Biomedical subjects

T Nechiporuk

Publications and source records attributed to T Nechiporuk.

10 recordsLinked to original sources

PubMed2000

ETL, a novel seven-transmembrane receptor that is developmentally regulated in the heart. ETL is a member of the secretin family and belongs to the epidermal growth factor-seven-transmembrane subfamily.

Using differential display of rat fetal and postnatal cardiomyocytes, we have identified a novel seven-transmembrane receptor, ETL. The cDNA-predicted amino acid sequence of ETL indicated that it encodes a 738-aa protein composed of a large extracellular domain with epidermal growth factor (EGF)-like repeats, a seven-transmembrane domain, and a short cytoplasmic tail. ETL belongs to the secretin family of G-protein-coupled peptide hormone receptors and the EGF-TM7 subfamily of receptors. The latter are characterized by a variable number of extracellular EGF and cell surface domains and conserved seven transmembrane-spanning regions. ETL mRNA expression is up-regulated in the adult rat and human heart. In situ hybridization analyses revealed expression in rat cardiomyocytes and abundant expression in vascular and bronchiolar smooth muscle cells. In COS-7 cells transfected with Myc-tagged rat ETL, rat ETL exists as a stable dimer and undergoes endoproteolytic cleavage of the extracellular domain. The proteolytic activity can be abolished by a specific mutation, T455A, in this domain. In transfected mammalian cells, ETL is associated with cell membranes and is also observed in cytoplasmic vesicles. ETL is the first seven-transmembrane receptor containing EGF-like repeats that is developmentally regulated in the heart.

Alternative Splicing↗

PubMed1998

Genomic structure of the human gene for spinocerebellar ataxia type 2 (SCA2) on chromosome 12q24.1.

Spinocerebellar ataxia type 2 (SCA2) is a member of a group of neurodegenerative diseases that are caused by instability of a DNA CAG repeat. We report the genomic structure of the SCA2 gene. Its 25 exons, encompassing approximately 130 kb of genomic DNA, were mapped onto the physical map of the region. Exonic sizes varied from 37 to 890 bp, and intronic sizes ranged from 323 bp to more than 15 kb. The CAG repeat was contained in the 5' coding region of the gene in exon 1. Determination of the splice junction sequences indicated the presence of only one deviation from the GT-AG rule at the donor splice site of intron 9, which contained a GC instead of a GT dinucleotide. Exon 10, immediately downstream from this rare splice donor site, was alternatively spliced. Alternative splicing does not affect the reading frame and is predicted to encode an isoform containing 70 amino acids less.

Alternative Splicing↗

PubMed1998

The mouse SCA2 gene: cDNA sequence, alternative splicing and protein expression.

Spinocerebellar ataxia type 2 (SCA2) is caused by expansion of a CAG trinucleotide repeat located in the coding region of the human SCA2 gene. Sequence analysis revealed that SCA2 is a novel gene of unknown function. In order to provide insights into the molecular mechanisms of pathogenesis of SCA2 and to identify conserved domains, we isolated and characterized the mouse homolog of the SCA2 gene. Sequence and amino acid analysis revealed 89% identity at the nucleotide and 91% identity at the amino acid level. However, there was no extended polyglutamine tract in the mouse SCA2 cDNA, suggesting that the normal function of SCA2 is not dependent on this domain. Northern blot analysis of different mouse tissues indicated that the mouse SCA2 gene was expressed in most tissues, but at varying levels. Alternative splicing seen in human SCA2 was conserved in the mouse. By northern blot analysis, SCA2 was expressed during embryogenesis as early as day 8 of gestation (E8). Immunohistochemical staining using affinity-purified antibodies demonstrated that ataxin 2 was expressed in the cytoplasm of Purkinje cells as well as in other neurons of the CNS.

Alternative Splicing↗

PubMed1997

A high-resolution PAC and BAC map of the SCA2 region.

The spinocerebellar ataxia type 2 (SCA2) gene has been localized to chromosome 12q24.1. To characterize this region and to aid in the identification of the SCA2 gene, we have constructed a 3.9-Mb physical map, which covers markers D12S1328 and D12S1329 known to flank the gene. The map comprises a contig of 84 overlapping yeast artificial chromosomes (YACs), P1 artificial chromosomes (PACs), and bacterial artificial chromosomes (BACs) onto which we placed 82 PCR markers. We localized eight genes and expressed sequence tags on this map, many of which had not been precisely mapped before. In contrast to YACs, which showed a high degree of chimerism and deletions in this region, PACs and BACs were stable. Only 1 in 65 PACs contained a small deletion, and 2 in 18 BACs were chimeric. The high-resolution physical map, which was used in the identification of the SCA2 gene, will be useful for the positional cloning of other disease genes mapped to this region.

Ataxins↗

PubMed1996

Identification of three new microsatellite markers in the spinocerebellar ataxia type 2 (SCA2) region and 1.2 Mb physical map.

Spinocerebellar ataxia type 2 (SCA2) is a neurodegenerative disease recently mapped to chromosome 12q close to the locus D12S84 by genetic linkage analysis. To generate additional genetic markers in the SCA2 region, we constructed a physical map of the region using yeast artificial chomosome (YAC), P1 artificial chromosome (PAC) and cosmid clones. The physical map was found to agree well with the genetic map. Three novel microsatellite markers were isolated and physically mapped. A novel approach to isolate CAG repeats directly from YAC DNAs is described.

Alleles↗

PubMed1996

Moderate expansion of a normally biallelic trinucleotide repeat in spinocerebellar ataxia type 2.

The gene for spinocerebellar ataxia type 2 (SCA2) has been mapped to 12q24.1. A 1.1-megabase contig in the candidate region was assembled in P1 artificial chromosome and bacterial artificial chromosome clones. Using this contig, we identified a CAG trinucleotide repeat with CAA interruptions that was expanded in patients with SCA2. In contrast to other unstable trinucleotide repeats, this CAG repeat was not highly polymorphic in normal individuals. In SCA2 patients, the repeat was perfect and expanded to 36-52 repeats. The most common disease allele contained (CAG)37, one of the shortest expansions seen in a CAG expansion syndrome. The repeat occurs in the 5'-coding region of SCA2 which is a member of a novel gene family.

Amino Acid Sequence↗

PubMed1996

Genetic mapping of the spinocerebellar ataxia type 2 gene on human chromosome 12.

The dominant spinocerebellar ataxias are a genetically heterogeneous group of diseases leading to premature death of neurons in the cerebellum and other parts of the nervous system. The mutation causing SCA1 is on human chromosome (CHR) 6p and SCA3 is on CHR 14q. To refine the location of the SCA2 gene on CHR 12q, we performed genetic linkage analysis between the SCA2 locus and nine Ioci (D12S58, D12S78, D12S317, D12S330, D12S353, D12S84, D12S105, D12S79, and PLA2) in three SCA2 families. The highest pairwise lod scores were obtained between SCA2 and D12S84/D12S105 and D12S79. We determined the best order and genetic distances among these loci in ten multigenerational families by multipoint linkage analysis and established the following order: D12S101-D12S58/IGF1- D12S78-D12S317-D12S330/D12S353-D12S84/D 12S105-D12S79-PLA2. Using this genetic map, multipoint linkage analysis placed SCA2 between D12S84/D12S105 and D12S79.

Chromosome Mapping↗

PubMed1996

Expression of neurofibromatosis 2 transcript and gene product during mouse fetal development.

Neurofibromatosis 2 (NF2) is an autosomal dominant inherited disorder that predisposes to benign tumors of the nervous system as well as a variety of ocular abnormalities. In contrast to NF1, NF2 is associated with only minor developmental abnormalities. The human NF2 gene encodes a tumor suppressor protein, termed schwannomin or merlin, which is a member of a superfamily of proteins thought to link cytoskeletal elements to cell membrane components. To determine the pattern of NF2 gene expression in mouse embryos, we sequenced the mouse NF2 gene and used in situ hybridization and antischwannomin antibodies to determine the developmental expression of the NF2 gene. Schwannomin was detected in most differentiated tissues but was undetectable in undifferentiated tissues. In particular, schwannomin was not detectable in mitotic neuroepithelial cells, the perichondrium, the liver, the neocortex, and the ventricular zone of the developing cerebral cortex. In the heart, expression was observed in all developmental stages beginning on embryonic day 8. In the eye, which shows developmental abnormalities in NF2 patients, expression was detected in the cells of the lens and in the pigment epithelium but weakly detected in retinal neurons. The most striking example of tightly regulated NF2 expression was observed in cells migrating from the ventricular zone to the cortical plate on embryonic days 15 and 16. Only cells in the intermediate zone expressed schwannomin, indicating that schwannomin may play an important role in cellular migration.

Amino Acid Sequence↗

PubMed1994

Differential expression and tissue distribution of type I and type II neurofibromins during mouse fetal development.

Mutations in the NF1 gene may cause developmental abnormalities and the formation of a variety of tumors of neural crest origin in humans. The NF1 gene codes for a large protein, neurofibromin (nf), which is structurally and functionally related to yeast and human ras-GTPase-activating proteins (ras-GAPs). Recently, two transcripts coding for type I and type II nf with different ras-GAP activity have been identified. Since ras proteins do not appear to be significantly regulated during mouse development, we examined if differential expression of neurofibromins may provide evidence for a role of nfs in regulating ras-mediated cell proliferation and differentiation. Nfs were expressed as early as E8. At E11 a marked increase of NF1 transcripts occurred and was associated with expression of nfs in all tissues. Type I and type II nfs each showed a different time course of expression and tissue localization, with type II nf present mainly from E8 through E10, although in the heart type II nf was present at E12. In some tissues such as heart and dorsal root ganglia rapid increases and decreases of nfs were detected related to differentiation of these tissues. These results are consistent with a role of nfs in regulating ras-mediated cell proliferation and differentiation during development and support distinct functional roles for type I and type II nfs.

Animals↗

PubMed1994

Alternative transcripts in the mouse neurofibromatosis type 2 (NF2) gene are conserved and code for schwannomins with distinct C-terminal domains.

Mutations in the neurofibromatosis type 2 (NF2) gene predispose individuals to the development of nervous system tumors and ocular abnormalities. The NF2 gene product, schwannomin, is a member of a superfamily of proteins thought to link cytoskeletal elements to cell membrane components. These proteins share significant homologies in the N-terminal and alpha-helical domains, but diverge in the C-terminus. During our efforts to characterize mouse NF2 transcripts, we identified four different transcripts by cDNA analysis and reverse-transcribed PCR that contained different sequences in the 3' end of the coding sequences. In human cell lines three isoforms encoding two distinct schwannomins were detected. The mouse and human transcripts containing 61 and 60 bp inserts, respectively, have not been previously described. The isoforms encode schwannomins with significantly altered C-termini and were expressed at different relative levels in adult mouse tissues and during mouse embryogenesis. These results suggest that schwannomin isoforms have distinct functional roles and predict the existence of human mutations involving the C-terminus of schwannomin.

Amino Acid Sequence↗