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PubMed2006

Adaptive evolution by mutations in the FLO11 gene.

In nature, Saccharomyces yeasts manifest a number of adaptive responses to overcome adverse environments such as filamentation, invasive growth, flocculation and adherence to solid surfaces. Certain Saccharomyces wild yeasts, namely "flor yeasts," have also acquired the ability to form a buoyant biofilm at the broth surface. Here we report that mutations in a single gene, identified as FLO11, separate these "floating" yeasts from their nonfloating relatives. We have determined that the capability to form a self-supporting biofilm at the liquid surface is largely dependent on two changes in the FLO11 gene. First, we identified a 111-nt deletion within a repression region of the FLO11 promoter that significantly increases FLO11 gene expression. Secondly, we found rearrangements within the central tandem repeat domain of the coding region that yield a more hydrophobic Flo11p variant. Together, these mutations result in dramatic increase in cell surface hydrophobicity, which in turn confers these yeasts the ability to float by surface tension, an adaptive mechanism to gain direct access to oxygen within oxygen-poor liquid environments.

Alleles↗

PubMed1996

Promoter region of the rat m4 muscarinic acetylcholine receptor gene contains a cell type-specific silencer element.

We describe here the characterization of the rat m4 muscarinic acetylcholine receptor gene and the identification of its regulatory region. Two 5'-noncoding exons are located approximately 5 kilobases upstream from the coding exon, and at least two alternatively spliced variants of m4 mRNA are expressed in the neuronal cell line PC12D. There are two transcription initiation sites. The promoter region is GC-rich, contains no TATA-box, but has two potential CAAT boxes and several putative binding sites for transcription factors Sp1 and AP-2. We assessed the m4 promoter activity functionally in transient expression assays using luciferase as a reporter. The proximal 435-base pair (bp) sequence of the 5'-flanking region produced luciferase activity in both m4-expressing neuronal cell lines (PC12D and NG108-15) and non-neuronal cell lines (L6 and 3Y1B). A longer fragment containing an additional 638-bp sequence produced luciferase activity only in m4-expressing neuronal cell lines. These data suggest that the proximal 435-bp sequence contains a constitutive promoter and that a 638-bp sequence farther upstream contains a cell type-specific silencer element. A consensus sequence for the neural-restrictive silencer element is found within this 638-bp segment.

Animals↗

PubMed2000

The AROM gene, spliced mRNAs encoding new DNA/RNA-binding proteins are transcribed from the opposite strand of the melanin-concentrating hormone gene in mammals.

Melanin-concentrating hormone (MCH) mRNA expression is induced by nerve growth factor and lithium in PC12 cells, whereas three large MCH RNA species are found in untreated cells. In this study, we investigated the structures, regulations of expression, and putative functions of these transcripts. Northern blot, rapid amplification of cDNA ends-polymerase chain reaction, reverse transcriptase-polymerase chain reaction, and sequencing experiments demonstrated that they are antisense RNAs complementary to the MCH gene. Two classes of antisense RNAs could be discriminated as follows: 1) non-coding unspliced RNAs that overlap mainly the coding part of the MCH gene; 2) spliced variant mRNAs complementary to the 3'-flanking end of the MCH gene and that encode putative proteins containing DNA/RNA binding domains. We named this new transcriptional unit AROM for antisense-RNA-overlapping-MCH gene. Spliced variant AROM mRNAs are expressed in a broad range of rat organs. Western blot and immunohistochemistry experiments revealed several proteins with cytoplasmic but also nuclear localization in PC12 cells. Time course studies during nerve growth factor and lithium treatment of PC12 cells indicated a reciprocal regulation of the MCH and AROM gene transcripts, reflected also at the level of AROM proteins. The major translational product is a 64-kDa protein (AROM-p64). Recombinant AROM-p64 displayed high binding to single-stranded DNA and poly(A) homopolymers suggesting that this protein could play a role in mRNA maturation/metabolism.

Amino Acid Sequence↗

PubMed2002

Human testis/sperm-specific histone H2B (hTSH2B). Molecular cloning and characterization.

Human sperm, unlike the sperm of other mammals, contain replacement histones with unknown biological functions. Here, we report the identification of the novel human gene coding for a testis/sperm-specific histone H2B (hTSH2B). This variant histone is 85% homologous to somatic H2B and has over 93% homology with the testis H2B of rodents. Using genomic PCR, two genetic alleles of hTSH2B were found in the human population. The hTSH2B gene is transcribed exclusively in testis, and the corresponding protein is also present in mature sperm. We expressed recombinant hTSH2B and identified this protein with a particular H2B subtype expressed in vivo. The subnuclear distribution of H2B variants in sperm was determined using biochemical fractionation and immunoblotting. The H2B variant associated with telomere-binding activity () was solubilized by Triton X-100 or micrococcal nuclease extraction, whereas hTSH2B was relatively tightly bound in nuclei. Immunofluorescence showed that hTSH2B was concentrated in spots located at the basal nuclear area of a subpopulation (20% of cells) of mature sperm. This fact may be of particular importance, because the hTSH2B "positive" and "negative" sperm cells may undergo significantly different decondensation processes following fertilization.

Amino Acid Sequence↗

PubMed1996

Stimulation of fibroblast growth factor receptor-1 occupancy and signaling by cell surface-associated syndecans and glypican.

The formation of distinctive basic FGF-heparan sulfate complexes is essential for the binding of bFGF to its cognate receptor. In previous experiments, cell-surface heparan sulfate proteoglycans extracted from human lung fibroblasts could not be shown to promote high affinity binding of bFGF when added to heparan sulfate-deficient cells that express FGF receptor-1 (FGFR1) (Aviezer, D., D. Hecht, M. Safran, M. Eisinger, G. David, and A. Yayon. 1994. Cell 79:1005-1013). In alternative tests to establish whether cell-surface proteoglycans can support the formation of the required complexes, K562 cells were first transfected with the IIIc splice variant of FGFR1 and then transfected with constructs coding for either syndecan-1, syndecan-2, syndecan-4 or glypican, or with an antisense syndecan-4 construct. Cells cotransfected with receptor and proteoglycan showed a two- to three- fold increase in neutral salt-resistant specific 125I-bFGF binding in comparison to cells transfected with only receptor or cells cotransfected with receptor and anti-syndecan-4. Exogenous heparin enhanced the specific binding and affinity cross-linking of 125I-bFGF to FGFR1 in receptor transfectants that were not cotransfected with proteoglycan, but had no effect on this binding and decreased the yield of bFGFR cross-links in cells that were cotransfected with proteoglycan. Receptor-transfectant cells showed a decrease in glycophorin A expression when exposed to bFGF. This suppression was dose-dependent and obtained at significantly lower concentrations of bFGF in proteoglycan-cotransfected cells. Finally, complementary cell-free binding assays indicated that the affinity of 125I-bFGF for an immobilized FGFR1 ectodomain was increased threefold when the syndecan-4 ectodomain was coimmobilized with receptor. Equimolar amounts of soluble syndecan-4 ectodomain, in contrast, had no effect on this binding. We conclude that, at least in K562 cells, syndecans and glypican can support bFGF-FGFR1 interactions and signaling, and that cell-surface association may augment their effectiveness.

Base Sequence↗

PubMed1998

Association of MSX1 and TGFB3 with nonsyndromic clefting in humans.

Nonsyndromic cleft lip with or without cleft palate (CL/P) and nonsyndromic cleft palate only (CPO) are common congenital anomalies with significant medical, psychological, social, and economic ramifications. Both CL/P and CPO are examples of complex genetic traits. There exists sufficient evidence to hypothesize that disease loci for CL/P and CPO can be identified by a candidate-gene linkage-disequilibrium (LD) strategy. Candidate genes for clefting, including TGFA, BCL3, DLX2, MSX1, and TGFB3, were screened for LD with either CL/P or CPO in a predominantly Caucasian population, with both case-control- and nuclear-family-based approaches. Previously reported LD for TGFA with both CL/P and CPO could not be confirmed, except in CL/P patients with a positive family history. Also, in contrast to previous studies, no LD was found between BCL3 and either CL/P or CPO. Significant LD was found between CL/P and both MSX1 and TGFB3 and between CPO and MSX1, suggesting that these genes are involved in the pathogenesis of clefting. In addition, a mutation search in the genes DLX2, MSX1, and TGFB3 was performed in 69 CPO patients and in a subset of the CL/P patients. No common mutations were found in the coding regions of these genes; however, several rare variants of MSX1 and TGFB3 were found that may alter the latters' normal function. These results form the basis for future research, including (a) mutation searches in the MSX1 and TGFB3 genes in Caucasian CL/P patients and (b) extension of the search for MSX1 mutations in CPO patients to the noncoding regions.

Amino Acid Substitution↗

PubMed2004

Isolated 3-methylcrotonyl-CoA carboxylase deficiency: evidence for an allele-specific dominant negative effect and responsiveness to biotin therapy.

Deficiency of 3-methylcrotonyl-CoA carboxylase (MCC) results in elevated excretion of 3-methylcrotonylglycine (3-MCG) and 3-hydroxyisovaleric acid (3-HIVA). MCC is a heteromeric mitochondrial enzyme comprising biotin-containing alpha subunits and smaller beta subunits, encoded by MCCA and MCCB, respectively. Mutations in these genes cause isolated MCC deficiency, an autosomal recessive disorder with a variable phenotype that ranges from severe neonatal to asymptomatic adult forms. No reported patients have responded to biotin therapy. Here, we describe two patients with a biochemical and, in one case, clinical phenotype of MCC deficiency, both of whom were responsive to biotin. The first patient presented at 3 months with seizures and progressive psychomotor retardation. Metabolic investigation at 2 years revealed elevated excretion of 3-MCG and 3-HIVA, suggesting MCC deficiency. High-dose biotin therapy was associated with a dramatic reduction in seizures, normalization of the electroencephalogram, and correction of the organic aciduria, within 4 weeks. MCC activity in fibroblasts was 25% of normal levels. The second patient, a newborn detected by tandem-mass-spectrometry newborn screening, displayed the same biochemical phenotype and remained asymptomatic with biotin up to the age of 18 months. In both patients, sequence analysis of the complete open reading frames of MCCA and MCCB revealed heterozygosity for MCCA-R385S and for the known polymorphic variant MCCA-P464H but revealed no other coding alterations. MCCA-R385S is unusual, in that it has a normal amount of MCC alpha protein but confers no MCC activity. We show that MCCA-R385S, but not other MCCA missense alleles, reduces the MCC activity of cotransfected MCCA-wild-type allele. Our results suggest that MCCA-R385S is a dominant negative allele and is biotin responsive in vivo.

Alleles↗

PubMed2006

A profile of alternative RNA splicing and transcript variation of CACNA1H, a human T-channel gene candidate for idiopathic generalized epilepsies.

Highly alternatively spliced genes may provide complex targets for disease mutations. Structural changes created by missense mutations may differentially affect the activity of alternative gene products, whereas missense, silent and non-coding mutations may alter developmental regulation of splice variant expression. CACNA1H is a human gene encoding Ca(v)3.2 low-voltage-activated, T-type calcium channels associated with bursting behavior in neurons and has been linked to more than 30 mutations apparently predisposing to childhood absence epilepsy (CAE) and other idiopathic generalized epilepsies (IGEs). Biophysical properties, including the effects of missense mutations, have been evaluated previously for a single splice form of Ca(v)3.2 expressed in transformed cell lines. We here show that CACNA1H is alternatively spliced at 12-14 sites, capable of generating both functional and non-functional transcripts. Variable cytoplasmic and extracellular protein domains point to likely differences in gating behavior, sensitivity to neuromodulation and interactions with extracellular matrix. Biophysical profiles of selected physiological Ca(v)3.2 forms reveal variations in kinetics and steady-state gating parameters, most likely to affect membrane firing. These were comparable to or larger than changes reported for previously studied mutations. Missense CAE and IGE mutations were clustered near segments associated with anomalous splicing. Missense and silent mutations were found to destroy, create or change the regulatory specificity of predicted exonic splicing enhancer sequences that may control splicing regulation. We discuss a paradigm for CACNA1H expression of Ca(v)3.2 subunits, which may influence future basic and clinical studies.

Alternative Splicing↗

PubMed1990

Computer prediction of the exon-intron structure of mammalian pre-mRNAs.

A novel approach to the problem of prediction of protein-coding regions is suggested. This approach combines the site prediction methods to predict splicing sites and the global coding region prediction methods to choose the best variant of spliced mRNA. One of the advantages of the suggested algorithm is that the resulting mRNA or protein sequence may then be immediately analyzed further. The true mRNA either coincides with the predicted one or ranks high in the list of variants. In the latter situation the predicted mRNA usually differs from the true one in only one or two of several exons. The combined approach allows the use of a priori information (e.g. the putative protein length or the number of exons). It is possible to use additional parameters not considered here, such as the preferred lengths of exons and introns, and particularly the preferred position of introns in the reading frame and the preferred codon position of exon termini.

Actins↗

PubMed1994

Identification of catalytically relevant amino acids of the extracellular Serratia marcescens endonuclease by alignment-guided mutagenesis.

By sequence alignment of the extracellular Serratia marcescens nuclease with three related nucleases we have identified seven charged amino acid residues which are conserved in all four sequences. Six of these residues together with four other partially conserved His or Asp residues were changed to alanine by site-directed PCR-mediated mutagenesis using a variant of the nuclease gene in which the coding sequence of the signal peptide was replaced by the coding sequence for an N-terminal affinity tag [Met(His)6GlySer]. Four of the mutant proteins showed almost no reduction in nuclease activity but five displayed a 10- to 1000-fold reduction in activity and one (His110Ala) was inactive. Based upon these results it is suggested that the S.marcescens nuclease employs a mechanism in which His110 acts in concert with a Mg2+ ion and three carboxylates (Asp107, Glu148 and Glu232) as well as one or two basic amino acid residues (Arg108, Arg152).

Amino Acid Sequence↗

PubMed1993

Mutations in zucchini yellow mosaic virus helper component protein associated with loss of aphid transmissibility.

Zucchini yellow mosaic virus (ZYMV) is a potyvirus transmitted by aphids in a non-persistent manner. Isolates having partially or totally lost their ability to be transmitted by aphids have been isolated and found to be affected in their helper component activities. We have sequenced the helper component coding region of poorly aphid-transmissible (PAT) variants of two strains of ZYMV, E15 and R5A. Mutations have been identified at the nucleotide level leading to two amino acid changes in the E15 PAT variant helper component and to one amino acid change located in the cysteine-rich region (well-conserved among potyviruses) in R5A PAT variant helper component. The mutation in the R5A variant changes the same amino acid as the one identified in potato virus C, a non-transmissible strain of potato virus Y.

Amino Acid Sequence↗

PubMed1987

Recombination rates and gene order for some serum alpha-protease inhibitors and immunoglobulin heavy-chain allotypes in pigs.

Linkage analysis between the genes coding for immunoglobulin heavy-chain allotypes and variants of some serum alpha-protease inhibitors produced lod scores above the significance limit of 3. The maximum likelihood estimate of the recombination fraction (theta) ranged from 0.15 to 0.20. Since this is the second report on this linkage group in pigs, the linkage is confirmed. Data from appropriate matings are consistent with a gene order of Pi1-Po1A-(Po1B)-Pi2-Igh1.

Animals↗

PubMed2006

Haplotypes of the human RET proto-oncogene associated with Hirschsprung disease in the Italian population derive from a single ancestral combination of alleles.

The RET proto-oncogene is the major gene involved in the complex genetics of Hirschsprung disease (HSCR), or aganglionic megacolon, showing causative loss-of-function mutations in 15-30% of the sporadic cases. Several RET polymorphisms and haplotypes have been described in association with the disease, suggesting a role for this gene in HSCR predisposition, also in the absence of mutations in the coding region. Finally, the presence of a functional variant in intron 1 has repeatedly been proposed to explain such findings. Here we report a case-control study conducted on 97 Italian HSCR sporadic patients and 85 population matched controls, using 13 RET polymorphisms distributed throughout the gene, from the basal promoter to the 3'UTR. Linkage disequilibrium and haplotype analyses have shown increased recombination between the 5' and 3' portions of the gene and an over-representation, in the cases studied, of two haplotypes sharing a common allelic combination that extends from the promoter up to intron 5. We propose that these two disease-associated haplotypes derive from a single founding locus, extending up to intron 19 and successively rearranged in correspondence with a high recombination rate region located between the proximal and distal portions of the gene. Our results suggests the possibility that a common HSCR predisposing variant, in linkage disequilibrium with such haplotypes, is located further downstream than the previously suggested interval encompassing intron 1.

Alleles↗

PubMed1981

Another Colicin V phenotype: in vitro adhesion of Escherichia coli to mouse intestinal epithelium.

Two assays were designed with which isogenic laboratory strains of Escherichia coli K-12 with and without ColV plasmids were compared for their ability to adhere in vitro to mouse intestinal epithelium. In both assays, disks of intestinal tissue were exposed to bacteria. In the first, disks were homogenized and the numbers of viable bacteria adherent to them were estimated from colony counts of plates inoculated with dilutions of the homogenates. In the second, bacteria were labeled with [(14)C]aspartic acid; the number of adherent cells per disk was estimated by liquid scintillation spectrometry. Data from each assay were compared by analysis of variance. In both assays, strains bearing the ColV plasmid adhered in two- to threefold-greater numbers than isogenic strains without the plasmid. These differences were highly significant statistically. A non-colicinogenic strain free of the ColV plasmid was selected by treatment of a ColV strain with sodium dodecyl sulfate. In the radioisotopic assay, the ColV strain associated with the epithelium in significantly greater numbers than the cured derivative. A ColV strain was created by conjugation; in the radioisotopic assay this strain bound to epithelium in significantly greater numbers than the recipient strain without the plasmid. The original ColV strain, when negatively stained and examined by electron microscopy, had pili that adsorbed male-specific bacteriophage, whereas its isogenic variant without ColV did not. Some such properties, coded by the plasmid, may increase the virulence of the bacteria.

Animals↗

PubMed1991

Chromosome structure: DNA nucleotide sequence elements of a subset of the minichromosomes of the protozoan Trypanosoma brucei.

The genome of the protozoan Trypanosoma brucei contains a set of about 100 minichromosomes of about 50 to 150 kb in size. The small size of these chromosomes, their involvement in antigenic variation, and their mitotic stability make them ideal candidates for a structural analysis of protozoan chromosomes and their telomeres. We show that a subset of the minichromosomes is composed predominantly of simple-sequence DNA, with over 90% of the length of the minichromosome consisting of a tandem array of 177-bp repeats, indicating that these molecules have limited protein-coding capacity. Proceeding from the tip of the telomere to a chromosome internal position, a subset of the minichromosomes contained the GGGTTA telomere repeat, a 29-bp telomere-derived repeat, a region containing 74-bp G + C-rich direct repeats separated by approximately 155 bp of A + T-rich DNA that has a bent character, and 50 to 150 kb of the 177-bp repeat. Several of the minichromosome-derived telomeres did not encode protein-coding genes, indicating that the repertoire of telomeric variant cell surface glycoprotein genes is restricted to some telomeres only. The telomere organization in trypanosomes shares striking similarities to the organization of telomeres and subtelomeres in humans, yeasts, and plasmodia. An electron microscopic analysis of the minichromosomes showed that they are linear molecules without abnormal structures in the main body of the chromosome. The structure of replicating molecules indicated that minichromosomes probably have a single bidirectional origin of replication located in the body of the chromosome. We propose a model for the structure of the trypanosome minichromosomes.

Animals↗

PubMed2005

CBLB variants in type 1 diabetes and their genetic interaction with CTLA4.

Type 1 diabetes (T1D) is a multifactorial disease with genetic and environmental components involved. Recent studies of an animal model of T1D, the Komeda diabetes-prone rat, have demonstrated that the Casitas-B-lineage lymphoma b (cblb) gene is a major susceptibility gene in the development of diabetes and other autoimmune features of this rat. As a result of the inhibitory role of Cbl-b in T cell costimulation, dysregulation of Cbl-b may also contribute to autoimmune diseases in man. Different isoforms of Cbl-b exist; we evaluated expression levels of two known transcript variants. Constitutive expression of both isoforms was demonstrated, as well as an increased expression, after cytokine exposure, of an isoform lacking exon 16, suggesting a possible role of this variant in the pathogenesis of autoimmunity. We screened coding regions of the human CBLB gene for mutations in a panel of individuals affected with several autoimmune diseases. Eight single nucleotide polymorphisms (SNPs) were detected. One SNP in exon 12 of the CBLB gene was significantly demonstrated to be associated to T1D in a large Danish T1D family material of 480 families. Evidence for common genetic factors underlying several autoimmune diseases has come from studies of cytotoxic T lymphocyte antigen 4 (CTLA4), which encodes another negatively regulatory molecule in the immune system. Gene-gene interactions probably play substantial roles in T1D susceptibility. We performed stratification of CBLB exon 12 SNP data, according to an established CTLA4 marker, CT60, and evidence for a genetic interaction between the CTLA4 and CBLB genes, involved in the same biological pathway of T cell receptor signaling, was observed.

Adaptor Proteins, Signal Transducing↗

PubMed1991

A study of schizophrenic outpatients with the Defense Mechanism Test.

Intrapsychic defensive strategies are allegedly expressed on the Defense Mechanism Test through the presentation, at increasing tachistoscopic exposure times, of a picture in which a central figure is threatened by a peripheral person. Several types of perceptual alterations of the stimulus configuration are coded as defenses in the subjective reports. A number of test variables have already been shown to differentiate significantly between nonclinical controls and nonpsychotic psychiatric patients. In the present study, after a review of the pertinent percept-genetic literature, nonpsychotic patients (n = 57) were compared with a group of schizophrenic outpatients in the active phase (n = 21). As predicted, significantly more schizophrenics than controls were coded for projection and regression and for certain variants of these signs. Four subcodings of repression, three of isolation, the sign of reaction formation, and several variants of identification differentiated in the same direction. Reports in which the central figure was too old and changed from a correct to an incorrect sex attribution were highly characteristic of the schizophrenic sample. No defensive variable was significantly linked with nonpsychotic pathology. Two variants, one of regression and one of identification, allowed a correct diagnostic placement for all schizophrenics and for 82.4% of the nonpsychotic patients. The findings, besides being a further clinical validation of the Defense Mechanism Test, provide a preliminary distinction between high- and low-level defenses. It is suggested that they are congruent with an hierarchical model which implies inclusive nonreflexive relationships between classes of mental disorders.

Adult↗

PubMed1997

A cell cycle-regulated histone H3 gene of alfalfa with an atypical promoter structure.

The control of cell cycle expression of histone genes in plants is incompletely understood. A new histone H3 gene was cloned from alfalfa (Medicago sativa) that codes for the replication-dependent histone H3.1 variant protein. Despite lacking all promoter sequence motifs that have been associated with cell cycle-dependent histone gene expression in plants, northern analysis of synchronized cells clearly linked gene expression to DNA replication. TTAATNA was recognized as a new sequence element in the 3' untranslated regions of this and all other cell cycle-dependent histone H3 genes of dicotyledonous plants. It is not found in the replication-independent histone H3 genes.

Amino Acid Sequence↗