Promoter region of the Escherichia coli O7-specific lipopolysaccharide gene cluster:structural and functional characterization of an upstream untranslated mRNA sequence

We report the identification of the promoter region of the Escherichia coli O7-specific lipopolysaccharide (LPS) gene cluster (wbEcO7). Typical -10 and -35 sequences were found to be located in the intervening region between galF and rlmB, the first gene of the wbEcO7 cluster. Data from RNase protection experiments revealed the existence of an untranslated leader mRNA segment of 173 bp, including the JUMPStart and two ops sequences. We characterized the structure of this leader mRNA by using the program Mfold and a combination of nested and internal deletions transcriptionally fused to a promoterless lac operon. Our results indicated that the leader mRNA may fold into a series of complex stem-loop structures, one of which includes the JUMPStart element. We have also found that one of the ops sequences resides on the predicted stem and the other resides on the loop region, and we confirmed that these sequences are essential for the RfaH-mediated regulation of the O polysaccharide cluster. A very similar stem-loop structure could be predicted in the promoter region of the LPS core operon encoding the waaQGPSBIJYZK genes. We observed another predicted stem-loop, located immediately downstream from the wbEcO7 transcription initiation site, which appeared to be involved in premature termination of transcription. This putative stem-loop is common to many other O polysaccharide gene clusters but is not present in core oligosaccharide genes. wbEcO7-lac transcriptional fusions in single copy numbers were also used to determine the effects of various environmental cues in the transcriptional regulation of O polysaccharide synthesis. No effects were detected with temperature, osmolarity, Mg2+ concentration, and drugs inducing changes in DNA supercoiling. We therefore conclude that the wbEcO7 promoter activity may be constitutive and that regulation takes place at the level of elongation of the mRNA in a RfaH-mediated manner.

Genetic analysis of the dTDP-rhamnose biosynthesis region of the Escherichia coli VW187 (O7:K1) rfb gene cluster:identification of functional homologs of rfbB and rfbA in the rff cluster and correct location of the rffE gene

The O-repeating unit of the Escherichia coli O7-specific lipopolysaccharide is made of galactose, mannose, rhamnose, 4-acetamido-4,6-dideoxyglucose, and N-acetyglucosamine. We have recently characterized the genes involved in the biosynthesis of the sugar precursor GDP-mannose occurring in the E. coli O7:K1 strain VW187 (C. L. Marolda and M. A. Valvano, J. Bacteriol. 175:148-158, 1993). In the present study, we identified and sequenced the rfbBDAC genes encoding the enzymes for the biosynthesis of another precursor, dTDP-rhamnose. These genes are localized on the upstream end of the rfbEcO7 region, and they are strongly conserved compared with similar genes found in various enteric and nonenteric bacteria. Upstream of rfbB we identified a DNA segment containing the rfb promoter and a highly conserved untranslated leader sequence also present in the promoter regions of other surface polysaccharide gene clusters. Also, we have determined that rfbB and rfbA have homologs, rffG (o355) and rffH (o292), respectively, located on the rff cluster, which is involved in the synthesis of enterobacterial common antigen. We provide biochemical evidence that rffG and rffH encode dTDP-glucose dehydratase and glucose-1-phosphate thymidylyltransferase activities, respectively, and we also show that rffG complemented the rfbB defect in the O7+ cosmid pJHCV32. We also demonstrate that rffG is distinct from rffE and map the rffE gene to the second gene of the rff cluster.

Construction and biochemical characterization of recombinant cytoplasmic forms of the IucD protein (lysine:N6-hydroxylase) encoded by the pColV-K30 aerobactin gene cluster

The aerobactin gene cluster in pColV-K30 consists of five genes (iucABCD iutA); four of these (iucABCD) are involved in aerobactin biosynthesis, whereas the fifth one (iutA) encodes the ferriaerobactin outer membrane receptor. iucD encodes lysine:N6-hydroxylase, which catalyzes the first step in aerobactin biosynthesis. Regardless of the method used for cell rupture, we have consistently found that IucD remains membrane bound, and repeated efforts to achieve a purified and active soluble form of the enzyme have been unsuccessful. To circumvent this problem, we have constructed recombinant IucD proteins with modified amino termini by creating three in-frame gene fusions of IucD to the amino-terminal amino acids of the cytoplasmic enzyme beta-galactosidase. Two of these constructs resulted in the addition to the iucD coding region of a hydrophilic leader sequence of 13 and 30 amino acids. The other construct involved the deletion of the first 47 amino acids of the IucD amino terminus and the addition of 19 amino acids of the amino terminus of beta-galactosidase. Cells expressing any of the three recombinant IucD forms were found to produce soluble N6-hydroxylysine. One of these proteins, IucD439, was purified to homogeneity from the soluble fraction of the cell lysates, and it was capable of participating in the biosynthesis of aerobactin, as determined in vitro by a cell-free system and in vivo by a cross-feeding bioassay. A medium ionic strength of 0.25 (250 mM NaCl) or higher was required to maintain the protein in a catalytically functional, tetrameric state.(ABSTRACT TRUNCATED AT 250 WORDS)

Small RNA-mediated chromatin silencing directed to the 3' region of the Arabidopsis gene encoding the developmental regulator, FLC.

Small RNA-mediated chromatin silencing is well characterized for repeated sequences and transposons, but its role in regulating single-copy endogenous genes is unclear. We have identified two small RNAs (30 and 24 nucleotides) corresponding to the reverse strand 3' to the canonical poly(A) site of FLOWERING LOCUS C (FLC), an Arabidopsis gene encoding a repressor of flowering. Genome searches suggest that these RNAs originate from the FLC locus in a genomic region lacking repeats. The 24-nt small RNA, which is most abundant in developing fruits, is absent in mutants defective in RNA polymerase IVa, RNA-DEPENDENT RNA POLYMERASE 2, and DICER-LIKE 3, components required for RNAi-mediated chromatin silencing. The corresponding genomic region shows histone 3 lysine 9 dimethylation, which was reduced in a dcl2,3,4 triple mutant. Investigations into the origins of the small RNAs revealed a polymerase IVa-dependent spliced, antisense transcript covering the 3' FLC region. Mutation of this genomic region by T-DNA insertion led to FLC misexpression and delayed flowering, suggesting that RNAi-mediated chromatin modification is an important component of endogenous pathways that function to suppress FLC expression.

Chick heart/hemangioblast progenitor 12 (chep 12: study of a novel gene expressed in the heart/hemangioblast percursors cells

Dissertação de mest., Ciências Biomédicas, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2009

Adenovirus-based exogenous gene expression in mammalian cells

Thesis (Ph.D.)--Brock University, 2010.

Cardiac myocyte gene expression profiling during H2O2-induced apoptosis

High levels of oxidative stress promote cardiac myocyte death, though lower levels are potentially cytoprotective/anabolic. We examined the changes in gene expression in rat neonatal cardiac myocytes exposed to apoptotic (0.2 mM) or nontoxic (0.04 mM) concentrations of H2O2 (2, 4, or 24 h) using Affymetrix microarrays. Using U34B arrays, we identified a ubiquitously expressed, novel H2O2-responsive gene [putative peroxide-inducible transcript 1 (Perit1)], which generates two alternatively spliced transcripts. Using 230 2.0 arrays, H2O2 (0.04 mM) promoted significant changes in expression of only 32 genes, all of which were seen with 0.2 mM H2O2. We failed to detect any increase in the rate of protein synthesis in cardiac myocytes exposed to <0.1 mM H2O2, further suggesting that global, low concentrations of H2O2 are not anabolic in this system. H2O2 (0.2 mM) promoted significant (P < 0.05, >1.75-fold) changes in expression of 649 mRNAs and 187 RNAs corresponding to no established gene. Of the mRNAs, 114 encoded transcriptional regulators including Krüppel-like factors (Klfs). Quantitative PCR independently verified the changes in Klf expression. Thus, H2O2-induced cardiac myocyte apoptosis is associated with dynamic changes in gene expression. The expression of these genes and their protein products potentially influences the progression of the apoptotic response.

Splice variants of the human ZC3H14 gene generate multiple isoforms of a zinc finger polyadenosine RNA binding protein

The human ZC3H14 gene encodes an evolutionarily conserved Cys(3)His zinc finger protein that binds specifically to polyadenosine RNA and is thus postulated to modulate post-transcriptional gene expression. Expressed sequence tag (EST) data predicts multiple splice variants of both human and mouse ZC3H14. Analysis of ZC3H14 expression in both human cell lines and mouse tissues confirms the presence of multiple alternatively spliced transcripts. Although all of these transcripts encode protein isoforms that contain the conserved C-terminal zinc finger domain, suggesting that they could all bind to polyadenosine RNA, they differ in other functionally important domains. Most of the alternative transcripts encode closely related proteins (termed isoforms 1, 2. 3, and 3short) that differ primarily in the inclusion of three small exons, 9, 10, and 11, resulting in predicted protein isoforms ranging from 82 to 64 kDa. Each of these closely related isoforms contains predicted classical nuclear localization signals (cNLS) within exons 7 and 11. Consistent with the presence of these putative nuclear targeting signals, these ZC3H14 isoforms are all localized to the nucleus. In contrast, an additional transcript encodes a smaller protein (34 kDa) with an alternative first exon (isoform, 4). Consistent with the absence of the predicted cNLS motifs located in exons 7 and 11, ZC3H14 isoform 4 is localized to the cytoplasm. Both EST data and experimental data suggest that this variant is enriched in testes and brain. Using an antibody that detects endogenous ZC3H14 isoforms 1-3 reveals localization of these isoforms to nuclear speckles. These speckles co-localize with the splicing factor, SC35, suggesting a role for nuclear ZC3H14 in mRNA processing. Taken together, these results demonstrate that multiple transcripts encoding several ZC3H14 isoforms exist in vivo. Both nuclear and cytoplasmic ZC3H14 isoforms could have distinct effects on gene expression mediated by the common Cys(3)His zinc finger polyadenosine RNA binding domain. (C) 2009 Elsevier B.V. All rights reserved.

Association of CSSM066 and ILSTS011 microsatellite markers and thyroglobulin gene SNP with backfat in Canchim cattle

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Sequence and Copy Number Analyses of HEXB Gene in Patients Affected by Sandhoff Disease: Functional Characterization of 9 Novel Sequence Variants

Sandhoff disease (SD) is a lysosomal disorder caused by mutations in the HEXB gene. To date, 43 mutations of HEXB have been described, including 3 large deletions. Here, we have characterized 14 unrelated SD patients and developed a Multiplex Ligation-dependent Probe Amplification (MLPA) assay to investigate the presence of large HEXB deletions. Overall, we identified 16 alleles, 9 of which were novel, including 4 sequence variation leading to aminoacid changes [c.626C>T (p.T209I), c.634C>A (p.H212N), c.926G>T (p.C309F), c.1451G>A (p.G484E)] 3 intronic mutations (c.1082+5G>A, c.1242+1G>A, c.1169+5G>A), 1 nonsense mutation c.146C>A (p.S49X) and 1 small in-frame deletion c.1260_1265delAGTTGA (p.V421_E422del). Using the new MLPA assay, 2 previously described deletions were identified. In vitro expression studies showed that proteins bearing aminoacid changes p.T209I and p.G484E presented a very low or absent activity, while proteins bearing the p.H212N and p.C309F changes retained a significant residual activity. The detrimental effect of the 3 novel intronic mutations on the HEXB mRNA processing was demonstrated using a minigene assay. Unprecedentedly, minigene studies revealed the presence of a novel alternative spliced HEXB mRNA variant also present in normal cells. In conclusion, we provided new insights into the molecular basis of SD and validated an MLPA assay for detecting large HEXB deletions.

The gene transformer-2 of Anastrepha fruit flies (Diptera, Tephritidae) and its evolution in insects

Abstract Background In the tephritids Ceratitis, Bactrocera and Anastrepha, the gene transformer provides the memory device for sex determination via its auto-regulation; only in females is functional Tra protein produced. To date, the isolation and characterisation of the gene transformer-2 in the tephritids has only been undertaken in Ceratitis, and it has been shown that its function is required for the female-specific splicing of doublesex and transformer pre-mRNA. It therefore participates in transformer auto-regulatory function. In this work, the characterisation of this gene in eleven tephritid species belonging to the less extensively analysed genus Anastrepha was undertaken in order to throw light on the evolution of transformer-2. Results The gene transformer-2 produces a protein of 249 amino acids in both sexes, which shows the features of the SR protein family. No significant partially spliced mRNA isoform specific to the male germ line was detected, unlike in Drosophila. It is transcribed in both sexes during development and in adult life, in both the soma and germ line. The injection of Anastrepha transformer-2 dsRNA into Anastrepha embryos caused a change in the splicing pattern of the endogenous transformer and doublesex pre-mRNA of XX females from the female to the male mode. Consequently, these XX females were transformed into pseudomales. The comparison of the eleven Anastrepha Transformer-2 proteins among themselves, and with the Transformer-2 proteins of other insects, suggests the existence of negative selection acting at the protein level to maintain Transformer-2 structural features. Conclusions These results indicate that transformer-2 is required for sex determination in Anastrepha through its participation in the female-specific splicing of transformer and doublesex pre-mRNAs. It is therefore needed for the auto-regulation of the gene transformer. Thus, the transformer/transfomer-2 > doublesex elements at the bottom of the cascade, and their relationships, probably represent the ancestral state (which still exists in the Tephritidae, Calliphoridae and Muscidae lineages) of the extant cascade found in the Drosophilidae lineage (in which tra is just another component of the sex determination gene cascade regulated by Sex-lethal). In the phylogenetic lineage that gave rise to the drosophilids, evolution co-opted for Sex-lethal, modified it, and converted it into the key gene controlling sex determination.

Cloning and characterization of a novel alternatively spliced transcript of the human CHD7 putative helicase

Abstract Background The CHD7 (Chromodomain Helicase DNA binding protein 7) gene encodes a member of the chromodomain family of ATP-dependent chromatin remodeling enzymes. Mutations in the CHD7 gene are found in individuals with CHARGE, a syndrome characterized by multiple birth malformations in several tissues. CHD7 was identified as a binding partner of PBAF complex (Polybromo and BRG Associated Factor containing complex) playing a central role in the transcriptional reprogramming process associated to the formation of multipotent migratory neural crest, a transient cell population associated with the genesis of various tissues. CHD7 is a large gene containing 38 annotated exons and spanning 200 kb of genomic sequence. Although genes containing such number of exons are expected to have several alternative transcripts, there are very few evidences of alternative transcripts associated to CHD7 to date indicating that alternative splicing associated to this gene is poorly characterized. Findings Here, we report the cloning and characterization by experimental and computational studies of a novel alternative transcript of the human CHD7 (named CHD7 CRA_e), which lacks most of its coding exons. We confirmed by overexpression of CHD7 CRA_e alternative transcript that it is translated into a protein isoform lacking most of the domains displayed by the canonical isoform. Expression of the CHD7 CRA_e transcript was detected in normal liver, in addition to the DU145 human prostate carcinoma cell line from which it was originally isolated. Conclusions Our findings indicate that the splicing event associated to the CHD7 CRA_e alternative transcript is functional. The characterization of the CHD7 CRA_e novel isoform presented here not only sets the basis for more detailed functional studies of this isoform, but, also, contributes to the alternative splicing annotation of the CHD7 gene and the design of future functional studies aimed at the elucidation of the molecular functions of its gene products.

Vergleichende Analyse der Gene und der Genomstruktur der geschlechtsbestimmenden Chromosomenregion von Chironomus und Camptochironomus

Im Rahmen der vorliegenden Arbeit wurde ein Bereich aus der geschlechtsbestimmenden Chromosomenregion, der „Contig SDR“, von C. tentans mit einer Größe von ~87 kb untersucht. Zur Erstellung des Contigs wurden 8 BAC-Klone aus C. tentans isoliert, teilweise subkloniert und sequenziert. Innerhalb des Contigs SDR konnten insgesamt 13 Gene sowie ein Teilbereich des Gens rpS5-like im distalen Bereich des Contigs SDR identifiziert werden. Hierbei handelt es sich um dieselben Gene, welche schon im Contig SDR von C. thummi identifiziert werden konnten. Ein Vergleich der beiden Contigs zeigt, dass die Abfolge der Gene zwischen den beiden Arten C. thummi und C. tentans identisch ist. Weiterhin konnten im Contig SDR von C. tentans sechs Bereiche lokalisiert werden, in denen repetitive oder transposable Elemente zu finden sind. Ein Vergleich der larvalen Transkripte (L4-Stadium) von 11 Genen des Contigs SDR aus C. thummi ♂ und C. thummi ♀ per RT-PCR und Hochdurchsatz-Sequenzierung zeigte mit Ausnahme der Gene luc7(p)-like sowie fs(1)K10-like bislang keine weiteren geschlechtsspezifischen Unterschiede. Im Gen luc7(p)-like konnte in C. thummi ♀ und C. piger ♀ ein alternativ gespleißtes Intron im eigentlichen Exon 2 identifiziert werden. Bei fs(1)K10-like konnte in C. thummi ♂ eine Duplikation des Gens nachgewiesen werden. Weiterhin wurden mit Hilfe des RACE-Verfahrens die 5’UTR- und 3’UTR-Bereiche der Transkripte analysiert. Hierdurch konnten differentielle Spleißprodukte identifiziert werden, welche jedoch nicht geschlechtsspezifisch auftreten. Die bioinformatische Bearbeitung der von den Genen der SDR kodierten Proteine auf konservierte Domänen zeigt vier Proteine, die möglicherweise als Transkriptions- oder Spleißfaktoren wirken können. Hierbei handelt es sich um die Proteine der Gene mi-er1-like, luc7(p)-like, polyhomeotic-like sowie rpn5-like. Für das auf Grund der männchenspezifischen Duplikation in C. thummi in den Fokus geratene Genprodukt von fs(1)K10-like konnten keine Domänen vorhergesagt werden. Somit kann nicht gesagt werden, ob das Protein im Rahmen der Geschlechtsdetermination eine Funktion haben könnte. Die Sequenzidentität der abgeleiteten AS-Sequenz liegt für alle Gene außer mi-er1-like (87,6 %) zwischen den beiden Arten C. tentans und C. thummi bei 93,3 %.

Mutations within the FGF5 gene are associated with hair length in cats

Hereditary hair length variability in mice and dogs is caused by mutations within the fibroblast growth factor 5 (FGF5) gene. The aim of this study was to evaluate the feline FGF5 orthologue as a functional candidate gene for the long hair phenotype in cats, which is recessive to short hair. We amplified the feline FGF5 cDNA and characterised two alternatively spliced transcripts by RT-PCR. Comparative cDNA and genomic DNA sequencing of long- and short-haired cats revealed four non-synonymous polymorphisms in the FGF5 coding sequence. A missense mutation (AM412646:c.194C>A) was found in the homozygous state in 25 long-haired Somali, Persian, Maine Coon, Ragdoll and crossbred cats. Fifty-five short-haired cats had zero or one copy of this allele. Additionally, we found perfect co-segregation of the c.194C>A mutation within two independent pedigrees segregating for hair length. A second FGF5 exon 1 missense mutation (AM412646:c.182T>A) was found exclusively in long-haired Norwegian Forest cats. The c.182T>A mutation probably represents a second FGF5 mutation responsible for long hair in cats. In addition to the c.194C>A mutation, a frameshift mutation (AM412646:c.474delT) was found with a high frequency in the long-haired Maine Coon breed. Finally, a missense mutation (AM412646:c.475A>C) was also associated with the long-haired phenotype in some breeds. However, as one short-haired cat was homozygous for this polymorphism, it is unlikely that it has a functional role in the determination of hair length.

Sendai virus RNA polymerase scanning for mRNA start sites at gene junctions

Mini-genomes expressing two reporter genes and a variable gene junction were used to study Sendai virus RNA polymerase (RdRp) scanning for the mRNA start signal of the downstream gene (gs2). We found that RdRp could scan the template efficiently as long as the initiating uridylate of gs2 (3' UCCCnnUUUC) was preceded by the conserved intergenic region (3' GAA) and the last 3 uridylates of the upstream gene end signal (ge1; 3' AUUCUUUUU). The end of the leader sequence (3' CUAAAA, which precedes gs1) could also be used for gene2 expression, but this sequence was considerably less efficient. Increasing the distance between ge1 and gs2 (up to 200 nt) led to the progressive loss of gene2 expression, in which half of gene2 expression was lost for each 70 nucleotides of intervening sequence. Beyond 200 nt, gene2 expression was lost more slowly. Our results suggest that there may be two populations of RdRp that scan at gene junctions, which can be distinguished by the efficiency with which they can scan the genome template for gs.