Expression of p53 synergistically augments caspases-mediated apoptosis induced by replication-competent adenoviruses in pancreatic carcinoma cells.

We examined cytotoxicity of replication-competent type 5 adenoviruses (Ad5) in human pancreatic carcinoma cells with a p53-defective genotype. The replication-competent Ad5 of which E1A gene was activated by exogenous transcriptional regulatory sequences, derived from the midkine and survivin genes, achieved cytotoxicity to the pancreatic carcinoma. These cells were susceptible to replication-incompetent Ad5 expressing the wild-type p53 gene. We also produced the replication-competent Ad5 bearing the same exogenous regulatory sequences and the type 35 Ad-derived fiber-knob region, and showed that the cytotoxicity was comparable to that of the replication-competent Ad5 prototype. We then investigated possible combinatory effects of the fiber-modified replication-competent Ad and Ad5 expressing the wild-type p53 gene, both of which did not interfere respective infections. The combination produced synergistic cytotoxic effects with enhanced cleavages of caspase-3 and PARP molecules, and with increased sub-G1 fractions and annexin V-positive populations although the viral production of the replication-competent Ad was rather suppressed by expressed p53. Pancreatic cells infected with both Ad showed increase of p53 and decrease of MDM2 and p21 levels, compared with those infected with Ad expressing the p53 gene. These data collectively indicated that replication-competent Ad augmented susceptibility of pancreatic cells to apoptosis through upregulated p53 expression.

Three-dimensional morphological characterization of the skin surface micro-topography using a skin replica and changes with age.

BACKGROUND/PURPOSE: Skin surface micro-topography (SSMT), consisting of pores, ridges and furrows, reflects the skin condition and is an important factor determining the aesthetics of the skin. Most previous studies evaluating SSMT have employed two-dimensional image analysis of magnified pictures captured by a video microscope. To improve the accuracy of SSMT analysis, we established a three-dimensional (3D) analysis method for SSMT and developed various parameters including the skin ridge number, and applied the method to study the age-dependent change in skin. METHODS: Confocal laser scanning microscopy was used for 3D measurement of the surface morphology of silicon replicas taken from the cheek. We then used these data to calculate the parameters that reflect the nature of SSTM including the skin ridge number using originally developed software. Employing a superscription technique, we investigated the variation in SSMT with age for replicas taken from the cheeks of 103 Japanese females (5-85 years old). RESULTS: The skin surface area and roughness, the area of pores, the area, length, depth and width of skin furrows and the number of skin ridges were examined. The surface roughness, the area of pores and the depth of skin furrows increased with age. The area and length of skin furrows and the number of skin ridges decreased with age. CONCLUSION: The method proposed to analyse SSMT three dimensionally is an effective tool with which to characterize the condition of the skin.

Measurement of two solvation regimes in water-ethanol mixtures using x-ray compton scattering.

Water-ethanol mixtures exhibit interesting anomalies in their macroscopic properties. Despite a lot of research, the origin of the anomalies and the microscopic structure itself is still far from completely known. We have utilized the synchrotron x-ray Compton scattering technique to elucidate the structure of aqueous ethanol from a new experimental perspective. The technique is uniquely sensitive to the local molecular geometries at the angstrom and subangstrom scales. The experiments reveal two distinct mixing regimes in terms of geometry: the dilute 5 mol % and the concentrated >15 mol % regimes. By comparing with pure liquids, the former regime is characterized by an intramolecular and the latter by an intermolecular change. The findings bring new light to evaluating the hypothesis of formation of clathratelike structures at the dilute concentrations.

Ilizarov external fixator for burst fracture of the lumbar spine: a case report.

A 50-year-old man presented with severe back pain and tenderness throughout the lumbar area after falling from a ladder. He had an unstable type-B burst fracture, with a spinal canal narrowing of 36% and an anterior height loss of 65%. His lower-limb neurological function was intact. An Ilizarov external spinal fixator was used; the pedicular half pins were inserted into the bilateral T11, T12, L2, and L3 pedicles; bilateral pedicular half pins were fixed at each level with external plates and rods. Postoperatively, the patient had a lordosis of 2 degrees and was able to walk 7 days later. The external fixator was removed at 10 weeks. Six years and 10 months after surgery, the patient had a kyphosis of 19 degrees that did not affect his activities of daily living.

Identification of human SEP1 as a glial cell line-derived neurotrophic factor-inducible protein and its expression in the nervous system.

Glial cell line-derived neurotrophic factor (GDNF) signals through multisubunit receptor complex consisting of RET tyrosine kinase and a glycosylphosphatidylinositol-anchored coreceptor called GDNF family receptor alpha1 (GFRalpha1). In the current study, we cloned a human SEP1 gene as a GDNF-inducible gene using human neuroblastoma cells that express RET and GFRalpha1. The induction of the SEP1 gene showed two peaks at 0.5-2 h and 24-48 h after GDNF stimulation by Northern blotting and quantitative real-time reverse transcriptase polymerase chain reaction. The late induction was also confirmed at protein levels by Western blotting with anti-SEP1 antibody. Immunostaining revealed that the expression of the SEP1 protein was detected in cell body, elongated neurites and growth cone-like structure of neuroblastoma cells treated with GDNF. In addition, we found a high level of SEP1 expression in neurons of the dorsal root and superior cervical ganglia and motor neurons of the spinal cord of mice in which RET is also expressed. SEP1 was co-immunoprecipitated with alpha- and beta-tubulins from the lysate of mouse brain. These results thus suggested that SEP1 is a GDNF-inducible and microtubule-associated protein that may play a role in the nervous system.

An archaeal protein homologous to mammalian SRP54 and bacterial Ffh recognizes a highly conserved region of SRP RNA.

The gene encoding the 54 kDa protein of signal recognition particle (SRP54) in the hyperthermophilic archaeon Pyrococcus furiosus has been cloned and sequenced. Recombinant P. furiosus SRP54 (pf-SRP54) and the N-terminal G-domain and C-terminal M-domain (pf-SRP54M) of pf-SRP54 with an amino-terminal addition of six histidine residues were expressed in Escherichia coli and subjected to binding experiments for SRP RNA, non-conserved 213-nucleotide RNA (helices 1, 2, 3, 4 and 5) and conserved 107-nucleotide RNA (helices 6 and 8) from SRP RNA. The RNA binding properties of the purified protein were determined by filter binding assays. The histidine-tagged pf-SRP54M bound specifically to the conserved 107-nucleotide RNA in the absence of pf-SRP19, unlike the eukaryotic homologue, with an apparent binding constant (K) of 18 nM.

Number of lymph node metastases determined by presurgical ultrasound and endoscopic ultrasound is related to prognosis in patients with esophageal carcinoma.

OBJECTIVE: To analyze the impact on prognosis of the number of lymph node metastases detected by ultrasound and endoscopic ultrasound in patients with esophageal carcinoma. SUMMARY BACKGROUND DATA: Ultrasound and endoscopic ultrasound are useful for diagnosing tumor depth and lymph node metastasis in patients with esophageal carcinoma. However, the clinical significance of the number of lymph node metastases before surgery has not been elucidated. METHODS: The authors evaluated lymph node metastases using preoperative ultrasound and endoscopic ultrasound in 329 consecutive patients who underwent esophagectomy with lymphadenectomy. TNM classification and one-to-one comparison of lymph node metastasis was performed between the preoperative and histologic diagnosis. The number of lymph node metastases was subdivided into four groups: zero, one to three, four to seven, and eight or more. RESULTS: The accuracy of preoperative ultrasound and endoscopic ultrasound diagnosis exceeded 70% in each category of TNM classification. The incidence of lymph node metastasis determined by preoperative and histologic diagnosis was 69.0% (234/339) and 59.3% (201/339), respectively. The correlation between preoperative and histologic diagnosis was significant (P <.0001). According to the subdivision of number of lymph node metastases, the accuracy rates associated with nodal involvement of zero, one to three, four to seven, and eight or more were 83.8%, 59.7%, 43.3%, and 96.0%, respectively. The clinical outcome between ultrasound and endoscopic ultrasound diagnosis and histologic diagnosis in stage grouping was almost similar. The 5-year survival rates of patients with zero, one to three, four to seven, and eight or more lymph node metastases determined by ultrasound and endoscopic ultrasound were 53.3%, 33.8% 17.0%, and 0%, respectively. The differences among groups were statistically significant. The survival curves associated with preoperative and histologic diagnosis were similar. CONCLUSIONS: Not only the stage grouping of TNM classification but also the number of lymph node metastases determined by ultrasound and endoscopic ultrasound before surgery may be useful for predicting prognosis in patients with esophageal carcinoma.

Immunological study on circulating murine osteoprotegerin/osteoclastogenesis inhibitory factor (OPG/OCIF): possible role of OPG/OCIF in the prevention of osteoporosis in pregnancy.

Osteoprotegerin (OPG)/osteoclastogenesis inhibitory factor (OCIF) is a soluble member of the tumor necrosis factor receptor family and plays a crucial role in the negative regulation of osteoclastic bone resorption. We have immunized OPG/OCIF knockout mice with murine rOPG/rOCIF and established a panel of hybridomas producing monoclonal antibodies (mAbs) to murine rOPG/rOCIF. Utilizing the mAbs, we developed enzyme-linked immunosorbent assay (ELISA) systems: one detecting both homodimeric and monomeric forms of murine OPG/OCIF and the other detecting only dimeric form of murine OPG/OCIF. With the aid of these ELISA systems we showed that OPG/OCIF is present mainly as a monomer in murine blood. The concentration of OPG/OCIF in normal mouse sera was approximately 500 pg/ml and there was no statistical difference in the serum concentration of OPG/OCIF among genders, age, and strains. Interestingly, the concentration of circulating OPG/OCIF in mouse markedly increased during pregnancy. The result indicated that circulating OPG/OCIF plays an important role in the protection of bone from excess resorption during pregnancy in mammals.

Effect of myocutaneous inflammatory changes caused by intra-arterial chemotherapy on the outcome of patients who undergo limb-saving surgery.

BACKGROUND: This study examined the effect of myocutaneous inflammatory changes caused by intra-arterial chemotherapy on the outcome of patients who undergo limb-saving surgery. METHODS: One hundred seven patients with malignant bone and soft tissue tumors were administered intra-arterial cisplatin and caffeine preoperatively with or without doxorubicin. Nine patients (8.4%) who had had myocutaneous inflammatory reactions were reviewed to determine the effects of this complication on limb-saving surgery. RESULTS: The patients complained of unbearable and continuous pain while undergoing intra-arterial infusion. Gait disturbances and muscle weakness were caused by their severe regional pain, resulting in the use of crutches even before surgery. Extended areas of necrotic skin and muscle were resected simultaneously with limb-saving surgery on four patients. Myocutaneous necrosis in one patient, indurations in two patients, and flares in two patients were not treated surgically. Pain relief was provided to eight patients at some point. Four patients with extensive myocutaneous necrosis around the knee joint had restricted range of motion and poor functional results. Radionuclide angiography using 99m-Technetium-macroaggregated albumins was performed to evaluate the blood flow to the affected muscle and tumor. It showed marked increase of the radioisotope perfusion in the affected muscles but little uptake in the tumor. These results may suggest that the affected muscles diminish the effects of anticancer drugs on the tumors. CONCLUSIONS: Myocutaneous inflammatory reactions should be prevented if possible to obtain good limb function and chemotherapeutic effects in patients who undergo intra-arterial chemotherapy. Resection of necrotic tissue is mandatory to relieve pain.

Molecular cloning of three genes encoding G protein alpha subunits in the white root rot fungus, Rosellinia necatrix.

Three genes encoding G protein alpha subunits were cloned from the white root rot fungus, Rosellinia necatrix, and characterized. Only one copy of each gene was present in the genome. The protein sequences of Rga1, Rga2, and Rga3 are very similar to those of MagA, MagB and MagC of Magnaporthe grisea, respectively. Moreover, Rga1 is similar to Mod-D which is closely related to vegetative incompatibility in Podospora anserina, which suggests that Rga1 is important in the vegetative incompatibility reaction in R. necatrix. Reverse transcription PCR (RT-PCR) analysis of Rga1, Rga2, and Rga3 mRNA expression showed that the three genes were all transcribed in R. necatrix cells.

Solvent effect on helicity induction of zinc bilinone bearing a chiral auxiliary at the helix terminal.

Solvent effects on helicity induction in zinc bilinone (ZnBL) derivatives bearing chiral auxiliaries at their 19-positions were investigated by using circular dichroic spectroscopy and (1)H NMR experiments. In ZnBLs 1 and 2, which possess (R)-2-methyl-1-phenylpropyloxy and (R)-1-phenylethyloxy groups at their 19-positions, respectively, the efficiency of helicity induction was significantly affected by employed solvents (78-95% de in 1 and 33-89% de in 2). The free energy changes of the P-M interconversion of 1 and 2 were linearly in proportion to reduction in polarizability of solvents: lower polarizability of solvents led to better efficiency of helicity induction in 1 and 2. With the support of the (1)H NMR study in addition to the molecular modeling previously reported, it was indicated that the solvophobic van der Waals interaction between the alkyl group in the chiral auxiliary and the A-ring of the bilinone skeleton in the preferred conformer plays a crucial role in determining the efficiency of helicity induction in 1 and 2.

DNA/RNA-dependent ATPase activity is associated with ATBF1, a multiple homeodomain-zinc finger protein.

The AT motif-binding factor 1 (ATBF1)-A is a large transcription factor containing four homeodomains and 23 zinc finger motifs. It has a number of motifs involved in transcriptional regulation, and in addition, several motifs found in enzymes, such as ATPases and helicases. In this study, we examined whether ATPase activity is associated with the ATBF1-A molecule. A 263-amino acid segment of the ATBF1-A molecule, termed AHZ, which contains the ATPase A-motif, homeodomain IV and zinc finger 21, was expressed in Escherichia coli in the form of glutathione S-transferase fusion protein and analyzed for ATPase activity. We found that AHZ was able to hydrolyze ATP with K(m) 10.6 microM and K(cat) 0.055 min(-1) at 5 mM Mg(2+) and pH 7.75. AHZ retained bacterial DNA and removal of the DNA resulted in 70% decrease in ATPase activity. The addition of double- or single-stranded DNAs restored 70-75% ATPase activity and that of RNA restored 50-55% activity. Site-directed mutagenesis of the A-motif resulted in 34% reduction of ATPase activity with no significant loss of bound DNA. In contrast, mutation of homeodomain IV and zinc finger 21 resulted in 90 and 80% reduction of ATPase, respectively, with the loss of the ability to bind to DNA and RNA. These results show that ATBF1 has at least one enzyme activity in addition to regulation of DNA transcription. The ATPase activity associated with ATBF1-A is DNA/RNA-dependent and unique in that it requires both homeodomain and zinc finger motifs.

Bilateral symmetrical cysts in the upper tibiae in a skeletally mature patient: might they be simple bone cysts?

A 55-year-old Japanese woman presented with right knee pain of 1-month duration. Radiological studies revealed bilateral mild osteoarthritic changes in the medial knee joint compartment and symmetrical cysts in the upper tibial metaphyses, extending to the epiphyses. Intraosseous ganglion was considered the most probable diagnosis. However, intraoperatively, serous fluid-filled cavities were recognized; these were curetted and filled with hydroxyapatite granules. Histopathological examination of the cyst wall revealed thin fibrous tissue formed of collagen fibers without a lining cell layer, with scattered lymphocytes, histiocytes, irregular masses of fibrin-like material, and periosteal osteocartilagenous callus formation; a picture compatible with simple bone cysts. Bilateral symmetrical cysts of the upper tibial metaphyses extending to the epiphyses are extremely rare. A literature review revealed that the age incidence, and bony locations of multiple and epiphyseal simple bone cysts are atypical in relation to the classic metaphyseal simple bone cysts. Also, multiple and epiphyseal simple bone cysts have a better prognosis than the classic metaphyseal ones. Four clinicoanatomic varieties of simple bone cysts are recognized; classic metaphyseal, nontubular, epiphyseal, and multiple.

5S rRNA binding proteins from the hyperthermophilic archaeon, Pyrococcus furiosus.

A determination was made of the nucleotide sequence of the 2719 bp region of a ribosomal protein gene cluster (PfeL32-PfeL19-PfL18-PfS5-PfL30) containing a 5S rRNA binding protein L18 homolog of hyperthermophilic archaea Pyrococcus furiosus. The organization of the archaeal ribosomal protein gene cluster is similar to that in the spc-operon of Escherichia coli (L6-L18-S5-L30-L15) but has two additional genes, namely those encoding PfeL32 and PfeL19, which were identified as extra proteins that are apparently not present in bacterial E. coli. Using an inducible expression system, P. furiosus mature PfL18 protein and a mutant PfL18 with the basic N-terminal amino acid region deleted were produced in large amounts in E. coli and Northwestern analysis showed the N-terminal region of PfL18, including the conserved arginine-rich region, to have a significant role in 5S rRNA-PfL18 interaction.

HSP70 and ribosomal protein L2: novel 5S rRNA binding proteins in Escherichia coli.

A Northwestern analysis of Escherichia coli total proteins with radiolabeled 5S rRNA identified two novel 5S rRNA interacting proteins, a 70 kDa and a 37 kDa protein, and three ribosomal proteins reported on already. The N-terminal sequencing of the 70 kDa protein separated by SDS-PAGE from the high-salt-washed fraction of crude ribosome led to the discovery of a polypeptide identical in its first 10 amino acid residues to E. coli heat shock protein 70. The N-terminal eight amino acid sequence of the 37 kDa protein extracted from the high-salt-washed ribosome is identical to that of the ribosomal protein L2. In addition, the interaction of these proteins with 5S rRNA has been confirmed with gel mobility shift assays.

Tumor necrosis factor alpha stimulates osteoclast differentiation by a mechanism independent of the ODF/RANKL-RANK interaction.

Osteoclast differentiation factor (ODF, also called RANKL/TRANCE/OPGL) stimulates the differentiation of osteoclast progenitors of the monocyte/macrophage lineage into osteoclasts in the presence of macrophage colony-stimulating factor (M-CSF, also called CSF-1). When mouse bone marrow cells were cultured with M-CSF, M-CSF-dependent bone marrow macrophages (M-BMM phi) appeared within 3 d. Tartrate-resistant acid phosphatase-positive osteoclasts were also formed when M-BMM phi were further cultured for 3 d with mouse tumor necrosis factor alpha (TNF-alpha) in the presence of M-CSF. Osteoclast formation induced by TNF-alpha was inhibited by the addition of respective antibodies against TNF receptor 1 (TNFR1) or TNFR2, but not by osteoclastogenesis inhibitory factor (OCIF, also called OPG, a decoy receptor of ODF/RANKL), nor the Fab fragment of anti-RANK (ODF/RANKL receptor) antibody. Experiments using M-BMM phi prepared from TNFR1- or TNFR2-deficient mice showed that both TNFR1- and TNFR2-induced signals were important for osteoclast formation induced by TNF-alpha. Osteoclasts induced by TNF-alpha formed resorption pits on dentine slices only in the presence of IL-1alpha. These results demonstrate that TNF-alpha stimulates osteoclast differentiation in the presence of M-CSF through a mechanism independent of the ODF/RANKL-RANK system. TNF-alpha together with IL-1alpha may play an important role in bone resorption of inflammatory bone diseases.

Basic fibroblast growth factor inhibits osteoclast formation induced by 1alpha,25-dihydroxyvitamin D(3) through suppressing the production of osteoclast differentiation factor.

Basic fibroblast growth factor (bFGF) inhibited osteoclast-like cell (OCL) formation in cocultures of mouse spleen cells with either osteoblasts or a stromal cell line, ST2, in the presence of 1alpha, 25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)]. bFGF directly acted on osteoblasts/stromal cells, but not osteoclast progenitors, to inhibit 1,25(OH)(2)D(3)-induced OCL formation. bFGF suppressed the mRNA expression of osteoclast differentiation factor (ODF) but did not affect that of osteoclastogenesis inhibitory factor (OCIF) in ST2 cells treated with 1,25(OH)(2)D(3) and dexamethasone. Enzyme-linked immunosorbent assay showed that bFGF hardly affected OCIF production in the treated ST2 cells. A genetically engineered soluble form of ODF, but not anti-OCIF neutralizing antibody, abolished bFGF-mediated inhibition of OCL formation. bFGF suppressed the binding of (125)I-labeled OCIF to both ST2 cells and osteoblasts treated with 1,25(OH)(2)D(3). These findings indicate that bFGF inhibits 1,25(OH)(2)D(3)-induced OCL formation via suppression of ODF production by osteoblasts/stromal cells.

Basic fibroblast growth factor induces osteoclast formation by reciprocally regulating the production of osteoclast differentiation factor and osteoclastogenesis inhibitory factor in mouse osteoblastic cells.

Basic fibroblast growth factor (bFGF) induced osteoclast formation in co-cultures of mouse spleen cells and osteoblasts. Osteoclastogenesis inhibitory factor (OCIF) and a selective cyclooxygenase-2 (COX-2) inhibitor, NS-398, abolished bFGF-induced osteoclast formation. bFGF did not affect spleen cells, but it did affect osteoblasts, to stimulate osteoclast formation. Northern blot analysis revealed that bFGF up-regulated the expression of osteoclast differentiation factor (ODF) and COX-2 and down-regulated the expression of OCIF in primary osteoblastic cells. NS-398 abolished the increase of ODF mRNA, but it had no effect on the decrease of OCIF mRNA. NS-398 suppressed the binding of (125)I-labeled OCIF to osteoblastic cells treated with bFGF. Enzyme-linked immunosorbent assay showed that bFGF inhibited OCIF production by osteoblastic cells, and the inhibition was not affected by NS-398. We conclude that bFGF induces osteoclast formation by stimulating ODF production through COX-2-mediated prostaglandin synthesis and by suppressing OCIF production through a mechanism independent of prostaglandin synthesis.

A novel molecular mechanism modulating osteoclast differentiation and function.

Osteoclasts, the multinucleated giant cells that resorb bone, develop from hematopoietic cells of the monocyte/ macrophage lineage. Osteoblasts, as well as bone marrow stromal cells, support osteoclast development through a mechanism of cell-to-cell interaction with osteoclast progenitors. We recently purified and molecularly cloned osteoclastogenesis inhibitory factor (OCIF), which was identical to osteoprotegerin (OPG). OPG/OCIF, a secreted member of the tumor necrosis factor (TNF) receptor family, inhibited differentiation and activation of osteoclasts. A single class of high-affinity binding sites for OPG/OCIF appeared on a mouse bone marrow stromal cell line, ST2, in response to 1alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3] and dexamethasone (Dex). When the binding sites were occupied by OPG/OCIF, ST2 cells failed to support the osteoclast formation from spleen cells. To identify an OPG/OCIF ligand, we screened a cDNA expression library of ST2 cells treated with 1,25(OH)2D3 and Dex using OPG/OCIF as a probe. The cloned molecule was found to be a member of the membrane-associated TNF ligand family, and it induced osteoclast formation from mouse and human osteoclast progenitors in the presence of macrophage colony-stimulating factor (M-CSF) in vitro. Expression of its gene in osteoblasts/stromal cells was up-regulated by osteotropic factors, such as 1,25(OH)2D3, prostaglandin E2 (P(GE2), parathyroid hormone (PTH), and interleukin (IL)-11. A polyclonal antibody against this protein, as well as OPG/OCIF, negated not only the osteoclastogenesis induced by the protein, but also bone resorption elicited by various osteotropic factors in a fetal mouse long bone culture system. These findings led us to conclude that the protein is osteoclast differentiation factor (ODF), a long sought-after ligand that mediates an essential signal to osteoclast progenitors for their differentiation into active osteoclasts. Recent analyses of ODF receptor demonstrated that RANK, a member of the TNF receptor family, is the signaling receptor for ODF in osteoclastogenesis, and that OPG/OCIF acts as a decoy receptor for ODF to compete against RANK. The discovery of ODF, OPG/OCIF, and RANK opens a new era in the investigation of the regulation of osteoclast differentiation and function.

Nucleotide sequences of genes for ribosomal protein L41 and tRNAThr(AGU) from Coprinus cinereus.

The nucleotide sequences of genes for the homolog in Coprinus cinereus of the eukaryotic ribosomal protein L41 and for tRNAThr(AGU) are reported. The gene for tRNAThr(AGU) was located upstream of the gene for the L41 ribosomal protein, and these genes were adjacent to each other but in opposite orientations. The deduced amino acid sequence of ribosomal protein L41 exhibited strong homology to those of L41 proteins of several yeasts. The 56th amino acid of the deduced protein was proline, as it is in the L41 protein of a cycloheximide-sensitive strain of yeast. The putative secondary structure of the tRNA gene resembled the characteristic cloverleaf structure of tRNAs. Elements resembling an A-box and a B-box were found in the gene for tRNAThr(AGU). These boxes are known as internal promoter elements in genes for eukaryotic tRNAs.