Presence Of Trans-acting Factors Research Articles

Complex translational regulation of BACE1 involves upstream AUGs and stimulatory elements within the 5' untranslated region

BACE1 is the protease responsible for the production of amyloid-β peptides that accumulate in the brain of Alzheimer's disease (AD) patients. BACE1 expression is regulated at the transcriptional, as well as post-transcriptional level. Very high BACE1 mRNA levels have been observed in pancreas, but the protein and activity were found mainly in brain. An up-regulation of the protein has been described in some AD patients without a change in transcript levels. The features of BACE1 5′ untranslated region (5′ UTR), such as the length, GC content, evolutionary conservation and presence of upstream AUGs (uAUGs), indicate an important regulatory role of this 5′ UTR in translational control. We demonstrate that, in brain and pancreas, almost all of the native BACE1 mRNA contains the full-length 5′ UTR. RNA transfection and in vitro translation show that translation is mainly inhibited by the presence of the uAUGs. We provide a mutational analysis that highlight the second uAUG as the main inhibitory element while mutations of all four uAUGs fully de-repress translation. Furthermore, we have evidence that a sequence within the region 222-323 of the BACE1 5′ UTR has a stimulatory effect on translation that might depend on the presence of trans-acting factors.

Cellular internal ribosome entry segments: structures, trans-acting factors and regulation of gene expression.

Initiation of translation in eukaryotic cells can occur by two distinct mechanisms, cap-dependent scanning and internal ribosome entry. The latter mechanism requires the formation of a complex RNA structural element termed an internal ribosome entry segment (IRES). IRESs are located in the 5' untranslated region of the message, and in the presence of trans-acting factors allow the ribosome to be recruited to a site that is a considerable distance from the cap structure. Many cellular mRNAs have now been shown to contain IRESs and it is likely that up to 10% of all mRNAs have the capability to initiate translation by this mechanism. The majority of IRESs that have been identified thus far are found in mRNAs whose protein products are associated with the control of cell growth and cell death, including many growth factors, proto-oncogenes and proteins required for apoptosis. In this review, we discuss the cellular situations when IRESs are required, the trans-acting factors that are necessary for IRES function and deregulation of IRES-mediated translation in tumorigenesis.

Cloning and Characterization of a Potential Transcriptional Activator of Human γ-Globin Genes

ABSTRACTHybrids produced by fusing human fetal erythroblasts (HFE) with mouse erythroleukemia (MEL) cells initially produce predominantly or exclusively human γ-globin and switch to human β globin expression as time in culture advances. One explanation for the initially predominant expression of γ-globin gene in these hybrids is the presence of trans-acting factors that activate γ-globin gene transcription. We used differential display of hybrids before and after the γ to β switch as well as fetal liver and adult erythroblasts to identify cDNAs that could be candidates for potential γ gene activators. Identically sized amplicons which were present in fetal liver erythroblasts and in the hybrids expressing only γ-globin but were absent in the adult erythroblasts and in the same hybrids after they had switched to β globin expression were cloned and sequenced. Fifty pairs of cDNAs fitting these criteria were chosen for further analysis. The sequences of the two members of 48 pairs differed from each other, revealing the low efficiency of this experimental approach. One clone pair coded for human proteosome subunit X. The second pair coded for a protein containing an acidic domain in the N-terminus and three consecutive CDC10/SW16/ankyrin repeats in the C-terminus. Transactivation assays in the yeast hybrid system and transient transfection assays in COS cells showed that a potent trans-activating domain resides in the N-terminus of this protein. Northern blot and RT-PCR assays showed that this gene is expressed in several fetal tissues but not in adult tissues. Stable transfection assays provided evidence that the product of this gene may increase the level of γ mRNA in HFE × MEL cell hybrids that undergo the γ to β switch, suggesting that this new gene encodes a protein that may function as γ gene activator.

Age-Related Changes in the Ovalbumin Gene of the Japanese Quail

Northern hybridization studies showed that the level of ovalbumin mRNA decreases in the oviduct of the Japanese quail after adulthood. In order to find out if this is due to changes in the conformation of the chromatin containing the promoter region of the gene, nuclei of the oviduct of young, adult and old birds were digested by DNaseI and micrococcal nuclease (MNase). Southern hybridization with the labelled promoter showed that this region is less sensitive to the two enzymes in the old birds. Both the endonucleases recognized the same hypersensitive sites. The results indicate that the chromatin containing the promoter is present in an open conformation in adult birds. Gel mobility shift assay using a 20-mer dsDNA containing the CAAT-box and nuclear extract of oviduct shows the presence oftrans-acting factors that bind to this region. The levels of these factors are lower in old birds. This may be the reason for the lower expression of the gene in old birds.

Analysis of the upstream regulatory region of human ventricular myosin light chain 1 gene

To explore the mechanisms regulating expression of ventricular myosin light chain 1, the human gene including 5′-flanking DNA was cloned and characterized by Southern blot and restriction mapping. A 2 kb 5′-flanking DNA was sequenced and linked to a chloramphenicol acetyltransferase reporter gene. The constructs then were transfected into cultured human and rat cardiomyocytes as well as rat aortic endothelial cells. Deletion analysis of constructs revealed that the basal promoter sequences, which were located within 62 base pairs of the cap site, could direct high levels of chloramphenicol acetyltransferase gene expression in the cardiomyocytes and endothelial cells. The region between −62 to −312 base pairs strongly repressed the chloramphenicol acetyltransferase gene expression in the cardiomyocytes and endothelial cells. Positive elements were found between −312 and −2000 base pairs of the cap site. These resuts are indicative, among other possibilities, that the human ventricular myosin light chain 1 gene is turned on in cardiomyocytes by the presence of trans-acting factors that are bound to upstream positive elements and is turned off in non-muscle cells by the presence of repressor-binding proteins. But this mechanism remains to be established.

Analysis of Aldoxn alleles isolated from natural populations of Drosophila melanogaster.

Aldox "null" alleles which were isolated from natural populations in Great Britain and North Carolina were analyzed for complementation. No complementation was observed between any combinations of "null" alleles for aldehyde oxidase (AO) specific activity in late third-instar larvae and newly emerged adults. AO immunologically cross-reacting material (AO-CRM) was quantitated in all homozygous stocks at both developmental stages as well as all allelic combinations in newly emerged adults. When the adult organism contains only Aldoxn alleles, the polypeptides are not immunologically recognizable or may be rapidly degraded. Larvae and adults have different abilities to degrade mutationally altered enzymatically inactive AO polypeptide or synthesize them differentially. This is indicated by easily measurable AO-CRM levels in late third-instar larvae of Aldoxn homozygotes, while newly emerged adult Aldoxn homozygotes have very little, if any, AO-CRM. Newly emerged adult heterozygotes of Aldoxn/Aldox+ do have increased AO-CRM, indicating that the Aldoxn alleles can code for a polypeptide which can be "rescued" if Aldox+ gene product is present. Heterozygotes containing an Aldox+ allele with a deficiency for the Aldox region produce 74.2% of the AO-CRM found in Aldox+ homozygotes. This may indicate the presence of trans-acting factors which serve to activate gene expression in a system in which each gene copy is not maximally expressed.