Identification of a putative estrogen response element in the gene encoding brain-derived neurotrophic factor.

We have been studying the role and mechanism of estrogen action in the survival and differentiation of neurons in the basal forebrain and its targets in the cerebral cortex, hippocampus, and olfactory bulb. Previous work has shown that estrogen-target neurons in these regions widely coexpress the mRNAs for the neurotrophin ligands and their receptors, suggesting a potential substrate for estrogen-neurotrophin interactions. Subsequent work indicated that estrogen regulates the expression of two neurotrophin receptor mRNAs in prototypic peripheral neural targets of nerve growth factor. We report herein that the gene encoding the neurotophin brain-derived neurotrophic factor (BDNF) contains a sequence similar to the canonical estrogen response element found in estrogen-target genes. Gel shift and DNA footprinting assays indicate that estrogen receptor-ligand complexes bind to this sequence in the BDNF gene. In vivo, BDNF mRNA was rapidly up-regulated in the cerebral cortex and the olfactory bulb of ovariectomized animals exposed to estrogen. These data suggest that estrogen may regulate BDNF transcription, supporting our hypothesis that estrogen may be in a position to influence neurotrophin-mediated cell functioning, by increasing the availability of specific neurotrophins in forebrain neurons.

Identification of a DNA element determining synaptic expression of the mouse acetylcholine receptor delta-subunit gene.

mRNAs for acetylcholine receptor genes are highly concentrated in the endplate region of adult skeletal muscle largely as a result of a transcription restricted to the subneural nuclei. To identify the regulatory elements involved, we employed a DNA injection of a plasmid containing a fragment of the acetylcholine receptor delta-subunit gene promoter (positions -839 to +45) linked to the reporter gene lacZ with a nuclear localization signal. Injection of the wild-type construct into mouse leg muscles yielded preferential expression of the reporter gene in the synaptic region. Analysis of various mutant promoters resulted in the identification of a DNA element (positions -60 to -49), referred to as the N box, that plays a critical role in subneural expression. Disruption of this 12-bp element in the context of a mouse delta-subunit promoter from positions -839 to +45 gives widespread expression of the reporter gene throughout the entire muscle fiber, indicating that this element is a silencer that represses delta-subunit gene transcription in extrajunctional areas. On the other hand, this element inserted upstream of a heterologous basal promoter preferentially enhances expression in the endplate region. This element therefore regulates the restricted expression of the delta-subunit gene both as an enhancer at the endplate level and as a silencer in extrajunctional areas. Furthermore, gel-shift experiments with mouse muscle extracts reveal an activity that specifically binds the 6-bp sequence TTCCGG of this element, suggesting that a transcription factor(s) controls the expression of the delta-subunit gene via this element.

Identification of a Drosophila muscle development gene with structural homology to mammalian early growth response transcription factors.

In Drosophila, stripe (sr) gene function is required for normal muscle development. Some mutations disrupt embryonic muscle development and are lethal. Other mutations cause total loss of only a single muscle in the adult. Molecular analysis shows that sr encodes a predicted protein containing a zinc finger motif. This motif is homologous to the DNA binding domains encoded by members of the early growth response (egr) gene family. In mammals, expression of egr genes is induced by intercellular signals, and there is evidence for their role in many developmental events. The identification of sr as an egr gene and its pattern of expression suggest that it functions in muscle development via intercellular communication.

Identification of an additional gene required for eukaryotic nonsense mRNA turnover.

Loss of function of any one of three UPF genes prevents the accelerated decay of nonsense mRNAs in Saccharomyces cerevisiae. We report the identification and DNA sequence of UPF3, which is present in one nonessential copy on chromosome VII. Upf3 contains three putative nuclear localization signal sequences, suggesting that it may be located in a different compartment than the cytoplasmic Upf1 protein. Epitope-tagged Upf3 (FLAG-Upf3) does not cofractionate with polyribosomes or 80S ribosomal particles. Double disruptions of UPF1 and UPF3 affect nonsense mRNA decay in a manner indistinguishable from single disruptions. These results suggest that the Upf proteins perform related functions in a common pathway.

Identification of planarian homeobox sequences indicates the antiquity of most Hox/homeotic gene subclasses.

The homeotic gene complex (HOM-C) is a cluster of genes involved in the anteroposterior axial patterning of animal embryos. It is composed of homeobox genes belonging to the Hox/HOM superclass. Originally discovered in Drosophila, Hox/HOM genes have been identified in organisms as distantly related as arthropods, vertebrates, nematodes, and cnidarians. Data obtained in parallel from the organization of the complex, the domains of gene expression during embryogenesis, and phylogenetic relationships allow the subdivision of the Hox/HOM superclass into five classes (lab, pb/Hox3, Dfd, Antp, and Abd-B) that appeared early during metazoan evolution. We describe a search for homologues of these genes in platyhelminths, triploblast metazoans emerging as an outgroup to the great coelomate ensemble. A degenerate PCR screening for Hox/HOM homeoboxes in three species of triclad planarians has revealed 10 types of Antennapedia-like genes. The homeobox-containing sequences of these PCR fragments allowed the amplification of the homeobox-coding exons for five of these genes in the species Polycelis nigra. A phylogenetic analysis shows that two genes are clear orthologues of Drosophila labial, four others are members of a Dfd/Antp superclass, and a seventh gene, although more difficult to classify with certainty, may be related to the pb/Hox3 class. Together with previously identified Hox/HOM genes in other flatworms, our analyses demonstrate the existence of an elaborate family of Hox/HOM genes in the ancestor of all triploblast animals.

Identification of a gene involved in the biosynthesis of cyclopropanated mycolic acids in Mycobacterium tuberculosis.

Mycolic acids represent a major constituent of the mycobacterial cell wall complex, which provides the first line of defense against potentially lethal environmental conditions. Slow-growing pathogenic mycobacteria such as Mycobacterium tuberculosis modify their mycolic acids by cyclopropanation, whereas fast-growing saprophytic species such as Mycobacterium smegmatis do not, suggesting that this modification may be associated with an increase in oxidative stress experienced by the slow-growing species. We have demonstrated the transformation of the distal cis double bond in the major mycolic acid of M. smegmatis to a cis-cyclopropane ring upon introduction of cosmid DNA from M. tuberculosis. This activity was localized to a single gene (cma1) encoding a protein that was 34% identical to the cyclopropane fatty acid synthase from Escherichia coli. Adjacent regions of the DNA sequence encode open reading frames that display homology to other fatty acid biosynthetic enzymes, indicating that some of the genes required for mycolic acid biosynthesis may be clustered in this region. M. smegmatis overexpressing the cma1 gene product significantly resist killing by hydrogen peroxide, suggesting that this modification may be an important adaptation of slow-growing mycobacteria to oxidative stress.

Identification of mutations in the Wiskott-Aldrich syndrome gene and characterization of a polymorphic dinucleotide repeat at DXS6940, adjacent to the disease gene.

The Wiskott-Aldrich syndrome (WAS) is an X-chromosome-linked recessive disease characterized by eczema, thrombocytopenia, and immunodeficiency. The disease gene has been localized to the proximal short arm of the X chromosome and recently isolated through positional cloning. The function of the encoded protein remains undetermined. In this study we have characterized mutations in 12 unrelated patients to confirm the identity of the disease gene. We have also revised the coding sequence and genomic structure for the WAS gene. To analyze further the transmittance of the disease gene, we have characterized a polymorphic microsatellite at the DXS6940 locus within 30 kb of the gene and demonstrate the inheritance of the affected alleles in families with a history of WAS.

Identification of the gene (SSU71/TFG1) encoding the largest subunit of transcription factor TFIIF as a suppressor of a TFIIB mutation in Saccharomyces cerevisiae.

Mutations in the Saccharomyces cerevisiae SSU71 gene were isolated as suppressors of a transcription factor TFIIB defect that confers both a cold-sensitive growth defect and a downstream shift in transcription start-site selection at the cyc1 locus. The ssu71-1 suppressor not only suppresses the conditional phenotype but also restores the normal pattern of transcription initiation at cyc1. In addition, the ssu71-1 suppressor confers a heat-sensitive phenotype that is dependent upon the presence of the defective form of TFIIB. Molecular and genetic analysis of the cloned SSU71 gene demonstrated that SSU71 is a single-copy essential gene encoding a highly charged protein with a molecular mass of 82,194 daltons. Comparison of the deduced Ssu71 amino acid sequence with the protein data banks revealed significant similarity to RAP74, the larger subunit of the human general transcription factor TFIIF. Moreover, Ssu71 is identical to p105, a component of yeast TFIIF. Taken together, these data demonstrate a functional interaction between TFIIB and the large subunit of TFIIF and that this interaction can affect start-site selection in vivo.

Identification of a DNA transformation gene required for com101A+ expression and supertransformer phenotype in Haemophilus influenzae.

DNA sequencing, RNA mapping, and protein expression experiments revealed the presence of a gene, tfoX+, encoding a 24.9-kDa polypeptide, that is transcribed divergently from a common promoter region with the Haemophilus influenzae rec-1+ gene. H. influenzae strains mutant for tfoX failed to bind transforming DNA and were transformation deficient. Primer extension experiments utilizing in vivo total RNA from precompetent and competent H. influenzae cells demonstrated that transcription of tfoX+ increased immediately upon competence induction, suggesting that tfoX+ is an early competence gene. Similar experiments showed that the expression of the late competence-specific gene, com101A+, was tfoX+ dependent. Moreover, expression of plasmid-borne tfoX+ in H. influenzae resulted in constitutive competence. The addition of cyclic adenosine monophosphate (cAMP) to strains carrying a tfoX::lacZ operon fusion resulted in an immediate increase in beta-galactosidase activity that correlated with an increase in genetic transformability. Collectively, our results suggest that TfoX may play a key role in the development of genetic competence by regulating the expression of late competence-specific genes.

Identification and cloning of the gene encoding BmpC: an outer-membrane lipoprotein associated with Brachyspira pilosicoli membrane vesicles

The intestinal spirochaete Brachyspira pilosicoli causes colitis in a wide variety of host species. Little is known about the structure or protein constituents of the B. pilosicoli outer membrane (OM). To identify surface-exposed proteins in this species, membrane vesicles were isolated from B. pilosicoli strain 95-1000 cells by osmotic lysis in dH(2)O followed by isopycnic centrifugation in sucrose density gradients. The membrane vesicles were separated into a high-density fraction (HDMV; p = 1.18 g CM-3) and a low-density fraction (LDMV; rho=1.12 g cm(-3)). Both fractions were free of flagella and soluble protein contamination. LDMV contained predominantly OM markers (lipo-oligosaccharide and a 29 kDa B. pilosicoli OM protein) and was used as a source of antigens to produce mAbs. Five B. pilosicoli-specific mAbs reacting with proteins with molecular masses of 23, 24, 35, 61 and 79 kDa were characterized. The 23 kDa protein was only partially soluble in Triton X-114, whereas the 24 and 35 kDa proteins were enriched in the detergent phase, implying that they were integral membrane proteins or lipoproteins. All three proteins were localized to the B. pilosicoli OM by immunogold labelling using specific mAbs. The gene encoding the abundant, surface-exposed 23 kDa protein was identified by screening a B. pilosicoli 95-1000 genome library with the mAb and was expressed in Escherichia coli. Sequence analysis showed that it encoded a unique lipoprotein, designated BmpC. Recombinant BmpC partitioned predominantly in the OM fraction of E. coli strain SOLR. The mAb to BmpC was used to screen a collection of 13 genetically heterogeneous strains of B. pilosicoli isolated from five different host species. Interestingly, only strain 95-1000 was reactive with the mAb, indicating that either the surface-exposed epitope on BmpC is variable between strains or that the protein is restricted in its distribution within B. pilosicoli.

Identification of two evolutionarily conserved and functional regulatory elements in intron 2 of the human BRCA1 gene

Cross-species comparative genomics is a powerful strategy for identifying functional regulatory elements within noncoding DNA. In this paper, comparative analysis of human and mouse intronic sequences in the breast cancer susceptibility gene (BRCA1) revealed two evolutionarily conserved noncoding sequences (CNS) in intron 2, 5 kb downstream of the core BRCA1 promoter. The functionality of these elements was examined using homologous-recombination-based mutagenesis of reporter gene-tagged cosmids incorporating these regions and flanking sequences from the BRCA1 locus. This showed that CNS-1 and CNS-2 have differential transcriptional regulatory activity in epithelial cell lines. Mutation of CNS-1 significantly reduced reporter gene expression to 30% of control levels. Conversely mutation of CNS-2 increased expression to 200% of control levels. Regulation is at the level of transcription and shows promoter specificity. Both elements also specifically bind nuclear proteins in vitro. These studies demonstrate that the combination of comparative genomics and functional analysis is a successful strategy to identify novel regulatory elements and provide the first direct evidence that conserved noncoding sequences in BRCA1 regulate gene expression. (c) 2005 Elsevier Inc. All rights reserved.

Identification of three gene candidates for multicellular resistance in colon carcinoma

Solid tumours display elevated resistance to chemo- and radiotherapies compared to individual tumour derived cells. This so-called multicellular resistance (MCR) phenomenon can only be partly explained by reduced diffusion and altered cell cycle status; even fast growing cells on the surface of solid tumours display MCR. Multicellular spheroids (MCS) recapture this phenomenon ex vivo and here we compare gene expression in exponentially growing MCS with gene expression in monolayer culture. Using an 18,664 gene microarray, we identified 42 differentially expressed genes and three of these genes can be linked to potential mechanisms of MCR. A group of interferon response genes were also up-regulated in MCS, as were a number of genes that that are indicative of greater differentiation in three-dimensional cultures.

Butyrylcholinesterase: Association with the metabolic syndrome and identification of 2 gene loci affecting activity

Background: Plasma cholinesterase activity is known to be correlated with plasma triglycerides, HDL- and LDL-cholesterol, and other features of the metabolic syndrome. A role in triglyceride metabolism has been proposed. Genetic variants that decrease activity have been studied extensively, but the factors contributing to overall variation in the population are poorly understood. We studied plasma cholinesterase activity in a sample of 2200 adult twins to assess covariation with cardiovascular risk factors and components of the metabolic syndrome, to determine the degree of genetic effects on enzyme activity, and to search for quantitative trait loci affecting activity. Methods and Results: Cholinesterase activity was lower in women than in men before the age of 50, but increased to activity values similar to those in males after that age. There were highly significant correlations with variables associated with the metabolic syndrome: plasma triglyceride, HDL- and LDL-cholesterol, apolipoprotein B and E, urate, and insulin concentrations; gamma-glutamyltransferase and aspartate and alanine aminotransferase activities; body mass index; and blood pressure. The heritability of plasma cholinesterase activity was 65%. Linkage analysis with data from the dizygotic twin pairs showed suggestive linkage on chromosome 3 at the location of the cholinesterase WHO gene and also on chromosome 5. Conclusions: Our results confirm and extend the connection between cholinesterase, cardiovascular risk factors, and metabolic syndrome. They establish a substantial heritability for plasma cholinesterase activity that might be attributable to variation near the structural gene and at an independent locus. (c) 2006 American Association for Clinical Chemistry.