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At least 253 records · Page 14Linked to original sources
PubMed2007

Large scale genetic screen identifies MAP17 as protein bypassing TNF-induced growth arrest.

Although activated macrophages destroy cancer cells more effectively than normal cells, the ability to escape activated macrophages is a characteristic of tumor cells. One of the mechanisms responsible for the specific killing of tumor cells by macrophages is the production of the cytokine tumor necrosis factor (TNF) alpha. Therefore, resistance to TNF may provide such cancer cells a selective advantage against host elimination. With the aim of identifying genes with these properties we undertook a large scale genetic screen to identify genes able to bypass TNF-induced G1 arrest. We identified MAP17, a small 17 kDa nonglycosylated membrane protein that localizes to the plasma membrane and the Golgi apparatus. Ectopic expression of MAP17 in tumor cells prevents TNF-induced G1 arrest by impairing p21waf1 induction. However, expression of MAP17 does not inhibit TNF-induced apoptosis in Me180-sensitive tumor cells. The inhibition of TNF is specific since MAP17 does not alter the response to other cytokines such as IFNgamma. As described in the Xenopus oocyte system, MAP17 increases the uptake of mannose in some cells, but this effect is not responsible for TNF bypass. We have also analyzed the expression of MAP17 mRNA in a panel of cell lines. MAP17 is expressed in 30% of cell lines of different origin. However, MAP17 mRNA expression did not correlate with TNF resistance. Our data indicates that although MAP17 expression might bypass TNF-induced growth arrest, it is not the only determinant of this response.

Carcinoma, Ductal, Breast↗
PubMed2003

A chemical genetic screen identifies inhibitors of regulated nuclear export of a Forkhead transcription factor in PTEN-deficient tumor cells.

The PI3K/PTEN/Akt signal transduction pathway plays a key role in many tumors. Downstream targets of this pathway include the Forkhead family of transcription factors (FOXO1a, FOXO3a, FOXO4). In PTEN null cells, FOXO1a is inactivated by PI3K-dependent phosphorylation and mislocalization to the cytoplasm, yet still undergoes nucleocytoplasmic shuttling. Since forcible localization of FOXO1a to the nucleus can reverse tumorigenicity of PTEN null cells, a high-content, chemical genetic screen for inhibitors of FOXO1a nuclear export was performed. The compounds detected in the primary screen were retested in secondary assays, and structure-function relationships were identified. Novel general export inhibitors were found that react with CRM1 as well as a number of compounds that inhibit PI3K/Akt signaling, among which are included multiple antagonists of calmodulin signaling.

Active Transport, Cell Nucleus↗
PubMed2001

Prenatal genetic screening in the Ashkenazi Jewish population.

The Ashkenazi Jewish community is a unique and ideal population in which to provide multiple disease screening because detection rates are high (> 95%) by testing a limited number of mutations. The residual risk that remains is very low. In addition, the lessons learned from carrier screening in this community indicate that only through genetic counseling and education can screening in the general population gain wide acceptance and provide maximum benefit.

Counseling↗
PubMed1999

Baculovirus-based genetic screen for antiapoptotic genes identifies a novel IAP.

The prototype baculovirus, Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) expresses p35, a potent anti cell-death gene that promotes the propagation of the virus by blocking host cell apoptosis. Infection of insect Sf-21 cells with AcMNPV lacking p35 induces apoptosis. We have used this pro-apoptotic property of the p35 null virus to screen for genes encoding inhibitors of apoptosis that rescue cells infected with the p35 defective virus. We report here the identification of Tn-IAP1, a novel member of the IAP family of cell death inhibitors. Tn-IAP1 blocks cell death induced by p35 null AcMNPV, actinomycin D, and Drosophila cell-death inducers HID and GRIM. Given the conserved nature of the cell death pathway, this genetic screen can be used for rapid identification of novel inhibitors of apoptosis from diverse sources.

Amino Acid Sequence↗
PubMed2006

Presenilin-based genetic screens in Drosophila melanogaster identify novel notch pathway modifiers.

Presenilin is the enzymatic component of gamma-secretase, a multisubunit intramembrane protease that processes several transmembrane receptors, such as the amyloid precursor protein (APP). Mutations in human Presenilins lead to altered APP cleavage and early-onset Alzheimer's disease. Presenilins also play an essential role in Notch receptor cleavage and signaling. The Notch pathway is a highly conserved signaling pathway that functions during the development of multicellular organisms, including vertebrates, Drosophila, and C. elegans. Recent studies have shown that Notch signaling is sensitive to perturbations in subcellular trafficking, although the specific mechanisms are largely unknown. To identify genes that regulate Notch pathway function, we have performed two genetic screens in Drosophila for modifiers of Presenilin-dependent Notch phenotypes. We describe here the cloning and identification of 19 modifiers, including nicastrin and several genes with previously undescribed involvement in Notch biology. The predicted functions of these newly identified genes are consistent with extracellular matrix and vesicular trafficking mechanisms in Presenilin and Notch pathway regulation and suggest a novel role for gamma-tubulin in the pathway.

Alleles↗
PubMed1995

Immunoglobulin mutant library genetically screened for folding stability exploiting bacterial signal transduction.

A model repertoire of variants of immunoglobulin kappa variable domain REIv with different folding stabilities was generated by oligonucleotide-directed randomization of position 29, a key conserved residue of hypervariable loop 1. Fused to ToxR', the membrane-anchored cytoplasmic domain of the Vibrio cholerae ToxR transcription activator, different members of the library induce different levels of transcription from the ctx promoter in Escherichia coli. Differences in transcription activation correlate positively with folding stabilities of the corresponding REIv domains. Since conformationally stabilized REIv derivatives elicit a dark red colony phenotype on EMB-lactose indicator plates, this procedure constitutes a genetic screen for immunoglobulin folding stability.

Amino Acid Sequence↗
PubMed2006

A genetic screen identifies genes essential for development of myelinated axons in zebrafish.

The myelin sheath insulates axons in the vertebrate nervous system, allowing rapid propagation of action potentials via saltatory conduction. Specialized glial cells, termed Schwann cells in the PNS and oligodendrocytes in the CNS, wrap axons to form myelin, a compacted, multilayered sheath comprising specific proteins and lipids. Disruption of myelinated axons causes human diseases, including multiple sclerosis and Charcot-Marie-Tooth peripheral neuropathies. Despite the progress in identifying human disease genes and other mutations disrupting glial development and myelination, many important unanswered questions remain about the mechanisms that coordinate the development of myelinated axons. To address these questions, we began a genetic dissection of myelination in zebrafish. Here we report a genetic screen that identified 13 mutations, which define 10 genes, disrupting the development of myelinated axons. We present the initial characterization of seven of these mutations, defining six different genes, along with additional characterization of mutations that we have described previously. The different mutations affect the PNS, the CNS, or both, and phenotypic analyses indicate that the genes affect a wide range of steps in glial development, from fate specification through terminal differentiation. The analysis of these mutations will advance our understanding of myelination, and the mutants will serve as models of human diseases of myelin.

Animals↗
PubMed1991

Genetic screening of newborns for sickle cell disease: correlation of DNA analysis with hemoglobin electrophoresis.

Although DNA analysis based on the polymerase chain reaction (PCR) offers potential advantages for screening newborns for sickle cell disease, few data are available concerning the reliability of PCR-based tests for such screening. We describe a protocol for detecting the A, S, and C alleles of the beta-globin gene in dried blood from phenylketonuria screening cards. This method is based on PCR and detection with allele-specific oligonucleotide probes. Results of a blind comparison of PCR analysis of the dried blood with hemoglobin electrophoresis of whole-blood samples agreed for 80 of 81 samples. The single discrepancy is probably not attributable to a failure of the PCR method, but rather to limitations of the electrophoresis method. The PCR method should be a highly accurate means of detecting beta-globin alleles in routine genetic screening with dried blood already collected for (e.g.) phenylketonuria screening.

Alleles↗
PubMed2006

Mycobacterium avium restriction fragment length polymorphism-IS IS1245 and the simple double repetitive element polymerase chain reaction typing method to screen genetic diversity in Brazilian strains.

Simple double repetitive element polymerase chain reaction (MaDRE-PCR) and Pvu II-IS1245 restriction fragment length polymorphism (RFLP) typing methods were used to type 41 Mycobacterium avium isolates obtained from 14 AIDS inpatients and 10 environment and animals specimens identified among 53 mycobacteria isolated from 237 food, chicken, and pig. All environmental and animals strains showed orphan patterns by both methods. By MaDRE-PCR four patients, with multiple isolates, showed different patterns, suggesting polyclonal infection that was confirmed by RFLP in two of them. This first evaluation of MaDRE-PCR on Brazilian M. avium strains demonstrated that the method seems to be useful as simple and less expensive typing method for screening genetic diversity in M. avium strains on selected epidemiological studies, although with limitation on analysis identical patterns except for one band.

Acquired Immunodeficiency Syndrome↗
PubMed2002

The minisequencing method: a simple strategy for genetic screening of MEN 2 families.

BACKGROUND: Multiple endocrine neoplasia type 2 is an autosomal dominant disorder. MEN 2A is characterized by medullary thyroid carcinoma, pheochromocytoma and hyperparathyroidism; MEN 2B by medullary thyroid carcinoma, pheochromocytoma and characteristic stigmata. Activating germline mutations of the RET proto oncogene are responsible for this hereditary syndrome. Codon 634 mutations are the most common mutations occurring in MEN 2A families whereas a specific mutation at codon 918 is observed in the great majority of MEN 2B families. Analysis of these codons will provide a final diagnosis in the great majority of affected families making unnecessary further studies. To specifically study the codons 634 and 918 we used a minisequencing method as an alternative method to complete sequencing. RESULTS: Using this mutation detection method we were able to reproduce in all cases, representative of 7 families, the information previously obtained by direct sequencing of PCR products. Depending on the number of primers used in the minisequencing reaction, we were able to interrogate either only one nucleotide of the target codon or the three nucleotides simultaneously. CONCLUSIONS: This technique appears as a simple, rapid and efficient method for genetic screening of MEN 2 families. It can be utilized to seek for unknown mutations at specific codons or to screen for previously identified mutations and is therefore of interest to study index cases or individuals at risk. Results suggest that complete sequencing is unnecessary.

Codon↗
PubMed2003

A genetic screen for hedgehog targets involved in the maintenance of the Drosophila anteroposterior compartment boundary.

The development of multicellular organisms requires the establishment of cell populations with different adhesion properties. In Drosophila, a cell-segregation mechanism underlies the maintenance of the anterior (A) and posterior (P) compartments of the wing imaginal disc. Although engrailed (en) activity contributes to the specification of the differential cell affinity between A and P cells, recent evidence suggests that cell sorting depends largely on the transduction of the Hh signal in A cells. The activator form of Cubitus interruptus (Ci), a transcription factor mediating Hh signaling, defines anterior specificity, indicating that Hh-dependent cell sorting requires Hh target gene expression. However, the identity of the gene(s) contributing to distinct A and P cell affinities is unknown. Here, we report a genetic screen based on the FRT/FLP system to search for genes involved in the correct establishment of the anteroposterior compartment boundary. By using double FRT chromosomes in combination with a wing-specific FLP source we screened 250,000 mutagenized chromosomes. Several complementation groups affecting wing patterning have been isolated, including new alleles of most known Hh-signaling components. Among these, we identified a class of patched (ptc) alleles exhibiting a novel phenotype. These results demonstrate the value of our setup in the identification of genes involved in distinct wing-patterning processes.

Animals↗
PubMed2000

A genetic screen for novel components of the Ras/Mitogen-activated protein kinase signaling pathway that interact with the yan gene of Drosophila identifies split ends, a new RNA recognition motif-containing protein.

The receptor tyrosine kinase (RTK) signaling pathway is used reiteratively during the development of all multicellular organisms. While the core RTK/Ras/MAPK signaling cassette has been studied extensively, little is known about the nature of the downstream targets of the pathway or how these effectors regulate the specificity of cellular responses. Drosophila yan is one of a few downstream components identified to date, functioning as an antagonist of the RTK/Ras/MAPK pathway. Previously, we have shown that ectopic expression of a constitutively active protein (yan(ACT)) inhibits the differentiation of multiple cell types. In an effort to identify new genes functioning downstream in the Ras/MAPK/yan pathway, we have performed a genetic screen to isolate dominant modifiers of the rough eye phenotype associated with eye-specific expression of yan(ACT). Approximately 190,000 mutagenized flies were screened, and 260 enhancers and 90 suppressors were obtained. Among the previously known genes we recovered are four RTK pathway components, rolled (MAPK), son-of-sevenless, Star, and pointed, and two genes, eyes absent and string, that have not been implicated previously in RTK signaling events. We also isolated mutations in five previously uncharacterized genes, one of which, split ends, we have characterized molecularly and have shown to encode a member of the RRM family of RNA-binding proteins.

Amino Acid Sequence↗
PubMed2002

Genetic screening for MEN1 mutations in families presenting with familial primary hyperparathyroidism.

A large number of families with familial isolated hyperparathyroidism (FIHP) have been reported. We wanted to determine if some of these families represent early manifestations of full-blown syndromes such as multiple endocrine neoplasia type 1 (MEN-1), as early identification may alter surgical and medical management. Four small families with a family history of hyperparathyroidism without clear-cut MEN-1 features were screened for a MEN1 mutation. The 10 exons of the MEN1 gene were amplified and analyzed by single-strand conformation analysis (SSCA). Abnormal SSCA shifts were then sequenced using an automated sequencer. Two germline mutations were found: R527X and P277H. The former was detected in three members of a family consisting of two children and a mother. At the time of testing the youngest son was normocalcemic and clinically normal but subsequently developed hyperparathyroidism (HPT). Since the initial testing, the family has been confirmed to be a MEN-1 family as the mother has developed abdominal pain and an elevated serum pancreatic polypeptide and the younger brother an anterior pituitary tumor and recurrent HPT. The latter P277H mutation was identified in two of three members tested from another family. Manifestations of MEN-1 syndrome have also developed. The father now has developed diarrhea and elevated serum gastrin; and the daughter has developed recurrent HPT. Genetic screening of families who clinically have FIHP is important and may influence the type of medical and surgical treatment and follow-up, as some have MEN-1 syndrome. Long-term screening for MEN syndromes should be included in this set of patients. Positive screening may predict disease and allow early detection and appropriate treatment before initiation of symptoms.

Adolescent↗
PubMed2001

A genetic screen for suppressors of Escherichia coli Tat signal peptide mutations establishes a critical role for the second arginine within the twin-arginine motif.

The Escherichia coli Tat protein export pathway transports folded proteins synthesized with N-terminal twin-arginine signal peptides. Twin-arginine signal sequences contain a conserved SRRxFLK "twin-arginine" amino acid sequence motif which is required for protein export by the Tat pathway. The E. coli trimethylamine N-oxide reductase (TorA) is a Tat-dependent periplasmic molybdoenzyme that facilitates anaerobic respiration with trimethylamine N-oxide as terminal electron acceptor. Here, we describe mutant strains constructed with modified TorA twin-arginine signal peptides. Substitution of the second arginine residue of the TorA signal peptide twin-arginine motif with either lysine or aspartate, or the simultaneous substitution of both arginines with lysine residues, completely abolished export. In each case, the now cytoplasmically localised TorA retained full enzymatic activity with the artificial electron donor benzyl viologen. However, the mutant strains were incapable of anaerobic growth with trimethylamine N-oxide and the non-fermentable carbon-source glycerol. The growth phenotype of the mutant strains was exploited in a genetic screen with the aim of identifying second-site suppressor mutations that allowed export of the modified TorA precursors.

Amino Acid Sequence↗
PubMed2006

A stochastic model to evaluate options for antenatal genetic screening.

Haemoglobinopathies are a group of genetic disorders which are particularly prevalent among certain risk groups such as ethnic groups. Antenatal screening of potential haemoglobinopathy carriers allows early diagnosis for affected fetuses, leading to therapeutic intervention or termination. However, it has drawbacks such as screening costs and possible miscarriage as a result of fetal testing. This paper describes a model that allows the outcomes of a screening programme to be estimated for different risk groups. The model has been implemented as a computer package that can be used to inform decisions made by health care planners.

Female↗