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1.
Oncogene ; 25(44): 6026-31, 2006 Sep 28.
Article in English | MEDLINE | ID: mdl-16702959

ABSTRACT

The tumor suppressor KLF6 is a member of the Krüppel-like family of transcription factors, which has been implicated in the pathogenesis of several human carcinomas. Uncovering the transcriptional targets relevant for its tumorigenic properties, including cellular proliferation and invasion, will be essential to understanding possible mechanisms by which KLF6 and its antagonistic splice form, KLF6-SV1, regulate this development. To begin defining possible metastatic-related pathways, we analysed the effect of KLF6 dysregulation on a recognized suppressor of cellular invasion, E-cadherin. Targeted KLF6 reduction in an ovarian cancer cell line, SKOV-3, resulted in a 50% reduction of E-cadherin expression (P<0.01) and conversely, KLF6-SV1 silencing upregulated E-cadherin approximately fivefold (P<0.0001). These changes resulted from KLF6 directly transactivating the E-cadherin promoter as demonstrated by luciferase promoter assay and chromatin immunoprecipitation (ChIP). KLF6-mediated changes in E-cadherin levels were accompanied by downstream changes in both the subcellular localization of beta-catenin and c-myc expression levels. Moreover, and consistent with these experimental findings, patient-derived epithelial ovarian tumors with low KLF6 and high KLF6-SV1 expression ratios had significantly decreased E-cadherin expression (P<0.0001). These combined findings highlight the E-cadherin pathway as a novel and functionally important mediator by which changes in KLF6 and KLF6-SV1 can directly alter ovarian tumor invasion and metastasis.


Subject(s)
Cadherins/biosynthesis , Cadherins/genetics , Gene Expression Regulation, Neoplastic , Kruppel-Like Transcription Factors/physiology , Ovarian Neoplasms/genetics , Ovarian Neoplasms/metabolism , Proto-Oncogene Proteins/physiology , Transcription, Genetic , Tumor Suppressor Proteins/physiology , 3' Untranslated Regions/genetics , Cadherins/physiology , Cell Line, Tumor , Female , Gene Expression Regulation, Neoplastic/physiology , Growth Inhibitors/genetics , Growth Inhibitors/physiology , HeLa Cells , Humans , Kruppel-Like Factor 6 , Neoplasm Invasiveness/genetics , Neoplasm Metastasis/genetics , Proto-Oncogene Proteins c-myc/biosynthesis , Proto-Oncogene Proteins c-myc/genetics , Signal Transduction/genetics , Subcellular Fractions/metabolism , beta Catenin/biosynthesis , beta Catenin/genetics , beta Catenin/metabolism
2.
Science ; 294(5551): 2563-6, 2001 Dec 21.
Article in English | MEDLINE | ID: mdl-11752579

ABSTRACT

Kruppel-like factor 6 (KLF6) is a zinc finger transcription factor of unknown function. Here, we show that the KLF6 gene is mutated in a subset of human prostate cancer. Loss-of-heterozygosity analysis revealed that one KLF6 allele is deleted in 77% (17 of 22) of primary prostate tumors. Sequence analysis of the retained KLF6 allele revealed mutations in 71% of these tumors. Functional studies confirm that whereas wild-type KLF6 up-regulates p21 (WAF1/CIP1) in a p53-independent manner and significantly reduces cell proliferation, tumor-derived KLF6 mutants do not. Our data suggest that KLF6 is a tumor suppressor gene involved in human prostate cancer.


Subject(s)
Genes, Tumor Suppressor , Mutation , Prostatic Neoplasms/genetics , Proto-Oncogene Proteins , Trans-Activators/genetics , Alleles , Amino Acid Substitution , Animals , Cell Division , Cell Line , Chromosome Mapping , Chromosomes, Human, Pair 10/genetics , Cyclin-Dependent Kinase Inhibitor p21 , Cyclins/genetics , Cyclins/metabolism , Genetic Heterogeneity , Humans , Kruppel-Like Factor 6 , Kruppel-Like Transcription Factors , Loss of Heterozygosity , Male , Mice , Microsatellite Repeats , Mutation, Missense , Proliferating Cell Nuclear Antigen/metabolism , Promoter Regions, Genetic , Trans-Activators/chemistry , Trans-Activators/physiology , Transcriptional Activation , Tumor Cells, Cultured , Up-Regulation , Zinc Fingers
3.
Am J Hum Genet ; 69(5): 1033-45, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11590545

ABSTRACT

May-Hegglin anomaly (MHA) and Fechtner (FTNS) and Sebastian (SBS) syndromes are autosomal dominant platelet disorders that share macrothrombocytopenia and characteristic leukocyte inclusions. FTNS has the additional clinical features of nephritis, deafness, and cataracts. Previously, mutations in the nonmuscle myosin heavy chain 9 gene (MYH9), which encodes nonmuscle myosin heavy chain IIA (MYHIIA), were identified in all three disorders. The spectrum of mutations and the genotype-phenotype and structure-function relationships in a large cohort of affected individuals (n=27) has now been examined. Moreover, it is demonstrated that MYH9 mutations also result in two other FTNS-like macrothrombocytopenia syndromes: Epstein syndrome (EPS) and Alport syndrome with macrothrombocytopenia (APSM). In all five disorders, MYH9 mutations were identified in 20/27 (74%) affected individuals. Four mutations, R702C, D1424N, E1841K, and R1933X, were most frequent. R702C and R702H mutations were only associated with FTNS, EPS, or APSM, thus defining a region of MYHIIA critical in the combined pathogenesis of macrothrombocytopenia, nephritis, and deafness. The E1841K, D1424N, and R1933X coiled-coil domain mutations were common to both MHA and FTNS. Haplotype analysis using three novel microsatellite markers revealed that three E1841K carriers--one with MHA and two with FTNS--shared a common haplotype around the MYH9 gene, suggesting a common ancestor. The two new globular-head mutations, K371N and R702H, as well as the recently identified MYH9 mutation, R705H, which results in DFNA17, were modeled on the basis of X-ray crystallographic data. Altogether, our data suggest that MHA, SBS, FTNS, EPS, and APSM comprise a phenotypic spectrum of disorders, all caused by MYH9 mutations. On the basis of our genetic analyses, the name "MYHIIA syndrome" is proposed to encompass all of these disorders.


Subject(s)
Genes, Dominant/genetics , Molecular Motor Proteins , Mutation/genetics , Myosin Heavy Chains/genetics , Nonmuscle Myosin Type IIA/genetics , Thrombocytopenia/genetics , Amino Acid Sequence , Chromosomes/genetics , DNA Mutational Analysis , Evolution, Molecular , Exons/genetics , Haplotypes/genetics , Humans , Microsatellite Repeats/genetics , Models, Molecular , Molecular Sequence Data , Myosin Heavy Chains/chemistry , Nephritis, Hereditary/genetics , Nephritis, Hereditary/physiopathology , Nonmuscle Myosin Type IIA/chemistry , Phenotype , Physical Chromosome Mapping , Protein Conformation , Sequence Alignment , Structure-Activity Relationship , Syndrome , Terminology as Topic , Thrombocytopenia/physiopathology
4.
Nat Genet ; 28(3): 261-5, 2001 Jul.
Article in English | MEDLINE | ID: mdl-11431697

ABSTRACT

The inherited osteolyses or 'vanishing bone' syndromes are a group of rare disorders of unknown etiology characterized by destruction and resorption of affected bones. The multicentric osteolyses are notable for interphalangeal joint erosions that mimic severe juvenile rheumatoid arthritis (OMIMs 166300, 259600, 259610 and 277950). We recently described an autosomal recessive form of multicentric osteolysis with carpal and tarsal resorption, crippling arthritic changes, marked osteoporosis, palmar and plantar subcutaneous nodules and distinctive facies in a number of consanguineous Saudi Arabian families. We localized the disease gene to 16q12-21 by using members of these families for a genome-wide search for homozygous-by-descent microsatellite markers. Haplotype analysis narrowed the critical region to a 1.2-cM region that spans the gene encoding MMP-2 (gelatinase A, collagenase type IV; (ref. 3). We detected no MMP2 enzymatic activity in the serum or fibroblasts of affected family members. We identified two family-specific homoallelic MMP2 mutations: R101H and Y244X. The nonsense mutation effects a deletion of the substrate-binding and catalytic sites and the fibronectin type II-like and hemopexin/TIMP2 binding domains. Based on molecular modeling, the missense mutation disrupts hydrogen bond formation within the highly conserved prodomain adjacent to the catalytic zinc ion.


Subject(s)
Arthritis/genetics , Matrix Metalloproteinase 2/genetics , Mutation , Osteolysis/genetics , Amino Acid Sequence , Arthritis/epidemiology , Female , Humans , Lod Score , Male , Molecular Sequence Data , Osteolysis/epidemiology , Osteolysis/pathology , Pedigree , Saudi Arabia/epidemiology , Sequence Homology, Amino Acid , Syndrome
5.
J Biol Chem ; 275(47): 36514-22, 2000 Nov 24.
Article in English | MEDLINE | ID: mdl-10969067

ABSTRACT

The metalloendopeptidase EC (EP24.15) is a neuropeptide-metabolizing enzyme expressed predominantly in brain, pituitary, and testis, and is implicated in several physiological processes and diseases. Multiple putative phosphorylation sites in the primary sequence led us to investigate whether phosphorylation effects the specificity and/or the kinetics of substrate cleavage. Only protein kinase A (PKA) treatment resulted in serine phosphorylation with a stoichiometry of 1.11 +/- 0.12 mol of phosphate/mol of recombinant rat EP24.15. Mutation analysis of each putative PKA site, in vitro phosphorylation, and phosphopeptide mapping indicated serine 644 as the phosphorylation site. Phosphorylation effects on catalytic activity were assessed using physiological (GnRH, GnRH(1-9), bradykinin, and neurotensin) and fluorimetric (MCA-PLGPDL-Dnp and orthoaminobenzoyl-GGFLRRV-Dnp-edn) substrates. The most dramatic change upon PKA phosphorylation was a substrate-specific, 7-fold increase in both K(m) and k(cat) for GnRH. In both rat PC12 and mouse AtT-20 cells, EP24.15 was serine-phosphorylated, and EP24.15 phosphate incorporation was enhanced by forskolin treatment, and attenuated by H89, consistent with PKA-mediated phosphorylation. Cloning of the full-length mouse EP24.15 cDNA revealed 96.7% amino acid identity to the rat sequence, and conservation at serine 644, consistent with its putative functional role. Therefore, PKA phosphorylation is suggested to play a regulatory role in EP24.15 enzyme activity.


Subject(s)
Metalloendopeptidases/metabolism , Amino Acid Sequence , Animals , Base Sequence , Cells, Cultured , Cloning, Molecular , Conserved Sequence , Cyclic AMP-Dependent Protein Kinases/metabolism , Hydrolysis , Metalloendopeptidases/genetics , Mice , Molecular Sequence Data , Mutagenesis, Site-Directed , Neuropeptides/metabolism , PC12 Cells , Phosphorylation , Pituitary Gland/enzymology , Rats , Sequence Alignment , Serine/metabolism
6.
Nat Genet ; 26(1): 103-5, 2000 Sep.
Article in English | MEDLINE | ID: mdl-10973259

ABSTRACT

The autosomal dominant, giant-platelet disorders, May-Hegglin anomaly (MHA; MIM 155100), Fechtner syndrome (FTNS; MIM 153640) and Sebastian syndrome (SBS), share the triad of thrombocytopenia, large platelets and characteristic leukocyte inclusions ('Döhle-like' bodies). MHA and SBS can be differentiated by subtle ultrastructural leukocyte inclusion features, whereas FTNS is distinguished by the additional Alport-like clinical features of sensorineural deafness, cataracts and nephritis. The similarities between these platelet disorders and our recent refinement of the MHA (ref. 6) and FTNS (ref. 7) disease loci to an overlapping region of 480 kb on chromosome 22 suggested that all three disorders are allelic. Among the identified candidate genes is the gene encoding nonmuscle myosin heavy chain 9 (MYH9; refs 8-10), which is expressed in platelets and upregulated during granulocyte differentiation. We identified six MYH9 mutations (one nonsense and five missense) in seven unrelated probands from MHA, SBS and FTNS families. On the basis of molecular modelling, the two mutations affecting the myosin head were predicted to impose electrostatic and conformational changes, whereas the truncating mutation deleted the unique carboxy-terminal tailpiece. The remaining missense mutations, all affecting highly conserved coiled-coil domain positions, imparted destabilizing electrostatic and polar changes. Thus, our results suggest that mutations in MYH9 result in three megakaryocyte/platelet/leukocyte syndromes and are important in the pathogenesis of sensorineural deafness, cataracts and nephritis.


Subject(s)
Blood Platelet Disorders/genetics , Leukocytes/pathology , Molecular Motor Proteins , Mutation , Myosin Heavy Chains/genetics , Alleles , Amino Acid Sequence , Animals , Blood Platelet Disorders/pathology , Cataract/genetics , Chickens , Chromosomes, Human, Pair 22 , Crystallography, X-Ray , Cytoplasm/metabolism , Genotype , Hearing Loss, Sensorineural/genetics , Humans , Models, Molecular , Molecular Sequence Data , Muscle, Smooth/metabolism , Mutation, Missense , Myosin Heavy Chains/chemistry , Myosins/chemistry , Myosins/genetics , Nephritis/genetics , Neutrophils/pathology , Neutrophils/ultrastructure , Phenotype , Protein Conformation , Protein Structure, Tertiary , Sequence Homology, Amino Acid , Syndrome , Thrombocytopenia/genetics
7.
Am J Med Genet ; 93(1): 11-8, 2000 Jul 03.
Article in English | MEDLINE | ID: mdl-10861676

ABSTRACT

The autosomal recessive multicentric osteolytic disorders of childhood-Torg, Winchester, and François syndromes-predominantly affect the carpal, tarsal, and interphalangeal joints, and their progressive bone loss and crippling arthritic deformities mimic severe juvenile rheumatoid arthritis. In a consanguineous Saudi Arabian family two affected sibs with facial anomalies and short stature displayed a distal arthropathy of the metacarpal, metatarsal, and interphalangeal joints starting in the first few months of life that eventually progressed to the proximal joints and resulted in crippling ankylosis and severe generalized osteopenia. Facial changes included proptosis, a narrow nasal bridge, bulbous nose, and micrognathia. In addition, they had large, painful fibrocollagenous palmar and plantar pads and mild body hirsutism. Affected individuals were of normal intelligence and had normal renal function. Routine hematologic, chemistry, and rheumatoid studies were within normal limits. Histologic examination of bone marrow and an interphalangeal joint biopsy were not informative. The autosomal recessive inheritance, clinical, and radiologic characteristics of the affected sibs suggested that they had a form of multicentric osteolysis most closely resembling the Torg syndrome, but with a unique facial appearance, fibrocollagenous pads, and body hirsutism not noted in the original description of the syndrome.


Subject(s)
Arthritis/genetics , Osteolysis, Essential/genetics , Abnormalities, Multiple/diagnostic imaging , Abnormalities, Multiple/genetics , Abnormalities, Multiple/pathology , Adult , Arthritis/diagnostic imaging , Arthritis/pathology , Consanguinity , Female , Humans , Male , Osteolysis, Essential/diagnostic imaging , Osteolysis, Essential/pathology , Pedigree , Radiography , Saudi Arabia , Syndrome
8.
Am J Hum Genet ; 66(4): 1449-54, 2000 Apr.
Article in English | MEDLINE | ID: mdl-10739770

ABSTRACT

The May-Hegglin anomaly (MHA) is an autosomal dominant platelet disorder of unknown etiology. It is characterized by thrombocytopenia, giant platelets, and leukocyte inclusion bodies, and affected heterozygotes are predisposed to bleeding episodes. The MHA gene has recently been localized, by means of linkage analysis, to a 13.6-cM region on chromosome 22, and the complete chromosome 22 sequence has been reported. We recently performed a genome scan for the MHA gene in 29 members of a large, multigenerational Italian family, and we now confirm that the MHA locus is on chromosome 22q12. 3-13.1. The maximal two-point LOD score of 4.50 was achieved with the use of marker D22S283, at a recombination fraction of.05. Haplotype analysis narrowed the MHA critical region to 6.6 cM between markers D22S683 and D22S1177. It is of note that the chromosome 22 sequence allowed all markers to be ordered correctly, identified all the candidate genes and predicted genes, and specifically determined the physical size of the MHA region to be 0. 7 Mb. These results significantly narrow the region in which the MHA gene is located, and they represent the first use of chromosome 22 data to positionally clone a disease gene.


Subject(s)
Blood Platelet Disorders/genetics , Chromosome Mapping , Chromosomes, Human, Pair 22/genetics , Blood Platelet Disorders/blood , Blood Platelet Disorders/pathology , Female , Genes, Dominant/genetics , Haplotypes/genetics , Humans , Italy , Lod Score , Male , Microsatellite Repeats/genetics , Pedigree , Polymorphism, Genetic/genetics , Software
9.
Carcinogenesis ; 21(2): 125-31, 2000 Feb.
Article in English | MEDLINE | ID: mdl-10657947

ABSTRACT

Regulation of cell proliferation by protein tyrosine phosphatases (PTPs) suggests that PTPs are important tumor suppressor genes. The gene encoding the leukocyte common-antigen-related (LAR) PTP receptor maps to chromosome 1p32-33, a region in which loss of heterozygosity is associated with human pheochromocytoma and other neuroectodermal tumors. The rat pheochromocytoma PC12 cell line was originally derived from the transplantable P259 tumor originating from the New England Deaconess Hospital (NEDH) line of Wistar inbred rats. Compared with their Wistar counterparts, 1-2-year-old NEDH rats exhibit a high incidence of spontaneous pheochromocytomas. This study investigates whether levels of LAR transcripts and protein are altered in NEDH adrenal tissue prior to tumor onset. In addition, alternative splicing of an LAR extracellular domain [LAR alternatively spliced element-c (LASE-c)], regulating LAR interaction with extracellular matrix components, was examined. These changes in LAR expression and alternative splicing were hypothesized to be more pronounced in tumor tissue and PC12 cells. Northern blot analysis demonstrated the presence of the approximately 5 kb LAR transcript in all cell lines examined, except PC12. In adrenal medulla tissue harvested from 2-3-month-old rats, LAR approximately 8 and approximately 5 kb transcript expression was decreased in NEDH compared with Wistar samples. RT-PCR demonstrated increased splicing of the LASE-c 27 bp alternatively spliced insert in the LAR extracellular domain in NEDH adrenal medulla tissue. Even greater LASE-c splicing was detected in adrenal medulla tumor tissue derived from 12-month-old NEDH rats and in PC12 cells. Western blot analysis demonstrated decreased levels of LAR protein and increased levels of LASE-c containing LAR protein isoforms in NEDH adrenal medulla tissue. These studies demonstrate that patterns of altered LAR expression present in PC12 cells and in pheochromocytoma tumor tissue are also present in adrenal tissue predisposed to a high incidence of spontaneous pheochromocytoma.


Subject(s)
Adrenal Gland Neoplasms/genetics , Cell Transformation, Neoplastic/genetics , Neoplasm Proteins/genetics , Nerve Tissue Proteins , Pheochromocytoma/genetics , Protein Isoforms/genetics , Protein Tyrosine Phosphatases , RNA, Messenger/biosynthesis , Rats, Wistar/genetics , Receptors, Cell Surface/genetics , Adrenal Cortex/enzymology , Adrenal Gland Neoplasms/enzymology , Adrenal Gland Neoplasms/pathology , Adrenal Medulla/enzymology , Alternative Splicing , Animals , Enzyme Induction , Female , Genetic Predisposition to Disease , Humans , Male , Neoplasm Proteins/biosynthesis , Neoplastic Syndromes, Hereditary/enzymology , Neoplastic Syndromes, Hereditary/genetics , PC12 Cells , Pheochromocytoma/enzymology , Pheochromocytoma/pathology , Protein Isoforms/biosynthesis , Rats , Receptor-Like Protein Tyrosine Phosphatases, Class 2 , Receptors, Cell Surface/biosynthesis , Reverse Transcriptase Polymerase Chain Reaction , Species Specificity
10.
Genes Chromosomes Cancer ; 27(2): 191-5, 2000 Feb.
Article in English | MEDLINE | ID: mdl-10612808

ABSTRACT

Hereditary cancers represent a unique opportunity to investigate the genetic etiology of their more common sporadic forms. We recently established genetic linkage for the rare autosomal-dominant bone dysplasia/cancer syndrome, diaphyseal medullary stenosis with malignant fibrous histiocytoma (DMS-MFH), to a 3-cM region on chromosome bands 9p21-22. This hereditary cancer syndrome is characterized by bone infarctions, cortical growth abnormalities, pathologic fractures, and painful debilitation. Most notably, 35% of affected individuals develop bone MFH, a sarcoma that, in its sporadic form, accounts for 6% of all bone cancers. To determine whether the hereditary and sporadic forms of bone MFH are genetically linked, we performed loss of heterozygosity (LOH) studies of the DMS-MFH critical region. In addition to the hereditary specimen, 71% (5/7) of informative sporadic bone MFH specimens displayed LOH for markers within that same region. Definition of the minimal region of LOH overlap effectively limited the DMS-MFH gene to a 2-cM region between markers D9S736 and D9S171. In summary, these studies suggest that a common genetic etiology underlies the autosomal-dominant and sporadic forms of this sarcoma and provide the basis for identifying the putative MFH tumor suppressor gene. Genes Chromosomes Cancer 27:191-195, 2000.


Subject(s)
Histiocytoma, Benign Fibrous/genetics , Adolescent , Adult , Aged , Bone Diseases, Developmental/genetics , Chromosomes, Human, Pair 9/genetics , DNA, Neoplasm/analysis , DNA, Neoplasm/genetics , Humans , Loss of Heterozygosity , Male , Microsatellite Repeats , Middle Aged , Neoplastic Syndromes, Hereditary/genetics
11.
J Hum Genet ; 44(5): 348-9, 1999.
Article in English | MEDLINE | ID: mdl-10496081

ABSTRACT

Alterations in chromosomal region 9p21-22 are among the most frequently encountered cytogenetic changes present in a number of human malignancies. In addition, the causative genes of a number of hereditary cancers have been genetically mapped to this region. We describe the isolation and precise localization of four novel polymorphic markers and a previously identified marker, D9S1846, from this region. Moreover, we have identified a retroposon-rich area within this oncogenic region containing a processed H3.3B pseudogene flanked by an L1 sequence and an Alu element. Together, these finely mapped and ordered reagents should prove useful for genetic mapping, sequencing, and loss of heterozygosity studies of the 9p21-22 region.


Subject(s)
Chromosomes, Human, Pair 9 , Polymorphism, Genetic , Pseudogenes , Base Sequence , Chromosome Mapping , DNA Primers , Genetic Markers , Humans , Polymerase Chain Reaction
12.
Am J Med Genet ; 84(4): 334-9, 1999 Jun 04.
Article in English | MEDLINE | ID: mdl-10340647

ABSTRACT

The finding of extensive lytic lesions in the mandible of a 19-year-old Ashkenazi Jewish woman led to the diagnosis of Type 1 Gaucher disease. She had extensive skeletal involvement, marked hepatosplenomegaly, and deficient acid beta-glucosidase activity. Mutation analysis identified heteroallelism for acid beta-glucosidase mutations N370S and P401L, the latter being a novel missense mutation in exon 9. Expression of the P401L allele resulted in an enzyme with a reduced catalytic activity (specific activity based on cross-reacting immunological material approximately 0.21), which was similar to that of the mild N370S mutant enzyme. The expression studies predicted a mild phenotype for the proposita's N370S/P401L genotype which was inconsistent with her severe diffuse skeletal disease and organ involvement. Since lytic mandibular lesions may be complicated by osteomyelitis, pathologic fracture, and tooth loss, regular dental assessments in Type 1 Gaucher patients should be performed.


Subject(s)
Gaucher Disease/genetics , Mandibular Diseases/genetics , beta-Glucosidase/genetics , Child , Female , Humans , Immunoblotting , Mutation, Missense/genetics
13.
Am J Hum Genet ; 64(3): 801-7, 1999 Mar.
Article in English | MEDLINE | ID: mdl-10053015

ABSTRACT

Diaphyseal medullary stenosis with malignant fibrous histiocytoma (DMS-MFH) is an autosomal dominant bone dysplasia/cancer syndrome of unknown etiology. This rare hereditary cancer syndrome is characterized by bone infarctions, cortical growth abnormalities, pathological fractures, and eventual painful debilitation. Notably, 35% of individuals with DMS develop MFH, a highly malignant bone sarcoma. A genome scan for the DMS-MFH gene locus in three unrelated families with DMS-MFH linked the syndrome to a region of approximately 3 cM on chromosome 9p21-22, with a maximal two-point LOD score of 5.49 (marker D9S171 at recombination fraction [theta].05). Interestingly, this region had previously been shown to be the site of chromosomal abnormalities in several other malignancies and contains a number of genes whose protein products are involved in growth regulation. Identification of this rare familial sarcoma-causing gene would be expected to simultaneously define the cause of the more common nonfamilial, or sporadic, form of MFH-a tumor that constitutes approximately 6% of all bone cancers and is the most frequently occurring adult soft-tissue sarcoma.


Subject(s)
Bone Diseases, Developmental/genetics , Chromosomes, Human, Pair 9/genetics , Histiocytoma, Benign Fibrous/genetics , Neoplastic Syndromes, Hereditary/genetics , Cell Line , Chromosome Mapping , Cloning, Molecular , Genes, Dominant , Genetic Linkage , Haplotypes , Humans , Lod Score , Pedigree , Polymorphism, Restriction Fragment Length
15.
Biochem Mol Med ; 59(2): 200-6, 1996 Dec.
Article in English | MEDLINE | ID: mdl-8986645

ABSTRACT

Cathepsin K(EC 3.4.22.38) is a lysosomal cysteine protease that is strongly implicated in bone resorption. The human cathepsin K gene is highly expressed in osteoclasts and gene mutations cause pycnodysostosis, an autosomal recessive skeletal dysplasia. To investigate the evolutionary relatedness of cathepsin K across species, the mouse cathepsin K gene was isolated. A mouse heart cDNA clone, pMCatKl, contained the 3' untranslated region, mature enzyme coding sequence, and most of the propeptide. The remainder of the gene was amplified from mouse melanocyte RNA using 5' rapid amplification of cDNA ends. The gene contained a 990-bp open reading frame, predicting a 329-amino-acid prepropolypeptide. The structure of the protein included a 15-amino-acid presignal, a 99-amino-acid proregion, and a 215-amino-acid mature enzyme. Two potential N-glycosylation sites were identified, one in the proregion and one in the mature enzyme. The 5' untranslated region was 135 bp. The 3' untranslated region was 470 bp including a 9-bp poly(A) tract and contained two polyadenylation signals. The mouse cathepsin K nucleotide and amino acid sequences were highly conserved with the human, rabbit, and chicken homologues across the proregion and mature enzyme. The mouse cathepsin K gene was isolated from an V129 genomic library, and characterization of its genomic structure and intron sizes revealed exons with the initiation ATG in exon 2 and termination TGA in exon 8, a genomic organization that was highly conserved with its human homologue. The availability of the mouse cathepsin K cDNA and genomic sequences will facilitate generation of a mouse model of cathepsin K deficiency by gene targeting.


Subject(s)
Cathepsins/genetics , Dysostoses/genetics , Amino Acid Sequence , Animals , Base Sequence , Cathepsin K , Cloning, Molecular , DNA, Complementary , Humans , Mice , Molecular Sequence Data , Sequence Homology, Amino Acid
16.
Pediatr Radiol ; 26(9): 675-7, 1996 Sep.
Article in English | MEDLINE | ID: mdl-8781110

ABSTRACT

Hardcastle syndrome is a rare, autosomally dominant inherited skeletal dysplasia, characterized by diaphyseal sclerosis, medullary stenosis, pathological fractures, bony infarction, and malignant transformation. A 19-year-old proband is presented and discussed, adding a fourth family to the world literature. Radiographic screening of family members is suggested from puberty onward. Thallium scanning is proposed as a more tumor-sensitive screening agent in affected individuals.


Subject(s)
Bone Neoplasms/diagnostic imaging , Camurati-Engelmann Syndrome/diagnostic imaging , Diaphyses/diagnostic imaging , Histiocytoma, Benign Fibrous/diagnostic imaging , Osteosclerosis/diagnostic imaging , Adult , Aged , Biopsy , Bone Neoplasms/complications , Camurati-Engelmann Syndrome/complications , Camurati-Engelmann Syndrome/genetics , Diaphyses/pathology , Female , Femur/diagnostic imaging , Fibula/diagnostic imaging , Histiocytoma, Benign Fibrous/complications , Humans , Magnetic Resonance Imaging , Male , Organotechnetium Compounds , Osteosclerosis/complications , Pedigree , Thallium Radioisotopes , Tibia/diagnostic imaging , Tomography, Emission-Computed
17.
Mol Cell Biol ; 15(3): 1642-50, 1995 Mar.
Article in English | MEDLINE | ID: mdl-7862155

ABSTRACT

Rodent BC1 RNA represents the first example of a neural cell-specific RNA polymerase III (Pol III) transcription product. By developing a rat brain in vitro system capable of supporting Pol III-directed transcription, we showed that the rat BC1 RNA intragenic promoter elements, comprising an A box element and a variant B box element, as well as its upstream region, containing octamer-binding consensus sequences and functional TATA and proximal sequence element sites, are necessary for transcription. The BC1 B box, lacking the invariant A residue found in the consensus B boxes of tRNAs, represents a functionally related and possibly distinct promoter element. The transcriptional activity of the BC1 B box element is greatly increased, in both a BC1 RNA and a chimeric tRNA(Leu) gene construct, when the BC1 5' flanking region is present and is appropriately spaced. Moreover, a tRNA consensus B-box sequence can efficiently replace the BC1 B box only if the BC1 upstream region is removed. These interactions, identified only in a homologous in vitro system, between upstream Pol II and intragenic Pol III promoters suggest a mechanism by which the tissue-specific BC1 RNA gene and possibly other Pol III-transcribed genes can be regulated.


Subject(s)
Cerebral Cortex/enzymology , DNA-Binding Proteins/biosynthesis , Neurons/enzymology , RNA Polymerase III/metabolism , Ribonucleoproteins, Small Cytoplasmic , Ribonucleoproteins/biosynthesis , Transcription, Genetic , Animals , Base Sequence , Chimera , DNA Primers , Escherichia coli/genetics , Exons , Introns , Kinetics , Molecular Sequence Data , Mutagenesis, Site-Directed , Organ Specificity , RNA Polymerase II/metabolism , RNA, Long Noncoding , RNA, Untranslated , Rats , Restriction Mapping , Sequence Homology, Nucleic Acid , Templates, Genetic
18.
Proc Natl Acad Sci U S A ; 91(9): 3607-11, 1994 Apr 26.
Article in English | MEDLINE | ID: mdl-8170955

ABSTRACT

ID elements are short interspersed repetitive DNA elements (SINEs) which have amplified in rodent genomes via retroposition, a process involving an RNA intermediate. BC1, an abundant ID-related transcript, is transcribed from a conserved, single-copy gene in rodents. The gene encoding BC1 RNA represents one of the earliest and possibly the first ID-containing sequence. Comparison of consensus sequences of each rodent ID with its corresponding BC1 RNA gene showed that the variations of BC1 RNA within rodents corresponded to specific changes within the ID consensus sequence for each rodent species. This supports the hypothesis that the BC1 gene is a master gene responsible for the amplification and evolution of ID elements. The rat ID family consists of at least four subfamilies, with the oldest subfamily having been derived from the BC1 RNA. The other three subfamilies appear to have been derived from a new master gene(s), which has been responsible for the large increase in ID element copy number within the rat genome. We have found that the guinea pig genome contains two copies of the BC1 gene, apparently the result of a DNA-mediated duplication event. Both of these guinea pig BC1 genes have a conserved TATA-like element in the 5' flanking region and have contributed to guinea pig ID amplifications.


Subject(s)
DNA-Binding Proteins/genetics , Repetitive Sequences, Nucleic Acid , Ribonucleoproteins, Small Cytoplasmic , Ribonucleoproteins/genetics , Rodentia/genetics , Animals , Base Sequence , Biological Evolution , Genes , Guinea Pigs , Mice , Molecular Sequence Data , Oligodeoxyribonucleotides/chemistry , RNA, Long Noncoding , RNA, Untranslated , Rats
19.
Proc Natl Acad Sci U S A ; 90(24): 11563-7, 1993 Dec 15.
Article in English | MEDLINE | ID: mdl-8265590

ABSTRACT

We demonstrate that the BC200 RNA gene, which encodes a neural small cytoplasmic RNA, is a member of the most prodigious family of interspersed repetitive DNA and that its product represents an example of a primate tissue-specific RNA polymerase III transcript. The BC200 RNA gene is an early monomeric member and one of the few postulated transcriptionally active Alu sequences in this family of nearly half a million retropositionally amplified elements dispersed throughout the human genome. Furthermore, the isolation of two pseudogenes, BC200 beta and BC200 gamma, demonstrates the gene's transpositional ability. Interestingly, the BC200 beta pseudogene may have been generated by a conversion-like event after the human/chimpanzee divergence, resulting in an exchange of the left arm of a dimeric Alu element with the BC200 RNA coding sequence. Our data on conserved features of the active BC200 alpha gene suggest that its RNA product has been "exapted" into a function of the primate brain and provides a selective advantage to the species.


Subject(s)
Brain/metabolism , Conserved Sequence , Genome, Human , Hominidae/genetics , Neurons/metabolism , RNA Polymerase III/genetics , RNA Polymerase III/metabolism , Animals , Base Sequence , Gene Conversion , Gene Expression , Genomic Library , Humans , Molecular Sequence Data , Oligodeoxyribonucleotides , Pan troglodytes/genetics , Primates/genetics , Pseudogenes , RNA Polymerase III/biosynthesis , Restriction Mapping , Sequence Homology, Nucleic Acid
20.
J Biol Chem ; 268(35): 26503-11, 1993 Dec 15.
Article in English | MEDLINE | ID: mdl-8253779

ABSTRACT

Receptor-linked tyrosine phosphatases regulate cell growth by dephosphorylating proteins involved in tyrosine kinase signal transduction. Within this gene family, the leukocyte common antigen-related (LAR) gene is of particular interest with respect to the nervous system because it has sequence similarity to the neural cell adhesion molecule N-CAM and is located in a chromosomal region (1p32-33) frequently deleted in neuroectodermal tumors. However, immunostaining has detected LAR in non-neural tissues, but not in the central nervous system, peripheral neurons, or adrenal medulla. In this study, rat brain cDNA library LAR clones corresponding to cytoplasmic and 3'-untranslated regions of human LAR were identified. Using probes derived from these clones, high stringency Northern blots revealed approximately 8 kilobase and variable length tissue- and cell-specific LAR transcripts in cortex, brainstem, cerebellum, spinal cord, peripheral tissues, and cultured neural, glial, and pheochromocytoma cells. In situ hybridization showed expression by brain and dorsal root ganglion neurons. LAR expression was developmentally regulated in a region-dependent manner. Changes in LAR expression were also found during nerve growth factor-induced PC12 pheochromocytoma cell differentiation and with contact-mediated inhibition of fibroblast growth. These observations and studies demonstrating neurotrophins functioning via tyrosine kinase receptors suggest that LAR represents an additional mechanism regulating neural development.


Subject(s)
Gene Expression Regulation , Glycoproteins/genetics , Protein Tyrosine Phosphatases/genetics , RNA, Messenger/metabolism , Receptors, Cell Surface , Animals , Base Sequence , Cells, Cultured , Cerebral Cortex/cytology , Cerebral Cortex/embryology , Cerebral Cortex/metabolism , Cloning, Molecular , DNA, Complementary , Glycoproteins/metabolism , Humans , Molecular Sequence Data , Neurons/metabolism , Phosphorylation , Protein Tyrosine Phosphatases/metabolism , RNA, Messenger/genetics , Rats , Rats, Sprague-Dawley , Receptor-Like Protein Tyrosine Phosphatases, Class 4 , Sequence Alignment
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