Phenotype
Latest Paper:
Katarzyna Borg,
Ewa Bocian,
Joanna Bernaciak,
Beata Nowakowska,
Katarzyna Derwińska,
Ewa Obersztyn,
Krzysztof Szczałuba,
Robert Smigiel,
Ewa Kostyk,
Tadeusz Mazurczak
Zakład Genetyki Medycznej, Instytut Matki i Dziecka, ul. Kasprzaka 17a, 01-211 Warszawa.
INTRODUCTION: In approximately 6% of balanced chromosomal rearrangements carriers, intellectual disability, dysmorphic features and congenital anomalies can be found. The abnormal phenotype might be the result of genomic imbalance or aberrant expression caused by direct breakage of a dosage sensitive gene. THE AIM OF THIS STUDY: To estimate the frequency and implication of the submicroscopic chromosomal aberrations on the abnormal phenotypes present in patients with balanced chromosomal rearrangements. Also an attempt was made to define the type of genetic defect and gene identification responsible for the intellectual disability and additional clinical features. MATERIAL AND METHODS: 22 patients with intellectual disability, congenital anomalies and dysmorphic features were analysed. Molecular karyotyping was performed in all patients using FISH with region-specific BAC clones, high resolution comparative genomic hybridization (HR-CGH) or array CGH (aCGH). A targeted or whole genome microarrays were applied. RESULTS: In 5 of 22 carriers 6 microdeletions and one duplication were found (7/22, 31.8%). Only two microdeletions were mapped at the chromosomal breakpoints. Three rearrangements had more complex structure than conventional methods demonstrated. In the chromosomal breakpoints of 21 patients the 24 genes, which functions suggest the relationship between abnormal gene expression and patients' intellectual disability, were mapped. CONCLUSIONS: We showed that in a considerable group of patients with balanced chromosomal rearrangements and abnormal phenotype the cryptic aberrations, unidentified by conventional methods, are present. These results confirmed the legitimacy of detailed analysis of the chromosomal breakpoints as well as the whole genome screening with the use of new cytogenetic methods.
Mesh-terms: Abnormalities, Multiple :: genetics; Chromosome Aberrations :: classification; Chromosome Mapping :: methods; Comparative Genomic Hybridization; Developmental Disabilities :: genetics; Female; Heterozygote; Humans; In Situ Hybridization, Fluorescence; Infant, Newborn; Male; Mental Retardation :: genetics; Phenotype; Translocation, Genetic;
Most cited papers:
Several single-base substitution mutations have been introduced into the lacZ alpha gene in cloning vector M13mp2, at 40-60% efficiency, in a rapid procedure requiring only transfection of the unfractionated products of standard in vitro mutagenesis reactions. Two simple additional treatments of the DNA, before transfection, produce a site-specific mutation frequency approaching 100%. The approach is applicable to phenotypically silent mutations in addition to those that can be selected. The high efficiency, approximately equal to 10-fold greater than that observed using current methods without enrichment procedures, is obtained by using a DNA template containing several uracil residues in place of thymine. This template has normal coding potential for the in vitro reactions typical of site-directed mutagenesis protocols but is not biologically active upon transfection into a wild-type (i.e., ung+) Escherichia coli host cell. Expression of the desired change, present in the newly synthesized non-uracil-containing covalently closed circular complementary strand, is thus strongly favored. The procedure has been applied to mutations introduced via both oligonucleotides and error-prone polymerization. In addition to its utility in changing DNA sequences, this approach can potentially be used to examine the biological consequences of specific lesions placed at defined positions within a gene.
A G Bodnar,
M Ouellette,
M Frolkis,
S E Holt,
C P Chiu,
G B Morin,
C B Harley,
J W Shay,
S Lichtsteiner,
W E Wright
Laboratory for Cell Biology and Genetics, The Rockefeller University, New York, NY 10021, USA. delange@rockvax.rockefeller.edu
Normal human cells undergo a finite number of cell divisions and ultimately enter a nondividing state called replicative senescence. It has been proposed that telomere shortening is the molecular clock that triggers senescence. To test this hypothesis, two telomerase-negative normal human cell types, retinal pigment epithelial cells and foreskin fibroblasts, were transfected with vectors encoding the human telomerase catalytic subunit. In contrast to telomerase-negative control clones, which exhibited telomere shortening and senescence, telomerase-expressing clones had elongated telomeres, divided vigorously, and showed reduced straining for beta-galactosidase, a biomarker for senescence. Notably, the telomerase-expressing clones have a normal karyotype and have already exceeded their normal life-span by at least 20 doublings, thus establishing a causal relationship between telomere shortening and in vitro cellular senescence. The ability to maintain normal human cells in a phenotypically youthful state could have important applications in research and medicine.
Mesh-terms: Biological Markers; Catalysis; Cell Aging; Cell Division; Cell Line; Cell Transformation, Neoplastic; Cloning, Molecular; Fibroblasts :: cytology; Homeostasis; Human; Karyotyping; Phenotype; Pigment Epithelium of Eye :: cytology; Proteins :: genetics; Proteins :: metabolism; RNA; RNA-Directed DNA Polymerase :: genetics; RNA-Directed DNA Polymerase :: metabolism; Stem Cells :: cytology; Stem Cells :: enzymology; Support, U.S. Gov't, P.H.S. ; Telomerase :: genetics; Telomerase :: metabolism; Telomere :: metabolism; Telomere :: physiology; Telomere :: ultrastructure; Transfection; Tumor Cells, Cultured; beta-Galactosidase :: metabolism;
Division of Molecular Carcinogenesis, Division of Tumor Biology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, Netherlands.
Mammalian genetic approaches to study gene function have been hampered by the lack of tools to generate stable loss-of-function phenotypes efficiently. We report here a new vector system, named pSUPER, which directs the synthesis of small interfering RNAs (siRNAs) in mammalian cells. We show that siRNA expression mediated by this vector causes efficient and specific down-regulation of gene expression, resulting in functional inactivation of the targeted genes. Stable expression of siRNAs using this vector mediates persistent suppression of gene expression, allowing the analysis of loss-of-function phenotypes that develop over longer periods of time. Therefore, the pSUPER vector constitutes a new and powerful system to analyze gene function in a variety of mammalian cell types.
Mesh-terms: Down-Regulation; Gene Silencing; Genes, p53; Genetic Techniques; Genetic Vectors; Humans; Ligases :: genetics; Mutation; Nucleic Acid Conformation; Phenotype; Protein Kinases :: genetics; Protein Kinases :: metabolism; Protein p53 :: metabolism; RNA, Messenger :: chemistry; RNA, Messenger :: genetics; RNA, Messenger :: metabolism; RNA, Small Interfering; RNA, Untranslated :: chemistry; RNA, Untranslated :: genetics; RNA, Untranslated :: metabolism; Research Support, Non-U.S. Gov't; Transfection; Tumor Cells, Cultured; Ubiquitin-Protein Ligase Complexes;
Laboratory of Human Carcinogenesis, National Cancer Institute, Bethesda, Maryland 20892.
Mesh-terms: Aflatoxins :: toxicity; Animals; Cell Transformation, Neoplastic :: genetics; Chromosome Deletion; Conserved Sequence; DNA Damage; DNA Mutational Analysis; DNA Repair; DNA Replication; Disease Models, Animal; Genes, p53 :: genetics; Genes, p53 :: physiology; Hepatitis B :: complications; Human; Mutation :: genetics; Neoplasms :: etiology; Neoplasms :: genetics; Neoplasms, Radiation-Induced :: genetics; Phenotype; Protein p53 :: chemistry; Protein p53 :: physiology; Sequence Analysis, DNA; Smoking :: adverse effects; Structure-Activity Relationship;
Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia 19104, USA.
Here, we show that the beta-chemokine receptor CKR-5 serves as a cofactor for M-tropic HIV viruses. Expression of CKR-5 with CD4 enables nonpermissive cells to form syncytia with cells expressing M-tropic, but not T-tropic, HIV-1 env proteins. Expression of CKR-5 and CD4 enables entry of a M-tropic, but not a T-tropic, virus strain. A dual-tropic primary HIV-1 isolate (89.6) utilizes both Fusin and CKR-5 as entry cofactors. Cells expressing the 89.6 env protein form syncytia with QT6 cells expressing CD4 and either Fusin or CKR-5. The beta-chemokine receptors CKR-3 and CKR-2b support HIV-1 89.6 env-mediated syncytia formation but do not support fusion by any of the T-tropic or M-tropic strains tested. Our results suggest that the T-tropic viruses characteristic of disease progression may evolve from purely M-tropic viruses prevalent early in virus infection through changes in the env protein that enable the virus to use multiple entry cofactors.
Mesh-terms: Acquired Immunodeficiency Syndrome :: virology; Base Sequence; Cell Fusion :: physiology; Chemokines :: physiology; HIV-1 :: physiology; Hela Cells :: physiology; Hela Cells :: virology; Human; Membrane Proteins :: physiology; Molecular Sequence Data; Phenotype; Receptors, CCR5; Receptors, CXCR4; Receptors, Chemokine; Receptors, Cytokine :: physiology; Receptors, HIV :: physiology; Support, Non-U.S. Gov't; Support, U.S. Gov't, P.H.S. ; Viral Envelope Proteins :: genetics;
Y Miki,
J Swensen,
D Shattuck-Eidens,
P A Futreal,
K Harshman,
S Tavtigian,
Q Liu,
C Cochran,
L M Bennett,
W Ding
Department of Medical Informatics, University of Utah Medical Center, Salt Lake City 84132.
A strong candidate for the 17q-linked BRCA1 gene, which influences susceptibility to breast and ovarian cancer, has been identified by positional cloning methods. Probable predisposing mutations have been detected in five of eight kindreds presumed to segregate BRCA1 susceptibility alleles. The mutations include an 11-base pair deletion, a 1-base pair insertion, a stop codon, a missense substitution, and an inferred regulatory mutation. The BRCA1 gene is expressed in numerous tissues, including breast and ovary, and encodes a predicted protein of 1863 amino acids. This protein contains a zinc finger domain in its amino-terminal region, but is otherwise unrelated to previously described proteins. Identification of BRCA1 should facilitate early diagnosis of breast and ovarian cancer susceptibility in some individuals as well as a better understanding of breast cancer biology.
Mesh-terms: Alleles; Alternative Splicing; Amino Acid Sequence; Animals; BRCA1 Protein; Breast Neoplasms :: genetics; Chromosomes, Human, Pair 17; Female; Genes, Tumor Suppressor; Genetic Predisposition to Disease; Germ-Line Mutation; Haplotypes; Human; Lod Score; Male; Molecular Sequence Data; Mutation; Neoplasm Proteins :: chemistry; Neoplasm Proteins :: genetics; Neoplasm Proteins :: physiology; Ovarian Neoplasms :: genetics; Pedigree; Phenotype; Support, Non-U.S. Gov't; Support, U.S. Gov't, P.H.S. ; Transcription Factors :: chemistry; Transcription Factors :: genetics; Transcription Factors :: physiology; Zinc Fingers;
Ravi S Kamath,
Andrew G Fraser,
Yan Dong,
Gino Poulin,
Richard Durbin,
Monica Gotta,
Alexander Kanapin,
Nathalie Le Bot,
Sergio Moreno,
Marc Sohrmann,
David P Welchman,
Peder Zipperlen,
Julie Ahringer
A principal challenge currently facing biologists is how to connect the complete DNA sequence of an organism to its development and behaviour. Large-scale targeted-deletions have been successful in defining gene functions in the single-celled yeast Saccharomyces cerevisiae, but comparable analyses have yet to be performed in an animal. Here we describe the use of RNA interference to inhibit the function of approximately 86% of the 19,427 predicted genes of C. elegans. We identified mutant phenotypes for 1,722 genes, about two-thirds of which were not previously associated with a phenotype. We find that genes of similar functions are clustered in distinct, multi-megabase regions of individual chromosomes; genes in these regions tend to share transcriptional profiles. Our resulting data set and reusable RNAi library of 16,757 bacterial clones will facilitate systematic analyses of the connections among gene sequence, chromosomal location and gene function in C. elegans.
Mesh-terms: Animals; Caenorhabditis elegans :: genetics; Computational Biology; Evolution, Molecular; Genes, Helminth :: genetics; Genome; Genomics; Helminth Proteins :: chemistry; Helminth Proteins :: genetics; Human; Multigene Family :: genetics; Phenotype; Protein Structure, Tertiary; RNA Interference; RNA, Helminth :: genetics; RNA, Helminth :: metabolism; Support, Non-U.S. Gov't; Support, U.S. Gov't, Non-P.H.S. ; Transcription, Genetic :: genetics; X Chromosome :: genetics;
We have reconstituted a matrix of basement membrane onto a filter in a Boyden chamber and assessed the ability of various malignant and nonmalignant cells to penetrate through the coated filter. Cells from all the malignant cell lines tested were able to cross the matrix in 5-6 h, whereas human fibroblasts as well as mouse 3T3 and 10T1/2 cell lines, which are not tumorigenic, were not invasive. In addition, normal primary prostate epithelial cells and benign prostatic hyperplasia cells were not invasive when tested in this assay, whereas malignant prostate carcinoma cells were highly invasive. Parallel experiments with these prostatic cells using the intrasplenic assay for metastasis detection in the nude mouse confirmed the benign behavior of the former cells and the metastatic phenotype of the latter ones. These results suggest that this in vitro test allows the rapid and quantitative assessment of invasiveness and a means to screen for drugs which alter the invasive phenotype of tumor cells.
Howard Hughes Medical Institute, Department of Developmental Biology, Beckman Center, Stanford University Medical Center, Stanford, California 94305-5323, USA.
Mesh-terms: Amino Acid Sequence; Animals; Caenorhabditis elegans :: genetics; Caenorhabditis elegans :: growth & development; Cell Adhesion :: genetics; Cytoskeletal Proteins :: metabolism; Drosophila Proteins; Embryonic Induction; Gene Expression Regulation, Developmental; Human; Membrane Proteins :: metabolism; Molecular Sequence Data; Phenotype; Proteoglycans :: metabolism; Proto-Oncogene Proteins :: genetics; Proto-Oncogene Proteins :: metabolism; Signal Transduction; Support, Non-U.S. Gov't; Support, U.S. Gov't, Non-P.H.S. ; Trans-Activators; Vertebrates :: genetics; Vertebrates :: growth & development;
