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PubMed2006

Universal external RNA controls for microbial gene expression analysis using microarray and qRT-PCR.

Gene expression analysis provides significant insight to understand regulatory mechanisms of biology, yet acquisition and reproduction of quality data, as well as data confirmation and verification remain challenging due to a lack of proper quality controls across different assay platforms. We present a set of six universal external RNA quality controls for microbial mRNA expression analysis that can be applied to both DNA oligo microarray and real-time qRT-PCR including using SYBR Green and TaqMan probe-based chemistry. This set of controls was applied for Saccharomyces cerevisiae and Pseudomonas fluorescens Pf-5 microarray assays and qRT-PCR for yeast gene expression analysis. Highly fitted linear relationships between detected signal intensity and mRNA input were described. Valid mRNA detection range, from 10 to 7000 pg and from 100 fg to 1000 pg were defined for microarray and qRT-PCR assay, respectively. Quantitative estimation of mRNA abundance was tested using randomly selected yeast ORF including function unknown genes using the same source of samples by the two assay platforms. Estimates of mRNA abundance by the two methods were similar and highly correlated in an overlapping detection range from 10 to 1000 pg. The universal external RNA controls provide a means to compare microbial gene expression data derived from different experiments and different platforms for verification and confirmation. Such quality controls ensure reliability and reproducibility of gene expression data, and provide unbiased normalization reference for validation, quantification, and estimate of variation of gene expression experiments. Application of these controls also improves efficiency and facilitates high throughput applications of gene expression analysis using the qRT-PCR assay.

Animals↗

PubMed1996

Transcriptional analysis of the candidate region for incontinentia pigmenti (IP2) in Xq28.

The hereditary form of incontinentia pigmenti (IP2) is a rare disorder characterized by abnormalities of the tissues and organs derived from the ectoderm and neuroectoderm and has been linked to Xq28 distal to the factor VIII gene (F8C). Four YAC clones covering the 1.1-Mb candidate region at the telomere of Xq28 were subjected to direct cDNA selection and Alu long-range PCR. The products of both methods were subsequently used to isolate 154 cosmid clones that were assembled into five cosmid contigs. This first-generation cosmid map covered the region almost entirely and was used as a basis for constructing a transcript map that was in turn integrated with the physical YAC and cosmid maps. To isolate specifically coding sequences, exon trapping and cDNA selection methods were combined. Exon trapping was carried out on YAC Alu-PCR products, YAC Alu long-range PCR products, and on pools of cosmids. The region-specific enriched cDNA library was then screened by using the exon trap products as complex probes. To ensure a more complete analysis, the products from cDNA selection experiments were also used to screen conventional oligo(dT) primed cDNA libraries. Twenty overlapping cDNA contigs were assembled and computer analyses were performed to identify EST hits, open reading frames, protein motifs, and protein sequence homologies. Five of the cDNA contigs corresponded to known sequences such as the factor VIII, c6.1A, and c6.1B. genes, and both distal copies of the factor VIII intron 22 repeat sequence. Expression patterns of the 15 new cDNA contigs were analyzed by Northern blot and RT-PCR studies and these data were integrated with expression data obtained from known EST sequences. Although a more detailed analysis of this 1.1-Mb region with respect to the structure and function of the genes will only ultimately be possible by a global sequencing approach, an analysis of all novel transcripts as candidate genes for incontinentia pigmenti is already in progress.

Adult↗

PubMed2006

Anchoring of a large set of markers onto a BAC library for the development of a draft physical map of the grapevine genome.

Five hundred and six EST-derived markers, 313 SSR markers and 26 BAC end-derived or SCAR markers were anchored by PCR on a subset of a Cabernet Sauvignon BAC library representing six genome equivalents pooled in three dimensions. In parallel, the 12,351 EST clusters of the grapevine UniGene set (build #11) from NCBI were used to design 12,125 primers pairs and perform electronic PCR on 67,543 nonredundant BAC-end sequences. This in silico experiment yielded 1,140 positive results concerning 638 different markers, among which 602 had not been already anchored by PCR. The data obtained will provide an easier access to the regulatory sequences surrounding important genes (represented by ESTs). In total, 1,731 islands of BAC clones (set of overlapping BAC clones containing at least one common marker) were obtained and 226 of them contained at least one genetically mapped anchor. These assigned islands are very useful because they will link the genetic map and the future fingerprint-based physical map and because they allowed us to indirectly place 93 ESTs on the genetic map. The islands containing two or more mapped SSR markers were also used to assess the quality of the integrated genetic map of the grapevine genome.

Chromosomes, Artificial, Bacterial↗

PubMed2004

The Aeromonas hydrophila LuxR homologue AhyR regulates the N-acyl homoserine lactone synthase, AhyI positively and negatively in a growth phase-dependent manner.

Aeromonas hydrophila is a pathogen of fish, amphibians and humans which produces N-acylhomoserine lactone quorum sensing signal molecules and possesses homologues of the Vibrio fischeri luxI and luxR quorum sensing genes termed ahyI and ahyR, respectively. The ahyI and ahyR genes of A. hydrophila comprise a divergon with a 62 bp intergenic region and control biofilm maturation and extracellular protease production. Stationary phase culture supernatants from an ahyR but not an ahyI mutant contain N-butanoylhomoserine lactone (C4-HSL) which is shown to be required for maximal ahyI expression. To determine whether AhyR regulates ahyI, the expression of AhyI was followed throughout growth by Western blot analysis. This revealed that AhyI can be detected in the exponential phase but appears to be degraded in stationary phase in the parent A. hydrophila strain. In an ahyR mutant however, the AhyI protein is only produced in stationary phase but production is sustained suggesting that AhyR controls the timing of AhyI production and turnover. By using RT-PCR, we mapped the transcriptional start site of ahyI which revealed that the 12 bp symmetrical lux-box like sequence present in the 62 bp ahyRI intergenic region overlaps with the -10 region of the ahyI promoter. To determine whether AhyR could bind to the ahyRI intergenic region, the ahyR gene was expressed and purified as a maltose binding protein (MalE) fusion. Electrophoretic mobility shift assays demonstrated that MalE-AhyR specifically bound to this sequence in both the presence and absence of N-butanoylhomoserine lactone (C4-HSL). Taken together, these data suggest that AhyR acts as both a negative and a positive regulator of ahyI and hence C4-HSL production in a growth phase dependent manner.

4-Butyrolactone↗

PubMedDate not supplied

A complete protein truncation test for BRCA1 and BRCA2.

The protein truncation test (PTT) is currently the fastest method in general use for detecting previously unidentified mutations in tumor suppressor genes. Greater than three kilobases of coding sequence can be screened by one PCR reaction, one coupled in vitro transcription/translation reaction, and one lane on an SDS-PAGE gel. The 16 kb of BRCA1/2 coding sequence can be screened with nine overlapping segments. Since 90% of BRCA1/2 mutations result in a truncated protein product, the theoretical false negative rate for a BRCA1/2 PTT screen should be 10%. In practice the false negative rate is much higher, especially when cDNA is used as template. However, the actual false negative rate for a given screen will depend on the details of how the test is performed. Design of the overlapping segments, gel parameters, and nonsense mediated mRNA decay can all influence the effectiveness of the screen. BRCA1/2 screening by PTT can be optimised by considering these variables. Furthermore, nonsense-mediated mRNA decay can be inhibited by blocking protein synthesis with cycloheximide.

Artifacts↗

PubMed2002

Foxa3 (HNF-3gamma) binds to and activates the rat proglucagon gene promoter but is not essential for proglucagon gene expression.

Members of the Forkhead box a (Foxa) transcription factor family are expressed in the liver, pancreatic islets and intestine and both Foxa1 and Foxa2 regulate proglucagon gene transcription. As Foxa proteins exhibit overlapping DNA-binding specificities, we examined the role of Foxa3 [hepatocyte nuclear factor (HNF)-3gamma] in control of proglucagon gene expression. Foxa3 was detected by reverse transcriptase PCR in glucagon-producing cell lines and binds to the rat proglucagon gene G2 promoter element in GLUTag enteroendocrine cells. Although Foxa3 increased rat proglucagon promoter activity in BHK fibroblasts, augmentation of Foxa3 expression did not increase proglucagon promoter activity in GLUTag cells. Furthermore, adenoviral Foxa3 expression did not affect endogenous proglucagon gene expression in islet or intestinal endocrine cell lines. Although Foxa3(-/-) mice exhibit mild hypoglycaemia during a prolonged fast, the levels of proglucagon-derived peptides and proglucagon mRNA transcripts were comparable in tissues from wild-type and Foxa3(-/-) mice. These findings identify Foxa3 as a member of the proglucagon gene G2 element binding-protein family that, unlike Foxa1, is not essential for control of islet or intestinal proglucagon gene expression in vivo.

Animals↗

PubMed2005

A computational and experimental approach to validating annotations and gene predictions in the Drosophila melanogaster genome.

Five years after the completion of the sequence of the Drosophila melanogaster genome, the number of protein-coding genes it contains remains a matter of debate; the number of computational gene predictions greatly exceeds the number of validated gene annotations. We have assembled a collection of >10,000 gene predictions that do not overlap existing gene annotations and have developed a process for their validation that allows us to efficiently prioritize and experimentally validate predictions from various sources by sequencing RT-PCR products to confirm gene structures. Our data provide experimental evidence for 122 protein-coding genes. Our analyses suggest that the entire collection of predictions contains only approximately 700 additional protein-coding genes. Although we cannot rule out the discovery of genes with unusual features that make them refractory to existing methods, our results suggest that the D. melanogaster genome contains approximately 14,000 protein-coding genes.

Animals↗

PubMed1994

Scallop striated and smooth muscle myosin heavy-chain isoforms are produced by alternative RNA splicing from a single gene.

We report here that the catch and striated adductor muscle myosin heavy-chain (MHC) isoforms of scallop (Argopecten irradians, previously Aequipecten irradians) are generated by alternative RNA splicing from a single gene. Scallop catch muscle cDNA and genomic DNA were amplified by PCR using primers based on the previously sequenced scallop striated muscle MHC cDNA. Mapping of the exon/intron borders and sequencing of a full-length catch muscle MHC in overlapping fragments revealed that the 24-kb gene encodes the MHC polypeptide in 27 exons and that four sets of tandem exon pairs are alternatively spliced into a striated and a catch MHC isoform. An additional alternative exon was identified in catch cDNA and is apparently spliced into a minor MHC isoform. The striated muscle-specific isoform is not expressed in other tissues, whereas the catch-type isoforms were also detected in various smooth muscles, but not in the striated one. Of the alternative exons, exons 5 and 6 encode part of the ATP-binding region and the 25-kDa/50-kDa proteolytic junction; exon 13 encodes part of one of the actin-binding regions and extends to the active site; exon 20 encodes the middle of the rod hinge region; exon 26 in the striated-specific sequence starts with the stop codon, whereas the catch-specific exon codes for an additional 10 residues. Differences between the alternative exons presumably determine the lower ATPase activity of smooth muscle myosin, contribute to the different structure of the striated and smooth muscle thick filaments, and may also be important for the molecular mechanism of the catch phenomenon.

Alternative Splicing↗

PubMed1998

Determination of gene organization in individual haplotypes by analyzing single DNA fragments from single spermatozoa.

To determine human Ig heavy chain variable region (VH) gene segment organization on individual homologous chromosomes, an efficient approach has been developed. Single spermatozoa were used as subjects for the study. Upon sperm lysis, VH regions in each sperm were randomly sheared into fragments by the random Brownian force. The fragments were separated from each other by aliquoting the lysate into a certain number of tubes. The gene segments in the VH1 and VH4 families in each tube were identified by denaturing gradient gel electrophoresis after PCR amplification. The polymorphic VH sequences were used to determine the parental origins of the analyzed sperm. VH segment organization in the parental haplotypes was determined by aligning the overlapping fragments from the spermatozoa with the corresponding haplotypes. Based on this comparison between the resulting haplotype maps and the composite map reported previously, the VH region on chromosome 14 could be subdivided into four portions. The numbers and compositions of the VH gene segments differ considerably among the maps in two portions, but are highly conserved in the other two. The data also indicate that the VH region on chromosome 15 may contain a large duplicated block with copy number varying among haplotypes. The approach used in the present study may be used to construct high-resolution haplotype maps without molecular cloning.

Chromosomes, Human, Pair 14↗

PubMed1998

Identification of scaffold/matrix attachment region in recurrent site of woodchuck hepatitis virus integration.

Scaffold or matrix attachment regions (S/MARs) are noncoding genomic DNA sequences displaying in vitro selective binding affinity for nuclear scaffold. They have been reported to be involved in the physical attachment of genomic DNA to the nuclear scaffold, and thus in the organization of the chromatin in functional loops or domains, and in the regulation of gene expression. In this work, we report the identification of an S/MAR in a woodchuck chromosomal locus, named b3n, previously described as a recurrent site of woodchuck hepatitis virus (WHV) DNA integration in woodchuck hepatocellular carcinoma (HCC). The 4.3-kb sequence of this locus contains several Alu-like repeats and a gag-like coding region with frameshift mutations. Computer analysis revealed the presence of a region with unusually high AT content, typical of most S/MARs, and of specific motifs (A boxes, T boxes, topoisomerase II sites, and unwinding elements) overlapping or in proximity to the region with high AT content, predicting that b3n might contain an S/MAR. Fragments of the b3n locus were isolated by conventional and inverse PCR techniques. In in vitro binding experiments with both heterologous and autologous scaffold preparations, a 592-bp fragment spanning the region rich in S/MAR features showed marked scaffold affinity, which was specific when autologous scaffolds were used. The presence of an S/MAR at the b3n locus and its nature as a recurrent WHV integration site in HCC suggest the involvement of S/MAR elements in some of the mechanisms leading to liver oncogenesis.

Amino Acid Sequence↗

PubMed1993

Functional imprinting and epigenetic modification of the human SNRPN gene.

The SNRPN gene encodes a small nuclear ribonucleoprotein subunit, SmN, thought to be involved in splicing of pre-mRNA. A closely related protein, SmB/B', is constitutively expressed in all tissues except the brain, where SmN is predominantly expressed. The mouse homolog of the SNRPN gene has been shown to be functionally imprinted in mouse brain, being expressed only from the paternally derived chromosome. SNRPN has been mapped to human chromosome 15q11-q13 within the shortest region of deletion overlap for the Prader-Willi syndrome. We have now demonstrated functional imprinting of the human SNRPN gene using reverse transcription followed by the polymerase chain reaction (RT-PCR). No expression was observed in cultured skin fibroblasts of Prader-Willi patients, but was found in all Angelman patients and normal controls examined. We have also demonstrated a parent-specific DNA methylation imprint within intron 5 of the SNRPN gene, which suggests an epigenetic mechanism by which parent-specific expression of this gene might be inherited. Our findings indicate that SNRPN is expressed only from the paternally derived chromosome 15 in humans and therefore may fulfill one major criterion for being involved in the pathogenesis of the Prader-Willi syndrome.

Angelman Syndrome↗

PubMed1999

New method for automatic identification identification and typing of single and multiple superimposed human papillomavirus sequences.

This study of specimens of human papillomaviruses (HPV) through HPV-specific polymerase chain reaction (PCR), followed by direct sequencing, resulted in 11% (38/354) superimposed HPV sequences, signifying coinfection with more than one HPV type. To address the diagnostic problem that these superimposed ("degenerated," overlapping) sequences pose, the authors created a papillomavirus database in Microsoft Excel (Microsoft Corporation, Redmond, WA, U.S.A.) and Corel Quattro Pro 9 (Corel Corporation, Ottawa, Ontario, Canada) formats, retrievable from http://www2.crosswinds.net/ -crosswindswatson/index.html. This sequence database is simultaneously a search and comparison tool for quick (several seconds) typing of HPV from regular and "degenerated" sequencing results. Some of the advantages of the method are as follows: (1) superimposed HPV sequences that differ in length could be readily identified from a single input; (2) the search is restricted to the currently known 127 PV types, which speeds up the typing; (3) the most common HPV sequencing artifacts are included for quick detection; (4) there is no proprietary code and the database could be easily improved; (5) HPV sequence identification does not require internet connection; and (6) new HPV types could be easily detected. This method allowed resolution of all but 1 of 354 HPV-positive specimens. From 38 superimposed HPV sequences, this method identified one known HPV type (3 specimens), two HPV types (30 specimens) and three HPV types (4 specimens).

DNA Primers↗

PubMed2006

Comparison of polymerase chain reaction systems for detection of different cdt genes in Escherichia coli strains.

AIMS: Cytolethal distending toxins (CDT) are tripartite toxins encoded by three adjacent or overlapping genes (cdtA, cdtB, cdtC) and found in multiple pathogens. The present knowledge regarding heterogeneity of cdt genes and our previous study revealed that the available polymerase chain reaction (PCR) systems lack adequate specificity. The detection of various cdt genes present in Escherichia coli strains, from different geographical regions demands further assays for wide-range coverage. On the basis of these observations, we were prompted to undertake the present study; hence the specificity of existing PCR systems was addressed using E. coli prototype strains with known cdt gene sequences. METHODS AND RESULTS: A multiplex PCR designed for the detection of E. coli cdt genes was found to be sensitive and specific enough for initial screening. However, for subtyping, the PCR systems yielded nonspecific products upon amplification. These primers are usually designed for sequences of the cdtB locus (the most conserved region of the gene), and since CDT-producing E. coli strains carry different cdt genes, none of the systems are really type specific. Furthermore, PCR systems with type-specific primers for other regions of the gene, i.e. ORF A or ORF C are found to be strain specific and their applications in different geographical regions have limitations. CONCLUSIONS: In conclusion, based on our observations, using these available primers, it seems that the existing PCR systems are not sufficiently accurate to differentiate between different types of cdt genes. SIGNIFICANCE AND IMPACT OF THE STUDY: The results obtained from this study revealed that so-far reported PCR systems are short in specificity. These PCR protocols were not found to be specific enough to detect various cdt genes and have a limited range of application. Moreover, due to similarities in cdt genes the cross-reaction between different sets of primers exists. Hence for epidemiological studies, some additional PCR protocols are required for screening clinical isolates for cdt genes.

Bacterial Toxins↗

PubMed2003

Comparative DNA microarray analysis of host cell transcriptional responses to infection by Coxiella burnetii or Chlamydia trachomatis.

DNA microarray analysis was conducted to investigate the transcriptional responses of the human monocytic leukemia cell line THP-1 to infection by Chlamydia trachomatis or Coxiella burnetii. RNA was isolated from mock infected cells and cells infected for 36 hours using TRIzol reagent. Biotinylated probes synthesized from RNA samples were hybridized to an Affymetrix U133A human genome chip consisting of 18,462 human gene probe sets. A total of 335 and 548 THP-1 genes were up- or downregulated at least twofold in cells infected with C. burnetii or C. trachomatis, respectively, when compared to uninfected cells. There was a high degree of overlap in transcriptional responses to infection with shared responses observed for 39 downregulated and 189 upregulated genes. Numerous pathogen-specific transcriptional responses were also observed. Quantitative RT-PCR and immunoblotting confirmed up- or down-regulation of a subset of THP-1 genes in response to infection by C. burnetii. This study provides insight into the host transcriptional responses to infection by Chlamydia and Coxiella that may affect the infectious cycle of each organism.

Base Sequence↗

PubMed1995

Mapping of the delayed-type hypersensitivity-inducing epitope of secreted protein MPT64 from Mycobacterium tuberculosis.

The gene encoding the immunogenic protein MPT64 found in culture filtrates of Mycobacterium tuberculosis H37Rv was expressed in Escherichia coli K-12 and purified as a recombinant protein. The purified recombinant MPT64 elicited delayed-type hypersensitivity (DTH) in outbred guinea pigs sensitized with Mycobacterium bovis BCG Tokyo. The skin reactions were comparable to those obtained with native MPT64. No skin reactions were observed when either recombinant MPT64 or native MPT64 was used in guinea pigs sensitized with M. bovis BCG Danish 1331. Amino- and carboxy-terminal deletion mutants of MPT64 were purified as fusion proteins for the mapping of DTH-inducing epitopes on recombinant MPT64 by use of the guinea pig skin test model. The part of the molecule responsible for the biological activity was located at the carboxy-terminal end. Further studies with overlapping synthetic peptides have pinpointed the biological activity at a single DTH-inducing epitope consisting of 15 residues between amino acids Gly-173 and Ala-187. Screening by PCR of 56 clinical isolates of M. tuberculosis from Danish and Tanzanian patients demonstrated the presence of mpt64 in all of the strains. These results point to MPT64 as a possible candidate for a skin test reagent specific for diagnosis of human tuberculosis.

Amino Acid Sequence↗

PubMed1998

The nucleotide sequence and spliced pol mRNA levels of the nonprimate spumavirus bovine foamy virus.

We have determined the complete nucleotide sequence of a replication-competent clone of bovine foamy virus (BFV) and have quantitated the amount of splice pol mRNA processed early in infection. The 544-amino-acid Gag protein precursor has little sequence similarity with its primate foamy virus homologs, but the putative nucleocapsid (NC) protein, like the primate NCs, contains the three glycine-arginine-rich regions that are postulated to bind genomic RNA during virion assembly. The BFV gag and pol open reading frames overlap, with pro and pol in the same translational frame. As with the human foamy virus (HFV) and feline foamy virus, we have detected a spliced pol mRNA by PCR. Quantitatively, this mRNA approximates the level of full-length genomic RNA early in infection. The integrase (IN) domain of reverse transcriptase does not contain the canonical HH-CC zinc finger motif present in all characterized retroviral INs, but it does contain a nearby histidine residue that could conceivably participate as a member of the zinc finger. The env gene encodes a protein that is over 40% identical in sequence to the HFV Env. By comparison, the Gag precursor of BFV is predicted to be only 28% identical to the HFV protein.

Alternative Splicing↗

PubMed2006

Molecular cloning and differential expression of three GnRH mRNAs in discrete brain areas and lymphocytes in red drum.

Three gonadotropin releasing hormones, seabream GnRH (GnRH-I), chicken GnRH-II (GnRH-II) and salmon GnRH (GnRH-III) cDNAs were isolated from the brain of the red drum, Sciaenops ocellatus. The GnRH cDNA sequences of red drum showed more similarity to those of Atlantic croaker, sea bass and sea bream. The real-time quantitative RT-PCR study revealed expression of GnRH-I and GnRH III mRNAs in the olfactory bulb plus telencephalon (OB+TEL), and preoptic-anterior hypothalamic area (POAH), indicating an overlap of the GnRH-I and GnRH-III systems in these forebrain regions. However, GnRH-II mRNA expression was detected only in the midbrain tegmentum (MT). The GnRH-I mRNA expression in the POAH in fish undergoing gonadal recrudescence was significantly higher than that in gonadally immature individuals, suggesting involvement of the POAH GnRH-I in gonadal maturation. On the other hand, GnRH-III mRNA expression was significantly higher in the OB+TEL region compared with that in the POAH. Moreover, the demonstration of GnRH-I mRNA and peptide expression in red drum lymphocytes indicates that the GnRH-I is synthesized in these cells of the immune system similar to the situation in mammals.

Animals↗

PubMed2002

Regulation and specificity of MHC2TA promoter usage in human primary T lymphocytes and cell line.

Although activated human T cells express MHC class II antigens, the regulation of these antigens in T cells is poorly understood. This study focuses on the control of the MHC2TA gene in these cells. MHC2TA encodes the transcriptional master regulator of MHC class II, the class II trans-activator (CIITA). It has at least three distinct promoters (PI, PIII, and PIV), each active in an overlapping subset of cell types and directing a slightly different product. This report used highly purified blood T cells prepared by negative immunoselection to analyze CIITA. Real-time PCR analysis indicates that resting T cells do not express detectable CIITA transcript, while activated T cells express the PIII CIITA form. Transient transfection of activated blood T cells using wild-type and mutant PIII promoter-reporter constructs shows that two promoter elements, activation response element-1 (ARE-1) and ARE-2, are important for PIII function. cAMP response element binding protein, a known activator of gene expression in activated T cells, activates PIII in primary T cells. However, an intact ARE-2 site is not required for this activation, indicating that cAMP response element binding protein does not activate via this site. EMSAs indicate that an activating transcription factor/cAMP response element binding protein/cAMP response element modulator family member, but not phosphorylated cAMP response element binding protein-1, binds to ARE-2. ARE-2 also forms a complex with an unidentified protein. The ARE-2 binding protein is constitutively expressed in a DR(+) T cell line, reflecting differences between the DR(+) cell line and primary blood lymphocytes. These results show that MHC2TA PIII is induced in activated T lymphocytes, and that the induced binding of ARE-2 is a crucial step in this process.

Acyltransferases↗