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E D Green

Publications and source records attributed to E D Green.

At least 37 records · Page 2Linked to original sources
PubMed2000

The SPCH1 region on human 7q31: genomic characterization of the critical interval and localization of translocations associated with speech and language disorder.

The KE family is a large three-generation pedigree in which half the members are affected with a severe speech and language disorder that is transmitted as an autosomal dominant monogenic trait. In previously published work, we localized the gene responsible (SPCH1) to a 5.6-cM region of 7q31 between D7S2459 and D7S643. In the present study, we have employed bioinformatic analyses to assemble a detailed BAC-/PAC-based sequence map of this interval, containing 152 sequence tagged sites (STSs), 20 known genes, and >7.75 Mb of completed genomic sequence. We screened the affected chromosome 7 from the KE family with 120 of these STSs (average spacing <100 kb), but we did not detect any evidence of a microdeletion. Novel polymorphic markers were generated from the sequence and were used to further localize critical recombination breakpoints in the KE family. This allowed refinement of the SPCH1 interval to a region between new markers 013A and 330B, containing approximately 6.1 Mb of completed sequence. In addition, we have studied two unrelated patients with a similar speech and language disorder, who have de novo translocations involving 7q31. Fluorescence in situ hybridization analyses with BACs/PACs from the sequence map localized the t(5;7)(q22;q31.2) breakpoint in the first patient (CS) to a single clone within the newly refined SPCH1 interval. This clone contains the CAGH44 gene, which encodes a brain-expressed protein containing a large polyglutamine stretch. However, we found that the t(2;7)(p23;q31.3) breakpoint in the second patient (BRD) resides within a BAC clone mapping >3.7 Mb distal to this, outside the current SPCH1 critical interval. Finally, we investigated the CAGH44 gene in affected individuals of the KE family, but we found no mutations in the currently known coding sequence. These studies represent further steps toward the isolation of the first gene to be implicated in the development of speech and language.

Base Sequence↗
PubMed2000

Definitive functional evidence for a tumor suppressor gene on human chromosome 7q31.1 neighboring the Fra7G site.

We have previously shown that loss of heterozygosity (LOH) on human chromosome (hchr) 7 at q31.1 is common in a variety of tumors of epithelial origin. Frequent LOH of a specific chromosomal marker is indicative of a closely linked tumor suppressor gene (TSG). However, recent reports have also indicated that such a high frequency of LOH could be due to the presence in this region of the second most common aphidicolin-inducible fragile site in the human genome (Fra7G). To address this controversy, we introduced single copies of hchr7 or hchr12 into a highly aggressive human prostate carcinoma cell line (PC3) by microcell-mediated transfer. The tumorigenicity of six clones of PC3/hchr7 hybrids and three clones of PCRhchr12 hybrids, obtained in four separate fusion experiments, were studied in BALB/c nude mice. All but one of the PC3/hchr7 hybrids increased tumor latency by at least twofold, whereas none of the PC3/hchr12 hybrids delayed tumor onset. No differences in the in vitro growth rate were observed among any of the cell lines assayed (parental and hybrids) suggesting that the observed tumor suppression was due to factors other than cell cycle regulation. Deletion mapping of the PC3/hchr7 tumors obtained after reversion to the malignant phenotype revealed a common region of loss centred around 7q31.1, supporting the TSG hypothesis. The smallest commonly deleted region was approximately 1.5 Mb in size and flanked by the markers D7S486 and D7S655.

Adenocarcinoma↗
PubMed2000

Comparative genomic sequence analysis of the human and mouse cystic fibrosis transmembrane conductance regulator genes.

The identification of the cystic fibrosis transmembrane conductance regulator gene (CFTR) in 1989 represents a landmark accomplishment in human genetics. Since that time, there have been numerous advances in elucidating the function of the encoded protein and the physiological basis of cystic fibrosis. However, numerous areas of cystic fibrosis biology require additional investigation, some of which would be facilitated by information about the long-range sequence context of the CFTR gene. For example, the latter might provide clues about the sequence elements responsible for the temporal and spatial regulation of CFTR expression. We thus sought to establish the sequence of the chromosomal segments encompassing the human CFTR and mouse Cftr genes, with the hope of identifying conserved regions of biologic interest by sequence comparison. Bacterial clone-based physical maps of the relevant human and mouse genomic regions were constructed, and minimally overlapping sets of clones were selected and sequenced, eventually yielding approximately 1.6 Mb and approximately 358 kb of contiguous human and mouse sequence, respectively. These efforts have produced the complete sequence of the approximately 189-kb and approximately 152-kb segments containing the human CFTR and mouse Cftr genes, respectively, as well as significant amounts of flanking DNA. Analyses of the resulting data provide insights about the organization of the CFTR/Cftr genes and potential sequence elements regulating their expression. Furthermore, the generated sequence reveals the precise architecture of genes residing near CFTR/Cftr, including one known gene (WNT2/Wnt2) and two previously unknown genes that immediately flank CFTR/Cftr.

Animals↗
PubMed2000

Molecular characterization of the mouse p47-phox (Ncf1) gene and comparative analysis of the mouse p47-phox (Ncf1) gene to the human NCF1 gene.

The cytosolic factor p47-phox (NCF1) is a key component of the phagocyte NADPH-oxidase system, critical for microbicidal activity. The human p47-phox gene has been well characterized and resides on chromosome 7q11. Here we describe the molecular characterization of the mouse ortholog (Ncf1), which maps to distal chromosome 5, and compare the structure of the genes, commenting on the degree of homology. The mouse and human genes contain the same number of exons and introns, but the mouse gene is more compact (7.8 kb versus 15.2 kb). A percentage identity plot analysis comparing the human and mouse genes indicates that sequence homology is generally restricted to exons and does not include any large segment of introns or the 5' flanking sequence. The mouse gene also contains notably fewer repetitive elements than its human counterpart (34% versus 50%). The start of transcription of the mouse gene has been localized to within 12 nucleotides of the translation start site, similar to the human ortholog. Our findings provide an important foundation for investigating the evolutionary history of the p47-phox gene, particularly as it relates to understanding the molecular basis of the p47-phox-deficient autosomal recessive form of chronic granulomatous disease.

5' Untranslated Regions↗
PubMed2000

ComboScreen facilitates the multiplex hybridization-based screening of high-density clone arrays.

MOTIVATION: The construction of physical maps based on bacterial clones [e.g. bacterial artificial chromosomes (BACs)] is valuable for a number of molecular genetics applications, including the high-resolution mapping of genomic regions of interest and the identification of clones suitable for systematic sequencing. A common approach for large-scale screening of bacterial clone libraries involves the hybridization of high-density arrays of immobilized, lysed colonies with collections of DNA probes. The use of a multiplex hybridization screening strategy, whereby pooled probes are analysed en masse, simplifies the effort by reducing the total number of parallel experiments required. However, this approach generates large amounts of hybridization-based data that must be carefully analysed, assimilated, and disambiguated in a careful but efficient manner. RESULTS: To facilitate the screening of high-density clone arrays by a multiplex hybridization approach, we have written a program called ComboScreen. This program provides an organizational framework and analytical tools required for the high-throughput hybridization screening of clone arrays with pools of probes. We have used this program extensively for constructing mouse sequence-ready BAC contig maps.

Chromosomes, Artificial, Bacterial↗
PubMed2000

The genomic region encompassing the nephropathic cystinosis gene (CTNS): complete sequencing of a 200-kb segment and discovery of a novel gene within the common cystinosis-causing deletion.

Nephropathic cystinosis is an autosomal recessive disorder caused by the defective transport of cystine out of lysosomes. Recently, the causative gene (CTNS) was identified and presumed to encode an integral membrane protein called cystinosin. Many of the disease-associated mutations in CTNS are deletions, including one >55 kb in size that represents the most common cystinosis allele encountered to date. In an effort to determine the precise genomic organization of CTNS and to gain sequence-based insight about the DNA within and flanking cystinosis-associated deletions, we mapped and sequenced the region of human chromosome 17p13 encompassing CTNS. Specifically, a bacterial artificial chromosome (BAC)-based physical map spanning CTNS was constructed by sequence-tagged site (STS)-content mapping. The resulting BAC contig provided the relative order of 43 STSs. Two overlapping BACs, which together contain all of the CTNS exons as well as extensive amounts of flanking DNA, were selected and subjected to shotgun sequencing. A total of 200,237 bp of contiguous, high-accuracy sequence was generated. Analysis of the resulting data revealed a number of interesting features about this genomic region, including the long-range organization of CTNS, insight about the breakpoints and intervening DNA associated with the common cystinosis-causing deletion, and structural information about five genes neighboring CTNS (human ortholog of rat vanilloid receptor subtype 1 gene, CARKL, TIP-1, P2X5, and HUMINAE). In particular, sequence analysis detected the presence of a novel gene (CARKL) residing within the most common cystinosis-causing deletion. This gene encodes a previously unknown protein that is predicted to function as a carbohydrate kinase. Interestingly, both CTNS and CARKL are absent in nearly half of all cystinosis patients (i.e., those homozygous for the common deletion). [The sequence data described in this paper have been submitted to the GenBank data library under accession nos. AF168787 and AF163573.]

Amino Acid Transport Systems, Neutral↗
PubMed2000

Comparative genome mapping in the sequence-based era: early experience with human chromosome 7.

The success of the ongoing Human Genome Project has resulted in accelerated plans for completing the human genome sequence and the earlier-than-anticipated initiation of efforts to sequence the mouse genome. As a complement to these efforts, we are utilizing the available human sequence to refine human-mouse comparative maps and to assemble sequence-ready mouse physical maps. Here we describe how the first glimpses of genomic sequence from human chromosome 7 are directly facilitating these activities. Specifically, we are actively enhancing the available human-mouse comparative map by analyzing human chromosome 7 sequence for the presence of orthologs of mapped mouse genes. Such orthologs can then be precisely positioned relative to mapped human STSs and other genes. The chromosome 7 sequence generated to date has allowed us to more than double the number of genes that can be placed on the comparative map. The latter effort reveals that human chromosome 7 is represented by at least 20 orthologous segments of DNA in the mouse genome. A second component of our program involves systematically analyzing the evolving human chromosome 7 sequence for the presence of matching mouse genes and expressed-sequence tags (ESTs). Mouse-specific hybridization probes are designed from such sequences and used to screen a mouse bacterial artificial chromosome (BAC) library, with the resulting data used to assemble BAC contigs based on probe-content data. Nascent contigs are then expanded using probes derived from newly generated BAC-end sequences. This approach produces BAC-based sequence-ready maps that are known to contain a gene(s) and are homologous to segments of the human genome for which sequence is already available. Our ongoing efforts have thus far resulted in the isolation and mapping of >3,800 mouse BACs, which have been assembled into >100 contigs. These contigs include >250 genes and represent approximately 40% of the mouse genome that is homologous to human chromosome 7. Together, these approaches illustrate how the availability of genomic sequence directly facilitates studies in comparative genomics and genome evolution.

Animals↗
PubMed2000

Divergent human and mouse orthologs of a novel gene (WBSCR15/Wbscr15) reside within the genomic interval commonly deleted in Williams syndrome.

Williams syndrome (WS) is a contiguous gene deletion disorder resulting in complex and intriguing clinical features. Detailed molecular characterization studies of the genomic segment on human chromosome 7q11.23 commonly deleted in WS have uncovered numerous genes, each of which is being actively studied for its possible role in the etiology of the syndrome. Our efforts have focused on the comparative mapping and sequencing of the WS region in human and mouse. In previous studies, we uncovered important differences in the long-range organization of these human and mouse genomic regions; in particular, the notable absence of large duplicated blocks of DNA in mouse that are present in human. Aided by available genomic sequence data, we have used a combination of gene-prediction programs and cDNA isolation to identify the human and mouse orthologs of a novel gene (WBSCR15 and Wbscr15, respectively) residing within the genomic segment commonly deleted in WS. Unlike the flanking genes, which are closely related in human and mouse, WBSCR15 and Wbscr15 are strikingly different with respect to their cDNA and corresponding protein sequences as well as tissue-expression pattern. Neither the WBSCR15- nor Wbscr15-encoded amino acid sequence shows a statistically significant similarity to any characterized protein. These findings reveal another interesting evolutionary difference between the human and mouse WS regions and provide an additional candidate gene to evaluate with respect to its possible role in the pathogenesis of WS.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Pendrin, the protein encoded by the Pendred syndrome gene (PDS), is an apical porter of iodide in the thyroid and is regulated by thyroglobulin in FRTL-5 cells.

Pendred syndrome is an autosomal recessive disorder characterized by congenital deafness and thyroid goiter. The thyroid disease typically develops around puberty and is associated with a mild organification defect, characterized by an inappropriate discharge of iodide upon perchlorate stimulation (a positive perchlorate discharge test). The gene (PDS) mutated in Pendred syndrome is expressed in thyroid and encodes a 780-amino acid protein (pendrin) that has recently been shown to function as an iodide/chloride transporter. We sought to establish the location of pendrin in the thyroid and to examine the regulatory network controlling its synthesis. Using peptide-specific antibodies for immunolocalization studies, pendrin was detected in a limited subset of cells within the thyroid follicles, exclusively at the apical membrane of the follicular epithelium. Interestingly, significantly greater amounts of pendrin were encountered in thyroid tissue from patients with Graves' disease. Using a cultured rat thyroid cell line (FRTL-5), PDS expression was found to be significantly induced by low concentrations of thyroglobulin (TG), but not by TSH, sodium iodide, or insulin. This is different from the established effect of TG, more typically a potent suppressor of thyroid-specific gene expression. Together, these results suggest that pendrin is an apical porter of iodide in the thyroid and that the expression and function of both the apical and basal iodide porters are coordinately regulated by follicular TG.

Amino Acid Sequence↗
PubMed1999

Expression pattern of the mouse ortholog of the Pendred's syndrome gene (Pds) suggests a key role for pendrin in the inner ear.

Pendred's syndrome is an autosomal-recessive disorder characterized by deafness and goiter. After our recent identification of the human gene mutated in Pendred's syndrome (PDS), we sought to investigate in greater detail the expression of the gene and the function of its encoded protein (pendrin). Toward that end, we isolated the corresponding mouse ortholog (Pds) and performed RNA in situ hybridization on mouse inner ears (from 8 days postcoitum to postnatal day 5) to establish the expression pattern of Pds in the developing auditory and vestibular systems. Pds expression was detected throughout the endolymphatic duct and sac, in distinct areas of the utricle and saccule, and in the external sulcus region within the cochlea. This highly discrete expression pattern is unlike that of any other known gene and involves several regions thought to be important for endolymphatic fluid resorption in the inner ear, consistent with the putative functioning of pendrin as an anion transporter. These studies provide key first steps toward defining the precise role of pendrin in inner ear development and elucidating the pathogenic mechanism for the deafness seen in Pendred's syndrome.

Amino Acid Sequence↗
PubMed1999

Identification and detection of the common 65-kb deletion breakpoint in the nephropathic cystinosis gene (CTNS).

The most common mutation in the cystinosis gene, CTNS, is a 65-kb deletion thought to have originated in Germany. Although homozygotes for this deletion are detectable by the absence of the D17S829 polymorphic marker, no method exists to identify heterozygotes. We identified the 65-kb deletion breakpoints and used flanking PCR primers to amplify a 423-bp fragment present only in the deletion alleles. Using this method, we determined that 121 of 216 (56%) cystinosis alleles examined bore the 65-kb deletion. We found no non-Europeans with the deletion, and the deletion size and breakpoints appeared identical in all patients studied, supporting the concept of a founder effect. The addition of D17S829 primers (266 bp apart) to the PCR created a multiplex PCR system useful for diagnosing cystinosis patients homozygous and heterozygous for the 65-kb deletion.

Alleles↗
PubMed1999

Construction of a high-resolution physical map of the approximate 1-Mb region of human chromosome 7q31.1-q31.2 harboring a putative tumor suppressor gene.

Reports of frequent loss of heterozygosity (LOH) of markers on human chromosome 7q in malignant myeloid disorders as well as breast, prostate, ovarian, colon, head and neck, gastric, pancreatic, and renal cell carcinomas suggest the presence of a tumor suppressor gene (TSG). Functional assays have demonstrated that the introduction of an intact copy of human chromosome 7 (hchr7) can restore senescence to immortalized human fibroblast cell lines having LOH of markers within 7q31-q32 and can inhibit the tumorigenic phenotype of a murine squamous cell carcinoma cell line. To facilitate the cloning of the putative TSG, we have constructed a high-resolution physical map of this region of hchr7, specifically that encompassing the markers D7S522 and D7S677 within 7q31.1-q31.2. By using a lower resolution yeast artificial chromosome-based map as a starting framework, we established complete clone coverage of the implicated critical region in bacterial-artificial chromosomes (BACs) and P1-derived artificial chromosomes (PACs). The resulting BAC/PAC-based contig map has provided suitable clones for the systematic sequencing of the entire interval. In addition, we have already identified 29 clusters of overlapping expressed-sequence tags (ESTs) and 4 known genes contained within these clones. Together, the physical map reported here coupled with the evolving sequence and gene maps should hasten the identification of the putative TSG residing within this region of hchr7.

Chromosome Mapping↗
PubMed1999

A family of mammalian anion transporters and their involvement in human genetic diseases.

Tremendous advances in human genetics have been made in recent years, as the fruits of the Human Genome Project are facilitating the identification of genes associated with myriad genetic diseases. Among the many triumphs in positional (and positional candidate) cloning are a number of cases where apparently unrelated diseases have been found to share common genetic origins. A vivid example of this has unfolded in the past few years with the identification of the genes causing diastrophic dysplasia, congenital chloride diarrhoea and Pendred syndrome. While these three disorders are clinically distinct, the associated genes ( DTDST, CLD and PDS, respectively) emanate from a well conserved family of genes that all encode anion transporters. Our current knowledge of these diseases coupled with new insights about the implicated genes and proteins illustrates the complex nature of mammalian genomes, especially with respect to the evolutionary subtleties of protein families and tissue-specific gene expression.

Animals↗