bone Marrow Stromal Cells: High-Throughput Expressed Sequence Tag Sequencing Analysis

“…This analysis entails comparing each EST with all others in a pairwise fashion, allowing the sequences to be grouped into clusters [32]. There are a total of 1860 gene sets (Fig.…”

“…Although incomplete and not error-free, ESTs remain an effective means for novel gene discovery and generating biologically informative probes for mapping genes to chromosomes as sequence-tagged sites (STSs), for identifying mutations leading to heritable diseases, and for full-length cDNA cloning [21,26]. The advantages of this approach are as follows: (1) it can be pursued as a relatively inexpensive and rapid way to access many of the expressed genes of a cell or tissue type [29,30]; and (2) with the advent of highthroughput sequencing technology and an increased interest in genome-wide studies, it became clear that ESTs could be generated in sufficient numbers to provide a rapid means of gene discovery [28,31], especially for those searching for human disease genes or constructing physical maps of the human genome [32].Based on the feasibility of EST analysis for gene discovery and pattern configuration in other cell types or organisms [31], we undertook a larger-scale EST project to sequence randomly isolated cDNAs from a HBMSC cDNA library. The FIG.…”

A Physical Map of Human Chromosome 7: An Integrated YAC Contig Map with Average STS Spacing of 79 kb

“…Since the onset of the Human Genome Project, we have focused on constructing a detailed physical map of human chromosome 7 by a YAC-based STScontent mapping strategy (Green and Green 1991;Green et al 1991aGreen et al , 1994Green et al , 1995Bouffard et al 1997). Specifically, our goals included (1) mapping an STS, on average, every 100 kb across the chromosome [a programmatic goal of the U.S. Human Genome Project (Collins and Galas 1993)]; (2) integration of the YAC-based physical map with the cytogenetic, genetic, and RH maps of the chromosome; (3) construction of a map that covered the majority of the chromosome, that established an unique order for most of the mapped STSs, and that provided large, contiguous stretches of redundant clone coverage.…”

“…l Presence of gene-and/or EST-specific STSs within the contig is indicated, along with the number of such STSs (in parenthesis). The chromosome 7-specific STSs can be broadly classified into four major categories: those derived from sequences corresponding to random segments of chromosome 7 DNA [e.g., isolated by flow-sorting, subcloning, or Alu-PCR amplification of human DNA from chromosome 7-containing monochromosomal hybrid cell lines (Green et al 1991a;Green 1993;Bouffard et al 1997)], YAC insert ends, genetic (microsatellite) markers, and genes and ESTs.…”

“…Although this effort resulted in the expansion of some contigs and merger of others, it also yielded four of the orphan contigs listed in Table 1. Third, we have purposely mapped numerous gene-and EST-specific STSs (Table 3), including a large set developed from a collection of chromosome 7-enriched ESTs (Touchman et al 1997). Undoubtedly, many of these STSs reside within GC-rich regions of the chromosome; in fact, several of the orphan contigs were created as a direct result of our gene/EST mapping effort and another 13 gene/EST-specific STSs failed to identify a positive YAC (Table 3).…”