Role of pulmonary macrophages in initiation of lung metastasis in anaplastic thyroid cancer.

Several clinical studies have demonstrated that increased macrophage infiltration into tumors confers metastatic potential and poor prognosis in cancer. Preclinical studies are needed to develop new strategies for countering metastasis. Our study was designed to investigate the impact of pulmonary macrophages on lung metastasis of anaplastic thyroid cancer (ATC). ATC (CAL-62) and macrophage (Raw264.7) were transfected with the effluc (CAL-62/effluc, Raw264.7/effluc). Coculture and migration assays were used to assess the effect of Raw264.7 or THP1 (human macrophage) (or conditioned medium) on the proliferation and/or migration of CAL-62/effluc cells in vitro. The effect of clodro-lipo or PBS-lipo on macrophage depletion was confirmed in vitro and in vivo. CAL-62/effluc cells (1 × 10(6) ) were intravenously injected into nude mice 24 h after clodro-lipo or PBS-lipo administration. Effect of clodro-lipo on the lung metastasis of CAL-62/effluc was assessed by bioluminescence imaging (BLI). Micro computed tomography (micro-CT) and histology. BLI signals of CAL-62/effluc and Raw264.7/effluc increased to cell number. Raw264.7 cells and THP1 cells promoted CAL-62/effluc proliferation, and conditioned medium of Raw264.7 cells promoted CAL-62/effluc migration. Clodro-lipo significantly depleted pulmonary macrophages in vitro and in vivo. Intensity of BLI signals in ATC lung metastasis was weaker in the clodro-lipo group than PBS-lipo control. Micro-CT imaging and hematoxylin/eosin staining revealed smaller tumor masses in the clodro-lipo group than PBS-lipo control. Our findings indicate that pulmonary macrophages have an important role in initiation of lung metastasis of ATC. New therapeutic strategies that preclude initiation of pulmonary metastasis could potentially be developed by targeting pulmonary macrophages.

Functional characterization of a new promoter isolated from torque teno sus virus 1.

Torque teno sus virus 1 (TTSuV1) has a non-enveloped, single-stranded, negative-sense circular DNA genome, and it is widely distributed in pigs. Open reading frame 1 (ORF1) of TTSuV1 can be transcribed into mRNA and then translated into protein; however, its promoter has not yet been identified. We used a dual-luciferase reporter system, involving pGL3-Basic and pRL-TK, to identify the promoter of TTSuV1 ORF1. Our results revealed that the sequence between nucleotides 196 and 525 promoted the transcription of the firefly luciferase gene. The core sequence of the promoter was between nucleotides 250 and 400. A comparison of the identified TTSuV1 ORF1 promoter with that from cytomegalovirus (CMV) suggested that the two promoters were similar in strength. Our findings provide new information regarding the molecular biology of TTSuV1 and have revealed a new promoter that can be used in plasmids for numerous applications.

Stabilized plasmid-based system for bioluminescent labeling of multiple streptococcal species.

OBJECTIVE: To determine if multiple streptococcal species can be easily labeled for biophotonic imaging using a toxin-antitoxin stabilized reporter plasmid containing the native firefly luciferase gene, originally developed for use in Group A Streptococcus. RESULTS: A number of streptococcal species including Group B Streptococcus, Group C Streptococcus, Group G Streptococcus, S. iniae, S. vestibularis, and S. salivarius were successfully transformed with the reporter plasmid. In absence of antibiotic selection, the plasmid had variable stability amongst the six strains. The expression of firefly luciferase was highest in Group B Streptococcus and S. iniae, as observed by the brightest signal and lowest detection limits in vitro. CONCLUSION: Multiple streptococcal species can be easily transformed with our toxin-antitoxin stabilized bioluminescent reporter plasmid. However, this plasmid shows variable stability and signal in different species, restricting its use for certain applications.

A cooled CCD camera-based protocol provides an effective solution for in vitro monitoring of luciferase.

Luciferase assay has become an increasingly important technique to monitor a wide range of biological processes. However, the mainstay protocols require a luminometer to acquire and process the data, therefore limiting its application to specialized research labs. To overcome this limitation, we have developed an alternative protocol that utilizes a commonly available cooled charge-coupled device (CCCD), instead of a luminometer for data acquiring and processing. By measuring activities of different luciferases, we characterized their substrate specificity, assay linearity, signal-to-noise levels, and fold-changes via CCCD. Next, we defined the assay parameters that are critical for appropriate use of CCCD for different luciferases. To demonstrate the usefulness in cultured mammalian cells, we conducted a case study to examine NFκB gene activation in response to inflammatory signals in human embryonic kidney cells (HEK293 cells). We found that data collected by CCCD camera was equivalent to those acquired by luminometer, thus validating the assay protocol. In comparison, The CCCD-based protocol is readily amenable to live-cell and high-throughput applications, offering fast simultaneous data acquisition and visual and quantitative data presentation. In conclusion, the CCCD-based protocol provides a useful alternative for monitoring luciferase reporters. The wide availability of CCCD will enable more researchers to use luciferases to monitor and quantify biological processes.

Aminoluciferins extend firefly luciferase bioluminescence into the near-infrared and can be preferred substrates over D-luciferin.

Firefly luciferase adenylates and oxidizes d-luciferin to chemically generate visible light and is widely used for biological assays and imaging. Here we show that both luciferase and luciferin can be reengineered to extend the scope of this light-emitting reaction. D-Luciferin can be replaced by synthetic luciferin analogues that increase near-infrared photon flux >10-fold over that of D-luciferin in live luciferase-expressing cells. Firefly luciferase can be mutated to accept and utilize rigid aminoluciferins with high activity in both live and lysed cells yet exhibit 10,000-fold selectivity over the natural luciferase substrate. These new luciferin analogues thus pave the way to an extended family of bioluminescent reporters.

In vivo brain delivery of v-myc overproduced human neural stem cells via the intranasal pathway: tumor characteristics in the lung of a nude mouse.

We aimed to monitor the successful brain delivery of stem cells via the intranasal route and to observe the long-term consequence of the immortalized human neural stem cells in the lungs of a nude mouse model. Stably immortalized HB1.F3 human neural stem cells with firefly luciferase gene (F3-effluc) were intranasally delivered to BALB/c nude mice. Bioluminescence images were serially acquired until 41 days in vivo and at 4 hours and 41 days ex vivo after intranasal delivery. Lungs were evaluated by histopathology. After intranasal delivery of F3-effluc cells, the intense in vivo signals were detected in the nasal area, migrated toward the brain areas at 4 hours (4 of 13, 30.8%), and gradually decreased for 2 days. The brain signals were confirmed by ex vivo imaging (2 of 4, 50%). In the mice with initial lung signals (4 of 9, 44.4%), the lung signals disappeared for 5 days but reappeared 2 weeks later. The intense lung signals were confirmed to originate from the tumors in the lungs formed by F3-effluc cells by ex vivo imaging and histopathology. We propose that intranasal delivery of immortalized stem cells should be monitored for their successful delivery to the brain and their tumorigenicity longitudinally.

An aggregation sensing reporter identifies leflunomide and teriflunomide as polyglutamine aggregate inhibitors.

Intracellular protein aggregation is a common pathologic feature in neurodegenerative diseases such as Huntington' disease, amyotrophic lateral sclerosis and Parkinson' disease. Although progress towards understanding protein aggregation in vitro has been made, little of this knowledge has translated to patient therapy. Moreover, mechanisms controlling aggregate formation and catabolism in cellulo remain poorly understood. One limitation is the lack of tools to quantitatively monitor protein aggregation and disaggregation. Here, we developed a protein-aggregation reporter that uses huntingtin exon 1 containing 72 glutamines fused to the N-terminal end of firefly luciferase (httQ72-Luc). httQ72-Luc fails to aggregate unless seeded by a non-luciferase-containing polyglutamine (polyQ) protein such as Q80-cfp. Upon co-aggregation, httQ72-luc becomes insoluble and loses its enzymatic activity. Using httQ72-Luc with Q80(CFP/YFP) as seeds, we screened the Johns Hopkins Clinical Compound Library and identified leflunomide, a dihydroorotate dehydrogenase inhibitor with immunosuppressive and anti-psoriatic activities, as a novel drug that prevents polyQ aggregation. Leflunomide and its active metabolite teriflunomide inhibited protein aggregation independently of their known role in pyrimidine biosynthesis, since neither uridine treatment nor other pyrimidine biosynthesis inhibitors affected polyQ aggregation. Inducible cell line and cycloheximide-chase experiments indicate that these drugs prevent incorporation of expanded polyQ into an aggregate. This study demonstrates the usefulness of luciferase-based protein aggregate reporters for high-throughput screening applications. As current trials are under-way for teriflunomide in the treatment of multiple sclerosis, we propose that this drug be considered a possible therapeutic agent for polyQ diseases.

Rapid in vivo assessment of drug efficacy against Mycobacterium tuberculosis using an improved firefly luciferase.

OBJECTIVES: In vivo experimentation is costly and time-consuming, and presents a major bottleneck in anti-tuberculosis drug development. Conventional methods rely on the enumeration of bacterial colonies, and it can take up to 4 weeks for Mycobacterium tuberculosis to grow on agar plates. Light produced by recombinant bacteria expressing luciferase enzymes can be used as a marker of bacterial load, and disease progression can be easily followed non-invasively in live animals by using the appropriate imaging equipment. The objective of this work was to develop a bioluminescence-based mouse model of tuberculosis to assess antibiotic efficacy against M. tuberculosis in vivo. METHODS: We used an M. tuberculosis strain carrying a red-shifted derivative of the firefly luciferase gene (FFlucRT) to infect mice, and monitored disease progression in living animals by bioluminescence imaging before and after treatment with the frontline anti-tuberculosis drug isoniazid. The resulting images were analysed and the bioluminescence was correlated with bacterial counts. RESULTS: Using bioluminescence imaging we detected as few as 1.7 × 10(3) and 7.5 × 10(4) reporter bacteria ex vivo and in vivo, respectively, in the lungs of mice. A good correlation was found between bioluminescence and bacterial load in both cases. Furthermore, a marked reduction in luminescence was observed in living mice given isoniazid treatment. CONCLUSIONS: We have shown that an improved bioluminescent strain of M. tuberculosis can be visualized by non-invasive imaging in live mice during an acute, progressive infection and that this technique can be used to rapidly visualize and quantify the effect of antibiotic treatment. We believe that the model presented here will be of great benefit in early drug discovery as an easy and rapid way to identify active compounds in vivo.

Optimized extract preparation methods and reaction conditions for improved yeast cell-free protein synthesis.

Cell-free protein synthesis (CFPS) has emerged as a powerful platform technology to help satisfy the growing demand for simple, affordable, and efficient protein production. In this article, we describe a novel CFPS platform derived from the popular bio-manufacturing organism Saccharomyces cerevisiae. By developing a streamlined crude extract preparation protocol and optimizing the CFPS reaction conditions we were able to achieve active firefly luciferase synthesis yields of 7.7 ± 0.5 µg mL(-1) with batch reactions lasting up to 2 h. This duration of synthesis is the longest ever reported for a yeast CFPS batch reaction. Furthermore, by removing extraneous processing steps and eliminating expensive reagents from the cell-free reaction, we have increased relative product yield (µg protein synthesized per $ reagent cost) over an alternative commonly used method up to 2000-fold from ∼2 × 10(-4) to ∼4 × 10(-1) µg $(-1) , which now puts the yeast CPFS platform on par with other eukaryotic CFPS platforms commercially available. Our results set the stage for developing a yeast CFPS platform that provides for high-yielding and cost-effective expression of a variety of protein therapeutics and protein libraries.

A rapid, inexpensive yeast-based dual-fluorescence assay of programmed--1 ribosomal frameshifting for high-throughput screening.

Programmed -1 ribosomal frameshifting (-1 PRF) is a mechanism that directs elongating ribosomes to shift-reading frame by 1 base in the 5' direction that is utilized by many RNA viruses. Importantly, rates of -1 PRF are fine-tuned by viruses, including Retroviruses, Coronaviruses, Flavivriuses and in two endogenous viruses of the yeast Saccharomyces cerevisiae, to deliver the correct ratios of different viral proteins for efficient replication. Thus, -1 PRF presents a novel target for antiviral therapeutics. The underlying molecular mechanism of -1 PRF is conserved from yeast to mammals, enabling yeast to be used as a logical platform for high-throughput screens. Our understanding of the strengths and pitfalls of assays to monitor -1 PRF have evolved since the initial discovery of -1 PRF. These include controlling for the effects of drugs on protein expression and mRNA stability, as well as minimizing costs and the requirement for multiple processing steps. Here we describe the development of an automated yeast-based dual fluorescence assay of -1 PRF that provides a rapid, inexpensive automated pipeline to screen for compounds that alter rates of -1 PRF which will help to pave the way toward the discovery and development of novel antiviral therapeutics.

Characterization of L1 retrotransposition with high-throughput dual-luciferase assays.

Recent studies employing genome-wide approaches have provided an unprecedented view of the scope of L1 activities on structural variations in the human genome, and further reinforced the role of L1s as one of the major driving forces behind human genome evolution. The rapid identification of novel L1 elements by these high-throughput approaches demands improved L1 functional assays. However, the existing assays use antibiotic selection markers or fluorescent proteins as reporters; neither is amenable to miniaturization. To increase assay sensitivity and throughput, we have developed a third generation assay by using dual-luciferase reporters, in which firefly luciferase is used as the retrotransposition indicator and Renilla luciferase is encoded on the same or separate plasmid for normalization. This novel assay is highly sensitive and has a broad dynamic range. Quantitative data with high signal-to-noise ratios can be obtained from 24- up to 96-well plates in 2-4 days after transfection. Using the dual-luciferase assays, we have characterized profiles of retrotransposition by various human and mouse L1 elements, and detailed the kinetics of L1 retrotransposition in cultured cells. Its high-throughput and short assay timeframe make it well suited for routine tests as well as large-scale screening efforts.

Measuring CREB activation using bioluminescent probes that detect KID-KIX interaction in living cells.

The cyclic adenosine monophosphate response element-binding protein (CREB) is a transcription factor that contributes to memory formation. The transcriptional activity of CREB is induced by its phosphorylation at Ser-133 and subsequent interaction with the CREB-binding protein (CBP)/p300. We designed and optimized firefly split luciferase probe proteins that detect the interaction of the kinase-inducible domain (KID) of CREB and the KIX domain of CBP/p300. The increase in the light intensity of the probe proteins results from the phosphorylation of the responsible serine corresponding to Ser-133 of CREB. Because these proteins have a high signal-to-noise ratio and are nontoxic, it has become possible for the first time to carry out long-term measurement of KID-KIX interaction in living cells. Furthermore, we examined the usefulness of the probe proteins for future high-throughput cell-based drug screening and found several herbal extracts that activated CREB.

Intracellular delivery of an antisense oligonucleotide via endocytosis of a G protein-coupled receptor.

Gastrin-releasing peptide receptor (GRPR), a member of the G protein-coupled receptor superfamily, has been utilized for receptor-mediated targeting of imaging and therapeutic agents; here we extend its use to oligonucleotide delivery. A splice-shifting antisense oligonucleotide was conjugated to a bombesin (BBN) peptide, and its intracellular delivery was tested in GRPR expressing PC3 cells stably transfected with a luciferase gene interrupted by an abnormally spliced intron. The BBN-conjugate produced significantly higher luciferase expression compared to unmodified oligonucleotide, and this increase was reversed by excess BBN peptide. Kinetic studies revealed a combination of saturable, receptor-mediated endocytosis and non-saturable pinocytosis for uptake of the conjugate. The K(m) value for saturable uptake was similar to the EC(50) value for the pharmacological response, indicating that receptor-mediated endocytosis was a primary contributor to the response. Use of pharmacological and molecular inhibitors of endocytosis showed that the conjugate utilized a clathrin-, actin- and dynamin-dependent pathway to enter PC3 cells. The BBN-conjugate partially localized in endomembrane vesicles that were associated with Rab7 or Rab9, demonstrating that it was transported to late endosomes and the trans-golgi network. These observations suggest that the BBN-oligonucleotide conjugate enters cells via a process of GRPR mediated endocytosis followed by trafficking to deep endomembrane compartments.

Quadruplex structures of muscle gene promoter sequences enhance in vivo MyoD-dependent gene expression.

Gene promoters are enriched in guanine clusters that potentially fold into quadruplex structures. Such quadruplexes were implicated in the regulation of gene expression, plausibly by interacting with transcription factors. We showed previously that homodimers of the myogenic transcription factor MyoD bound in vitro most tightly bimolecular quadruplexes of promoter sequences of muscle-specific genes. By contrast, MyoD-E47 heterodimers formed tighter complexes with d(CANNTG) E-box motifs that govern muscle gene expression. Here, we show that DNA quadruplexes enhance in vivo MyoD and E-box-driven expression of a firefly luciferase (FL) reporter gene. HEK293 cells were transfected with FL expressing p4RTK-FL vector alone or together with MyoD expressing pEMSV-MyoD plasmid, with quadruplexes of alpha7 integrin or sarcomeric mitochondrial creatine kinase (sMtCK) muscle gene promoters or with a combination thereof. Whereas MyoD elevated by approximately 10-fold the levels of FL mRNA and protein, the DNA quadruplexes by themselves did not affect FL expression. However, together with MyoD, quadruplex DNA increased by approximately 35-fold the amounts of FL mRNA and protein. Without affecting its expression, DNA quadruplexes bound MyoD in the cells. Based on these results, we propose models for the regulation of muscle gene transcription by direct interaction of MyoD with promoter quadruplex structures.

Time-dependent increase in ribosome processivity.

We created a novel tripartite reporter RNA to separately and simultaneously examine ribosome translation rates at the 5'- and 3'-ends of a large open reading frame (ORF) in vitro in HeLa cell lysates. The construct contained Renilla luciferase (RLuc), ß-galactosidase and firefly luciferase (FLuc) ORFs linked in frame and separated by a viral peptide sequence that causes cotranslational scission of emerging peptide chains. The length of the ORF contributed to low ribosome processivity, a low number of initiating ribosomes completing translation of the entire ORF. We observed a time-dependent increase in FLuc production rate that was dependent on a poly(A) tail and poly(A)-binding protein, but was independent of eIF4F function. Stimulation of FLuc production occurred earlier on shorter RNA templates. Cleavage of eIF4G at times after ribosome loading on templates occurred did not cause immediate cessation of 5'-RLuc translation; rather, a delay was observed that shortened when shorter templates were translated. Electron microscopic analysis of polysome structures in translation lysates revealed a time-dependent increase in ribosome packing and contact that correlated with increased processivity on the FLuc ORF. The results suggest that ORF transit combined with PABP function contribute to interactions between ribosomes that increase or sustain processivity on long ORFs.

The 5'-untranslated region of the mouse mammary tumor virus mRNA exhibits cap-independent translation initiation.

In this study, we demonstrate the identification of an internal ribosome entry site (IRES) within the 5'-untranslated region (5'-UTR) of the mouse mammary tumor virus (MMTV). The 5'-UTR of the full-length mRNA derived from the infectious, complete MMTV genome was cloned into a dual luciferase reporter construct containing an upstream Renilla luciferase gene (RLuc) and a downstream firefly luciferase gene (FLuc). In rabbit reticulocyte lysate, the MMTV 5'-UTR was capable of driving translation of the second cistron. In vitro translational activity from the MMTV 5'-UTR was resistant to the addition of m(7)GpppG cap-analog and cleavage of eIF4G by foot-and-mouth disease virus (FMDV) L-protease. IRES activity was also demonstrated in the Xenopus laevis oocyte by micro-injection of capped and polyadenylated bicistronic RNAs harboring the MMTV-5'-UTR. Finally, transfection assays showed that the MMTV-IRES exhibits cell type-dependent translational activity, suggesting a requirement for as yet unidentified cellular factors for its optimal function.

Mechanism of PTC124 activity in cell-based luciferase assays of nonsense codon suppression.

High-throughput screening (HTS) assays used in drug discovery frequently use reporter enzymes such as firefly luciferase (FLuc) as indicators of target activity. An important caveat to consider, however, is that compounds can directly affect the reporter, leading to nonspecific but highly reproducible assay signal modulation. In rare cases, this activity appears counterintuitive; for example, some FLuc inhibitors, acting through posttranslational Fluc reporter stabilization, appear to activate gene expression. Previous efforts to characterize molecules that influence luciferase activity identified a subset of 3,5-diaryl-oxadiazole-containing compounds as FLuc inhibitors. Here, we evaluate a number of compounds with this structural motif for activity against FLuc. One such compound is PTC124 {3-[5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl]benzoic acid}, a molecule originally identified in a cell-based FLuc assay as having nonsense codon suppression activity [Welch EM, et al., Nature (2007) 447:87-91]. We find that the potency of FLuc inhibition for the tested compounds strictly correlates with their activity in a FLuc reporter cell-based nonsense codon assay, with PTC124 emerging as the most potent FLuc inhibitor (IC(50) = 7 +/- 1 nM). However, these compounds, including PTC124, fail to show nonsense codon suppression activity when Renilla reniformis luciferase (RLuc) is used as a reporter and are inactive against the RLuc enzyme. This suggests that the initial discovery of PTC124 may have been biased by its direct effect on the FLuc reporter, implicating firefly luciferase as a molecular target of PTC124. Our results demonstrate the value of understanding potential interactions between reporter enzymes and chemical compounds and emphasize the importance of implementing the appropriate control assays before interpreting HTS results.

Lymphoid-tissue-specific homing of bone-marrow-derived dendritic cells.

Because of their potent immunoregulatory capacity, dendritic cells (DCs) have been exploited as therapeutic tools to boost immune responses against tumors or pathogens, or dampen autoimmune or allergic responses. Murine bone marrow-derived DCs (BM-DCs) are the closest known equivalent of the blood monocyte-derived DCs that have been used for human therapy. Current imaging methods have proven unable to properly address the migration of injected DCs to small and deep tissues in mice and humans. This study presents the first extensive analysis of BM-DC homing to lymph nodes (and other selected tissues) after intravenous and intraperitoneal inoculation. After intravenous delivery, DCs accumulated in the spleen, and preferentially in the pancreatic and lung-draining lymph nodes. In contrast, DCs injected intraperitoneally were found predominantly in peritoneal lymph nodes (pancreatic in particular), and in omentum-associated lymphoid tissue. This uneven distribution of BM-DCs, independent of the mouse strain and also observed within pancreatic lymph nodes, resulted in the uneven induction of immune response in different lymphoid tissues. These data have important implications for the design of systemic cellular therapy with DCs, and in particular underlie a previously unsuspected potential for specific treatment of diseases such as autoimmune diabetes and pancreatic cancer.

Sequential bioluminescence resonance energy transfer-fluorescence resonance energy transfer-based ratiometric protease assays with fusion proteins of firefly luciferase and red fluorescent protein.

We report here the preparation of ratiometric luminescent probes that contain two well-separated emission peaks produced by a sequential bioluminescence resonance energy transfer (BRET)-fluorescence resonance energy transfer (FRET) process. The probes are single soluble fusion proteins consisting of a thermostable firefly luciferase variant that catalyze yellow-green (560nm maximum) bioluminescence and a red fluorescent protein covalently labeled with a near-infrared fluorescent dye. The two proteins are connected by a decapeptide containing a protease recognition site specific for factor Xa, thrombin, or caspase 3. The rates of protease cleavage of the fusion protein substrates were monitored by recording emission spectra and plotting the change in peak ratios over time. Detection limits of 0.41nM for caspase 3, 1.0nM for thrombin, and 58nM for factor Xa were realized with a scanning fluorometer. Our results demonstrate for the first time that an efficient sequential BRET-FRET energy transfer process based on firefly luciferase bioluminescence can be employed to assay physiologically important protease activities.

Coupled termination/reinitiation for translation of the downstream open reading frame B of the prototypic hypovirus CHV1-EP713.

Cryphonectria hypovirus 1 (CHV1), associated with the picorna-like superfamily, infects the chestnut blight fungus and attenuates the virulence of the host fungus. The genomic RNA of the virus has two continuous open reading frames, A and B, separated by the pentanucleotide UAAUG. We present here evidence suggesting that ORF B is translated from genome-sized virus mRNA by a coupled termination/reinitiation mechanism mediated by the pentamer. In the coupled translation, the overlapping UAA and AUG triplets serve as the stop codon of ORF A and the initiator of ORF B, respectively. This was established by the use of a luciferase assay with a basic construct containing the ORF A sequence and the firefly luciferase gene while retaining the pentamer between the two coding sequences. The proportion of ribosomes reinitiating translation after terminating was determined to be 2.5-4.4% by three independent assay systems in fungal and insect cells. Use of a series of mutant constructs identified two sequence elements, the pentamer and the p40 sequence, that affect the efficiency of coupled translation and virus replication. Together, these results provide the first example of coupled translation facilitated by the pentanucleotide UAAUG in the kingdom Fungi. The mechanism by which the preceding p40-coding sequence promotes reinitiation is discussed.