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At least 991 records · Page 55Linked to original sources
PubMed1998

Demonstration that the TyrR protein and RNA polymerase complex formed at the divergent P3 promoter inhibits binding of RNA polymerase to the major promoter, P1, of the aroP gene of Escherichia coli.

In previous studies, we have identified three promoters (P1, P2, and P3) in the regulatory region of the Escherichia coli aroP gene (P. Wang, J. Yang, and A. J. Pittard, J. Bacteriol. 179:4206-4212, 1997). Both P1 and P2 can direct mRNA synthesis for aroP expression, whereas P3 is a divergent promoter which overlaps with P1. The repression of transcription from the major promoter, P1, has been postulated to involve the activation of the divergent promoter, P3, by the TyrR protein (P. Wang, J. Yang, B. Lawley, and A. J. Pittard, J. Bacteriol. 179:4213-4218, 1997). In the present study, we confirmed the proposed mechanism of P3-mediated repression of P1 transcription by studying the binding of RNA polymerase to the promoters P1 and P3 in vitro in the presence and absence of TyrR protein and its cofactors. Our results show that (i) only one RNA polymerase molecule can bind to the DNA fragment carrying the aroP regulatory region, (ii) RNA polymerase has a higher affinity for P1 than for either P2 or P3 and binds to P1 in the absence of TyrR protein, (iii) in the presence of TyrR protein and its cofactor, phenylalanine or tyrosine, RNA polymerase preferentially binds to P3, and (iv) RNA polymerase does not respond to the activation-defective mutant TyrR protein TyrR-RQ10 and remains bound to P1 in the presence of TyrR-RQ10 and either of the cofactors.

Amino Acid Transport Systems↗
PubMed1989

Psychoanalysis and psychotherapy: an historical perspective.

I have reviewed the historical unfolding of psychoanalytic (or dynamic) psychotherapy as a therapeutic application of psychoanalytic theory directed towards patients not considered appropriate for proper psychoanalysis. I have divided this history into three phases. The first phase that I call the prehistory of dynamic psychotherapy is the period of Freud's development of psychoanalysis as a distinctive therapy for mental illness and its clear differentiation from the varieties of suggestive therapies in vogue at the time. The second phase (from the 40s to the late 70s) is the period of the development of psychoanalytically-based expressive and supportive psychotherapies related to but clearly separate from proper psychoanalysis in terms of specific techniques, goals, and the portions of the psychopathological spectrum against which each is directed. I call it the period of psychoanalytic consensus. The third current phase, from the late 70s to date, I call the period of fragmented consensus. The leading protagonists in the forging of the second phase consensus, brought to its fullest expression in the landmark publications of 1954, have now diverged widely in their perspectives on these issues as expressed in a 1979 Symposium, 25 years after.

Europe↗
PubMed2006

Differential regulation of TIMP-1, -2, and -3 mRNA and protein expressions during mouse incisor development.

Tissue inhibitors of metalloproteinases (TIMPs) possess multiple functions, in addition to their matrix metalloproteinase (MMP) inhibitory activity. The continuously growing incisor of mouse possesses a stem cell compartment at the apical end of the epithelium (the apical loop) and thus provides an excellent tool to analyze the mechanisms of organogenesis and cytodifferentiation. To understand the functions of TIMPs in tooth development, we have analyzed the gene expression and protein localization of TIMP-1, -2, and -3 during mouse incisor development, from embryonic day 13 (E13) to postnatal day 3 (P3). TIMP-1 was present on the basement membrane during early developmental stages. At P2, TIMP-1 was strongly detected along the apical loop, transiently disappeared from the basement membrane in the cytodifferentiation zone, and later reappeared at the distal end of functional ameloblasts. Expression of TIMP-2 protein was restricted to the outer part of the apical loop throughout the examined stages. At P2, TIMP-2 was present on the basement membrane at the outer part of the apical loop. The dental follicle also expressed Timp-2, and the corresponding protein was abundant within the extracellular matrix. Timp-3 mRNA was highly expressed in the mesenchyme surrounding the apical loop. During matrix formation, Timp-3 was expressed by subodontoblasts, and the protein was detected in this layer and between odontoblasts. Distinct temporal and spatial expression patterns of TIMPs suggest divergent functions of these factors in incisor organogenesis.

Animals↗
PubMed2005

Different expression patterns of duplicated PHANTASTICA-like genes in Lotus japonicus suggest their divergent functions during compound leaf development.

Recent studies on leaf development demonstrate that the mechanism on the adaxial-abaxial polarity pattern formation could be well conserved among the far-related species, in which PHANTASTICA (PAHN)-like genes play important roles. In this study, we explored the conservation and diversity on functions of PHAN-like genes during the compound leaf development in Lotus japonicus, a papilionoid legume. Two PHAN-like genes in L. japonicus, LjPHANa and LjPHANb, were found to originate from a gene duplication event and displayed different expression patterns during compound leaf development. Two mutants, reduced leaflets1 (rel1) and reduced leaflets3 (rel3), which exhibited decreased adaxial identity of leaflets and reduced leaflet initiation, were identified and investigated. The expression patterns of both LjPHANs in rel mutants were altered and correlated with abnormalities of compound leaves. Our data suggest that LjPHANa and LjPHANb play important but divergent roles in regulating adaxial-abaxial polarity of compound leaves in L. japonicus.

Amino Acid Sequence↗
PubMed1999

Evolutionary alterations of the minimal promoter for notochord-specific Brachyury expression in ascidian embryos.

The Brachyury genes of two divergent ascidians, As-T of Halocynthia roretzi and Ci-Bra of Ciona intestinalis, are expressed exclusively in notochord precursor cells. A previous study showed that the notochord-specific expression of Ci-Bra is controlled by a minimal promoter that is composed of three distinct regions: a region responsible for repression of expression in non-notochord mesoderm cells, a region for activation of expression in notochord cells, and a region for activation of expression in non-notochord mesoderm cells, distal to proximal to the transcription initiation site, respectively. We examined various deletion constructs of the As-T/lacZ fusion gene and demonstrate that a module between -289 and -250 bp of the 5'-flanking region is responsible for notochord-specific expression of the reporter gene. Gel-shift assays suggested the binding of nuclear protein(s) to this module. The 5'-flanking region of As-T contains a potential T-binding motif (-ACCTAGGT-) around -160 bp. Deletion of this motif from the p(-289)As-T/lacZ diminished the reporter gene expression. In addition, coinjection of p(-289)As-T/lacZ and synthetic As-T mRNA resulted in ectopic expression of lacZ in non-notochord cells, suggesting that the T-binding motif is responsible for autoactivation of the gene. These findings revealed striking differences between the minimal promoters of As-T and Ci-Bra so far revealed, with respect to their notochord-specific expression. Furthermore, reciprocal injections of reporter gene constructs, namely As-T/lacZ into Ciona eggs and Ci-Bra/lacZ into Halocynthia eggs, suggest alterations in the cis-regulatory elements and trans-activation factors that have occurred during evolution of the two ascidian species.

Animals↗
PubMed1986

Nucleotide sequence and in vivo expression of the ilvY and ilvC genes in Escherichia coli K12. Transcription from divergent overlapping promoters.

The ilvC gene of Escherichia coli K12 encodes acetohydroxy acid isomeroreductase, the second enzyme in the parallel isoleucine-valine biosynthetic pathway. Previous data have shown that transcription of the ilvC gene is induced by the acetohydroxy acid isomeroreductase substrates, acetohydroxybutyrate or acetolactate, and that this substrate induction of ilvC expression is mediated by a positive activator encoded by the ilvY gene. We report here the isolation and complete nucleotide sequence of the ilvY and ilvC genes. The ilvY and ilvC genes encode polypeptides of Mr 33,200 and 54,000, respectively. In vitro transcription-translation of these gene templates results in the synthesis of gene products of these identical molecular weights. The ilvC gene is transcribed in the same direction as the genes of the adjacent ilvGMEDA operon. The ilvY gene is transcribed in a direction opposite to the ilvC and ilvGMEDA genes. The in vivo transcriptional initiation sites of the ilvY and ilvC genes have been determined by S1 nuclease protection experiments. These transcriptional initiation sites are 45 nucleotides apart, and transcription of the ilvY and ilvC genes is initiated via divergent overlapping promoters. The nucleotide sequence of the ilvY and ilvC promoters and 5'-coding regions of Salmonella typhimurium LT2 have been determined. A comparison of these sequences with E. coli K12 suggests regions important in the promotion, regulation, and translation of the ilvY and ilvC genes. A model is presented in which the ilvY-encoded activator binds to an operator site in the overlapping promoter region and reciprocally regulates the transcription of the ilvY and ilvC genes. The carboxyl-terminal amino acid sequence of threonine deaminase encoded by the ilvA gene of the ilv-GMEDA operon of E. coli K12 has been identified by homology with the previously deduced threonine deaminase amino acid sequence encoded by the ilv1 gene of Saccharomyces cerevisiae. Based on the deduced amino acid sequences of the ilvA and ilvY genes, the translational termination codons for both genes are shown to be separated by 52 nucleotides. The proximity of the ilvA and ilvY genes suggests that the 3'-ends of these transcripts overlap.

Alcohol Oxidoreductases↗
PubMed2000

Vnd/nkx, ind/gsh, and msh/msx: conserved regulators of dorsoventral neural patterning?

Expression of vnd in ventral, ind in intermediate, and msh in dorsal columns of fly neurectoderm, and of homologous gene families in corresponding domains of vertebrate neurectoderm, suggests that elements of dorsoventral neural patterning have been evolutionarily conserved. However, upstream signaling pathways regulating this columnar gene expression pattern appear to have diverged significantly throughout evolution. In addition, while recent loss-of-function studies in flies and mice indicate that these three genes may have a conserved role in regional specification, there is no obvious conservation of the particular cell fates deriving from corresponding domains. The three-column expression pattern may thus represent a developmental mechanism that is more resistant to evolutionary changes than genetic events upstream or downstream of it.

Animals↗
PubMed1989

Effect of mutations in the cyclic AMP receptor protein-binding site on araBAD and araC expression.

Maximum expression of the adjacent but divergently transcribed araBAD operon and araC gene requires the presence of cyclic AMP (cAMP) and the cAMP receptor protein (CRP). DNase I protection studies have previously revealed a high-affinity CRP-binding site in the ara regulatory region. Deletion mutations introduced into this site resulted in reduced expression of araBAD and araC. However, other experiments have demonstrated that spacing changes in the ara regulatory region may have multiple effects due to disruption of a DNA loop. Thus, the deletions could have destroyed the CRP-binding site, the ability to form a loop, or both. In the present study, substitution mutations were introduced into the CRP site in order to avoid creating spacing changes. We found that a 3-base-pair substitution resulted in a 30% reduction in araBAD expression, whereas a 6-base-pair substitution resulted in an 80% reduction. Both of these substitution mutations reduced araC expression threefold. We conclude that CRP bound to this site regulates expression in both directions. We found that a spacing change in the CRP site does not alter araBAD expression any more than does a substitution mutation.

Arabinose↗
PubMed2000

Highly diverged homologs of Saccharomyces cerevisiae mitochondrial mRNA-specific translational activators have orthologous functions in other budding yeasts.

Translation of mitochondrially coded mRNAs in Saccharomyces cerevisiae depends on membrane-bound mRNA-specific activator proteins, whose targets lie in the mRNA 5'-untranslated leaders (5'-UTLs). In at least some cases, the activators function to localize translation of hydrophobic proteins on the inner membrane and are rate limiting for gene expression. We searched unsuccessfully in divergent budding yeasts for orthologs of the COX2- and COX3-specific translational activator genes, PET111, PET54, PET122, and PET494, by direct complementation. However, by screening for complementation of mutations in genes adjacent to the PET genes in S. cerevisiae, we obtained chromosomal segments containing highly diverged homologs of PET111 and PET122 from Saccharomyces kluyveri and of PET111 from Kluyveromyces lactis. All three of these genes failed to function in S. cerevisiae. We also found that the 5'-UTLs of the COX2 and COX3 mRNAs of S. kluyveri and K. lactis have little similarity to each other or to those of S. cerevisiae. To determine whether the PET111 and PET122 homologs carry out orthologous functions, we deleted them from the S. kluyveri genome and deleted PET111 from the K. lactis genome. The pet111 mutations in both species prevented COX2 translation, and the S. kluyveri pet122 mutation prevented COX3 translation. Thus, while the sequences of these translational activator proteins and their 5'-UTL targets are highly diverged, their mRNA-specific functions are orthologous.

5' Untranslated Regions↗
PubMed2003

The zebrafish annexin gene family.

The Annexins (ANXs) are a family of calcium- and phospholipid-binding proteins that have been implicated in many cellular processes, including channel formation, membrane fusion, vesicle transport, and regulation of phospholipase A2 activity. As a first step toward understanding in vivo function, we have cloned 11 zebrafish anx genes. Four genes (anx1a, anx2a, anx5,and anx11a) were identified by screening a zebrafish cDNA library with a Xenopus anx2 fragment. For these genes, full-length cDNA sequences were used to cluster 212 EST sequences generated by the Zebrafish Genome Resources Project. The EST analysis revealed seven additional anx genes that were subsequently cloned. The genetic map positions of all 11 genes were determined by using a zebrafish radiation hybrid panel. Sequence and syntenic relationships between zebrafish and human genes indicate that the 11 genes represent orthologs of human anx1,2,4,5,6,11,13,and suggest that several zebrafish anx genes resulted from duplications that arose after divergence of the zebrafish and mammalian genomes. Zebrafish anx genes are expressed in a wide range of tissues during embryonic and larval stages. Analysis of the expression patterns of duplicated genes revealed both redundancy and divergence, with the most similar genes having almost identical tissue-specific patterns of expression and with less similar duplicates showing no overlap. The differences in gene expression of recently duplicated anx genes could explain why highly related paralogs were maintained in the genome and did not rapidly become pseudogenes.

Amino Acid Sequence↗
PubMed1997

Four conserved promoter motifs regulate transcription of the gene encoding human complement component C2.

The early complement components of the classical activation pathway of the complement cascade include the components C1, C4, C2, and C3. These components act in concert to opsonize bacteria, clear immune complexes, and produce inflammatory mediators. They are not structurally homologous nor are they coordinately regulated. The expression of the early complement components is divergent in terms of cytokine responsiveness and tissue specificity. The only pattern of expression shared by the early complement components is inducibility by gamma-IFN and expression in cells of hepatic or monocytic lineage. Nevertheless, four novel conserved promoter motifs were identified in the 5' flanking region of multiple early complement component promoters. Mutation of these four motifs in the C2 promoter decreased transcription in hepatoma cells in transient transfection analyses, and a synthetic promoter consisting of just the four motifs supported transcription in hepatoma cells. Electrophoretic mobility shift assays demonstrate that three of the four conserved elements bind DNA-binding proteins in a tissue-specific manner. One DNA-binding protein is expressed ubiquitously, but the other three are restricted to cells of monocytic or hepatic lineage. Two of the DNA-binding proteins appear to be members of the zinc-finger family of transcription factors. Therefore, these four motifs appear to bind DNA-binding proteins that may function in the tissue-specific expression of C2. Conservation of these four motifs in multiple early complement component genes suggests that these may represent a conserved transcriptional strategy.

Carcinoma, Hepatocellular↗
PubMed2006

Cloning and developmental expression of the Xenopus homeobox gene Xvsx1.

In contrast to the high degree of evolutionary conservation of the Vsx2/Chx10 gene family, vertebrate orthologues of Vsx1 display more divergent sequences and spatio-temporal expression patterns. Here, we report the cloning and expression pattern of Xenopus laevis Vsx1. Differently from the mouse and zebrafish orthologues, Xvsx1 transcription is activated at early neurula both in the evaginating eye vesicles and in the presumptive spinal cord. Compared to other retinal homeobox genes, such as Xrx1, Xsix3 and Xpax6, Xvsx1 is activated at a later stage; in addition, its anterior expression appears to be more specifically restricted to the retina. At tail bud stage, Xvsx1 expression in retinal progenitors persists, and its neural tube expression, which in the spinal cord corresponds to interneurons, progressively expands anteriorly reaching the midbrain-hindbrain boundary. During retinal neurogenesis, Xvsx1 expression is maintained in retinal progenitors and in a peripheral region of the ciliary marginal zone, while in the central retina, it becomes restricted to differentiated bipolar cells.

Amino Acid Sequence↗
PubMed2003

Coexpression of neighboring genes in Caenorhabditis elegans is mostly due to operons and duplicate genes.

In many eukaryotic species, gene order is not random. In humans, flies, and yeast, there is clustering of coexpressed genes that cannot be explained as a trivial consequence of tandem duplication. In the worm genome this is taken a step further with many genes being organized into operons. Here we analyze the relationship between gene location and expression in Caenorhabditis elegans and find evidence for at least three different processes resulting in local expression similarity. Not surprisingly, the strongest effect comes from genes organized in operons. However, coexpression within operons is not perfect, and is influenced by some distance-dependent regulation. Beyond operons, there is a relationship between physical distance, expression similarity, and sequence similarity, acting over several megabases. This is consistent with a model of tandem duplicate genes diverging over time in sequence and expression pattern, while moving apart owing to chromosomal rearrangements. However, at a very local level, nonduplicate genes on opposite strands (hence not in operons) show similar expression patterns. This suggests that such genes may share regulatory elements or be regulated at the level of chromatin structure. The central importance of tandem duplicate genes in these patterns renders the worm genome different from both yeast and human.

Animals↗
PubMed2003

Identification and differential expression of two thioredoxin h isoforms in germinating seeds from pea.

The NADPH/NADP-thioredoxin (Trx) reductase (NTR)/Trx system (NTS) is a redox system that plays a posttranslational regulatory role by reducing protein targets involved in crucial cellular processes in microorganisms and animals. In plants, the system includes several h type Trx isoforms and has been shown to intervene in reserve mobilization during early seedling growth of cereals. To determine whether NTS was operational during germination of legume seeds and which Trx h isoforms could be implicated, Trx h isoforms expression was monitored in germinating pea (Pisum sativum cv Baccara) seeds, together with the amount of NTR and NADPH. Two new isoforms were identified: Trx h3, similar to the two isoforms already described in pea but not expressed in seeds; and the more divergent isoform, Trx h4. Active recombinant proteins were produced in Escherichia coli and used to raise specific antibodies. The expression of new isoforms was analyzed at both mRNA and protein levels. The lack of correlation between mRNA and protein abundances suggests the occurrence of posttranscriptional regulation. Trx h3 protein amount remained constant in both axes and cotyledons of dry and imbibed seeds but then decreased 2 d after radicle protrusion. In contrast, Trx h4 was only expressed in axes of dry and imbibed seeds but not in germinated seeds or in seedlings, therefore appearing as closely linked to germination. The presence of NTR and NADPH in seeds suggests that NTS could be functional during germination. The possible role of Trx h3 and h4 in this context is discussed.

Amino Acid Sequence↗
PubMed1986

Promoter-probe vectors for the analysis of divergently arranged promoters.

A series of plasmid-based promoter-probe vectors has been constructed which are particularly useful for the analysis of divergent control regions. Each vector contains a pair of divergently oriented indicator genes whose expression can be monitored over a wide range by simple assay methods. These genes are separated by different polylinkers. Specifically, the beta-galactosidase gene (lacZ) was employed in combination with either the galactokinase gene (galK) or the alkaline phosphatase gene (phoA). In all cases translational stop codons are present in all three reading frames upstream from the initiation codon. The vectors permit direct detection of promoters--independent of insert orientation--on indicator plates after transformation. Using this vector system, we further characterized the divergent tet control regions of transposon Tn10 and plasmid pBR322.

Alkaline Phosphatase↗
PubMed1984

Comparison of the beta-like globin gene families of rabbits and humans indicates that the gene cluster 5'-epsilon-gamma-delta-beta-3' predates the mammalian radiation.

The members of the rabbit and human beta-like globin gene families have been compared both by a computer-generated dot matrix graphical analysis of each entire gene and by calculating divergences in the coding regions. The rabbit-human gene pairs beta 4-epsilon, beta 3-gamma, psi beta 2-delta, and beta 1-beta were identified as orthologous on the basis of sequence similarities found in flanking and intervening sequences as well as by quantitative divergence calculations. The orthologous genes are in the same order on the chromosome in each species, which suggests that an ancestral family with the arrangement 5'-epsilon-gamma-delta-beta-3' preceded the mammalian radiation. Descendants of ancestral epsilon have diverged more slowly than other beta-like genes and are expressed only in embryonic life. Descendants of ancestral gamma and beta diverged at a higher rate and are expressed at wider range of developmental times. Descendants of delta have undergone nonreciprocal recombination at a high frequency and are often pseudogenes. Paralogous comparisons among the rabbit beta-like globin genes show that the beta 4-beta 3 and psi beta 2-beta 1 pairs are most similar and that beta 4 and beta 3 are more closely related to beta 1 than to psi beta 2. This fits with a branching pattern where the primordial beta split into ancestral epsilon/gamma and delta/beta genes, which later split into epsilon and gamma or delta and beta, respectively. Rabbit genes beta 4 and beta 1 acquired similar 3' untranslated regions after the epsilon/gamma split but prior to the mammalian radiation, presumably via a gene conversion event. The 5' end of beta 2 apparently converted with beta 1 after the radiation, and afterward it became a pseudogene.

Animals↗
PubMed2006

Validation of candidate bovine reference genes for use with real-time PCR.

Accurate quantification with real-time PCR requires the use of stable endogenous controls. Recently, there has been much debate concerning the stability of commonly used reference or housekeeping genes. To address this concern, a number of statistical approaches have been designed to analyse data and assist in determining the most appropriate reference genes for experimental comparisons. In this study, three programs, BestKeeper, Norm Finder, and geNorm were used to assess four candidate reference genes: 18S rRNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), acidic ribosomal protein large (RPLP0) and beta-actin, for use in expression profiling of individuals from divergent cattle genotypes subject to parasitic challenge with the cattle tick Boophilus microplus. Results demonstrated beta-actin and GAPDH were the most suitable reference genes in blood and could be used either individually or combined as an index to normalise data. RPLP0 was identified as the least stable gene, while 18S rRNA was omitted as being too highly expressed. As the recommendations on the most suitable reference genes varied between the programs, it is recommended that more than one should be utilised, to ensure the most robust experimental tools are selected.

Actins↗