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PubMed2002

Cloning and functional analysis of the mouse 5-lipoxygenase promoter.

5-lipoxygenase (ALOX5), an enzyme essential for the formation of all leukotrienes, is highly regulated at multiple levels, including gene transcription. The human ALOX5 promoter sequence has been cloned and is well characterized. Several important cis-acting elements have been identified including a G+C-rich sequence approximately 145-179 base pairs (bp) upstream from the ATG start codon. This region contains consensus-binding sites for the transcription factor serum protein 1, a zinc-finger transcription factor (SP1) and early growth-response protein 1, a zinc-finger transcription factor (EGR-1) and is unique in that functionally significant polymorphisms alter these sequences. To further understand the significance of these polymorphisms and other regulatory sequences in the promoter we cloned approximately 2,000 bp of the mouse promoter sequence from a 129/SvJ BAC library for direct comparison with the human gene. Like the human promoter, the mouse Alox5 promoter lacks a TATA box and has multiple start sites. The first 292 bp immediately upstream of the translational start site function as a core promoter that is capable of mediating high basal transcription in RAW cells but not 3T3 cells. There are vast differences in the distribution of consensus cis elements between human and mouse genes; however, three areas of strong homology exist and they contain consensus-binding sites for the SP1, GATA, GGAGA, and ETS family of transcription factors. We show that Sp1/Sp3 is essential for constitutive promoter-reporter activity.

3T3 Cells↗

PubMedDate not supplied

Detection of infectious bronchitis virus by real-time reverse transcriptase-polymerase chain reaction and identification of a quasispecies in the Beaudette strain.

In this report, we describe a real-time reverse transcriptase-polymerase chain reaction (RRT-PCR) diagnostic test for infectious bronchitis virus (IBV) with the use of fluorescence resonance energy transfer (FRET) technology. Two primers that amplify a 383-base pair product between nucleotide positions 703 and 1086 relative to the start codon for the S1 gene of the Massachusetts 41 virus were designed and used to amplify the Beaudette, Massachusetts 41, Florida 18288, Connecticut, Iowa 97, Arkansas DPI, CA/NE95/99, DE/072/ 92, and GA/0470/98 strains of IBV. The primers were specific and did not amplify New Castle disease virus, Mycoplasma spp., or infectious laryngotracheitis virus. For RRT-PCR by FRET, an anchor probe conjugated to fluorescein and a detection probe conjugated to a red fluorophore were designed to anneal to a hypervariable region within the 383-base pair product. The level of sensitivity was 1 x 10(4) RNA molecules used as starting template. After amplification, a melting curve analysis was conducted to specifically identify IBV types. Because of sequence differences in the annealing position of the detection probe, the Arkansas, Connecticut, Beaudette, and Massachusetts 41 strains could be differentiated. No fluorescence was observed for the DE/072/ 92 and GA/0470/98 viruses with the anchor and detection probes. When the Beaudette strain was examined, two melting peaks were observed at 44 C and 51 C, indicating a quasispecies in that laboratory strain of IBV. Routine typing of vaccine strains of IBV was possible with this technology, but high standard deviations associated with the melting curve analysis of the FRET probes described herein made it difficult to use this test reliably for routine typing of IBV field isolates.

Animals↗

PubMed1993

Identification of a novel operon in Lactococcus lactis encoding three enzymes for lactic acid synthesis: phosphofructokinase, pyruvate kinase, and lactate dehydrogenase.

The discovery of a novel multicistronic operon that encodes phosphofructokinase, pyruvate kinase, and lactate dehydrogenase in the lactic acid bacterium Lactococcus lactis is reported. The three genes in the operon, designated pfk, pyk, and ldh, contain 340, 502, and 325 codons, respectively. The intergenic distances are 87 bp between pfk and pyk and 117 bp between pyk and ldh. Plasmids containing pfk and pyk conferred phosphofructokinase and pyruvate kinase activity, respectively, on their host. The identity of ldh was established previously by the same approach (R. M. Llanos, A. J. Hillier, and B. E. Davidson, J. Bacteriol. 174:6956-6964, 1992). Each of the genes is preceded by a potential ribosome binding site. The operon is expressed in a 4.1-kb transcript. The 5' end of the transcript was determined to be a G nucleotide positioned 81 bp upstream from the pfk start codon. The pattern of codon usage within the operon is highly biased, with 11 unused amino acid codons. This degree of bias suggests that the operon is highly expressed. The three proteins encoded on the operon are key enzymes in the Embden-Meyerhoff pathway, the central pathway of energy production and lactic acid synthesis in L. lactis. For this reason, we have called the operon the las (lactic acid synthesis) operon.

Amino Acid Sequence↗

PubMed1995

The smaller of two overlapping cheA gene products is not essential for chemotaxis in Escherichia coli.

The cheA locus of Escherichia coli encodes two similar proteins, CheAL (654 amino acids) and CheAS (557 amino acids), which are made by initiating translation from different in-frame start sites [start(L) and start(S)]. CheAL plays an essential role in chemotactic signaling. It autophosphorylates at a histidine residue (His-48) and then donates this phosphate to response regulator proteins that modulate flagellar rotation and sensory adaptation. CheAS lacks the first 97 amino acids of CheAL, including the phosphorylation site at His-48. Although it is unable to autophosphorylate, CheAS can form heterodimers with mutant CheAL subunits to restore kinase function and chemoreceptor control of autophosphorylation activity. To determine whether these or other activities of CheAS are important for chemotaxis, we constructed cheA lesions that abrogated CheAS expression. Mutants in which the CheAS start codon was changed from methionine to isoleucine (M98I) or glutamine (M98Q) retained chemotactic ability, ranging from 50% (M98Q) to 80% (M98I) of wild-type function. These partial defects could not be alleviated by supplying CheAS from a specialized transducing phage, indicating that the lesions in CheAL--not the lack of CheAS--were responsible for the reduced chemotactic ability. In other respects, the behavior of the M98I mutant was essentially normal. Its flagellar rotation pattern was indistinguishable from wild type, and it exhibited wild-type detection thresholds and peak positions in capillary chemotaxis assays. The lack of any substantive defect in this start(S) mutant argues that CheAS makes a negligible contribution to chemotactic ability in the laboratory. Whether it has functional significance in other settings remains to be seen.

Bacterial Proteins↗

PubMed1995

Specific inhibition of hepatitis C viral gene expression by antisense phosphorothioate oligodeoxynucleotides.

The inhibitory effect of antisense phosphorothioate oligodeoxynucleotides (S-ODN) on hepatitis C viral gene expression and analyzed in an in vitro test system and in cell culture. S-ODN were directed against different stem loop structures in the 5'noncoding region (NCR) of the hepatitis C virus, (HCV) RNA and against a nucleotide stretch, including the start codon of the polyprotein precursor. The inhibitory effect of these S-ODN was quantified employing a viral RNA consisting of the first 407 nucleotides of a HCV type 1b genome fused to the coding sequence of the firefly luciferase gene. For in vitro assays this RNA was generated by in vitro transcription and used as a template in a rabbit reticulocyte lysate in vitro translation system. The production of active luciferase in the absence or presence of S-ODN was monitored using an enzymatic assay. The best results were obtained with S-ODN 4 directed against nucleotides 326 to 348, comprising the start AUG of the polyprotein coding sequence. With this oligonucleotide, a specific and dose-dependent effect was observed with a maximal inhibition of 96 +/- 1% at a S-ODN concentration of 4.14 mumol/L. For cell culture experiments, the hepatoblastoma cell line HepG2 was transfected with a plasmid expressing the HCV-luciferase fusion RNA. In this assay system S-ODN 2, complementary to nucleotides 264 to 282 of the HCV RNA, and S-ODN 4 were most efficient and reduced the viral translation by 96 +/- 0.4% and 94 +/- 0.7%, respectively, at a concentration of 0.3 mumol/L. The inhibition was specific (1) because the expression of the HCV-luciferase fusion RNA was not significantly impaired by the control S-ODN and (2) because the expression of an unrelated messenger RNA was not or only slightly downregulated. These data suggest that HCV gene expression can be inhibited effectively by antisense S-ODN. Therefore, this approach represents a promising perspective for the treatment of hepatitis C.

Base Sequence↗

PubMed1995

Mouse aggrecan, a large cartilage proteoglycan: protein sequence, gene structure and promoter sequence.

Seven genomic clones for mouse aggrecan core protein have been isolated including 3 kb of 5'- and 7 kb of 3'-flanking sequences. All exon sequences and their intron boundary sequences in these clones were identified and mapped by DNA sequencing. The gene spans at least 61 kb and contains 18 exons. Exon 1 encodes 5'-untranslated sequence and exon 2 contains a translation start codon, methionine. The coding sequence is 6545 bp for a 2132-amino-acid protein with calculated M(r) = 259,131 including an 18-amino-acid signal peptide. There is a strong correlation between structural domains and exons. Notably, the chondroitin sulphate domain consisting of 1161 amino acids is encoded by a single exon of 3.6 kb. Although link protein has similar structural domains and subdomains, the sequence identity and the organization of exons encoding the subdomains B and B' of G1 and G2 domains revealed a strong similarity of mouse aggrecan to both human versican and rat neurocan. Primer extension analysis identified four transcription start sites which are close together. The promoter sequence showed high G/C content (65%) and contained several consensus binding motifs for transcription factors including Sp-1 and the glucocorticoid receptor. There are stretches of sequences similar to the promoter region of both the type-II collagen and link protein genes. These sequences may be important for cartilage gene expression.

Aggrecans↗

PubMed2003

Versatile EGFP reporter plasmids for cellular localization of recombinant gene products in filamentous fungi.

The recent development of variants of the green fluorescent protein (GFP) with altered codon composition facilitated the efficient expression of this reporter protein in a number of fungal species. In this report, we describe the construction and application of a series of plasmids, which support the expression of an enhanced gfp (egfp) gene in filamentous fungi and assist the study of diverse developmental processes. Included were a promoterless egfp vector for monitoring the expression of cloned promoters/enhancers in fungal cells and vectors for creating translation fusions to the N-terminus of EGFP. The vectors were further modified by introducing a variant hygromycin B phosphotransferase (hph) gene, lacking the commonly found NcoI site. Instead, this site, which contained an ATG start codon, was placed in front of the egfp gene and thus was made suitable for the cloning of translational fusions. The applicability of these vectors is demonstrated by analyzing transcription regulation and protein localization and secretion in two ascomycetes, Acremonium chrysogenum and Sordaria macrospora. In the latter, the heterologous egfp gene is stably inherited during meiotic divisions, as can easily be seen from fluorescent ascospores.

Acremonium↗

PubMed1999

The Bradyrhizobium japonicum nolA gene encodes three functionally distinct proteins.

Examination of nolA revealed that NolA can be uniquely translated from three ATG start codons. Translation from the first ATG (ATG1) predicts a protein (NolA1) having an N-terminal, helix-turn-helix DNA-binding motif similar to the DNA-binding domains of the MerR-type regulatory proteins. Translation from ATG2 and ATG3 would give the N-terminally truncated proteins NolA2 and NolA3, respectively, lacking the DNA-binding domain. Consistent with this, immunoblot analyses of Bradyrhizobium japonicum extracts with a polyclonal antiserum to NolA revealed three distinct polypeptides whose molecular weights were consistent with translation of nolA from the three ATG initiation sites. Site-directed mutagenesis was used to produce derivatives of nolA in which ATG start sites were sequentially deleted. Immunoblots revealed a corresponding absence of the polypeptide whose ATG start site was removed. Translational fusions of the nolA mutants to a promoterless lacZ yielded functional fusion proteins in both Escherichia coli and B. japonicum. Expression of NolA is inducible upon addition of extracts from 5-day-old etiolated soybean seedlings but is not inducible by genistein, a known inducer of the B. japonicum nod genes. The expression of both NolA2 and NolA3 requires the presence of NolA1. NolA1 or NolA3 is required for the genotype-specific nodulation of soybean genotype PI 377578.

5' Untranslated Regions↗

PubMed1989

The extended promoter of the gene encoding ribosomal protein S33 in yeast consists of multiple protein binding elements.

At least 4 different, protein binding cis-acting elements are present in the upstream region of the S33-gene. The major protein binding site is situated between positions -148 and -163 relative to the ATG start codon. It binds a trans-acting factor designated SUF (S33 Upstream Factor). When yeast cells are growing on glucose, deletion of this site results in a decrease of transcription of 50%. Using ethanol as a carbon-source, deletion of the SUF-responsive site lowers the transcription as much as 80%. A second protein binding site is found between positions -85 and -105. Only extracts from glucose-grown cells contain a factor that is able to bind to this site in vitro. A third protein binding site was found using a protein extract from ethanol-grown cells. This site, which is located quite close to the transcriptional start site, is probably responsible for the 20% residual transcription when the SUF binding site is removed. Finally, a site far upstream was found, which binds a protein from both glucose-grown and ethanol-grown cells. This site may function as an upstream repression site which is only functional when a non-fermentable carbon-source is used. Taking these findings into account, we present a model for the carbon-source dependent transcription activation of the gene encoding S33.

Base Sequence↗

PubMed1996

The 5' coding region of Paramecium surface antigen genes controls mutually exclusive transcription.

Paramecium tetraurelia stock 51 can express at least 11 different types of surface antigens, yet only a single type is expressed on the surface of an individual cell at any one time. The differential expression of stock 51 type A and B surface antigen genes (51A and 51B) is regulated at the level of transcription. Previously, we reported that nucleotide sequences upstream of position -26 (relative to the start of translation) in the 51A and 51B surface antigen genes are necessary for transcriptional activity but are not sufficient to direct differential transcriptional control. In this report we demonstrate that at least some of the critical elements necessary for differential transcription of the 51A and 51B genes lie within the 5' coding region. A hybrid gene that contains 51B upstream sequences (-475 to +1) attached to the ATG start codon of 51A is not cotranscribed with the 51B gene. In contrast, further substitution with 51B sequences (-1647 to +885) allows the chimeric gene to be coexpressed with 51B. A different hybrid gene containing a substitution of 51B sequence from -26 to +885 in the 51A gene is also coexpressed with 51B, revealing that the critical elements within the coding region of 51B do not require 51B upstream sequences for their effect. Coinjection of the 51A gene with the chimeric gene that contains 51B up to +885 showed that the same sequences that allow coexpression with 51B prevent cotranscription with 51A. Together, these results demonstrate that a region downstream of the transcriptional start site between nucleotide positions +1 and +885 (relative to translational start) is necessary to control differential transcriptional activity.

Animals↗

PubMed2000

Expression, purification, and characterization of recombinant S-adenosylhomocysteine hydrolase from the thermophilic archaeon Sulfolobus solfataricus.

S-Adenosylhomocysteine hydrolase from Sulfolobus solfataricus was expressed in Escherichia coli by inserting the genomic fragment containing the gene encoding for S-adenosylhomocysteine hydrolase downstream the isopropyl-beta-d-thiogalactoside-inducible promoter of pTrc99A expression vector. An ATG positioned 25 bp upstream of the gene which is in frame with a stop codon was utilized as the initiation codon. This construct was used to transform E. coli RB791 and E. coli JM105 strains. The recombinant protein, purified by a fast and efficient two-step procedure (yield of 0.4 mg of enzyme per gram of cells), does not appear homogeneous on SDS-PAGE because of the presence of a protein contaminant corresponding to a "truncated" S-adenosylhomocysteine hydrolase subunit lacking the first 24 amino acid residues. The recombinant enzyme shows the same molecular mass, optimum temperature, and kinetic features of S-adenosylhomocysteine hydrolase isolated from S. solfataricus but it is less thermostable. To construct a vector which presents a correct distance between the ribosome-binding site and the start codon of S-adenosylhomocysteine hydrolase gene, a NcoI site was created at the translation initiation codon using site-directed mutagenesis. The expression of the homogeneous mutant S-adenosylhomocysteine hydrolase was achieved at high level (1.7 mg of mutant protein per gram of cells). The mutant S-adenosylhomocysteine hydrolase and the native one were indistinguishable in all physicochemical and kinetic properties including thermostability, indicating that the interactions involving the NH(2)-terminal sequence of the protein play a role in the thermal stability of S. solfataricus S-adenosylhomocysteine hydrolase.

Adenosylhomocysteinase↗

PubMed1996

Hepatitis B virus carriers without precore mutations in hepatitis B e antigen-negative stage show more severe liver damage.

Hepatitis B e antigen (HBeAg) is considered to be a major target for the immune response in chronic hepatitis B. The G-->A mutation at nucleotide 1896 may mediate viral escape by creating a TAG stop codon in the precore region, thus preventing HBeAg production. This mutation frequently evolves during HBe seroconversion if thymine, but rarely if cytosine, is present in position 1858. Applying a combination of polymerase chain reaction (PCR) and restriction enzyme action, we have studied the relation of the TAG mutation and the nucleotide (nt) 1858 variants to liver damage assessed by histology activity index (HAI) scoring in 175 chronic hepatitis B virus (HBV) carriers. A TAG mutation was found in 68 of 71 (96%) of HBeAg-negative carriers infected with a T-1858 strain, but not in any of 33 carriers infected exclusively with a C-1858 strain. Four patients showed a mutation of the precore start codon, and 2 had a TAA stop mutation at codon 2. HBeAg-positive infection with a mixture of wild-type and TAG mutant virus indicated active liver damage, because 8 of 9 (89%) of such patients had a Knodell HAI greater than or equal to 8. In HBeAg-negative stage, both inflammation and fibrosis were more pronounced in carriers infected with wild-type HBV compared with precore mutants. C-1858 strains were associated with more inflammation and fibrosis compared with T-1858 strains. C-1858 strains were found in 71% of northern European, 17% of southern European, 31% of African, 2% of Middle Eastern, and 10% of Far Eastern carriers. Analysis of these variants and mutants may prove useful for clinical evaluation and choice of therapy, and may be facilitated by the methods described.

Adolescent↗

PubMed1998

The various Sendai virus C proteins are not functionally equivalent and exert both positive and negative effects on viral RNA accumulation during the course of infection.

Recombinant Sendai viruses were prepared which cannot express their Cprime, C, or Cprime plus C proteins due to mutation of their respective start codons ([Cprime-minus], [C-minus] and [double mutant], respectively). The [Cprime-minus] and [C-minus] stocks were similar to that of wild-type (wt) virus in virus titer and plaque formation, whereas the double-mutant stock had a much-reduced PFU or 50% egg infective dose/particle ratio and produced very small plaques. Relative to the wt virus infection, the [Cprime-minus] and [C-minus] infections of BHK cells resulted in significantly greater accumulation of viral RNAs, consistent with the known inhibitory effects of the Cprime and C proteins. The double-mutant infection, in contrast, was delayed in its accumulation of viral RNAs; however, once accumulation started, overaccumulation quickly occurred, as in the single-mutant infections. Our results suggest that the Cprime and C proteins both provide a common positive function early in infection, so that only the double mutant undergoes delayed RNA accumulation and exhibits the highly debilitated phenotype. Later in infection, the same proteins appear to act as inhibitors of RNA accumulation. In infections of mice, [Cprime-minus] was found to be as virulent as wt virus whereas [C-minus] was highly attenuated. These results suggest that the Cprime and C proteins cannot be functionally equivalent, since C can replace Cprime for virulence in mice whereas Cprime cannot replace C.

Animals↗

PubMed1982

Intronless human dihydrofolate reductase genes are derived from processed RNA molecules.

Three groups of recombinant bacteriophage containing coding sequences for dihydrofolate reductase (DHFR; tetrahydrofolate dehydrogenase; 5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase, EC 1.5.1.3) were isolated from two human DNA clone libraries. One recombinant (lambda hDHFR-1) contains three exons that encode the COOH-terminal portion of human DHFR. The other two human DHFR genes (hDHFR-psi 1 and hDHRF-psi 2) lack introns. hDHFR-psi 2 contains several in-phase termination codons and is only 93% homologous to the normal human DHFR coding sequences, whereas hDHFR-psi 1 has an open reading frame and is virtually identical to the coding sequence of the normal DHFR gene. The region of DNA sequence homology between each intronless gene and the normal DHFR gene extends 2.9 kilobases beyond the end of the coding sequences. At the 3' end of this homologous sequence, each intronless gene has an A-rich tract. The lack of introns and the presence of the 3' A-rich tract suggest that hDHFR-psi 1 and hDHFR-psi 2 were derived from processed RNA molecules. A short DNA sequence, 60 nucleotides 5' to the ATG start codon in lambda hDHFR-psi 2, is directly repeated immediately after the 3' A-rich tract; such terminal direct repeats also flank integrated proretroviruses and transposable DNA elements and are thought to be the hallmark of inserted DNA sequences.

Base Sequence↗

PubMed1997

Structural and functional characterization of the human alpha3 nicotinic subunit gene promoter.

We describe the structural and functional features of the human alpha3 nicotinic receptor subunit promoter. A 0.35-kb region immediately upstream of the start codon was identified that when transfected in human neuroblastoma cells was able to drive the expression of the luciferase reporter gene with a strength comparable to that of the well-characterized simian virus 40 promoter/enhancer. This region displayed the features of a multistart-site, GC-rich, TATA-less, and CAAT-less promoter, containing many overlapping Sp1 and AP-2 putative binding sites. Further dissections of the 0.35-kb fragment revealed that its 3' region, specifying the 5' UT of the mRNA, plays a relevant positive effect in determining the strength of the promoter. This region contains putative cis-acting elements for AP-2, nuclear factor-kappaB, and the recently described multiple-start site element downstream-1. By mutation analysis, we showed that these sites are functional and when combined increase the promoter activity by 4-fold. The 0.35-kb promoter was found to be under the negative control of upstream sequences that include a modern Alu repeat. The alpha3 Alu repeat works as a composite region, containing both positive and negative elements that control the activity of the downstream promoter. Finally, we investigated the tissue-specific activity of the human alpha3 gene 5' regulatory sequences, showing that they are able to drive the expression of the reporter gene preferentially in neuronal cells.

Base Sequence↗

PubMed1990

Putidaredoxin reductase and putidaredoxin. Cloning, sequence determination, and heterologous expression of the proteins.

The oxidation of camphor by cytochrome P-450cam requires the participation of a flavoprotein, putidaredoxin reductase, and an iron-sulfur protein, putidaredoxin, to mediate the transfer of electrons from NADH to P-450 for oxygen activation. A 2.2-kilobase pair BamHI-StuI fragment from whole cell DNA of camphor-grown Pseudomonas putida has been cloned and sequenced. Translation of the sequence revealed two open reading frames that could code for putidaredoxin reductase and putidaredoxin. In the case of putidaredoxin, the translated sequence matched the published sequence (Tanaka, M., Haniu, M., Yasunobu, K. T., Dus, K., and Gunsalus, I. C. (1974) J. Biol. Chem. 249, 3689-3701) with the exception of one amino acid. Codon usage in these proteins, like the proteins of other Pseudomonads, is strongly biased to G + C in the third nucleotide. A potential transcription termination site was found 3' to the putidaredoxin coding region. The "FAD-binding" amino acid consensus sequence, present in other flavoproteins, was found in putidaredoxin reductase beginning at residue 11 and a second occurrence of this sequence was found beginning with amino acid 156. The second sequence could represent the NAD-binding site. The regions encoding putidaredoxin reductase and putidaredoxin were subcloned and independently expressed in Escherichia coli at the level of 0.4 and 4.8 mg of enzymatically active protein/g wet weight of cells, respectively. Site-directed mutagenesis was used to change the rare start codon, GTG, of putidaredoxin reductase to ATG which resulted in an 18-fold increase in the level of expression of this protein to 7.4 mg/g wet weight of cells. The construction of these two clones, which express these important proteins, will facilitate studies of their interaction with each other and with P-450cam.

Amino Acid Sequence↗

PubMed2001

The human gastrin-releasing peptide receptor gene structure, its tissue expression and promoter.

The human gastrin-releasing peptide receptor (hGRP-R) is aberrantly expressed in cancers of the colon, lung and prostate and mediates signals of cellular proliferation. However, the underlying mechanisms of aberrant and/or activation of hGRP-R expression are unknown. Therefore, a genomic clone is identified, the hGRP-R gene is characterized, and the hGRP-R promoter is defined. The protein coding region is divided into three exons and exon/intron splice sites occur in the proximal 2nd and distal 3rd intracellular loops of the receptor molecule. The hGRP-R locus extends over more than 27 kb and is assigned to the chromosomal band Xp22 by fluorescence in situ hybridization. With primer extension experiments, we demonstrate two major transcription start sites in gastrointestinal and breast cancer cells, located 43 and 36 bp downstream of a TTTAAA motif which is identified 407 to 402 bp upstream of the ATG start codon. The hGRP-R is found most abundantly expressed in the normal human pancreas, where four gene-specific transcripts can be detected by Northern blot analysis, whereas only two transcripts are detected in the human stomach and, very weakly, in the adrenal cortex and the brain. In contrast, the human GRP-R is not expressed in the normal human colon, lung, and prostate. Steady state hGRP-R mRNA can also be detected in some cultured cells from breast, lung, and duodenal cancer. Robust hGRP-R promoter activity is demonstrated in a duodenal carcinoma cell line that natively expresses the functional hGRP-R. Truncation studies suggest a CRE motif, located 112 bp upstream of the major transcription start site, is required to confer basal hGRP-R promoter activity in duodenal cancer cells. These studies provide the necessary data to further elucidate molecular mechanisms of aberrant hGRP-R expression in human cancers.

Blotting, Northern↗

PubMed1999

Sequences surrounding the transcription initiation site of the Arabidopsis enoyl-acyl carrier protein reductase gene control seed expression in transgenic tobacco.

The NADH-specific enoyl-acyl carrier protein (ACP) reductase, which catalyses the last reducing step during the fatty acid biosynthesis cycle, is encoded in Arabidopsis thaliana encoded by a single housekeeping gene (ENR-A) which is differentially expressed during plant development. To identify elements involved in its tissue-specific transcriptional control, a fragment comprising the 1470 bp region directly upstream of the ATG start codon of the ENR-A gene was fused to the uidA (GUS) reporter gene and analysed in transgenic Nicotiana tabacum plants. GUS activity found during development of the transgenic plants was similar to endogenous ENR protein levels found in both tobacco and Arabidopsis plants, except for developing flowers. In floral tissue the promoter fragment showed very little activity in contrast to the relatively high level of endogenous ENR expression. Successive deletions from the 5' and 3' regions of the promoter fragment revealed the presence of at least three elements which control GUS expression in different stages of development in the transgenic tobacco plants. First, expression in young developing leaves required both the presence of sequences between -329 to -201 relative to the transcription start and part of the untranslated leader comprising the first intron. Second, root-specific GUS expression was still observed after deletion of the 5'-upstream sequences up to 19 bp of the transcription initiation site. Further, the additional removal of the intron from the untranslated leader increased root-specific expression by ca. 4- to 5-fold. Third, high expression in seeds was still observed with the minimal upstream promoter segment of 19 bp. This seed expression level was found to be independent of the presence or absence of the intron in the untranslated leader. Finally, 3' deletion of the leader sequence up to 17 bp of the transcription start greatly impaired GUS activity during all stages of plant development, suggesting that the deleted sequence of the leader either functions as an enhancer for transcription initiation or stabilizes the mRNA.

5' Untranslated Regions↗