Developmental genomics and its relation to aging.
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Aging↗
Biomedical subjects
D Schlessinger
Publications and source records attributed to D Schlessinger.
Developmental genomics and its relation to aging
Explore the source record for details and available documents.
Journal Article↗
YAC/STS map of 15Mb of Xp21.3-p11.3, at 100kb resolution, with refined comparisons of genetic distances and DMD structure.
The 15 Mb region between DXS997 and DXS8054 in Xp21.3-p11.3 has been mapped at seven-fold average coverage in yeast artificial chromosomes (YACs) and 100 kb inter-sequence tagged site (STS) distance. YACs from six different collections show self-consistent maps. The STSs include 18 (CA) repeat and one tetranucleotide repeat marker that detect polymorphism, as well as eight well-studied genes, a second site for MXS1 sequences, and three expressed sequence tags (ESTs). One of the ESTs maps to intron 7 of Duchenne muscular dystrophy, and seems to be a processed intronic sequence with a poly(A) tail.
Base Sequence↗
Multiple Sp1 sites efficiently drive transcription of the TATA-less promoter of the human glypican 3 (GPC3) gene.
Simpson-Golabi-Behmel Syndrome (SGBS) is an X-linked disease characterized by pre- and postnatal overgrowth. Recently, we have shown that mutations in the glypican family gene, GPC3, cause SGBS. This gene is predominantly expressed in the same mesoderm-derived tissues that overgrow in its absence. To investigate the basis for promoter function, 3.3kb of GC-rich DNA 5' of the transcribed region were fused to a luciferase cDNA, transfected into Caco-2 and NT2 cells, and assayed for activity. Deletion analysis identified a 218-bp fragment upstream of the transcription start site that conferred more than 80% of maximal reporter gene activation. This fragment contains five putative Sp1 binding sites, three of which (centered at nt -14, -34, and -92) were active when assessed by DNaseI footprinting and gel shift/supershift assays. Additionally, Sp1 specifically transactivated transcription in Sp1-deficient Drosophila SL2 cells, demonstrating the functionality of Sp1 on the GPC3 promoter. A full-length promoter construct was also highly active in HeLa cells, which do not express endogenous GPC3. These results indicate that the GPC3 promoter is dependent on Sp1 for proper activation, but tissue-specific repression in non-expressing cells must involve either DNA that lies outside the region tested or auxiliary structural features of chromatin.
Animals↗
Localization of 67 exons on a YAC contig spanning 1.5 Mb around the multidrug resistance gene region of human chromosome 7q21.1.
A contig of 21 nonchimeric yeast artificial chromosomes (YACs) was previously assembled across 1.5 Mb of the multidrug resistance (MDR) gene (PGY1 and PGY3) region of human chromosome 7q21.1. This region of the human genome has now been subjected to exon amplification to detect the presence of additional genes. Exon trapping was performed directly on the YACs. Sixty-seven gene fragments were isolated and characterized by sequence analysis and comparison with public databases. The localization of these exons in the 1.5-Mb region was determined by hybridization to YAC clones, and they were localized in 11 subregions of YAC contigs. The exon collection includes 21 exons that were identical to known cDNA sequences of PGY1, PGY3, sorcin (SRI), the cDNA similar to the delta subunit of the human amiloride-sensitive Na- channel (SCNED), and 4 cDNAs with unknown function; 43 exons that showed homology/similarity to known cDNA sequences of mouse DMP1, rat COT, mouse and human NADHD, human MDC, 3 cDNAs encoding possible membrane proteins, and 21 other cDNAs; and 3 exons that shared no homology/similarity with any sequence in public databases. The nucleotide sequences of all the PGY1 and PGY3 exons were identical to the corresponding cDNA sequences previously determined, and these exons were localized to the expected positions on the appropriate YAC clones. No other member of the MDR gene family thus appeared to be present in the 1.5-Mb region. The integrated physical and exon maps should prove valuable for both fine mapping and determination of a complete gene map of this segment of the genome.
ATP Binding Cassette Transporter, Subfamily B↗
Cloning and expression of an immunoglobulin superfamily gene (IGSF1) in Xq25.
We have isolated a novel full-length cDNA for a gene (IGSF1) located in distal Xq25. This transcript is highly expressed in adult testis and fetal liver but is undetectable in adult liver. A smaller alternate form is highly expressed in adult heart. The gene encodes a protein of 1327 amino acids with several recognizable functional domains. The protein has a putative signal peptide and transmembrane region, 15 potential sites for N-linked glycosylation, and 12 C2-type immunoglobulin (Ig)-like domains. All of the Ig-like domains contain the two conserved cysteine residues that form intradomain disulfide bonds typical of this superfamily. These features are consistent with a possible role for this molecule in cell surface recognition or cell-cell interaction.
Amino Acid Sequence↗
Gpc3 expression correlates with the phenotype of the Simpson-Golabi-Behmel syndrome.
Interest in glypican-3 (GPC3), a member of the glypican-related integral membrane heparan sulfate proteoglycans (GRIPS) family, has increased with the finding that it is mutated in the Simpson-Golabi-Behmel overgrowth syndrome (Pilia et al. [1996] Nat. Genet. 12:241-247). The working model suggested that the membrane-bound protein acts locally to limit tissue and organ growth and that it may function by interacting with insulin-like growth factor 2 (IGF2) to limit its local effective level. Here we have tested two predictions of the model. In situ hybridization with the mouse gene cDNA was used to study the expression pattern during embryonic and fetal development. In agreement with predictions, the gene is expressed in precisely the organs that overgrow in its absence; and the patterns of expression of Gpc3 and those reported for Igf2 are strictly correlated.
Abnormalities, Multiple↗
Pathological consequences of sequence duplications in the human genome.
As large-scale sequencing accumulates momentum, an increasing number of instances are being revealed in which genes or other relatively rare sequences are duplicated, either in tandem or at nearby locations. Such duplications are a source of considerable polymorphism in populations, and also increase the evolutionary possibilities for the coregulation of juxtaposed sequences. As a further consequence, they promote inversions and deletions that are responsible for significant inherited pathology. Here we review known examples of genomic duplications present on the human X chromosome and autosomes.
Gene Duplication↗
Localisation of X linked recessive idiopathic hypoparathyroidism to a 1.5 Mb region on Xq26-q27.
X linked recessive idiopathic hypoparathyroidism (HPT) has been observed in two kindreds from Missouri, USA. Affected subjects, who are males, suffer from infantile onset of epilepsy and hypocalcaemia, which appears to be the result of an isolated congenital defect of parathyroid gland development; females are not affected and are normocalcaemic. The gene causing HPT has been previously mapped to a 7 cM interval, flanked centromerically by F9 and telomerically by DXS98, in Xq26-q27, and an analysis of mitochondrial DNA has established a common ancestry for these two kindreds. In order to define further the map location of HPT and thereby facilitate its isolation, we have undertaken linkage studies using polymorphic loci whose order has been established as Xcen - DXS1001 - DXS294 - DXS102 - F9 - DXS1232 - DXS984 - CDR1 - DXS105 - DXS1205 - DXS1227 - DXS98 - DXS52 - Xqter, within this region. Our results established linkage (lod score > 3) between HPT and eight of these 12 loci and indicated that the most likely location of HPT was within a 1.5 Mb interval flanked centromerically by F9 and telomerically by DXS984. Thus, the results of this study have helped to refine the map location of HPT, and this will facilitate the identification of this putative developmental gene and its role in the embryological formation of the parathyroids.
Chromosome Mapping↗
The gene defective in anhidrotic ectodermal dysplasia is expressed in the developing epithelium, neuroectoderm, thymus, and bone.
Anhidrotic ectodermal dysplasia (EDA) is characterized by defects in the development of teeth, hair, and sweat glands. To study the expression of the human gene defective in EDA in human fetal development (Weeks 6-23 of gestational age) and in adult tissues, in situ hybridization and immunohistochemistry were used. First signs of expression were detected at Week 8 in epidermis and in neuroectodermal cells. Starting at Week 12, osteoblasts and thymus were positive for EDA mRNA. Hair follicles expressed EDA mRNA from 18 weeks. The presence of the EDA protein coincided with mRNA expression in the tissues examined. The expression pattern of the EDA gene is consistent with typical involvement of the skin in the syndrome. However, the expression is not limited to the ectodermal tissues and many sites of expression are not obviously reflected in the clinical features of the syndrome.
Adult↗
Mutational analysis of PHEX gene in X-linked hypophosphatemia.
Hypophosphatemic rickets is commonly an X-linked dominant disorder (XLH or HYP) associated with a renal tubular defect in phosphate transport and bone deformities. The XLH gene, referred to as PHEX, or formerly as PEX (phosphate regulating gene with homologies to endopeptidases on the X-chromosome), encodes a 749-amino acid protein that putatively consists of an intracellular, transmembrane, and extracellular domain. PHEX mutations have been observed in XLH patients, and we have undertaken studies to characterize such mutations in 46 unrelated XLH kindreds and 22 unrelated patients with nonfamilial XLH by single stranded conformational polymorphism and DNA sequence analysis. We identified 31 mutations (7 nonsense, 6 deletions, 2 deletional insertions, 1 duplication, 2 insertions, 4 splice site, 8 missense, and 1 within the 5' untranslated region), of which 30 were scattered throughout the putative extracellular domain, together with 6 polymorphisms that had heterozygosity frequencies ranging from less than 1% to 43%. Single stranded conformational polymorphism was found to detect more than 60% of these mutations. Over 20% of the mutations were observed in nonfamilial XLH patients, who represented de novo occurrences of PHEX mutations. The unique point mutation (a-->g) of the 5'untranslated region together with the other mutations indicates that the dominant XLH phenotype is unlikely to be explained by haplo-insufficiency or a dominant negative effect.
Amino Acid Sequence↗
Impact and implications of yeast and human artificial chromosomes.
Artificial chromosomes have been developed in the last 10 years to sustain genome mapping and, more recently, to begin initiating functional studies and some approaches to gene therapy. The use of yeast artificial chromosomes (YACs) in mapping the human X chromosome is reported as an example. The requirements which have postponed the development of human artificial chromosomes have now been relatively met, and some prospects are previewed here.
Chromosome Mapping↗
Duplication and distribution of repetitive elements and non-unique regions in the human genome.
Genome mapping efforts and the initial sequencing of large segments of human DNA permit ongoing assessment of the patterns and extent of sequence duplication and divergence in the human genome. Initial sequence data indicate that the most highly repetitive sequences show isochore-related enrichment and clustering produced by successive insertional recombination and local duplication of particular repetitive elements. Regional duplication is also observed for a number of otherwise unique genomic sequences and thereby makes these segments become repetitive. The consequences of these duplication events are: (1) clustering of related genes, along with a variety of coregulatory mechanisms; and (2) recombinations between the nearby homologous sequences, which can delete genes in individuals and account for a significant fraction of human genetic disease.
Genetic Diseases, Inborn↗
The Tabby phenotype is caused by mutation in a mouse homologue of the EDA gene that reveals novel mouse and human exons and encodes a protein (ectodysplasin-A) with collagenous domains.
Mouse Tabby (Ta) and X chromosome-linked human EDA share the features of hypoplastic hair, teeth, and eccrine sweat glands. We have cloned the Ta gene and find it to be homologous to the EDA gene. The gene is altered in two Ta alleles with a point mutation or a deletion. The gene is expressed in developing teeth and epidermis; no expression is seen in corresponding tissues from Ta mice. Ta and EDA genes both encode alternatively spliced forms; novel exons now extend the 3' end of the EDA gene. All transcripts recovered have the same 5' exon. The longest Ta cDNA encodes a 391-residue transmembrane protein, ectodysplasin-A, containing 19 Gly-Xaa-Yaa repeats. The isoforms of ectodysplasin-A may correlate with differential roles during embryonic development.
Alternative Splicing↗
Differential expression of XAP5, a candidate disease gene.
We have isolated a full-length cDNA corresponding to the XAP5 gene in Xq28. An unusual feature of the cDNA is that it contains runs of CCG repeats in the 5' untranslated region, typical of genes that exhibit anticipation. It has a striking pattern of differential expression and is greatly enhanced in various fetal tissues. This predicted protein encodes a unique 339-amino-acid polypeptide that contains a large percentage of highly charged residues and a possible nuclear localization signal. A comparison to genomic sequence shows that XAP-5 comprises 13 exons spanning 6.5 kb. An examination of the human population indicates that the longest CCG run is polymorphic and varies in length from 8 to 12 repeats.
Amino Acid Sequence↗
Construction of a pilot human YAC library in a recombination-defective yeast strain.
Using high-molecular-weight DNA fragments from a human lymphoblastoid cell line, a pilot collection of 2500 YACs was constructed in YKK115, a recombination-deficient strain of Saccharomyces cerevisiae carrying mutations in both the rad51 and rad52 genes. Analysis of 520 clones from the current library by pulsed-field gel electrophoresis revealed more than 97% single YACs with an insert size averaging 340 kb. Fluorescent in situ hybridization (FISH) performed with 37 clones on metaphase chromosomes suggested a high proportion mapping at centromeric (7) or telomeric (4) locations. The results are consistent with the stabilization of YACs in strains disarmed in recombination functions [Kohno, K., Oshiro, T., Kishine, H., Wada, M., Takeda, H., Ihara, N., Imamoto, F., Kano, Y. and Schlessinger, D. (1997) Human YACs unstable in a rad52 single mutant strain become stable in rad51rad52 double mutant. Gene, 000, 000-000 (GENE 10429)], and further suggest that the YACs may include regions that have been difficult to clone in other strains.
Cell Line↗
Construction and characterization of a rad51rad52 double mutant as a host for YAC libraries.
RAD52 or RAD51 recombination-deficient yeast strains stabilize otherwise unstable YACs containing ribosomal DNA or the human color vision locus (Kohno et al., 1994). Thus the RAD52RAD51 pathways(s) are apparently involved in the instability of YACs containing tandem repeat loci, presumably by promoting recombination-based deletion formation. Some other genomic loci are still unstable or unrecoverable in those strains, but we now find that greater stability is observed in a rad51rad52 double mutant strain that we have newly constructed. YACs containing a highly unstable region around DXS49 or centromeric regions throw off a variety of products in single mutants, but are much more stable in the rad51rad52 strain, which could therefore provide a better host for library construction and maintenance.
Chromosomes, Artificial, Yeast↗
Mapping of 59 EST gene markers in 31 intervals spanning the human X chromosome.
The positioning of Expressed Sequence Tags (ESTs) constitutes an important step towards a functional map of the human genome, including candidate genes for human genetic disorders that have been localized by linkage analysis. We localized 59 ESTs on the human X chromosome, including 44 derived from infant brain and 15 from adult muscle cDNA libraries. Localizations by a somatic cell hybrid panel were refined for five cDNAs by mapping them in yeast artificial chromosome (YAC) contigs.
Animals↗