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At least 73 records · Page 4Linked to original sources
PubMed2003

Functional genetic selection of the decoding center in E. coli 16S rRNA.

Ribosomal RNAs (rRNAs) play crucial roles in protein biosynthesis. The decoding center of 16S rRNA in 30S subunit affords a place for interaction between mRNA and tRNA, and contributes to the fidelity of the decoding by monitoring the codon-anticodon base pairing. The helices 18 and 44 in 16S rRNA are known to be major components of the decoding center. To investigate functional role of the conserved sequence in rRNAs, we employed a new genetic method that allows us to identify and select from randomized E. coli rRNA libraries those rRNA sequences absolutely required for the ribosome function. Functional consensus sequences were identified in both helices, providing us with a new insight into the decoding mechanism.

Escherichia coli↗
PubMed1982

Serologic identification of the human secondary B cell antigens. Correlations between function, genetics, and structure.

The secondary B cell (SB) antigens are polymorphic HLA-linked antigens on human B cells and macrophages that are identified by primed T cell responses but are genetically distinct from the HLA-DR, MB, and MT antigens. Serologic identification of the SB molecule, using the monoclonal antibody ILR1, now makes it possible to correlate the function of these determinants in human T cell recognition with an Ia-like molecular structure and a genetic locus that marks a new HLA subregion. Three lines of evidence indicate that the ILR1 molecule identifies an epitope on some alleles of the SB gene: (a) the polymorphism of ILR1 -reactivity in the population correlates with SB2 SB3; (b) T cell proliferative response to SB2 and SB3 are specifically inhibited by ILR1; and (c) ILR1 reactivity is exactly concordant with the expression of SB2 in a panel of HLA-deletion mutant lymphoblastoid cell line. Together with previous studies, these results indicate that the SB antigens are on Ia-like molecules. Furthermore, the serologic studies of HLA-deletion mutant cell lines demonstrate that there are two HLA regions centromeric to HLA-B controlling expression of Ia-like molecules: a region toward HLA-B that controls expression of HLA-DR, and a region toward GLO that controls expression of SB.

Antibodies, Monoclonal↗
PubMed2006

Functional genetic diversity in the high-affinity carnitine transporter OCTN2 (SLC22A5).

Systemic carnitine deficiency (SCD) is a rare autosomal recessive disease resulting from defects in the OCTN2 (SLC22A5) gene, which encodes the high-affinity plasma membrane carnitine transporter. Although OCTN2 is fairly well studied in its relationship with SCD, little is known about the carrier frequency of disease-causing alleles of OCTN2, or of more common functional polymorphisms in this gene. To address these issues, we screened for genetic variants in the OCTN2 coding region by direct sequencing of the exons and flanking intronic region of OCTN2 in a large sample (n = 276) of ethnically diverse subjects. In addition, we established lymphoblastoid cell lines from subjects homozygous for either allele of the previously identified promoter region variant, -207G>C. We found eight amino acid sequence variants of OCTN2, of which three (Phe17Leu, Leu144Phe, and Pro549Ser) were polymorphic in at least one ethnic group. When assayed for functional activity by expression in human embryonic kidney 293 cells, using as probes both the endogenous substrate (l-carnitine) and the organic cation tetraethylammonium, three variants showed functional differences from the reference OCTN2 (Phe17Leu, Tyr449Asp, Val481Phe; p < 0.05). Further studies of the Phe17Leu polymorphism showed a reduced V(max) for l-carnitine transport to approximately 50% of the reference OCTN2. Confocal microscopy studies using an OCTN2-GFP fusion protein showed that Phe17Leu had distinct subcellular localization from the reference OCTN2, with diffuse cytoplasmic retention of Phe17Leu, in contrast to reference OCTN2, which localized specifically to the plasma membrane. Lymphoblasts from subjects homozygous for the -207G allele showed increased l-carnitine transport compared with the -207C/C homozygotes (p < 0.05). This study suggests that although loss-of-function mutations in OCTN2 are likely to be rare, common variants of OCTN2 found in healthy populations may contribute to variation in the disposition of carnitine and some clinically used drugs.

Amino Acid Sequence↗
PubMed2003

Functional genetic dissection of nuclear receptor signalling in obesity, diabetes and liver regeneration using spatiotemporally controlled somatic mutagenesis in the mouse.

The mouse is an excellent animal model for defining human diseases. The null allele mutations (knockouts, KO) have already provided valuable information about their functions, but have also revealed major complications and difficulties: (1) an early embryonic lethality, (2) temporal effect (developmental stage or adult stage), (3) functional redundancy and (4) spatio-effect (cell-autonomous or non-autonomous). To overcome these limitations, spatio-temporally controlled somatic mutagenesis, Cre-ER(T)/LoxP system, was established. The nuclear receptors (NRs) play central roles in development, organogenesis, metabolism and energy homeostasis through their ability to transduce hormonal signals into modulation of gene activity. Obesity, excess energy storage in adipose tissue, has a strong link to diabetes. Among NRs, retinoid X receptor alpha (RXRalpha)-peroxisome proliferator-activated receptor gamma (PPARgamma) heterodimers can mediate adipocyte differentiation and obesity which has been demonstrated with in vitro cell culture systems and RXR- and PPARgamma-specific ligand studies. Therefore an adipocyte-specific temporally controlled somatic mutagenesis system was established and analysed. Furthermore, the functional roles of NRs to control liver regeneration were also studied with similar system in hepatocytes.

Adipocytes↗
PubMed2006

Exaggerated behavioral phenotypes in Fmr1/Fxr2 double knockout mice reveal a functional genetic interaction between Fragile X-related proteins.

Individuals affected by Fragile X syndrome (FXS) experience cognitive impairment, hyperactivity, attention deficits, social anxiety and autistic-like behaviors. FXS results from the loss of expression of the Fragile X mental retardation (FMR1) gene, whose protein product FMRP is thought to play an important role in neuronal function and synaptic plasticity. Two paralogs of FMRP, FXR1P and FXR2P, have been identified, forming the Fragile X-related (FXR) family of proteins. Although the functions of FXR1P and FXR2P are not well understood, there are similarities among all three FXR proteins in gene structure, amino acid sequence, expression pattern and cellular functions. Mouse models have been described for loss of Fmrp, Fxr1p and Fxr2p, the mouse homologs of FMRP, FXR1P and FXR2P. In earlier studies, we found that Fmr1 knockout (KO) mice, which do not express Fmrp, and Fxr2 KO mice, which do not express Fxr2p, show similarities in some behavioral responses such as hyperactivity. To better understand the functional relationship between FMRP and FXR2P, we generated Fmr1 KO, Fxr2 KO, Fmr1/Fxr2 double KO and wild-type control mice as littermates on the same genetic background and examined them in several behavioral assays. Results show that Fmr1/Fxr2 double KO mice have exaggerated behavioral phenotypes in open-field activity, prepulse inhibition of acoustic startle response and contextual fear conditioning when compared with Fmr1 KO mice, Fxr2 KO mice or wild-type littermates. Our findings suggest that Fmr1 and Fxr2 genes contribute in a cooperative manner to pathways controlling locomotor activity, sensorimotor gating and cognitive processes.

Acoustic Stimulation↗
PubMed2005

Identification of functional genetic variants in cyclooxygenase-2 and their association with risk of esophageal cancer.

BACKGROUND & AIMS: Overexpression of cyclooxygenase-2 (COX-2) is implicated in many steps of cancer development. Single nucleotide polymorphisms (SNPs) in the COX-2 promoter might contribute to differential COX-2 expression and subsequent interindividual variability in susceptibility to cancer. This study sought to identify functional SNPs in the COX-2 promoter and evaluated their effects on the risk of developing esophageal squamous cell carcinoma (ESCC). METHODS: Thirty individual DNA samples were sequenced to search for SNPs, and the function of the SNPs was examined by a set of biochemical assays. Genotypes and haplotypes were analyzed in 1026 patients and 1270 controls, and odds ratios and 95% confidence intervals (CIs) were estimated by logistic regression. RESULTS: Three SNPs, -1290A-->G, -1195G-->A, and -765G-->C, were identified; the frequencies of variant alleles were 0.04, 0.51, and 0.02, respectively. The -1195G-->A change creates a c-MYB binding site and displays a higher promoter activity. The -1195A-containing haplotypes had significantly increased luciferase expression and COX-2 messenger RNA levels in esophageal tissues compared with the -1195G-containing counterparts. A case-control analysis showed a 1.72-fold (95% CI, 1.35-2.20) and 2.24-fold (95% CI, 1.59-3.16) excess risk of developing ESCC for the -1195AA or -765CC genotype carriers compared with noncarriers. A greater risk of developing ESCC was observed for A(-1195)-C(-765)-containing haplotypes compared with G(-1195)-G(-765)-containing haplotypes, suggesting an interaction between the -1195G-->A and -765G-->C polymorphisms in the context of haplotype. CONCLUSIONS: These findings indicate that genetic variants in COX-2 may play a role in mediating susceptibility to esophageal cancer.

Adult↗
PubMed2005

Association study of functional genetic variants of innate immunity related genes in celiac disease.

BACKGROUND: Recent evidence suggest that the innate immune system is implicated in the early events of celiac disease (CD) pathogenesis. In this work for the first time we have assessed the relevance of different proinflammatory mediators typically related to innate immunity in CD predisposition. METHODS: We performed a familial study in which 105 celiac families characterized by the presence of an affected child with CD were genotyped for functional polymorphisms located at regulatory regions of IL-1alpha, IL-1beta, IL-1RN, IL-18, RANTES and MCP-1 genes. Familial data was analysed with a transmission disequilibrium test (TDT) that revealed no statistically significant differences in the transmission pattern of the different genetic markers considered. RESULTS: The TDT analysis for IL-1alpha, IL-1beta, IL-1RN, IL-18, and MCP-1 genes genetic variants did not reveal biased transmission to the affected offspring. Only a borderline association of RANTES promoter genetic variants with CD predisposition was observed. CONCLUSION: Our results suggest that the analysed polymorphisms of IL-1alpha, IL-1beta, IL-1RN, IL-18, RANTES and MCP-1 genes do not seem to play a major role in CD genetic predisposition in our population.

Celiac Disease↗
PubMed2004

Functional genetic screen for genes involved in senescence: role of Tid1, a homologue of the Drosophila tumor suppressor l(2)tid, in senescence and cell survival.

We performed a genetic suppressor element screen to identify genes whose inhibition bypasses cellular senescence. A normalized library of fragmented cDNAs was used to select for elements that promote immortalization of rat embryo fibroblasts. Fragments isolated by the screen include those with homology to genes that function in intracellular signaling, cellular adhesion and contact, protein degradation, and apoptosis. They include mouse Tid1, a homologue of the Drosophila tumor suppressor gene l(2)tid, recently implicated in modulation of apoptosis as well as gamma interferon and NF-kappaB signaling. We show that GSE-Tid1 enhances immortalization by human papillomavirus E7 and simian virus 40 T antigen and cooperates with activated ras for transformation. Expression of Tid1 is upregulated upon cellular senescence in rat and mouse embryo fibroblasts and premature senescence of REF52 cells triggered by activated ras. In accordance with this, spontaneous immortalization of rat embryo fibroblasts is suppressed upon ectopic expression of Tid1. Modulation of endogenous Tid1 activity by GSE-Tid1 or Tid1-specific RNA interference alleviates the suppression of tumor necrosis factor alpha-induced NF-kappaB activity by Tid1. We also show that NF-kappaB sequence-specific binding is strongly downregulated upon senescence in rat embryo fibroblasts. We therefore propose that Tid1 contributes to senescence by acting as a repressor of NF-kappaB signaling.

Animals↗
PubMed2006

Mapping of an internal protease cleavage site in the Ssy5p component of the amino acid sensor of Saccharomyces cerevisiae and functional characterization of the resulting pro- and protease domains by gain-of-function genetics.

Ssy5p is a 77-kDa protein believed to be a component of the SPS amino acid sensor complex in the plasma membrane of Saccharomyces cerevisiae. Ssy5p has been suggested to be a chymotrypsin-like serine protease that activates the transcription factor Stp1p upon exposure of the yeast to extracellular amino acid. Here we overexpressed and partially purified Ssy5p to improve our understanding of its structure and function. Antibodies against Ssy5p expressed in Escherichia coli were isolated and used to detect Ssy5p processing in S. cerevisiae cells. Partial purification and N-terminal sequencing of processed Ssy5p revealed in vivo cleavage of Ssy5p between amino acids 381 and 382. We also isolated constitutively signaling SSY5 mutants and quantified target promoter activation and Stp1p processing. One mutant contained an amino acid substitution in the prodomain, whereas three others harbored amino acid substitutions in the protease domain. Dose-response analysis indicated that all four mutants exhibited increased basal levels of Stp1p processing. Interestingly, whereas the three constitutive mutants mapping to the protease domain of Ssy5p exhibited the decreased 50% effective concentration (EC(50)) characteristic of constitutive mutations previously found in Ssy1p, Ptr3p, and Ssy5p, the EC(50) of the mutation that maps to the prodomain of Ssy5p remained essentially unchanged. In a model of Ssy5p derived from its similarities with alpha-lytic protease from Lysobacter enzymogenes, the sites corresponding to the mutations in the protease domain are clustered in a region facing the prodomain, suggesting that this region interacts with the prodomain and participates in the conformational dynamics of sensing.

Amino Acid Sequence↗
PubMed2006

The functional genetic variant Ile646Val located in the kinase binding domain of the A-kinase anchoring protein 10 is associated with familial breast cancer.

Overexpression of cAMP-dependent protein kinase A (PKA) is a hallmark of the great majority of human cancers including breast cancer. A-kinase anchoring proteins (AKAPs) coordinate the specificity of PKA signalling by localizing the kinase to its subcellular sites. We tested the hypothesis whether the functional amino acid exchange Ile646Val, located in the kinase-binding domain of AKAP10, is a low-penetrance familial breast cancer risk factor. Ile646Val alters the binding of AKAP10 to PKA and is associated with morbidity. The analysis of 787 BRCA1/2 mutation-negative familial breast cancer patients and 993 controls revealed an association of the AKAP10 Ile646Val polymorphism with increased familial breast cancer risk [odds ratio (OR)=1.25, 95% confidence interval (CI) 1.03-1.51, P=0.024]. Our previous study has shown that AKAP13 Lys526Gln is associated with familial breast cancer (OR=1.58). Here, we discovered that carriers of both variants, AKAP10 Ile646Val and AKAP13 Lys526Gln, are at a further enhanced breast cancer risk (OR=2.41, 95% CI 1.30-4.46, P=0.005). PKA is a major target of therapeutic anticancer strategies. Phosphorylation of the estrogen receptor (ER) alpha by PKA induces resistance against the anti-estrogen tamoxifen. Our results indicate for the first time the importance of AKAP10 Ile646Val for familial breast cancer susceptibility. Due to the impact of Ile646Val on the subcellular localization of PKA, it will be interesting to investigate whether this polymorphism influences the effectiveness of PKA and tamoxifen based therapeutic anticancer concepts.

A Kinase Anchor Proteins↗
PubMed2025

Functional genetics of rice PISTILLATA genes reveals new roles and target genes in flowering time, female fertility, and parthenocarpy.

Floral organ identity is controlled largely by the combinatorial action of MADS domain homeotic transcription factors. Lodicules are specialized plant organs in cereals and grasses that are involved in floret opening and facilitate pollination and fertility in rice (Oryza sativa L.). To understand the mechanisms underlying the specification of the rice lodicule, we investigated the developmental functions of the rice PISTILLATA (PI) paralogs, OsMADS2, and OsMADS4. Null osmads2 mutants reiterated OsMADS2 nonredundant lodicule specification roles and revealed new roles in flowering time and floral organ number and fate. Doubly perturbed osmads2 osmads4kd florets had severe abnormalities, were female infertile, yet could initiate parthenocarpy. Ubiquitous OsMADS4 overexpression rescued osmads2 abnormalities. We also utilized genome-wide binding analyses and transcriptome profiling to identify putative target genes contributing to OsMADS2 functions. In osmads2d8/d8 null mutant, we observed deregulated genes in a plethora of processes including lodicule and stamen development, floral organ number, and cell wall development. Some examples are cell division regulators (Cyclin D6, Cyclin-P4-1-like), an aquaporin (PIP1A), a peptide transporter, a vascular developmental regulator (HOX1), and a cell wall modulator (GH9B16). The deregulation of these genes may be associated with the disrupted cell division, tissue differentiation, and physiology of the malformed lodicules in osmads2 and osmads2 osmads4kd florets. Altogether, we reveal novel roles for the rice PI paralogs in flowering time, panicle exsertion, and embryo sac differentiation, identify gene targets for lodicule development, and provide mechanistic insights on the functional diversification of rice PI paralogs.

Oryza↗
PubMed2001

[Role of morpho-chemical plasticity in the genetic-functional organization of the brain in animals].

Quantitative cytochemistry has revealed that emotional stress-sensitive rats (August) differ from emotional stress-resistant ones (Wistar) in the exchange of neurotransmitters and in the level of protein in the neurons. The differences were found at the level of some brain regions (sensomotor cortex, hippocamp, caudate and adjacent nuclei) and various cells (neurons of the cortex and subcortical structures). It has been concluded that animals of genetically different lines differ not only in behavioral characteristics, but in metabolism in some brain structures.

Acetylcholinesterase↗
PubMed2006

A functional genetic screen identifies TFE3 as a gene that confers resistance to the anti-proliferative effects of the retinoblastoma protein and transforming growth factor-beta.

The helix-loop-helix transcription factor TFE3 has been suggested to play a role in the control of cell growth by acting as a binding partner of transcriptional regulators such as E2F3, SMAD3, and LEF-1. Furthermore, translocations/TFE3 fusions have been directly implicated in tumorigenesis. Surprisingly, however, a direct functional role for TFE3 in the regulation of proliferation has not been reported. By screening retroviral cDNA expression libraries to identify cDNAs that confer resistance to a pRB-induced proliferation arrest, we have found that TFE3 overrides a growth arrest in Rat1 cells induced by pRB and its upstream regulator p16(INK4A). In addition, TFE3 expression blocks the anti-mitogenic effects of TGF-beta in rodent and human cells. We provide data supporting a role for endogenous TFE3 in the direct regulation of CYCLIN E expression in an E2F3-dependent manner. These observations establish TFE3 as a functional regulator of proliferation and offer a potential mechanism for its involvement in cancer.

Animals↗
PubMed2000

A tissue-specific coactivator of steroid receptors, identified in a functional genetic screen.

Steroid receptors mediate responses to lipophilic hormones in a tissue- and ligand-specific manner. To identify nonreceptor proteins that confer specificity or regulate steroid signaling, we screened a human cDNA library in a steroid-responsive yeast strain. One of the identified cDNAs, isolated in the screen as ligand effect modulator 6, showed no homology to yeast or Caenorhabditis elegans proteins but high similarity to the recently described mouse coactivator PGC-1 and was accordingly termed hPGC-1. The hPGC-1 DNA encodes a nuclear protein that is expressed in a tissue-specific manner and carries novel motifs for transcriptional regulators. The expression of hPGC-1 in mammalian cells enhanced potently the transcriptional response to several steroids in a receptor-specific manner. hPGC-1-mediated enhancement required the receptor hormone-binding domain and was dependent on agonist ligands. Functional analysis of hPGC-1 revealed two domains that interact with steroid receptors in a hormone-dependent manner, a potent transcriptional activation function, and a putative dimerization domain. Our findings suggest a regulatory function for hPGC-1 as a tissue-specific coactivator for a subset of nuclear receptors.

Animals↗