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PubMed2005

Upstream sample processing facilitates PCR detection of Listeria monocytogenes in mayonnaise-based ready-to-eat (RTE) salads.

Sample pretreatment to reduce volume and concentrate cells of the target organism(s) prior to molecular detection offers a useful supplement or alternative to cultural enrichment. The purpose of this study was to develop an upstream processing method to facilitate the detection of Listeria monocytogenes in ready-to-eat (RTE) salads by PCR. Potato salad, a model RTE commodity, was seeded with L. monocytogenes and processed by two alternative upstream sample processing methods (designated one-step and two-step centrifugation), followed by DNA extraction, PCR amplification, and Southern hybridization. The two-step method resulted in 1,000-fold improvements in the PCR detection limit, from 10(6) Cfu/g (no sample processing) to 10(3) Cfu/g. The two-step method was applied for upstream sample processing of four representative deli salad items artificially inoculated with L. monocytogenes at levels ranging from 10(1)-10(6) Cfu/g. Following DNA extraction, PCR amplification, and Southern hybridization, detection was achieved at input levels of 10(5) Cfu/g for chicken salad, 10(4) Cfu/g for macaroni salad, and 10(3) Cfu/g for potato and seafood salads. The two-step method reported here facilitates the production of a final sample concentrate of reduced volume and improved purity which was compatible with PCR amplification. This approach offers further progress in our efforts to reduce or eliminate cultural enrichment in an effort to speed time to results when applying molecular methods to the detection of pathogens in foods.

Blotting, Southern↗
PubMed2005

DNA polymorphisms and haplotypes in the 5'-upstream region of the ABO blood group gene.

We investigated the polymorphisms in the 5'-upstream region between nucleotide position (nt) -9600 and nt-4105 of the ABO blood group gene using PCR and direct sequencing methods. We found 16 single nucleotide polymorphisms, two insertion-deletion polymorphisms and two sequence polymorphisms. One of the insertion-deletion polymorphisms was found at nts from -9605 to -9204, and the alleles of that locus were named ABORR*L (non-deletion) and ABORR*S (52-base-deletion). There were two haplotypes constructed from 14 polymorphisms in the region between nt-9600 and nt-7565; they were tentatively named ABORR*L-associated and ABORR*S-associated haplotypes. The ABORR*L-associated haplotype may link with ABO*O(A) (also known as ABO*O201), and the ABORR*S-associated haplotype links with the other common alleles. This indicates the existence of two major lineages of the Japanese ABO alleles in the 5'-upstream region from nt-7565. In contrast to these findings, we observed six haplotypes in the region between nt-6371 and nt-4105, and we assume that the sequence in that region is variable as compared with those in the other 5'-upstream regions. We examined the generation of two sequence polymorphisms found in the present study. In the both cases, the formation of a hairpin loop caused by palindrome in a single-stranded DNA molecule may play an important role in generating the polymorphism.

5' Flanking Region↗
PubMed2005

Upstream regions of the estrogen receptor alpha proximal promoter transcript regulate ER protein expression through a translational mechanism.

Estrogen receptor alpha (ER) mRNA is primarily transcribed from two promoters, the two transcripts share identical sequence encoding the same ER protein but differ in upstream regions. The 5' region of the two transcripts contain upstream open reading frames (uORFs) encoding potential peptides of 20 and 18 amino acids. The peptides have five C-terminal residues in common. These studies were undertaken to determine if the uORFs and encoded peptides differentially affected expression of ER. Expression of green fluorescent protein (GFP) reporter constructs containing upstream proximal promoter transcript sequences with the first 18 codons of ER fused to GFP was tested in HeLa cells. The cells expressed reduced levels of GFP as compared to the pEGFP-N1 parent vector; the effect was dependent on the presence of an intact proximal ER transcript uORF. Similar regulation by the uORF was seen in transfected MCF-7, MDA MB-231 and Ishikawa cells. Only protein expression was affected by eliminating the uORF; RNA levels were unchanged. This indicates the mechanism is translational rather than being an effect of the introduced point mutations on either mRNA stability or transcription. Eliminating the uORF did not significantly increase expression from similar distal promoter transcript ER-GFP constructs. However, study of in-frame fusions of GFP to the entire proximal and distal uORFs and to their translational start motifs showed that the translational start region of the distal uORF was inherently better at initiating translation than the AUG environment of the proximal promoter transcript uORF. The data indicate there are regulatory properties suppressing expression from the ER translation start which are specific to the unique regions of the ER proximal promoter transcript and these are likely associated with the proximal transcript uORF peptide product.

5' Untranslated Regions↗
PubMed1992

Analysis of upstream sequences of the human estrogen receptor gene.

An upstream region of the human estrogen receptor gene (ER) spanning from -2776 to -38 in relation to a previously described exon 1 was sequenced and analyzed for the possible presence of open reading frames (ORF) and regulatory motifs. We identified a long 459 bp ORF beginning at -2301. Most of the transcriptional regulatory motifs present within the ER upstream sequence are distributed either upstream or downstream of this long ORF which displays a TATA box, a CACC motif and a canonical cap site in the 5' flanking region.

Base Sequence↗
PubMed2002

The binding of synthetic analogs of the upstream, terminal residue of the O-polysaccharides (O-PS) of Vibrio cholerae O:1 serotypes Ogawa and Inaba to two murine monoclonal antibodies (MAbs) specific for the Ogawa lipopolysaccharide (LPS).

The binding of nineteen analogues of the upstream, terminal, monosaccharide residue of each of the O-polysaccharide (O-PS) of Vibrio cholerae O:1, serotype Ogawa and Inaba, with two murine monoclonal IgG antibodies both specific for the Ogawa LPS were measured using fluorescence spectroscopy. The use of the deoxy and the deoxyfluoro analogs allowed further refinement of the hydrogen-bonding pattern involved in the binding. Based on the binding characteristics observed for some of the ligands in the Inaba series, the binding of the monosaccharide that represents the upstream, terminal unit of the O-PS of V. cholerae O:1 serotype Inaba was redefined. We show for the first time that the upstream, terminal monosaccharide of the Inaba O-PS shows weak binding with these two anti-Ogawa antibodies. The results obtained allow further rationalization of the structural basis for the binding of V. cholerae O:1 antigens to their homologous antibodies.

Animals↗
PubMed2001

Three different stable human breast adenocarcinoma sublines that overexpress ALDH3A1 and certain other enzymes, apparently as a consequence of constitutively upregulated gene transcription mediated by transactivated EpREs (electrophile responsive elements) present in the 5'-upstream regions of these genes.

ALDH3A1 catalyzes the detoxification of cyclophosphamide, mafosfamide, 4-hydroperoxycyclophosphamide and other oxazaphosphorines. Constitutive ALDH3A1 levels, as well as those of certain other drug-metabolizing enzymes, e.g. NQO1 and CYP1A1, are relatively low in cultured, relatively oxazaphosphorine-sensitive, human breast adenocarcinoma MCF-7 cells. However, transient cellular insensitivity to the oxazaphosphorines can be brought about in these cells by transiently elevating ALDH3A1 levels in them as a consequence of transient exposure to: (1) electrophiles such as catechol that induce the transcription of a battery of genes, e.g. ALDH3A1 and NQO1, having in common an electrophile responsive element (EpRE) in their 5'-upstream regions; or (2) Ah-receptor agonists, e.g. indole-3-carbinol and polycyclic aromatic hydrocarbons such as 3-methylcholanthrene, that induce the transcription of a battery of genes, e.g. ALDH3A1, NQO1 and CYP1A1, having in common a xenobiotic responsive element (XRE) in their 5'-upstream regions. Further, MCF-7 sublines that are constitutively, i.e. when grown in the absence of the original selecting pressure, relatively oxazaphosphorine-insensitive as a consequence of constitutively relatively elevated cellular ALDH3A1 levels evolved when MCF-7 cells were: (1) continuously exposed for several months to gradually increasing concentrations of 4-hydroperoxycyclophosphamide or benz(a)pyrene; or (2) briefly exposed (once for 30 min) to a high concentration (1 mM) of mafosfamide. Each of these three stable sublines is constitutively relatively cross-insensitive to benz(a)pyrene and other polycyclic aromatic hydrocarbons. Cellular levels of NQO1, but not of CYP1A1, are also constitutively relatively elevated in each of the three sublines. RT-PCR-based experiments established that ALDH3A1 mRNA levels are constitutively elevated ( approximately 5- to 8-fold) in each of the three sublines. The elevated ALDH3A1 mRNA levels are not the consequence of gene amplification, hypomethylation of a relevant regulatory element, or ALDH3A1 mRNA stabilization. Collectively, these observations suggest that constitutively elevated levels of ALDH3A1 and certain other enzymes in the three stable sublines are probably the consequence of a constitutive change in the cellular concentration of a key component of the EpRE signaling pathway, such that the cellular concentration of the relevant ultimate transactivating factor is constitutively elevated, i.e. gene transcription promoted by transactivated EpREs is constitutively upregulated. Further, constitutively upregulated gene transcription mediated by transactivated EpREs can be relatively easily induced, whereas that mediated by transactivated XREs cannot, at least in MCF-7 cells. Still further, the three sublines may facilitate study of the signaling pathway that leads to transactivation of the EpREs present in the 5'-upstream regions of ALDH3A1, NQO1 and other gene loci.

Aldehyde Dehydrogenase↗
PubMed1999

The 25 kDa protein recognizing the rat curved region upstream of the origin of the L-strand replication is the rat homologue of the human mitochondrial transcription factor A.

Mass spectrometry matrix-assisted laser desorption ionization (MALDI) analysis and N-terminus sequencing as well as immunoblotting experiments using human and mouse antibodies have allowed us to identify the 25 kDa protein, previously isolated from rat liver using magnetic beads coated with a rat liver mitochondrial (mt) DNA region upstream of the Ori-L, as the homologue of human mt transcription factor A (mtTFA). We can therefore identify this DNA binding protein as the rat mtTFA. Furthermore, since we previously showed that the 25 kDa protein purified from rat liver was able to bind the curved mtDNA region upstream of the Ori-L as well as the curved mtDNA in the D-loop region, the results here reported lead us to state, for the first time, that mtTFA binds both the curved regions of mtDNA upstream of the two replication origins.

Amino Acid Sequence↗
PubMed2002

FIS modulates the kinetics of successive interactions of RNA polymerase with the core and upstream regions of the tyrT promoter.

We have applied laser UV photo-footprinting to characterise kinetically complexes involving the activator protein FIS, RNA polymerase and the tyrT promoter of Escherichia coli. FIS photo-footprints strongly to three binding sites upstream of the core promoter. The polymerase photo-footprints in the near-consensus -35 hexamer on the non-template strand of DNA in a fashion similar to that of stable complexes involving the lacUV5 promoter. The kinetics of the interactions of polymerase alone with the tyrT promoter differ from those observed previously at the lacUV5 promoter. In the absence of FIS, we observe an upstream polymerase-induced signal at -122 within FIS site III that occurs subsequent to changes in the core promoter region and is strongly dependent on negative supercoiling. These observations support the proposal that the upstream region of the promoter is wrapped around the polymerase. We propose that the wrapped DNA allows the polymerase to overcome, at least in part, the barrier to DNA untwisting imparted by the G+C-rich discriminator. We further suggest that FIS plays a similar role and may facilitate polymerase escape.

Base Sequence↗
PubMed1999

Conditions providing enhanced transfection efficiency in rat pheochromocytoma PC12 cells permit analysis of the activity of the far-upstream and proximal promoter of the brain creatine kinase gene.

While brain creatine kinase (CKB) is expressed at highest levels in the brain, where it functions in regenerating ATP, the gene elements and protein factors regulating CKB transcription in neuronal and glial cells have not been identified. To investigate the regulation of CKB in neuronal cells, we examined the expression of the promoter proximal and 5' far-upstream regions of the rat CKB gene transiently transfected into rat PC12 pheochromocytoma cells. Initially, these experiments were hampered by the extremely low transfection efficiency of PC12 cells. We increased efficiency by greater than 200-fold by employing CaPO4-precipitated DNA transfection into PC12 cells which were optimized for transient transfection by: (i) culturing cells in polylysine-coated dishes to insure attachment throughout transfection; (ii) exposing cells to transfected DNA for an optimal time and employing a glycerol shock; and, most importantly, (iii) dissociating the characteristic self-adhesive clumps of PC12 into mostly single cells. Use of the plasmid expressing green fluorescent protein allowed identification of the transfected cells that averaged 10-20% of the total. Analyses of CKB promoter-CAT gene constructs showed that in PC12 cells expression of the proximal (0.2 kb) CKB promoter was low while expression of the 1.4 kb promoter was three fold higher and the 2.9 kb promoter was ten fold higher, suggesting the presence of at least two upstream cis-acting, positive regulatory elements. In agreement, the steady-state CKB mRNA level was higher in PC12 than in other neuronal cell lines examined, possibly reflecting the effects of positive upstream factors. The results are discussed in relation to how this economical and straightforward transfection procedure may be useful in identify factors regulating the transcription of CKB and other genes expressed in neuronal cells.

Animals↗
PubMed2000

Repetitive sequences upstream of the pfg27/25 gene determine polymorphism in laboratory and natural lines of Plasmodium falciparum.

The structure of the genomic region located upstream of the gametocyte-specific gene pfg27/25 of Plasmodium falciparum was analysed in laboratory lines and field isolates of the parasite. The gene is located in a subtelomeric region of chromosome 13 in parasite clones 3D7 and HB3. Analysis of laboratory lines and field isolates of P. falciparum indicated that polymorphism upstream of pfg27/25 is mainly due to the structure of a repetitive DNA region located at about half a kilobase from the pfg27/25 coding sequence. Different types of repetitive sequences are present in this region, whose copy number is variable in different parasite lines. In addition a GC-rich sequence element contained in this region, which is proposed to be the startpoint of pfg27/25 mRNA, presents either a direct or a reverse orientation in different parasite lines. Genomic deletions upstream of the pfg27/25 gene are also described in two laboratory lines of the parasite, which eliminate two newly identified malaria genes. orf P and orf Gap, from the genome of these parasites. One of them, orf Gap, deleted from the reference parasite clone 3D7, is abundantly expressed as mature mRNA in asexual parasites. PCR analysis on 64 field isolates of P. falciparum indicated that orf P and orf Gap sequences are present in all tested samples of naturally propagating parasites.

Animals↗
PubMed2001

Transcription of the Escherichia coli dcw cluster: evidence for distal upstream transcripts being involved in the expression of the downstream ftsZ gene.

Escherichia coli strains VIP596 and VIP597 have been constructed to compare the amount of transcription of the ftsZ gene derived from proximal promoters in the ddlB-ftsZ region with that originating in the upstream regions of the dcw cluster. Both strains have in common a beta-galactosidase reporter fusion located at the ddlB locus, but differ in that VIP597 has a transcription terminator Omega interposon located downstream from lacZ. In addition, these strains have the ddlB, ftsQ, ftsA and ftsZ genes under the control of the IPTG-inducible promoter P(tac), allowing to control artificially ftsZ expression for normal cell division to take place. When beta-galactosidase activity was measured in VIP596 and VIP597 and compared to the levels measured in strain VIP407, in which the lacZ reporter fusion is located in the ftsZ gene, they were found to account for nearly 66% of the total transcription entering into ftsZ. This result indicates that the reduction in ftsZ transcription observed when the promoters in the ddlB-ftsA region are disconnected from the upstream sequences of the dcw cluster (as observed by Flärdh et al., Mol. Microbiol. 30 (1998) 305-316) in strain VIP490) is the direct consequence of the interruption in the transcription originated upstream and not due to the effect of such sequences on the promoters proximal to ftsZ.

Bacterial Proteins↗
PubMed2003

The 62-kb upstream region of Bombyx mori fibroin heavy chain gene is clustered of repetitive elements and candidate matrix association regions.

We sequenced an 80 kb DNA region containing the complete sequence of the silkworm Bombyx mori fibroin gene and its flanking, especially the upstream, regions (-62 kb). About 30% of the 62 kb upstream region is composed of repetitive elements including short interspersed elements Bm1, long interspersed elements L1Bm and mariner-like elements Bmmar1 which are widespread over the silkworm genome. This 62 kb region is also enriched of commonly considered matrix association region (MAR) motifs. A total of 25 individual MAR recognition signatures (MRSs) were identified, with 24 at the upstream and one at the downstream region. Combining two newly developed MAR prediction programs (MAR-finder and Chrclass), ten candidate MARs were predicted, with five containing MRS and seven related to the repetitive elements. The wide distribution of nested repetitive elements, candidate MARs, DNase I hypersensitive sites and other potential regulatory factors recognition sites indicates this region is probably a unique huge cis-acting element contributing to the regulation of the spatial and temporal specificity and efficiency of fibroin gene expression.

5' Flanking Region↗
PubMed1997

The 5'-upstream region of the rat phospholipase C-beta 3 gene contains two critical Sp1 sites and an HIV Inr-like element.

The 5'-upstream region of the rat phospholipase C-beta 3 gene (PLC-beta 3) has been cloned and characterized. Sequence analysis of the 5'-upstream region showed that it contains a GC-rich region (-166 to +1: 79%) and multiple binding sites for the transcription factors Sp1, AP-1 and AP-2, but does not contain a canonical TATA box. Primer extension analysis of total RNA isolated from rat glial cell C6Bul revealed that single transcription start point (tsp) is located at an initiator (Inr) element similar to that found in the HIV promoter. Gel mobility shift and competitive mobility shift assays indicated that this Inr element forms a DNA-protein complex with the HIV Inr-binding protein, LBP-1/CP2 or a homologue. In order to localize functional elements of the 5'-upstream region of the rat PLC-beta 3 gene, 5'-deletion fragments were cloned into a chloramphenicol acetyltransferase (CAT) reporter vector. Transient transfection analyses of the 5'-deletion mutants identified a crucial promoter element located at -128 to -14. Supershift mobility assays, site-directed mutagenesis and DNase I footprints indicated that Sp1 binds to three GC boxes within the sequence between -128 and -14 of the PLC-beta 3 promoter. Transient transfection analyses of promoter constructs containing site-specific mutation(s) of these three GC boxes demonstrated that two GC boxes, located proximal to the tsp, are important elements for normal promoter activity.

Animals↗
PubMed1997

Upstream genomic sequence of the human connexin26 gene.

Human connexin 26 (Cx26) has been considered to be a candidate suppressor gene in mammary epithelial cells. To gain insight into the transcriptional regulation of this gene, we have cloned and sequenced the 5' portion of the gene, which extends 4.8 kb upstream from the ATG translation start site. The 3' end of the non-coding exon 1 (160 bp) is located at 3149 bp upstream from the 5' end of exon 2. Comparison between the human Cx26 gene and the mouse gene reveals a highly conserved promoter region with 81% homology. In addition to six GC boxes and two GT boxes, a TTAAAA box is located at -24 to -19 bp upstream of the transcription start point. Analogous to the mouse beta-casein gene, the promoter region of the human Cx26 gene also contains a YY1-like binding site and a consensus mammary gland factor binding site.

Animals↗
PubMed2001

A distant upstream region of the rat multipartite Na(+)-Ca(2+) exchanger NCX1 gene promoter is sufficient to confer cardiac-specific expression.

The Na(+)-Ca(2+) exchanger (NCX) regulates intracellular calcium homeostasis. We report on an upstream region of the rat NCX1 multipartite promoter that is active in cardiac myocytes. Although inactive in most non-cardiac cell lines, its activity can be rescued by cotransfection with GATA-4 and -6, but not GATA-5 transcription factors. In transgenic mice and similar to endogenous NCX1 mRNA expression, the upstream promoter region directs uniform beta-galactosidase expression in cardiac myocytes from approximately 7.75dpc. In adult mouse hearts, promoter activity is, however, significantly reduced and heterogeneous, except in the conduction system (sinoatrial and atrioventricular node, atrioventricular bundles). The upstream NCX1 promoter region thus directs appropriate spatial and temporal control of cardiac expression throughout development.

Animals↗
PubMed2002

Structured RNA upstream of insect cap distal iron responsive elements enhances iron regulatory protein-mediated control of translation.

Iron regulatory protein (IRP) blocks ribosomal assembly by binding to an iron responsive element (IRE) located proximal (<60 nts) to the mRNA cap, thereby repressing translation. Constructs with IREs located 60-100 nts from the cap permit ribosomal assembly but the ribosomes pause at IRE/IRP complexes resulting in partial repression of translation. However, insect ferritin mRNAs have cap-distal IREs located 90-156 nts from the cap. Because iron can be toxic, it seems unlikely that insects would be unable to fully regulate ferritin synthesis at the level of translation. Calpodes ferritin consists of two subunits, S and G. In vitro translation of Calpodes ferritin and IRP1 from fat body mRNA yields only G subunits suggesting that IRP1 more efficiently represses translation of the S subunit than the G. When repression is removed by the addition of IRE competitor RNA, the synthesis of both subunits is greatly increased. S and G ferritin mRNAs have identical IREs in similar far cap-distal positions. While both ferritin mRNAs are predicted to have stem-loops between the IRE and the RNA cap, in general insect S mRNAs have more cap-proximal RNA structure than G mRNAs. Therefore, we examined the effect of upstream secondary structure on ribosomal assembly onto S ferritin mRNA constructs using sucrose gradient analysis of translation initiation complexes. We found no evidence for ribosomal assembly on wild type Calpodes S ferritin mRNA in the presence of IRP1 while constructs lacking the wild type secondary structure showed ribosomal pausing. Constructs with wild type secondary structure preceded by an unstructured upstream leader assemble ribosomes in the presence or absence of IRP1. Sequence and RNA folding analyses of other insect ferritins with cap-distal IREs failed to identify any common sequences or IRE-like structures that might bind to IRP1 with lower affinity or to another RNA binding protein. We propose that stem-loops upstream from the IRE act like pleats that shorten the effective distance between the IRE and cap and allow full translational repression by IRP1. In this way some cap-distal IREs may function like cap-proximal ones.

5' Untranslated Regions↗
PubMed1991

Sequences more than 500 base pairs upstream of the human U3 small nuclear RNA gene stimulate the synthesis of U3 RNA in frog oocytes.

Small nuclear RNA (snRNA) genes contain strong promoters capable of initiating transcription once every 4 s. Studies on the human U1 snRNA gene, carried out in other laboratories, showed that sequences within 400 bp of the 5' flanking region are sufficient for maximal levels of transcription both in vivo and in frog oocytes [reviewed in Dahlberg and Lund (1988)]. We studied the expression of a human U3 snRNA gene by injecting 5' deletion mutants into frog oocytes. The results show that sequences more than 500 bp upstream of the U3 snRNA gene have a 2-3-fold stimulatory effect on the U3 snRNA synthesis. These results indicate that the human U3 snRNA gene is different from human U1 snRNA gene in containing regulatory elements more than 500 bp upstream. The U3 snRNA gene upstream sequences contain an AluI homologous sequence in the -1200 region; these AluI sequences were transcribed in vitro and in frog oocytes but were not detectable in HeLa cells.

Animals↗
PubMed1983

Chromatin structure differs between coding and upstream flanking sequences of the yeast 35S ribosomal genes.

Staphylococcal nuclease (EC 3.1.4.7) and DNase I (EC 3.1.4.5) digestion analysis of the nuclear chromatin structure of the yeast 35S rDNA gene shows the presence of typical and homogeneous nucleosome patterns across the coding sequence. These nucleosomal patterns change abruptly, around the site of transcription initiation and upstream in the 5'-flanking sequences, to a unique pattern with both nucleosomal and nonnucleosomal character. The mix arises, at least partly, from heterogeneity within the population of upstream regions; some regions are nucleosomal, but the majority are nonnucleosomal. The nonnucleosomal set of upstream regions appears to be nucleoprotein associated and, in fact, may be an altered nucleosome structure rather than totally restructured. The abruptness of the transition from nucleosome to other structure suggests restricted nucleosome locations in the region around the transcription initiation site of this gene.

Base Sequence↗