Luciferase Open Reading Frame Research Articles

Implications of differential transcription start site selection on chronic myeloid leukemia and prostate cancer cell protein expression.

The relevance of minor transcription start sites in broad promoters is not well understood. We have studied AGAP2 expression in prostate cancer and chronic myeloid leukemia (CML), showing transcription is initiated from alternative transcription start sites (TSSs) within a single TSS cluster, producing cancer-type-specific AGAP2 mRNAs with small differences in their 5' UTR length. Interestingly, in the CML cell lines where the 5' UTR is longer, AGAP2 protein levels are lower. We demonstrate that the selection of an upstream TSS involved the formation of a G quadruplex in the 5' UTR, decreasing polysome formation. After developing a bioinformatics pipeline to query data from the FANTOM project and the NCl-60 human tumor cell lines screen, we found HK1 expression can also be regulated by the same mechanism. Overall, we present compelling data supporting TSS selection within a TSS cluster play a role in protein expression and should not be ignored.

Cryptic promoter activation occurs by at least two different mechanisms in the Arabidopsis genome.

In gene-trap screening of plant genomes, promoterless reporter constructs are often expressed without trapping of annotated gene promoters. The molecular basis of this phenomenon, which has been interpreted as the trapping of cryptic promoters, is poorly understood. Here, we found that cryptic promoter activation occurs by at least two different mechanisms using Arabidopsis gene-trap lines in which a firefly luciferase (LUC) open reading frame (ORF) without an apparent promoter sequence was expressed from intergenic regions: one mechanism is 'cryptic promoter capturing', in which the LUC ORF captured pre-existing promoter-like chromatin marked by H3K4me3 and H2A.Z, and the other is 'promoter de novo origination', in which the promoter chromatin was newly formed near the 5' end of the inserted LUC ORF. The latter finding raises a question as to how the inserted LUC ORF sequence is involved in this phenomenon. To examine this, we performed a model experiment with chimeric LUC genes in transgenic plants. Using Arabidopsis psaH1 promoter-LUC constructs, we found that the functional core promoter region, where transcription start sites (TSSs) occur, cannot simply be determined by the upstream nor core promoter sequences; rather, its positioning proximal to the inserted LUC ORF sequence was more critical. This result suggests that the insertion of the coding sequence alters the local distribution of TSSs in the plant genome. The possible impact of the two types of cryptic promoter activation mechanisms on plant genome evolution and endosymbiotic gene transfer is discussed.

The transcriptome of the Bermuda fireworm Odontosyllis enopla (Annelida: Syllidae): A unique luciferase gene family and putative epitoky-related genes.

The Bermuda fireworm Odontosyllis enopla exhibits an extremely tight circalunar circadian behavior that results in an impressive bioluminescent mating swarm, thought to be due to a conventional luciferase-mediated oxidation of a light-emitting luciferin. In addition, the four eyes become hypertrophied and heavily pigmented, and the nephridial system is modified to store and release gametes and associated secretions. In an effort to elucidate transcripts related to bioluminescence, circadian or circalunar periodicity, as well as epitoky-related changes of the eyes and nephridial system, we examined the transcriptomic profile of three female O. enopla during a bioluminescent swarm in Ferry Reach, Bermuda. Using the well-characterized luciferase gene of the Japanese syllid Odontosyllis undecimdonta as a reference, a complete best-matching luciferase open reading frame (329 amino acids in length) was found in all three individuals analyzed in addition to numerous other paralogous sequences in this new gene family. No photoproteins were detected. We also recovered a predicted homolog of 4-coumarate-CoA ligase (268 amino acids in length) that best matched luciferase of the firefly Luciola with the best predicted template being the crystal structure of luciferase for Photinus pyralis, the common eastern firefly. A wide variety of genes associated with periodicity were recovered including predicted homologs of clock, bmal1, period, and timeless. Several genes corresponding to putative epitoky-related changes of the eyes were recovered including predicted homologs of a phototransduction gene, a retinol dehydrogenase and carotenoid isomerooxygenase as well as a visual perception related retinal rod rhodopsin-sensitive cGMP 3',5'-cyclic phosphodiesterase. Genes correlating to putative epitoky-related changes of the nephridia included predicted homologs of nephrocystin-3 and an egg-release sex peptide receptor.

Heterologous Matrix Metalloproteinase Gene Promoter Activity Allows In Vivo Real-time Imaging of Bleomycin-Induced Lung Fibrosis in Transiently Transgenized Mice.

Idiopathic pulmonary fibrosis is a very common interstitial lung disease derived from chronic inflammatory insults, characterized by massive scar tissue deposition that causes the progressive loss of lung function and subsequent death for respiratory failure. Bleomycin is used as the standard agent to induce experimental pulmonary fibrosis in animal models for the study of its pathogenesis. However, to visualize the establishment of lung fibrosis after treatment, the animal sacrifice is necessary. Thus, the aim of this study was to avoid this limitation by using an innovative approach based on a double bleomycin treatment protocol, along with the in vivo images analysis of bleomycin-treated mice. A reporter gene construct, containing the luciferase open reading frame under the matrix metalloproteinase-1 promoter control region, was tested on double bleomycin-treated mice to investigate, in real time, the correlation between bleomycin treatment, inflammation, tissue remodeling and fibrosis. Bioluminescence emitted by the lungs of bleomycin-treated mice, corroborated by fluorescent molecular tomography, successfully allowed real time monitoring of fibrosis establishment. The reporter gene technology experienced in this work could represent an advanced functional approach for real time non-invasive assessment of disease evolution during therapy, in a reliable and translational living animal model.

Abstract 1927: Myc-induced suppression of the RNA-binding protein ZAR2 in breast cancer cells

Abstract Our objective is to verify the hypothesis that c-Myc-regulated oncomiR miR-130a-5p suppresses the level of the RNA-binding tumor suppressor protein ZAR2 in the cMyc-high aggressive breast cancer cells. We experimentally validated the binding of miR130a-5p miRISC to ZAR2 mRNA in vivo and inhibition of the translation of this mRNA by this binding. We used three commercially available c-Myc-high ZAR2-low breast cancer cell lines BT549, MDA-MB-453 and MDA-MB-231 and two c-Myc-low ZAR2-high breast cancer cells MCF7 and HCC38 for our studies. We cloned the full length 3′-UTR (179 bp) of human ZAR2 mRNA immediately downstream of the firefly luciferase open reading frame sequence contained in the reporter plasmid. We also have created different mutants of the 3’-UTR, each having mutation in each of the putative miR130a-5p binding sites. We transiently transfected the recombinant plasmid and miR130a-5p miRNA mimic into breast cancer cells and assessed luciferase activity or fluorescence 24-48 hours after transfection. We also have transfected just the reporter construct into breast cancer cells which express the endogenous miR130a-5p miRNAs, along with vectors which express mutant versions of the miRNA binding sites. In this experimental system the wild-type reporters had less activity than their respective mutants. To evaluate the functional importance of miR130a-5p miRNA/ZAR2 mRNA pair, we transiently over-expressed a miR130a-5p miRNA mimic in the cMyc-low breast cancer breast cancer cells and subsequently analyzed the level of ZAR2 protein levels. Our data showed that ZAR2 protein levels are decreased by the mimic increasing the in vitro invasiveness of these cells. Our results thus reveal a novel mechanism for the induction of aggressiveness by cMyc in the breast cancer cells. This research is supported in part by DOD-CDMRP IDEA Expansion Grant# BC103645 and NIH/NCI grant 1R21CA181920-01 to GC and NIH grant 1U54RR026140 TO SM Citation Format: Smita Misra, Gautam Chaudhuri. Myc-induced suppression of the RNA-binding protein ZAR2 in breast cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1927.

Constructing a p53-fused dual luciferase reporter and verifying its function

To construct a p53-fused dual luciferase reporter and to test whether this reporter can mimic wild-type p53 activities in a high-throughput screen. A restriction endonuclease site was added to each terminus and the stop codon of the wild-type full-length p53 open reading frame (ORF) was removed by PCR. A restriction endonuclease site was added to each terminus and the start codon of the firefly luciferase ORF was removed by PCR. The two modified ORFs were inserted upstream of the IRES-induced renilla luciferase ORF in a CMV-derived vector. The p53 fusion protein was expressed in cells to test its MDM2-mediated degradation, subcellular localization, and induction of p53-responsive promoter. The p53-fused dual luciferase reporter was successfully constructed. After transfection into the host cells, the reporter expressing the p53 fusion protein that was degraded by oncoprotein MDM2, was mainly located inside the nucleus, and induced the p53-responsive promoter, respectively. The p53-fused dual luciferase reporter (p53FL/IRES/RL) can identify modulators of P53 protein level in a high-throughput screen of genetic or chemical libraries.

Abstract 1959: Identification of microRNAs that target Id-1 in esophageal cancer.

Abstract Inhibitor of DNA binding-1 (Id-1) belongs to a family of helix-loop-helix (HLH) proteins that lack the fundamental domain for DNA binding. Since Id-1 is overexpressed in esophageal squamous cell carcinoma (ESCC), and its expression level is associated with cancer invasion and metastasis, suppression of Id-1 may have therapeutic significance. MicroRNAs (miRNAs), which constitute non-coding small RNAs of around 22-nucleotides and negatively regulate gene expression as post-transcriptional regulator, were reported to be involved in carcinogenesis. However, there is limited information about the miRNAs that regulate Id-1. This project aims to identify miRNAs that can target Id-1 and exert tumor suppressive functions. In this study, several bioinformatics software programs, including TargetScan, RegRNA, MicroRNA and EMBL-EBI, were used to predict miRNAs that can directly bind to the 3’UTR of Id-1 mRNA. The miRNAs which have high scores and are predicted to target Id-1 by at least 3 different target prediction algorithms were chosen as candidates for this study. These miRNA candidates are: miR-29 family (miR-29a, miR-29b and miR-39c), miR-330, miR-300, miR-593, miR-654, miR-338, miR-346, miR-632. Plasmids which can over-express these miRNAs were designed and constructed using BLOCK-iT™ Pol II miR RNAi Expression system (Invitrogen®). Successful over-expression of the miRNAs in ESCC cell lines were verified by qRT-PCR. The transient and stable miRNA over-expressing ESCC cells were harvested and subjected to western blot analysis for Id-1 protein expression level. Direct binding of miRNAs to the 3’UTR of Id-1 was analyzed by 3’UTR luciferase assay. The pGL3-Id-1 3’UTR luciferase reporter vector was constructed by cloning the Id-1 3’ UTR downstream of the firefly luciferase open reading frame and the luciferase activities were measured using the Dual Luciferase Assay System. The cell invasion ability of miRNA over-expressing ESCC cells was analyzed by using BD BioCoat™ Matrigel™ Invasion Chamber. Cell proliferation and colony formation assays were performed to investigate the anti-proliferation effect of these miRNAs. In this study, ten miRNA over-expressing plasmids were constructed and over-expression of several miRNAs was achieved. We found that miR-29c could directly target the 3’UTR of Id-1 and down-regulate Id-1 protein level. miR-29c exerts tumor suppressing effects by reducing the ESCC cell invasion ability. Further study using miR-29c inhibitors in western blot assay, Id-1 3’UTR luciferase assay and cell invasion assay will be conducted. In summary, this study shows that miR-29c can suppress Id-1 and inhibit cell invasion in ESCC cells. This study is supported by General Research Funds from Research Grants Council of the Hong Kong SAR (Project Nos. HKU762610M and HKU763111M) Citation Format: L Han, B Li, SW Tsao, ALM Cheung. Identification of microRNAs that target Id-1 in esophageal cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1959. doi:10.1158/1538-7445.AM2013-1959

Insulin growth factor 2 mRNA binding protein 1 (IGF2BP1) regulates translation of the multidrug resistance protein 2 (MRP2) by binding to its 5′‐untranslated region (5′UTR)

MRP2 is a member of the ATP binding cassette superfamily (ABC) of efflux transporters and mediates the ATP‐dependent efflux of glucuronide and glutathione conjugates from liver, kidney and intestinal cells. The MRP2 5′UTR was investigated for its regulation of downstream gene expression. Both transient gene expression in HepG2 cells and in vitro translation assays of MRP2 5′UTR‐luciferase constructs showed that a 35 nt RNA motif in the MRP2 5′UTR stimulates downstream open reading frame (ORF) translation. One, 2, 3 and 5 copies of the 35 nt motif were inserted between renilla and firefly luciferase ORFs in bicistronic constructs and transfected into HepG2 cells, where analysis of gene expression showed that the 35 nt motif enhanced translation of the 3′ cistron without affecting its mRNA expression. In vitro translation assays of the bicistronic transcripts showed that the 35 nt motif increased translation of the 3′ cistron, consistent with potential Internal Ribosome Entry Site (IRES) activity of the 35 nt RNA motif. Using RNA EMSA, affinity purification and Mass Spectrometry, IGF2BP1 was identified as a protein that bound to the 35 nt RNA motif, while over‐expression of IGF2BP1 in HepG2 cells increased MRP2 translation. Thus, IGF2BP1 stimulates cap‐dependent and cap‐independent translation initiation through binding to the MRP2 5′UTR 35 nt motif.

MiR-331-3p Regulates ERBB-2 Expression and Androgen Receptor Signaling in Prostate Cancer

MicroRNAs (miRNAs) are short, non-coding RNAs that regulate gene expression and are aberrantly expressed in human cancer. The ERBB-2 tyrosine kinase receptor is frequently overexpressed in prostate cancer and is associated with disease progression and poor survival. We have identified two specific miR-331-3p target sites within the ERBB-2 mRNA 3'-untranslated region and show that miR-331-3p expression is decreased in prostate cancer tissue relative to normal adjacent prostate tissue. Transfection of multiple prostate cancer cell lines with miR-331-3p reduced ERBB-2 mRNA and protein expression and blocked downstream phosphatidylinositol 3-kinase/AKT signaling. Furthermore, miR-331-3p transfection blocked the androgen receptor signaling pathway in prostate cancer cells, reducing activity of an androgen-stimulated prostate-specific antigen promoter and blocking prostate-specific antigen expression. Our findings provide insight into the regulation of ERBB-2 expression in cancer and suggest that miR-331-3p has the capacity to regulate signaling pathways critical to the development and progression of prostate cancer cells.

Arabidopsis mutants reveal multiple singlet oxygen signaling pathways involved in stress response and development

Shortly after the release of singlet oxygen ((1)O(2)) in chloroplasts drastic changes in nuclear gene expression occur in the conditional flu mutant of Arabidopsis that reveal a rapid transfer of signals from the plastid to the nucleus. Factors involved in this retrograde signaling were identified by mutagenizing a transgenic flu line expressing a (1)O(2)-responsive reporter gene. The reporter gene consisted of the luciferase open reading frame and the promoter of an AAA-ATPase gene (At3g28580) that was selectively activated by (1)O(2) but not by superoxide or hydrogen peroxide. A total of eight second-site mutants were identified that either constitutively activate the reporter gene and the endogenous AAA-ATPase irrespectively of whether (1)O(2) was generated or not (constitutive activators of AAA-ATPase, caa) or abrogated the (1)O(2)-dependent up-regulation of these genes as seen in the transgenic parental flu line (non-activators of AAA-ATPase, naa). The characterization of the mutants strongly suggests that (1)O(2)-signaling does not operate as an isolated linear pathway but rather forms an integral part of a signaling network that is modified by other signaling routes and impacts not only stress responses of plants but also their development.

Identification of Adjacent Binding Sites for the YY1 and E4BP4 Transcription Factors in the Ovine PrP (Prion) Gene Promoter

The PrP gene encodes the cellular isoform of the prion protein (PrP(c)) which has been shown to be crucial to the development of transmissible spongiform encephalopathies (TSEs). PrP knock-out mice, which do not express endogenous PrP(c), exhibit resistance to TSE disease. The regulation of PrP gene expression represents, therefore, a crucial factor in the development of TSEs. Two sequence motifs in the PrP promoter (positions -287 to -263 from transcriptional start) were previously reported as being highly conserved, and it was suggested that they represent binding sites for as yet unidentified transcription factors. To test this hypothesis, binding of nuclear proteins was analyzed by electrophoretic mobility shift assays using ovine or murine cells and tissues with radiolabeled DNA probes containing the conserved motif sequences. Specific binding was observed to both motifs, and polymorphic variants of these motifs exhibited differential binding. Two proteins bound to these motifs were identified as the Yin Yang 1 (YY1) (motif 1) and E4BP4 (motif 2) transcription factors. Functional promoter analysis of four different promoter variants revealed that motif 1 (YY1) was associated with inhibitory activity in the context of the PrP promoter, whereas motif 2 (E4BP4) was linked to a slight enhancing activity. This represents the first demonstration of binding of nuclear factors to two highly conserved DNA sequence motifs within mammalian PrP promoters. The action of these factors on the PrP promoter is haplotype-specific, leading us to propose that the prion protein expression pattern and, with it, the distribution of TSE infectivity may be associated with PrP promoter genotype.

Regulation of Epidermal Growth Factor Receptor Signaling in Human Cancer Cells by MicroRNA-7

The epidermal growth factor receptor (EGFR) is frequently overexpressed in cancer and is an important therapeutic target. Aberrant expression and function of microRNAs have been associated with tumorigenesis. Bioinformatic predictions suggest that the human EGFR mRNA 3'-untranslated region contains three microRNA-7 (miR-7) target sites, which are not conserved across mammals. We found that miR-7 down-regulates EGFR mRNA and protein expression in cancer cell lines (lung, breast, and glioblastoma) via two of the three sites, inducing cell cycle arrest and cell death. Because miR-7 was shown to decrease EGFR mRNA expression, we used microarray analysis to identify additional mRNA targets of miR-7. These included Raf1 and multiple other genes involved in EGFR signaling and tumorigenesis. Furthermore, miR-7 attenuated activation of protein kinase B (Akt) and extracellular signal-regulated kinase 1/2, two critical effectors of EGFR signaling, in different cancer cell lines. These data establish an important role for miR-7 in controlling mRNA expression and indicate that miR-7 has the ability to coordinately regulate EGFR signaling in multiple human cancer cell types.

The Biflavonoid Isoginkgetin Is a General Inhibitor of Pre-mRNA Splicing

Membrane-permeable compounds that reversibly inhibit a particular step in gene expression are highly useful tools for cell biological and biochemical/structural studies. In comparison with other gene expression steps where multiple small molecule effectors are available, very few compounds have been described that act as general inhibitors of pre-mRNA splicing. Here we report construction and validation of a set of mammalian cell lines suitable for the identification of small molecule inhibitors of pre-mRNA splicing. Using these cell lines, we identified the natural product isoginkgetin as a general inhibitor of both the major and minor spliceosomes. Isoginkgetin inhibits splicing both in vivo and in vitro at similar micromolar concentrations. It appears to do so by preventing stable recruitment of the U4/U5/U6 tri-small nuclear ribonucleoprotein, resulting in accumulation of the prespliceosomal A complex. Like two other recently reported general pre-mRNA splicing inhibitors, isoginkgetin has been previously described as an anti-tumor agent. Our results suggest that splicing inhibition is the mechanistic basis of the anti-tumor activity of isoginkgetin. Thus, pre-mRNA splicing inhibitors may represent a novel avenue for development of new anti-cancer agents.

A Protective Role for the Human SMG-1 Kinase against Tumor Necrosis Factor-α-induced Apoptosis

The human suppressor of morphogenesis in genitalia-1 (hSMG-1) protein kinase plays dual roles in mRNA surveillance and genotoxic stress response pathways in human cells. Here, we report that small interfering RNA-mediated depletion of hSMG-1, but not ATM, ATR, hUpf1, or hUpf2, in human U2OS osteosarcoma cells markedly increases the magnitude and accelerates the rate of apoptosis induced by tumor necrosis factor-alpha (TNFalpha) stimulation. The increase in TNFalpha-mediated cell killing observed in hSMG-1-depleted cells is not related to the suppression of nonsense-mediated mRNA decay or to the inhibition of TNFalpha-induced NF-kappaB activation. Rather, we observed that loss of hSMG-1 accelerates the degradation of the long form of the FLICE-inhibitory protein (FLIP(L)), an inhibitor of death-inducing signaling complex-mediated caspase-8 activation, in TNFalpha-treated cells. These results suggest that hSMG-1 plays an important role in cell survival during TNFalpha-induced stress.

Human copper transporter 2 is localized in late endosomes and lysosomes and facilitates cellular copper uptake

High-affinity cellular copper uptake is mediated by the CTR (copper transporter) 1 family of proteins. The highly homologous hCTR (human CTR) 2 protein has been identified, but its function in copper uptake is currently unknown. To characterize the role of hCTR2 in copper homoeostasis, epitope-tagged hCTR2 was transiently expressed in different cell lines. hCTR2-vsvG (vesicular-stomatitis-virus glycoprotein) predominantly migrated as a 17 kDa protein after imunoblot analysis, consistent with its predicted molecular mass. Chemical cross-linking resulted in the detection of higher-molecular-mass complexes containing hCTR2-vsvG. Furthermore, hCTR2-vsvG was co-immunoprecipitated with hCTR2-FLAG, suggesting that hCTR2 can form multimers, like hCTR1. Transiently transfected hCTR2-eGFP (enhanced green fluorescent protein) was localized exclusively to late endosomes and lysosomes, and was not detected at the plasma membrane. To functionally address the role of hCTR2 in copper metabolism, a novel transcription-based copper sensor was developed. This MRE (metal-responsive element)-luciferase reporter contained four MREs from the mouse metallothionein 1A promoter upstream of the firefly luciferase open reading frame. Thus the MRE-luciferase reporter measured bioavailable cytosolic copper. Expression of hCTR1 resulted in strong activation of the reporter, with maximal induction at 1 muM CuCl2, consistent with the K(m) of hCTR1. Interestingly, expression of hCTR2 significantly induced MRE-luciferase reporter activation in a copper-dependent manner at 40 and 100 microM CuCl2. Taken together, these results identify hCTR2 as an oligomeric membrane protein localized in lysosomes, which stimulates copper delivery to the cytosol of human cells at relatively high copper concentrations. This work suggests a role for endosomal and lysosomal copper pools in the maintenance of cellular copper homoeostasis.

A Ferritin-responsive Internal Ribosome Entry Site Regulates Folate Metabolism

Cytoplasmic serine hydroxymethyltransferase (cSHMT) enzyme levels are elevated by the expression of the heavy chain ferritin (H ferritin) cDNA in cultured cells without corresponding changes in mRNA levels, resulting in enhanced folate-dependent de novo thymidylate biosynthesis and impaired homocysteine remethylation. In this study, the mechanism whereby H ferritin regulates cSHMT expression was determined. cSHMT translation is shown to be regulated by an H ferritin-responsive internal ribosome entry site (IRES) located within the cSHMT mRNA 5'-untranslated region (5'-UTR). The cSHMT 5'-UTR exhibited IRES activity during in vitro translation of bicistronic mRNA templates, and in MCF-7 and HeLa cells transfected with bicistronic mRNAs. IRES activity was depressed in H ferritin-deficient mouse embryonic fibroblasts and elevated in cells expressing the H ferritin cDNA. H ferritin was shown to interact with the mRNA-binding protein CUGBP1, a protein known to interact with the alpha and beta subunits of eukaryotic initiation factor eIF2. Small interference RNA-mediated depletion of CUGBP1 decreased IRES activity from bicistronic templates that included the cSHMT 3'-UTR in the bicistronic construct. The identification of this H ferritin-responsive IRES represents a mechanism that accounts for previous observations that H ferritin regulates folate metabolism.

DNA Repair of Clustered Uracils in HeLa Cells

Two or more base damages, abasic sites or single-strand breaks (SSBs) within two helical turns of the DNA form a multiply damaged site (MDS) or clustered lesion. Studies in vitro and in bacteria indicate that attempts to repair two closely opposed base lesions can potentially form a lethal double-strand break (DSB). Ionizing radiation and chemotherapeutic agents introduce complex lesions, and the inability of a cell to repair MDSs is believed to contribute to the lethality of these treatments. The goal of this work was to extend the in vitro studies by examining MDS repair in mammalian cells under physiological conditions. Here, two opposing uracil residues separated by 3, 5, 7, 13 or 29 base-pairs were chosen as model DNA lesions. Double-stranded oligonucleotides containing no damage, a single uracil residue or the MDS were introduced into a non-replicating mammalian construct within the firefly luciferase open reading frame, or at the 5' or 3' end of the luciferase expression cassette. Following transient transfection into HeLa cells, luciferase activity was measured or plasmid DNA was re-isolated from the cells. Formation of a DSB was expected to decrease luciferase expression. However, certain single uracil residues as well as the MDSs decreased luciferase activity, which suggested that the reduction in activity was not due to DSB formation. In fact, Southern analysis of the re-isolated plasmid did not show the presence of linear DNA and demonstrated that none of the constructs was destroyed during repair. Further analysis of the re-isolated DNA demonstrated that only a small percentage of molecules originally carrying a single lesion or an MDS contained deletions. This work indicates that the majority of the clustered lesions were not converted to DSBs and that repair systems in mammalian cells may have established mechanisms to avoid the accumulation of SSB-repair intermediates.

Genetic and phylogenetic characterization of the type II cyclobutane pyrimidine dimer photolyases encoded by Leporipoxviruses

Shope fibroma virus and myxoma virus encode proteins predicted to be Type II photolyases. These are enzymes that catalyze light-dependent repair of cyclobutane pyrimidine dimers (CPDs). When the Shope fibroma virus S127L gene was expressed in an Escherichia coli strain lacking functional CPD repair pathways, the expressed gene protected the bacteria from 70–75% of the ultraviolet (UV) light-induced cytotoxic DNA damage. This proportion suggests that Leporipoxvirus photolyases can only repair CPDs, which typically comprise ∼70% of the damage caused by short wavelength UV light. To test whether these enzymes can protect virus genomes from UV, we exposed virus suspensions to UV-C light followed by graded exposure to filtered visible light. Viruses encoding a deletion of the putative photolyase gene were unable to photoreactivate UV damage while this treatment again eliminated 70–90% of the lethal photoproducts in wild-type viruses. Western blotting detected photolyase protein in extracts prepared from purified virions and it can be deduced that the poxvirion interior must be fluid enough to permit diffusion of this ∼50-kDa DNA-binding protein to the sites where it catalyzes photoreactivation. Photolyase promoters are difficult to categorize using bioinformatics methods, as they do not obviously resemble any of the known poxvirus promoter motifs. By fusing the SFV promoter to DNA encoding a luciferase open reading frame, the photolyase promoter was found to exhibit very weak late promoter activity. These data show that the genomes of Leporipoxviruses, similar to that of fowlpox virus, encode catalytically active photolyases. Phylogenetic studies also confirmed the monophyletic origin of poxviruses and suggest an ancient origin for these genes and perhaps poxviruses.

A quantitative analysis of intron effects on mammalian gene expression.

In higher eukaryotes, intron-containing and intronless versions of otherwise identical genes can exhibit dramatically different expression profiles. Introns and the act of their removal by the spliceosome can affect gene expression at many different levels, including transcription, polyadenylation, mRNA export, translational efficiency, and the rate of mRNA decay. However, the extent to which each of these steps contributes to the overall effect of any one intron on gene expression has not been rigorously tested. Here we report construction and initial characterization of a luciferase-based reporter system for monitoring the effects of individual introns and their position within the gene on protein expression in mammalian cells. Quantitative analysis of constructs containing human TPI intron 6 at two different positions within the Renilla luciferase open reading frame revealed that this intron acts primarily to enhance mRNA accumulation. Spliced mRNAs also exhibited higher translational yields than did intronless transcripts. However, nucleocytoplasmic mRNA distribution and mRNA stability were largely unaffected. These findings were extended to two other introns in a TCR-beta minigene.

Use of an in Vivo Reporter Assay to Test for Transcriptional and Translational Fidelity in Yeast

Eukaryotic RNA polymerase II and Escherichia coli RNA polymerase possess an intrinsic ribonuclease activity that is stimulated by the polymerase-binding proteins SII and GreB, respectively. This factor-activated hydrolysis of nascent RNA has been postulated to be involved in transcription elongation as well as removal of incorrect bases misincorporated into RNA. Little is known about the frequency of misincorporation by RNA polymerases in vivo or about the mechanisms involved in improving RNA polymerase accuracy. Here we have developed a luciferase reporter system in an effort to assay for base misincorporation in living Saccharomyces cerevisiae. The assay employs a luciferase open reading frame that contains a premature stop codon. The inactive truncated enzyme would become active if misincorporation by RNA polymerase II took place at the stop triplet. Yeast lacking SII did not display a significant change in reporter activity when compared with wild-type cells. We estimate that under our assay conditions, mRNAs with a misincorporation at the test site could not exceed 1 transcript per 500 cells. The reporter assay was very effective in detecting the previously described process of nonsense suppression (translational read-through) by ribosomes, making it difficult to determine an absolute level of basal (SII-independent) misincorporation by RNA polymerase II. Although these data cannot exclude the possibility that SII is involved in proofreading, they make it unlikely that such a contribution is physiologically significant, especially relative to the high frequency of translational errors.