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A deletion that removes one repetitive extragenic palindromic sequence downstream of the structural gene of Escherichia coli glutamate dehydrogenase, reduces twofold the half-life of gdhA mRNA.
We isolated the 3'-downstream part of the human aortic smooth muscle alpha-actin (SM alpha A)-encoding gene and determined the nucleotide sequence, including the ninth (last) exon and 3'-untranslated (UT) region. From the comparison of the human 3'-UT region with rat and chicken 3'-UT regions, its homology is lower than those in 3'-UT regions of other actin isoforms such as cardiac alpha-actin and cytoskeletal beta-actin. Therefore, by using the 3'-UT region of the human SM alpha A gene as an actin isoform-specific probe, this gene was detected as a single copy only in the human genome, which expressed the 1.7-kb RNA transcript in an aortic tissue-specific manner.
We have examined in detail the effects of various induction temperatures on the expression of a heterologous fusion gene controlled by the bacteriophage lambda PL promoter in a heat-inducible Escherichia coli expression system which utilizes the CIts857 repressor. Experiments performed over a temperature range spanning 29-42 degrees C indicate that, under our conditions, temperatures as low as 29 degrees C may be required to fully repress the CI857-controlled transcription from PL, and that the highest protein yields are obtained after induction at 36 degrees C for 6 h. We cloned the cat reporter gene downstream from a heterologous gene controlled by PL and found that cat expression at a low induction temperature permits the monitoring of productive transcription through the heterologous gene and thus aids in selecting transformants that are capable of producing the heterologous protein in E. coli.
The nucleotide sequence of the downstream flanking region of the mreD gene of Escherichia coli was determined. Two open reading frames (ORFs) were found, a 591-bp orfE and a 1467-bp orfF. Based on the sequence, it is suggested that the three mre genes (encoding the murein pathway), mreB, mreC and mreD, and these two ORFs possibly form an operon.
The nucleotide sequence of the 2218-bp T4 DNA fragment encompassing gene 31 and five complete open reading frames (ORFs) is presented. We show here that one of these ORFs, ORF31.-1, located downstream from gene 31, is the rIII gene. The position of the gene was established by comparison with the sequences of the rIII gene mutants, r67, rES40 and rBB9. The ORF corresponding to the rIII gene encodes a basic protein of 82 amino acids with an M(r) of 9323 and a pI of 9.28. According to the Chou and Fasman [Adv. Enzymol. 47 (1978) 45-148] secondary structure prediction, the rIII protein has a relatively high helical content. In addition, discrepancies with the overlapping sequences determined by other authors in this region are indicated.
The genetic structure and regulation of the c-myc gene was comprehensively studied for the first time in Burkitt's lymphoma with t(2;8) translocation. In a Burkitt's lymphoma cell line, KOBK101, the immunoglobulin kappa-encoding gene on chromosome 2, accompanied by its enhancer, was translocated to the pvt-1 locus located about 250 kb downstream from c-myc on chromosome 8. Only the c-myc allele on the translocated chromosome carried aberrant SalI and KpnI sites in the first intron, so the two c-myc alleles and their transcripts were analyzed separately. The c-myc allele on the untranslocated chromosome conserved the normal c-myc sequence and was transcriptionally silent. In contrast, the c-myc allele on the translocated chromosome was actively transcribed at three- to fivefold higher levels, as compared with non-malignant B-cell lines. Additionally, it carried predominant multiple mutations consisting of 64 nucleotide substitutions, three short deletions, and a one-base insertion, most of which clustered in the first exon and intron. The 24-base deletion in the first intron completely overlapped the binding site of a putative negative transcriptional factor of the 138-kDa phosphoprotein, MIF. Thus, the multiple mutations and the deregulated, allele-specific expression of c-myc were associated with the chromosomal translocation in cis. Together activation by the long-distance immunoglobulin kappa enhancer, and the alleviation of negative regulation by the mutations, seemed to cause the allele-specific activation of c-myc.
Transposon Tn5tac1 can generate conditional mutations by virtue of an outward-facing tac promoter, which is regulated by the lac repressor and isopropyl-beta-D-thiogalactopyranoside (IPTG). We report here on a Tn5tac1 insertion in Escherichia coli that results in a conditional (IPTG-elicited) folA mutant phenotype: During aerobic growth, IPTG caused decreased synthesis of dihydrofolate reductase (DHFR; encoded by the folA gene) and hypersensitivity to trimethoprim (a DHFR inhibitor); during anaerobic growth, IPTG elicited auxotrophy that was satisfied by thymine or glycine or threonine. The Tn5tac1 insertion was downstream from folA, with the tac promoter pointing into the gene (antisense direction). Complementation tests indicated that the conditional folA deficiency was a cis effect of transcription from the tac promoter, perhaps due to head-to-head collision between converging RNA polymerases.
The immunoglobulin kappa locus (Ig kappa) is active only in the B-lymphocyte cell lineage. By exon-trapping we found a gene situated downstream from the murine Ig kappa locus. This gene encodes a protein with 53% sequence identity to the ribose 5-phosphate isomerase A (RPI-A) of Escherichia coli and is therefore likely to be the murine homologue (mRPI) of this enzyme. We confirmed this assumption by showing that a glutathione S-transferase (GST)::mRPI fusion protein has enzymatic activity and that an anti-mRPI antibody detects a protein of the predicted mass of RPI (33 kDa). Cloning and sequencing of the human counterpart show that the RPI gene is evolutionarily conserved. The expression of mRPI is not influenced by the rearrangement status of the Ig kappa locus in B cells and mRPI is expressed in all tissues. We thus show that two genes with very different expression patterns, a housekeeping gene and a gene expressed in a tissue-specific manner, can be located on a chromosome in close proximity to each other.
The FLT3 gene encodes a subclass-III receptor tyrosine kinase (RTKIII). We have determined the structural organization of the downstream part of the human FLT3 gene (also designated dsp-FLT3) that corresponds to the intracellular region of the protein. The coding region is spread over twelve exons spanning 10 kb of genomic DNA. Exon sizes range from 83 to 154 bp, while intron sizes range from 86 bp to more than 1.9 kb. Comparison with the corresponding domain of other RTKIII genes (KIT and FMS) shows that these genes share the same number of exons, which are highly conserved in size, sequence and exon/intron boundary positions. In addition, the intron phase of equivalent introns of FLT3, KIT and FMS are all identical. Our results reinforce our hypothesis based initially only on the KIT and FMS comparison showing that RTKIII genes share a common structural organization and have evolved from a common ancestor gene by cis and trans duplication. Comparison of the genomic organization of the intracellular-encoding part of RTKIII genes with that of RTKI, II and IV genes shows that subclasses III and IV are the most closely related.
The P9 stem-loop is one of the conserved structural elements found in all group-I introns. Using two deletion mutants in this region of the Tetrahymena thermophilia large ribosomal subunit intron, we show that removal of the P9 element, either alone, or together with the non-conserved downstream P9.1 and P9.2 elements, results in an intron incapable of the first step of the splicing reaction at a low concentration of Mg2+. The mutant introns also require high concentrations of Mg2+ for the second step in splicing, as well as hydrolysis reactions, suggesting that P9, as well as P9.1 and P9.2, are important structural elements in the final folded form of the intron. In addition, RNase-T1-mediated-structure-probing experiments demonstrated that the loss of P9, P9.1 and P9.2 changes the structural context of the region binding the 5' splice site. The deletions lead to less efficient recognition of the 3' splice site and an accumulation of unligated exons. These observations support the view that the P9, P9.1 and P9.2 stem-loops play an important role in the binding of the 3' splice site.
The nirC and nirF genes were identified downstream from nirSM, the structural genes encoding nitrite reductase (NIR) and cytochrome c-551 from Pseudomonas aeruginosa (Pa). The nirC gene encodes a probable c-type cytochrome with a signal sequence for membrane translocation. The nirF gene codes for a protein of 392 amino acids. A nirF mutant of Pa, constructed by marker exchange mutagenesis, synthesized an inactive NIR protein whose activity was restored by adding purified heme d1. The mutant strain produced an active NIR, when it was transformed by a broad-host-range plasmid carrying nirF. These results showed that the product of nirF was essential for the biosynthesis of heme d1 in Pa.
In the protozoan parasite, Babesia bovis, the glutamine-dependent carbamoyl phosphate synthetase-encoding gene (CPSII) contains contiguous amidotransferase- and synthetase-encoding sequences. Unlike the organisation in most eukaryotes, the gene is not fused with other genes encoding enzymes of pyrimidine biosynthesis de novo. The nucleotide sequences immediately upstream and downstream from the gene contain motifs which may be involved in regulating its expression.
In synaptic transmission, vesicles are proposed to dock at presynaptic active zones by the association of synaptobrevin (v-SNARE) with syntaxin (t-SNARE). We test this hypothesis in Drosophila strains lacking neural synaptobrevin (n-synaptobrevin) or syntaxin. We showed previously that loss of either protein completely blocks synaptic transmission. Here, we attempt to establish the level of this blockade. Ultrastructurally, vesicles are still targeted to the presynaptic membrane and dock normally at specialized release sites. These vesicles are mature and functional since spontaneous vesicle fusion persists in the absence of n-synaptobrevin and since vesicle fusion is triggered by hyperosmotic saline in the absence of syntaxin. We conclude that the SNARE hypothesis cannot fully explain the role of these proteins in synaptic transmission. Instead, both proteins play distinct roles downstream of docking.
Asymmetric cell divisions allow a sensory organ precursor (SOP) cell to generate a neuron and its support cells in the Drosophila PNS. We demonstrate a role of tramtrack (ttk), previously identified as a zinc finger-containing putative transcription factor, in the determination of different daughter cell fates. Both loss of function and overexpression of ttk affect the fates of the SOP progeny. Whereas loss of ttk function transforms support cells to neurons, ttk overexpression results in the reverse transformation. ttk is expressed in support cells but not in neurons. It has been shown that numb, a membrane-associated protein asymmetrically distributed during the SOP division, confers different daughter cell fates. Loss of ttk or numb function results in reciprocal cell fate transformation. Epistatic studies suggest that ttk acts downstream of numb. We propose that ttk executes the command dictated by asymmetrically localized numb to specify distinct daughter cell fates during multiple asymmetric divisions.
Damage to blood corpuscles seems to be related to the magnitude and exposure time of the turbulent shear stresses (TSS). According to in vitro studies the critical TSS level for lethal erythrocyte and thrombocyte damage is 150-400 N/m2, for exposure times within physiological ranges. To study the distribution of TSS in the human ascending aorta, a hot-film anemometer needle probe was used to register blood velocities at 41 evenly distributed measuring points in the cross-sectional area 5-6 cm downstream of the aortic annulus. Measurements were made in the ascending aorta after normal aortic valves (prior to coronary bypass surgery), after stenotic aortic valves, and after implantation of either St. Jude Medical or Starr Edwards Silastic Ball valves. Three-dimensional visualization of velocity profiles were performed and Reynolds normal stresses (RNS) were calculated within 50-ms overlapping time windows in systole. By coordinating the mean RNS for each time window and for all 41 measuring points, 2-dimensional color-coded mapping of the RNS distribution was made. Based on the velocity profiles and the RNS distribution a relative blood damage index (RBDI) was calculated to incorporate the magnitude and exposure time for RNS in the entire cross-sectional area into one parameter. Turbulent shear stresses were estimated by using a previously determined correlation equation between RNS and TSS. After normal aortic valves, RNS was below 4 N/m2.(ABSTRACT TRUNCATED AT 250 WORDS)
The human reduced folate carrier (hRFC) is ubiquitously but differentially expressed in human tissues and its levels are regulated by up to six alternatively spliced non-coding regions (designated A1/A2, A, B, C, D, and E) and by at least four promoters. By transient transfections of HepG2 human hepatoma cells with 5' and 3' deletion constructs spanning 2883 bp of upstream sequence, a transcriptionally important region was localized to within 177 bp flanking the transcriptional start sites for exon C. By gel shift and chromatin immunoprecipitation assays, Sp1 and C/EBP beta transcription factors were found to bind consensus elements (GC-box, CCAAT-box) within this region. The functional importance of these elements was confirmed by transient tranfections of HepG2 cells with hRFC-C reporter constructs in which these elements were mutated, and by co-transfections of Drosophila SL-2 cells with wild-type hRFC-C promoter and expression constructs for Sp1 and C/EBP beta. Whereas both Sp1 and C/EBP beta transactivated hRFC-C promoter activity, C/EBP alpha and gamma were transcriptionally inert. Sp1 combined with C/EBP beta resulted in a synergistic transactivation. In HepG2 cells, transfections with Sp1 and C/EBP beta both increased endogenous levels of hRFC-C transcripts. By 3' deletion analysis, a repressor sequence was localized to within 71 bp flanking the minimal promoter. On gel shifts, a novel transcriptional repressor was localized to within 30 bp. Collectively, these results identify transcriptionally important regions in the hRFC-C minimal promoter that include a GC-box and CCAAT-box, and suggest that cooperative interactions between Sp1 and C/EBP beta are essential for hRFC-C transactivation. Another possible factor in the tissue-specific regulation of the hRFC-C region involves the downstream repressor flanking the minimal promoter.
The high incidence of overexpression of some members of the Rho family of GTPases in human tumors suggests that (1) these proteins are involved in cancer onset, and (2) they are potential candidates for a therapeutic intervention. In recent years, the characterization of downstream effectors to Rho GTPases has provided crucial clues on the general cellular effects that permit aberrant proliferation and adhesiveness of tumor cells. The activation of many of these effector proteins in turn results in the modulation of the activity of several transcription factors that play an important role at various levels of Rho signaling. The precise mechanisms by which Rho GTPases participate in carcinogenesis are still not fully understood. However, it is becoming more evident that the specific role of Rho overexpression in tumor initiation, progression and metastasis, as well as the nature and cause of such overexpression in specific human tumors (i.e., transient or stable; tumor environment-regulated; genetic or epigenetic) may be linked to the activation of specific signaling pathways that result in transcriptional regulation. In this review, we summarize the functions of Rho proteins in the regulation of several transcription factors and their relationship to tumor biology.
Colorectal cancer is a major cause of mortality and whilst up to 80% of sporadic colorectal tumours are considered preventable, trends toward increasing obesity suggest the potential for a further increase in its worldwide incidence. Novel methods of colorectal cancer prevention and therapy are therefore of considerable importance. Non-steroidal anti-inflammatory drugs (NSAIDs) are chemopreventive against colorectal cancer, mainly through their inhibitory effects on the cyclooxygenase isoform COX-2. COX enzymes represent the committed step in prostaglandin biosynthesis and it is predominantly increased COX-2-mediated prostaglandin-E2 (PGE2) production that has a strong association with colorectal neoplasia, by promoting cell survival, cell growth, migration, invasion and angiogenesis. COX-1 and COX-2 inhibition by traditional NSAIDs (for example, aspirin) although chemopreventive have some side effects due to the role of COX-1 in maintaining the integrity of the gastric mucosa. Interestingly, the use of COX-2 selective NSAIDs has also shown promise in the prevention/treatment of colorectal cancer while having a reduced impact on the gastric mucosa. However, the prolonged use of high dose COX-2 selective inhibitors is associated with a risk of cardiovascular side effects. Whilst COX-2 inhibitors may still represent viable adjuvants to current colorectal cancer therapy, there is an urgent need to further our understanding of the downstream mechanisms by which PGE2 promotes tumorigenesis and hence identify safer, more effective strategies for the prevention of colorectal cancer. In particular, PGE2 synthases and E-prostanoid receptors (EP1-4) have recently attracted considerable interest in this area. It is hoped that at the appropriate stage, selective (and possibly combinatorial) inhibition of the synthesis and signalling of those prostaglandins most highly associated with colorectal tumorigenesis, such as PGE2, may have advantages over COX-2 selective inhibition and therefore represent more suitable targets for long-term chemoprevention. Furthermore, as COX-2 is found to be overexpressed in cancers such as breast, gastric, lung and pancreatic, these investigations may also have broad implications for the prevention/treatment of a number of other malignancies.