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1.
Mol Cell Biol ; 9(3): 1277-83, 1989 Mar.
Article in English | MEDLINE | ID: mdl-2725498

ABSTRACT

The influence of DNA repair on the molecular nature of mutations induced by UV light (254 nm) was investigated in UV-induced hprt mutants from UV-sensitive Chinese hamster cells (V-H1) and the parental line (V79). The nature of point mutations in hprt exon sequences was determined for 19 hprt mutants of V79 and for 17 hprt mutants of V-H1 cells by sequence analysis of in vitro-amplified hprt cDNA. The mutation spectrum in V79 cells consisted of single- and tandem double-base pair changes, while in V-H1 cells three frameshift mutations were also detected. All base pair changes in V-H1 mutants were due to GC----AT transitions. In contrast, in V79 all possible classes of base pair changes except the GC----CG transversion were present. In this group, 70% of the mutations were transversions. Since all mutations except one did occur at dipyrimidine sites, the assumption was made that they were caused by UV-induced photoproducts at these sites. In V79 cells, 11 out of 17 base pair changes were caused by photoproducts in the nontranscribed strand of the hprt gene. However, in V-H1 cells, which are completely deficient in the removal of pyrimidine dimers from the hprt gene and which show a UV-induced mutation frequency enhanced seven times, 10 out of 11 base pair changes were caused by photoproducts in the transcribed strand of the hprt gene. We hypothesize that this extreme strand specificity in V-H1 cells is due to differences in fidelity of DNA replication of the leading and the lagging strand. Furthermore, we propose that in normal V79 cells two processes determine the strand specificity of UV-induced mutations in the hprt gene, namely preferential repair of the transcribed strand of the hprt gene and a higher fidelity of DNA replication of the nontranscribed strand compared with the transcribed strand.


Subject(s)
DNA/radiation effects , Amino Acid Sequence , Animals , Base Sequence , Cell Line , DNA Repair/radiation effects , Hypoxanthine Phosphoribosyltransferase/genetics , Mutation , Pyrimidine Dimers/metabolism , Pyrimidine Dimers/radiation effects , Transcription, Genetic , Ultraviolet Rays
2.
Oncogene ; 10(8): 1521-8, 1995 Apr 20.
Article in English | MEDLINE | ID: mdl-7731706

ABSTRACT

We have isolated a gene, called MN1, which resides on chromosome 22 and which was found to be disrupted by a balanced translocation (4;22) in meningioma 32. The MN1 gene spans about 70 kb and consists of at least two large exons of approximately 4.7 kb and 2.8 kb. The MN1 cDNA codes for a protein of 1319 amino acids when the first methionine in the open reading frame is used. The MN1 cDNA contains two CAG repeats, one of which codes for a string of 28 glutamines. The t(4;22) disrupts the 5'-exon within the open reading frame. In meningioma 32 no expression of the MN1 mRNA is observed. These results suggest that inactivation of the MN1 gene in this tumour may contribute to its pathogenesis.


Subject(s)
Chromosomes, Human, Pair 22 , Genes, Tumor Suppressor , Meningeal Neoplasms/genetics , Meningioma/genetics , Translocation, Genetic , Amino Acid Sequence , Base Sequence , Blotting, Northern , Blotting, Southern , Chromosomes, Human, Pair 4 , Cloning, Molecular , DNA, Complementary/chemistry , Humans , Molecular Sequence Data
3.
J Neuropathol Exp Neurol ; 54(2): 224-35, 1995 Mar.
Article in English | MEDLINE | ID: mdl-7876890

ABSTRACT

In a series of 126 meningiomas, tumor and patient characteristics were investigated and statistically analyzed. A combined cytogenetic and molecular genetic approach was used to study chromosomal abnormalities and loss of markers on chromosome 22q. This approach was successfully applied to 93 meningiomas. In 66 cases, complete or partial loss of chromosome 22 was observed and in at least 12 of them this chromosome was involved in structural aberrations. In addition to chromosome 22 changes, chromosomes 1, 6, 11, 13, 14, 18, 19, X, and Y were also frequently involved in structural and numerical aberrations. Statistical analysis revealed a significant association between the number of chromosomal abnormalities and tumor grade. Complex karyotypes predominated in the group of grade II/III meningiomas. Furthermore, other variables showed statistically (or marginally statistically) significant differences. Meningiomas from the convexity were more often grade II/III, displayed predominantly (partial) loss of chromosome 22 and had complex karyotypes more often. These features were frequently found in meningiomas from males. Base meningiomas, on the other hand, occurred more often in females; they were usually grade I, showed loss of (parts of) chromosome 22 less often and displayed fewer additional chromosomal abnormalities.


Subject(s)
Chromosome Aberrations/genetics , Chromosome Deletion , Chromosomes, Human, Pair 22/genetics , Meningeal Neoplasms/genetics , Meningeal Neoplasms/pathology , Meningioma/genetics , Meningioma/pathology , Adult , Aged , Aged, 80 and over , Chromosome Disorders , Female , Heterozygote , Humans , In Situ Hybridization , Karyotyping , Male , Middle Aged
4.
EMBO J ; 8(9): 2503-7, 1989 Sep.
Article in English | MEDLINE | ID: mdl-2479553

ABSTRACT

We have determined the primary structure of a mutant insulin receptor of a leprechaun patient born from a consanguineous marriage. A characteristic feature of leprechaunism is an extreme resistance to insulin. In this patient the insulin resistance seems to result from an observed lack of insulin binding to intact cells. Solubilization of cells in non-ionic detergents leads to the appearance of insulin receptors which can bind insulin. However, the insulin-stimulated autophosphorylation of the receptor's beta subunit is markedly reduced. Cloning and sequencing of cDNA derived from insulin receptor mRNA of this patient revealed a leucine-to-proline mutation at position 233 in the alpha subunit. By means of DNA amplification we found that the patient is homozygous for this mutation and that the parents and two grandparents from the consanguineous line are heterozygous. The heterozygous individuals all show decreased insulin binding to cultured fibroblasts. In addition, they are mildly insulin resistant in vivo. These observations show a linkage between the leucine-to-proline mutation and the observed insulin resistance in this family. We therefore conclude that the mutation in the homozygous form is responsible for the extreme insulin resistance in the leprechaun patient. The mutation for the first time characterizes a region in the insulin receptor which seems to be involved in transmitting the insulin binding signal to the tyrosine kinase domain.


Subject(s)
Insulin Resistance/genetics , Receptor, Insulin/genetics , Cells, Cultured , Cloning, Molecular , Deoxyribonuclease HpaII , Deoxyribonucleases, Type II Site-Specific , Fibroblasts/metabolism , Genotype , Humans , Leucine , Male , Mutation , Pedigree , Proline , Radioligand Assay
5.
Am J Hum Genet ; 48(4): 783-90, 1991 Apr.
Article in English | MEDLINE | ID: mdl-2014801

ABSTRACT

Cytogenetic analysis of meningioma cells from one particular patient (MN32) displayed the stem-line karyo-type 45, XY, -1, 4p+, 22q-, 22q+, which thus had rearrangements of both chromosomes 22. The 22q+ marker appeared as a dicentric: 22 pter----q11::1p11----qter. The reciprocal product of this translocation has presumably been lost because it lacked a centromere. The 22q- chromosome also appeared to have lost sequences distal to band q11. We assumed that this marker could have been the result of a reciprocal translocation between chromosomes 4 and 22. To investigate the 4p+ and 22q- chromosomes in more detail, human-hamster somatic cell hybrids were constructed that segregated the 22q- and 4p+ chromosomes. Southern blot analysis with DNA from these hybrids showed that sequences from 22q were indeed translocated to 4p+ and that reciprocally sequences from 4p were translocated to 22q-, demonstrating a balanced t(4;22)(p16;q11). On the basis of these results we presume that in this tumor a tumor-suppressor gene is deleted in the case of the 22q+ marker and that the t(4;22) disrupts the second allele of this gene. The latter translocation was mapped between D22S1 and D22S15, a distance of 1 cM on the linkage map of this chromosome. The area in which we have located the translocation is within the region where the gene predisposing to neurofibromatosis 2 has been mapped.


Subject(s)
Chromosomes, Human, Pair 22/ultrastructure , Chromosomes, Human, Pair 4/ultrastructure , Genes, Tumor Suppressor , Meningioma/genetics , Translocation, Genetic , Alleles , Animals , Chromosome Banding , Cricetinae , DNA/chemistry , Genetic Linkage , Genetic Markers , Humans , Hybrid Cells/ultrastructure , Tumor Cells, Cultured
6.
Lab Invest ; 77(1): 85-92, 1997 Jul.
Article in English | MEDLINE | ID: mdl-9251681

ABSTRACT

To identify new molecular markers for differentiation of normal and neoplastic colon epithelium, we have studied changes in gene expression during the in vitro differentiation of the HT29-D4 colon carcinoma cell line. Using a modified differential display procedure, we cloned a novel cDNA, designated differentiation-related gene 1 (Drg1). Drg1 mRNA has a length of approximately 3 kb and is induced approximately 20-fold during in vitro differentiation of the colon carcinoma cell lines HT29-D4 and Caco-2. The absence of Drg1 induction in growth-inhibited A431 epidermoid carcinoma cells indicates that Drg1 up-regulation in colon carcinoma cells is not a result of decreased proliferation. The Drg1 cDNA contains an open-reading frame of 1182 bp that encodes a protein with a predicted molecular weight of 43 kd. Drg1 mRNA is expressed most prominently in placental membranes and prostate, kidney, small intestine, and ovary tissues. Compared to normal colon mucosa, Drg1 mRNA expression is decreased in colon adenomas and adenocarcinomas. An antiserum raised against recombinant Drg1 protein detected a band of the expected size in Western blots. Immunohistochemistry showed that in normal colon Drg1 protein is expressed in the cytoplasm and basolateral membranes of surface epithelial cells that border the gut lumen, indicating that Drg1 protein is expressed late during differentiation, just before apoptosis and shedding of cells into the colon lumen.


Subject(s)
Cell Cycle Proteins , Colon/cytology , Colorectal Neoplasms/genetics , Down-Regulation , Intestinal Mucosa/cytology , Proteins/genetics , Up-Regulation , Amino Acid Sequence , Animals , Base Sequence , Cell Differentiation , Colon/metabolism , Humans , Immune Sera , Intracellular Signaling Peptides and Proteins , Molecular Sequence Data , RNA, Messenger/metabolism , Rats , Tumor Cells, Cultured
7.
Genes Chromosomes Cancer ; 9(2): 124-8, 1994 Feb.
Article in English | MEDLINE | ID: mdl-7513542

ABSTRACT

We describe a patient who developed multiple meningiomas but had no clear evidence of neurofibromatosis type 2. Four of the tumors, derived from three different sites, were analyzed cytogenetically and/or at the DNA level using chromosome 22 specific probes. All four tumors showed loss of the same copy of chromosome 22. On the chromosome that was retained in the tumors, we found two constitutional aberrations, a 1.5 kb deletion and a point mutation. The patient had inherited both alterations from her father. The father has not developed any meningiomas so far but he has been treated for a well-differentiated adenocarcinoma of the lung and a brain metastasis from this tumor. The mother and 75 unrelated individuals did not show any of the chromosome 22 alterations. The multiple tumors found in the patient suggest that she has a predisposing gene for the development of meningiomas. The finding that all investigated tumors lost the same, constitutionally normal copy of chromosome 22 could indicate that the predisposing gene resides on chromosome 22 and was affected by the constitutional mutations.


Subject(s)
Chromosome Aberrations/genetics , Chromosome Deletion , Chromosomes, Human, Pair 22 , DNA, Neoplasm/genetics , Meningeal Neoplasms/genetics , Meningioma/genetics , Neoplasms, Multiple Primary/genetics , Point Mutation , Sequence Deletion , Adenocarcinoma/genetics , Adenocarcinoma/secondary , Adult , Brain Neoplasms/genetics , Brain Neoplasms/secondary , Chromosome Disorders , Chromosomes, Human, Pair 22/ultrastructure , DNA Mutational Analysis , Female , Genetic Markers , Humans , Lung Neoplasms/genetics , Male , Middle Aged
8.
Am J Hum Genet ; 54(6): 1022-9, 1994 Jun.
Article in English | MEDLINE | ID: mdl-7911002

ABSTRACT

The gene for the hereditary disorder neurofibromatosis type 2 (NF2), which predisposes for benign CNS tumors such as vestibular schwannomas and meningiomas, has been assigned to chromosome 22 and recently has been isolated. Mutations in the NF2 gene were found in both sporadic meningiomas and vestibular schwannomas. However, so far only 6 of the 16 exons of the gene have been analyzed. In order to extend the analysis of an involvement of the NF2 gene in the sporadic counterparts of these NF2-related tumors, we have used reverse transcriptase-PCR amplification followed by SSCP and DNA sequence analysis to screen for mutations in the coding region of the NF2 gene. Analysis of the NF2 gene transcript in 53 unrelated patients with meningiomas and vestibular schwannomas revealed mutations in 32% of the sporadic meningiomas (n = 44), in 50% of the sporadic vestibular schwannomas (n = 4), in 100% of the tumors found in NF2 patients (n = 2), and in one of three tumors from multiple-meningioma patients. Of the 18 tumors in which a mutation in the NF2 gene transcript was observed and the copy number of chromosome 22 could be established, 14 also showed loss of (parts of) chromosome 22. This suggests that in sporadic meningiomas and NF2-associated tumors the NF2 gene functions as a recessive tumor-suppressor gene. The mutations detected resulted mostly in frameshifts, predicting truncations starting within the N-terminal half of the putative protein.


Subject(s)
Genes, Neurofibromatosis 2/genetics , Meningeal Neoplasms/genetics , Meningioma/genetics , Mutation/genetics , Neurofibromatosis 2/genetics , Base Sequence , Chromosome Deletion , Chromosomes, Human, Pair 22 , DNA Mutational Analysis , Humans , In Situ Hybridization, Fluorescence , Karyotyping , Molecular Sequence Data , Polymerase Chain Reaction , Polymorphism, Restriction Fragment Length , RNA, Messenger/analysis , RNA, Neoplasm/analysis , Transcription, Genetic
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