Structure of the leukemia-associated human CBFB gene.
Explore the source record for details and available documents.
Retraction Notice↗
Biomedical subjects
F S Collins
Publications and source records attributed to F S Collins.
Failure of embryonic hematopoiesis and lethal hemorrhages in mouse embryos heterozygous for a knocked-in leukemia gene CBFB-MYH11.
The fusion oncogene CBFB-MYH11 is generated by a chromosome 16 inversion in human acute myeloid leukemia subtype M4Eo. Mouse embryonic stem (ES) cells heterozygous for this oncogene were generated by inserting part of the human MYH11 cDNA into the mouse Cbfb gene through homologous recombination (knock-in). Chimeric mice were leukemia free, but the ES cells with the knocked-in Cbfb-MYH11 gene did not contribute to their hematopoietic tissues. Mouse embryos heterozygous for Cbfb-MYH11 lacked definitive hematopoiesis and developed multiple fatal hemorrhages around embryonic day 12.5. This phenotype is very similar to that resulting from homozygous deletions of either Cbfb or Cbfa2 (AML1), consistent with a dominant negative function of the Cbfb-MYH11 fusion oncogene. An impairment of primitive hematopoiesis was also observed, however, suggesting a possible additional function of Cbfb-MYH11.
Amino Acid Sequence↗
Allelic deletions on chromosome 11q13 in multiple tumors from individual MEN1 patients.
Familial multiple endocrine neoplasia type 1 is an autosomal dominant hereditary disorder characterized by multiple parathyroid, pancreatic, duodenal, and pituitary tumors. The parathyroid tumors may arise as diffuse areas of hyperplasia, whereas the pancreatic and duodenal tumors usually form as discrete nodules. Except for a single report, tumor loss of heterozygosity (LOH) mapping of the putative MEN1 suppressor gene on chromosome 11q13 in the past has been restricted by analysis of a single tumor from individual patients and somatic cellular contamination. For this reason, it has not been possible to analyze the clonality of the emerging MEN1 neoplasms. Furthermore, it has been previously unknown whether the LOH pattern varies between individual MEN1 tumors in a given patient or among tumors of different histological origins within unrelated patients. To address these previous limitations, the present study introduces a refinement in microdissection in which endothelial cells are stained and selectively excluded. Tissue microdissection was applied to study LOH patterns on chromosome 11q13 using 8 polymorphic DNA markers in 44 different MEN1 tumors from parathyroid, pancreas, and duodenum in nine unrelated patients. In addition, X-chromosome inactivation clonal analysis was applied to 16 individual microdissected regions from seven parathyroid glands in three female patients. The LOH rates of parathyroid lesions (100%) and endocrine tumors of the pancreas (83%) were strikingly different from the LOH rate of gastrinomas (21%), suggesting that the mechanism that drives LOH may be influenced by the tissue context. Moreover, combined LOH and X-chromosome inactivation scoring of the same microdissected region revealed that parathyroid MEN1 neoplasms can consist of more than one clone. In this study, the centromeric boundary of the putative MEN1 gene was PYGM. Analysis of differential LOH patterns in multiple microdissected tumors in the same patient constitutes a novel approach to suppressor gene mapping.
Adult↗
Characterization of EZH1, a human homolog of Drosophila Enhancer of zeste near BRCA1.
Recent transcription mapping efforts within chromosome 17q21 have led to the identification of a human homolog of the Drosophila gene Enhancer of zeste, E(z). A member of the Polycomb group (Pc-G) of proteins, Drosophila E(z) acts as a negative regulator of the segment identity genes of the Antennapedia and Bithorax complexes. Here we report the full-length protein coding sequence of human EZH1 (Enhancer of zeste homolog 1) and compare the respective protein sequences in both species. EZH1 encodes a protein of 747 amino acids that displays 55% amino acid identity overall (70% similarity) with Drosophila E(z). The strongest homology was noted (79% identity, 89% similarity) within the carboxy-terminal 245 amino acids, including the SET domain, a region of E(z) also conserved in other Drosophila proteins with roles in development and/or chromatin structure. A large Cysrich region with a novel spatial pattern of cysteine residues was also conserved in both EZH1 and E(z). The strong sequence conservation suggest potential roles for EZH1 in human development as a transcriptional regulator and as a component of protein complexes that stably maintain heterochromatin. EZH1 is expressed as two major transcripts in all adult and fetal human tissues surveyed; comparison of cloned cDNAs suggests that alternative splicing may account for at least part of the transcript size difference. Analysis of one cDNA revealed an unusual splicing event involving EZH1 and a tandemly linked gene GPR2 and suggests a potential mechanism for modifying the EZH1 protein in the conserved C-terminal domain. The sequence and isolated cDNAs will provide useful reagents for determining the function of EZH1 and the importance of the evolutionarily conserved domains.
Adult↗
A 500-kb physical map and contig from the Harvey ras-1 gene to the 11p telomere.
A contiguous physical map was constructed from the Harvey ras-1 (HRAS1) gene to the 11p telomere. The contig spans approximately 500 kb and is minimally composed of a telomere-containing YAC and P1 and cosmid clones. Included in the contig are 11 sequence-tagged sites derived from P1 and cosmid ends. Three genes were placed on the contig in the following order: telomere-ribonuclease/angiogenin inhibitor (RNH)-Harvey ras-1 (HRAS1)-HRAS1-related complex (HRC). Two novel tetranucleotide repeats (heterozygosity of 66 and 68%) and a complex CA repeat (heterozygosity of 78%) were isolated and characterized.
Base Sequence↗
Identification and chromosomal localization of Atm, the mouse homolog of the ataxia-telangiectasia gene.
Atm, the mouse homolog of the human ATM gene defective in ataxia-telangiectasia (A-T), has been identified. The entire coding sequence of the Atm transcript was cloned and found to contain an open reading frame encoding a protein of 3066 amino acids with 84% overall identity and 91% similarity to the human ATM protein. Variable levels of expression of Atm were observed in different tissues. Fluorescence in situ hybridization and linkage analysis located the Atm gene on mouse chromosome 9, band 9C, in a region homologous to the ATM region on human chromosome 11q22-q23.
Amino Acid Sequence↗
A human gene (DDX10) encoding a putative DEAD-box RNA helicase at 11q22-q23.
A human gene encoding a putative RNA helicase, designated DDX10, was identified 400 kb telomeric to the ataxia-telangiectasia gene at chromosome 11q22-q23. The predicted amino acid sequence shows very high similarity to a subgroup of DEAD-box RNA helicases involved in ribosome biogenesis. This novel gene encodes a 3.2-kb transcript in a variety of human tissues. A processed pseudogene of DDX10 was detected at chromosome 9q21-q22. We observed a rare trinucleotide repeat length polymorphism within the coding sequence of DDX10.
Amino Acid Sequence↗
Core binding factor beta-smooth muscle myosin heavy chain chimeric protein involved in acute myeloid leukemia forms unusual nuclear rod-like structures in transformed NIH 3T3 cells.
Patients with the M4Eo subtype of acute myeloid leukemia almost invariably are found to have an inversion of chromosome 16 in their leukemic cells, which results in a gene fusion between the transcription factor called core binding factor beta (CBFbeta) on 16q and a smooth muscle myosin heavy chain (SMMHC) gene on 16p. Subcellular localizations of the wild-type CBFbeta and the CBFbeta-SMMHC fusion protein were determined by immunofluorescence of NIH 3T3 cells that overexpress wild-type or fusion protein. Normal CBFbeta showed an unexpected perinuclear pattern consistent with primary localization in the Golgi complex. The CBFbeta-SMMHC fusion protein had a very different pattern. Nuclear staining included rod-like crystalline structures as long as 11 microm. The heterodimeric partner of CBFbeta, CBFalpha, formed part of this complex. Cytoplasmic staining included stress fibers that colocalized with actin, probably as a consequence of the myosin heavy chain component of the fusion protein. Deletion of different regions of the CBFbeta portion of the fusion protein showed that binding to CBFalpha was not required for nuclear translocation. However, deletion of parts of the SMMHC domain of the fusion protein involved in myosin-mediated filament formation resulted in proteins that did not form rod-like structures. These observations confirm previous indirect evidence that the CBFbeta-SMMHC fusion protein is capable of forming macromolecular nuclear aggregates and suggests possible models for the mechanism of leukemic transformation.
3T3 Cells↗
Isolation of tetranucleotide repeat polymorphisms flanking the BRCA1 gene.
Large pools of cosmids from the BRCA1 region of human chromosome 17 were screened for tetranucleotide repeat polymorphisms by hybridizing shotgun subcloned pools with a mixture of 25 oligonucleotides. Identified subclones were PCR amplified and directly sequenced to design PCR primers for short tandem repeat polymorphism (STRP) analysis of family DNAs. With the identification of the BRCA1 gene and the observation that most mutations in this > 100-kb gene are unique, haplotyping and linkage analysis may play a significant role in diagnosis and carrier detection of BRCA1-associated breast and ovarian cancers. We report the characterization of 15 new STRPs flanking the BRCA1 locus.
BRCA1 Protein↗
BRCA1--lots of mutations, lots of dilemmas.
Explore the source record for details and available documents.
BRCA1 Protein↗
Novel missense mutation (G314R) in a cystic fibrosis patient with hepatic failure.
Explore the source record for details and available documents.
Adolescent↗
Identification of the chimeric protein product of the CBFB-MYH11 fusion gene in inv(16) leukemia cells.
An expressed gene formed by fusion between the CBFB transcription factor gene and the smooth muscle myosin heavy chain gene MYH11 is consistently detected by reverse transcription polymerase chain reaction (RT-PCR) in patients who have acute myeloid leukemia (AML) subtype M4Eo with an inversion of chromosome 16. We have previously shown that a CBFB-MYH11 cDNA construct can produce a chimeric protein and transform NIH 3T3 cells. However, the presence of the chimeric protein in patient cells has not been demonstrated previously. Here, we show that such chimeric proteins can be identified in vivo, primarily in the nuclei of the leukemic cells, by use of antibodies against the C-terminus of the smooth muscle myosin heavy chain and the fusion junction peptide. A very high molecular weight protein/DNA complex is generated when nuclear extracts from patient cells are used in electrophoretic mobility shift assays, as seen in NIH 3T3 cells transfected with the CBFB-MYH11 cDNA. Immunofluorescence staining shows that the proteins are organized in vivo into novel structures within cell nuclei. One isoform of the transcript of the CBFB-MYH11 fusion gene, containing the MHC204 C-terminus, was the predominant from in all five cases studied.
3T3 Cells↗
Transforming properties of the leukemic inv(16) fusion gene CBFB-MYH11.
Explore the source record for details and available documents.
Animals↗
Chromosomal localization of 15 ion channel genes.
Several human Mendelian diseases, including the long-QT syndrome, malignant hyperthermia, and episodic ataxia/myokymia syndrome, have recently been demonstrated to be due to mutations in ion channel genes. Systematic mapping of ion channel genes may therefore reveal candidates for other heritable disorders. In this study, the GenBank and dbEST databases were used to identify members of several ion channel families (voltage-gated calcium and sodium, cardiac chloride, and all classes of potassium channels). Genes and ESTs without prior map localization were identified based on GDB and OWL database information and 15 genes and ESTs were selected for mapping. Of these 15, only the serotonin receptor 5HT3R had been previously mapped to a chromosome. A somatic cell hybrid panel (SCH) was screened with an STS from each gene and, if necessary the results verified by a second SCH panel. For three ESTs, rodent derived PCR products of the same size as the human STS precluded SCH mapping. For these three, human P1 clones were isolated and the genomic location was determined by metaphase FISH. These genes and ESTs can now be further evaluated as candidate genes for inherited cardiac, neuromuscular and psychiatric disorders mapped to these chromosomes. Furthermore, the ESTs developed in this study can be used to isolate genomic clones, enabling the determination of each transcript's genomic structure and physical map location. This approach may also be applicable to other gene families and may aid in the identification of candidate genes for groups of related heritable disorders.
Animals↗
Detection of heterozygous mutations in BRCA1 using high density oligonucleotide arrays and two-colour fluorescence analysis.
The ability to scan a large gene rapidly and accurately for all possible heterozygous mutations in large numbers of patient samples will be critical for the future of medicine. We have designed high-density arrays consisting of over 96,600 oligonucleotides 20-nucleotides (nt) in length to screen for a wide range of heterozygous mutations in the 3.45-kilobases (kb) exon 11 of the hereditary breast and ovarian cancer gene BRCA1. Reference and test samples were co-hybridized to these arrays and differences in hybridization patterns quantitated by two-colour analysis. Fourteen of fifteen patient samples with known mutations were accurately diagnosed, and no false positive mutations were identified in 20 control samples. Eight single nucleotide polymorphisms were also readily detected. DNA chip-based assays may provide a valuable new technology for high-throughput cost-efficient detection of genetic alterations.
BRCA1 Protein↗
CFTR: the nucleotide binding folds regulate the accessibility and stability of the activated state.
The functional roles of the two nucleotide binding folds, NBF1 and NBF2, in the activation of the cystic fibrosis transmembrane conductance regulator (CFTR) were investigated by measuring the rates of activation and deactivation of CFTR Cl- conductance in Xenopus oocytes. Activation of wild-type CFTR in response to application of forskolin and 3-isobutyl-1-methylxanthine (IBMX) was described by a single exponential. Deactivation after washout of the cocktail consisted of two phases: an initial slow phase, described by a latency, and an exponential decline. Rate analysis of CFTR variants bearing analogous mutations in NBF1 and NBF2 permitted us to characterize amino acid substitutions according to their effects on the accessibility and stability of the active state. Access to the active state was very sensitive to substitutions for the invariant glycine (G551) in NBF1, where mutations to alanine (A), serine (S), or aspartic acid (D) reduced the apparent on rate by more than tenfold. The analogous substitutions in NBF2 (G1349) also reduced the on rate, by twofold to 10-fold, but substantially destabilized the active state as well, as judged by increased deactivation rates. In the putative ATP-binding pocket of either NBF, substitution of alanine, glutamine (Q), or arginine (R) for the invariant lysine (K464 or K1250) reduced the on rate similarly, by two- to fourfold. In contrast, these analogous substitutions produced opposite effects on the deactivation rate. NBF1 mutations destabilized the active state, whereas the analogous substitutions in NBF2 stabilized the active state such that activation was prolonged compared with that seen with wild-type CFTR. Substitution of asparagine (N) for a highly conserved aspartic acid (D572) in the ATP-binding pocket of NBF1 dramatically slowed the on rate and destabilized the active state. In contrast, the analogous substitution in NBF2 (D1370N) did not appreciably affect the on rate and markedly stabilized the active state. These results are consistent with a hypothesis for CFTR activation that invokes the binding and hydrolysis of ATP at NBF1 as a crucial step in activation, while at NBF2, ATP binding enhances access to the active state, but the rate of ATP hydrolysis controls the duration of the active state. The relatively slow time courses for activation and deactivation suggest that slow processes modulate ATP-dependent gating.
1-Methyl-3-isobutylxanthine↗
KVLQT1 mutations in three families with familial or sporadic long QT syndrome.
Congenital long QT syndrome (LQTS) is a heterogeneous group of heritable disorders characterized by prolongation of the QT interval on the electrocardiogram, ventricular arrhythmias and sudden death. At least four genes can, when mutated, produce this phenotype. Of these genes, the recently identified KVLQT1 potassium channel is thought to be the one most commonly responsible. In this study, we used single strand conformational polymorphism (SSCP) analysis to screen two large and nine small LQTS families for mutations of the KVLQT1 potassium channel gene. We identified a novel missense mutation in two unrelated families which substitutes a serine for a conserved glycine in the putative pore region of the KVLQT1 channel. In a third family, a new alanine to valine mutation at a CpG dinucleotide resulted in the spontaneous occurrence of the long QT syndrome in monozygotic twin offspring of unaffected parents. Mutations at this same nucleotide have been observed in eight of the 19 LQTS families determined to have KVLQT1 mutations, suggesting this is a mutational hot spot. Both of these mutations alter the amino acid sequence in, or adjacent to, the pore of the channel and many diminish the channel's ability to conduct a repolarizing potassium current. To date, all KVLQT1 mutations determined to cause the LQTS are missense mutations. These data confirm the role of KVLQT1 in the LQTS and suggest that mutant KVLQT1 proteins may exert a dominant negative effect on repolarizing potassium currents by forming multimers with normal potassium channel protein subunits, dramatically reducing the number of fully-functional KVLQT1 channels.
Amino Acid Sequence↗