Cloverleaf Model Research Articles

Discovery of a tRNA-like base sequence in the coronavirus genome and possible mechanism of action.

BackgroundThe question of whether the coronavirus genome contain as-yetununderstood genetic component.Purpose (Objective)Elucidate the novel functions of the discovered tRNA-like base sequence and lead to the development of novel therapeutic agents.MethodsA novel tRNA-like base sequence was found in the sequences complementary to the genomes of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and SARS-CoV. By comparing mutations in the tRNA-like base sequences of these two viruses, it was found that base pairing in the cloverleaf model of SARS-CoV-2 was more robust than that of SARS-CoV.ResultsThe results of homology search between a short sequence of the coronavirus tRNA-like base sequence and human genes suggest that the molecule produced by this novel tRNA-like sequence may be involved in the splicing of human messenger RNA.ConclusionsExperimental molecular evidence of the tRNA-like base sequence discovered in this study is urgently needed.Supplementary InformationThe online version contains supplementary material available at 10.1007/s13258-022-01299-w.

The cloverleaf model of cognitive behaviour therapy as experiential learning: implications for case formulation, therapeutic practice and practitioner development

Abstract Cognitive behaviour therapy (CBT) is an evidence-based psychotherapy for a wide variety of psychological problems. While the exact working mechanisms of CBT remain unknown, its mode of action might usefully be conceptualised as facilitated experiential learning. An adapted ‘cloverleaf’ version of Borton’s ‘what, so what, now what’ learning model is presented to elaborate some of the potential benefits of taking an experiential learning perspective on CBT. These include conceptualising the maintenance of client problems as inhibited experiential learning and the CBT therapeutic process as the cultivation of more effective experiential learning. An experiential learning perspective might also provide an accessible way for trainee and early-career CBT therapists to understand more clearly the learning methodology that underlies CBT’s distinctive approach to psychotherapy. The model is also intended to create an overarching conceptual bridge between reflective practice, the therapist’s experiential learning in the client role, and the client’s experience of CBT as facilitated experiential learning. Key learning aims (1) To introduce a modified ‘cloverleaf’ experiential learning process model that can be applied to the conceptualisation of client difficulties, CBT therapeutic processes, and practitioner development. (2) To demonstrate how the model can be used to develop cross-sectional and descriptive maintenance formulations of client problems and client wellbeing. (3) To show how the model can be used as a conceptual and practical tool to help formulate both the therapeutic process and challenges and obstacles to that process. (4) To help practitioners make links between the process of personal and professional development and client change processes.

The complete mitochondrial genome of the Anabas testudineus (Perciformes, Anabantidae) and its comparison with other related fish species

In the present study, the complete mitochondrial genome sequence of Anabas testudineusis reported using PGM sequencer (Ion Torrent, Life Technologies, La Jolla, CA). The complete mitogenome of climbing perch, A. testudineusis obtained by the de novo sequences assembly of genomic reads using the Torrent Mapping Alignment Program (TMAP), which is 16 603 bp in length. The mitogenome of A. testudineus composed of 13 protein- coding genes, two rRNA, and 22 tRNAs. Here, 20 tRNAs genes showed typical clover leaf model, and D-Loop as the control region along with gene order and organization, being closely similar to Osphronemidae and most of other Perciformes fish mitogenomes of NCBI databases. The mitogenome in the present study has 99% similarity to the complete mitogenome sequence of earlier reported A. testudineus. The phylogenetic analysis of Anabantidae depicted that their mitogenomes are closely related to each other. The complete mitogenome sequence of A. testudineus would be helpful in understanding the population genetics, phylogenetics, and evolution of Anabantidae.

From Clinical-Developmental Theory to Assessment: The Holistic Student Assessment Tool

A description and test of the Holistic Student Assessment Tool (HSA), an assessment tool to measure children’s and adolescents’ resiliencies in relation to externalizing and internalizing problem behaviors. The HSA is based on the authors’ research-based clinical-developmental Clover Leaf Model of resilience and psychopathology, and is one of the first attempts at closing the gap between risk and resilience approaches in developmental assessment. The HSA was tested in a cross-sectional sample of 423 children and adolescents. The results lend support to the HSA as a valid measure of children’s and adolescents’ resiliencies. Furthermore, the resilience scales mostly exhibited the theoretically expected convergent and divergent relationships with the psychopathology scales. In addition, we show how the resilience scales predict adolescents’ externalizing and internalizing symptoms. We contend that evidence-based intervention to address youth aggression needs to be based on sound developmental assessment.

Vitamin D Receptor: Key Roles in Bone Mineral Pathophysiology, Molecular Mechanism of Action, and Novel Nutritional Ligands

The vitamin D hormone, 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], binds with high affinity to the nuclear vitamin D receptor (VDR), which recruits its retinoid X receptor (RXR) heterodimeric partner to recognize vitamin D responsive elements (VDREs) in target genes. 1,25(OH)(2)D(3) is known primarily as a regulator of calcium, but it also controls phosphate (re)absorption at the intestine and kidney. Fibroblast growth factor 23 (FGF23) is a phosphaturic hormone produced in osteoblasts that, like PTH, lowers serum phosphate by inhibiting renal reabsorption through Npt2a/Npt2c. Real-time PCR and reporter gene transfection assays were used to probe VDR-mediated transcriptional control by 1,25(OH)(2)D(3). Reporter gene and mammalian two-hybrid transfections, plus competitive receptor binding assays, were used to discover novel VDR ligands. 1,25(OH)(2)D(3) induces FGF23 78-fold in osteoblasts, and because FGF23 in turn represses 1,25(OH)(2)D(3) synthesis, a reciprocal relationship is established, with FGF23 indirectly curtailing 1,25(OH)(2)D(3)-mediated intestinal absorption and counterbalancing renal reabsorption of phosphate, thereby reversing hyperphosphatemia and preventing ectopic calcification. Therefore, a 1,25(OH)(2)D(3)-FGF23 axis regulating phosphate is comparable in importance to the 1,25(OH)(2)D(3)-PTH axis that regulates calcium. 1,25(OH)(2)D(3) also elicits regulation of LRP5, Runx2, PHEX, TRPV6, and Npt2c, all anabolic toward bone, and RANKL, which is catabolic. Regulation of mouse RANKL by 1,25(OH)(2)D(3) supports a cloverleaf model, whereby VDR-RXR heterodimers bound to multiple VDREs are juxtapositioned through chromatin looping to form a supercomplex, potentially allowing simultaneous interactions with multiple co-modulators and chromatin remodeling enzymes. VDR also selectively binds certain omega3/omega6 polyunsaturated fatty acids (PUFAs) with low affinity, leading to transcriptionally active VDR-RXR complexes. Moreover, the turmeric-derived polyphenol, curcumin, activates transcription of a VDRE reporter construct in human colon cancer cells. Activation of VDR by PUFAs and curcumin may elicit unique, 1,25(OH)(2)D(3)-independent signaling pathways to orchestrate the bioeffects of these lipids in intestine, bone, skin/hair follicle, and other VDR-containing tissues.

5S RNA. An analysis of possible base pairing schemes.

Possible ways of arranging base paris which are compatible with the primary structure of 5 S RNA from Escherichia coli and KB cells are discussed. If only A·U and G·C base pairs are allowed, the maximum helical content of clover leaf models with up to four leaves is 68%. In multihelical models with helical regions consisting of a minimum of four base pairs the maximum belical content is 60%. With helical regions consisting of three base pairs, at least some of which are A·U base pairs, the helical content can be raised to 73%. It is unlikely that higher helical contgents are possible unless shorter helices are included. The structures with a given helical content are never unique so that if the secondary structure of 5 S RNA is unique there must be other factors in operation.

Development of a Technology Readiness Assessment Measure: The Cloverleaf Model of Technology Transfer

Successful technology transfer begins with the identification of appropriate candidate technologies for transfer. Technology transfer managers have few reliable tools to guide the process of determining when and which technologies are likely to be successful in the commercialization process. This paper describes a multi-stage process which incorporated ideas from the literature, materials in use, pretesting of a model, and the collective wisdom of practitioners to devise a technology transfer readiness tool, dubbed the “Cloverleaf Model” Tool for Technology Transfer Assessment, because of its identification of the four requirements for success. The tool has been customized for different environments. It can be used over time to compare technologies and adapted to the unique situation of users.

Residual tRNA secondary structure in ‘denaturing‘ 8M urea/TBE polyacrylamide gels: effects on electrophoretic mobility and dependency on prior chemical modification of the tRNA

Fifteen individual species of tRNA were treated with the chemical modifiers diethylpyrocarbonate, 50% aqueous hydrazine or hydrazine/3 M NaCl. Following purification of the chemically modified material on polyacrylamide gels containing 8 M urea, variant minor bands, in addition to the expected main band, were observed for 12 of the 15 tRNAs. Characterization of the content of chemically altered bases in material recovered from such bands indicated that tRNAs containing modified nucleotides in base-paired stems were excluded from the main band and present, often in enhanced amounts, in the minor variant bands. The persistence of residual secondary structure on 8 M urea gels run at 45 degrees C and the ability of chemically modified bases to alter electrophoretic mobilities warrant caution in designing and interpreting experiments in which chemically modified RNA is isolated on gels prior to further analysis. tRNA(Val) (VAC) was unique in that modified bases in non base-paired regions, according to the cloverleaf model of secondary structure, caused exclusion from the main band. Consequently, we propose a secondary structure for partially denatured tRNA(Val) (VAC), in which these bases are located in double stranded regions of the molecule.

Genes for tRNAAsp, tRNAPro, tRNATyr and two tRNAsSer in wheat mitochondrial DNA

We have begun a systematic search for potential tRNA genes in wheat mtDNA, and present here the sequences of regions of the wheat mitochondrial genome that encode genes for tRNA(Asp) (anticodon GUC), tRNA(Pro) (UGG), tRNA(Tyr) (GUA), and two tRNAs(Ser) (UGA and GCU). These genes are all solitary, not immediately adjacent to other tRNA or known protein coding genes. Each of the encoded tRNAs can assume a secondary structure that conforms to the standard cloverleaf model, and that displays none of the structural aberrations peculiar to some of the corresponding mitochondrial tRNAs from other eukaryotes. The wheat mitochondrial tRNA sequences are, on average, substantially more similar to their eubacterial and chloroplast counterparts than to their homologues in fungal and animal mitochondria. However, an analysis of regions ∼ 150 nucleotides upstream and ∼ 100 nucleotides downstream of the tRNA coding regions has revealed no obvious conserved sequences that resemble the promoter and terminator motifs that regulate the expression of eubacterial and some chloroplast tRNA genes. When restriction digests of wheat mtDNA are probed with (32)P-labelled wheat mitochondrial tRNAs, <20 hybridizing bands are detected, whether enzymes with 4 bp or 6 bp recognition sites are used. This suggests that the wheat mitochondrial genome, despite its large size, may carry a relatively small number of tRNA genes.

Suppressor and novel mutants of bacteriophage T4 tRNA Gly

We have isolated a weak UGA suppressor of phage T4 tRNA Gly in which the anticodon is changed from UCC to UCA. Two secondary mutants lacking suppressor activity are atypical in accumulating tRNA Gly. Both mutations change the T stem of the cloverleaf model. One involves a G to A change at the 5′ base position of the middle base-pair; the second involves a C to U change at a constant base position next to the T loop. The precursor RNAs of the mutants were cleaved in vitro with the catalytic RNA subunit of RNase P. Relative to normal precursor RNA, the precursor mutated at the middle base-pair position of the T stem was cleaved more rapidly, whereas the precursor mutated at the base-pair position next to the T loop was cleaved more slowly.

Equilibrium and kinetics of thermal unfolding of yeast 5.8S ribosomal RNA in aqueous salt solutions

Equilibrium and kinetics of thermal melting of yeast 5.8S ribosomal RNA in aqueous NaCl were investigated by differential thermal melting and temperature jump methods. Two peaks were observed in each of the melting curves at 1 mM-1 M Na + and linearity between each melting temperature T m and log[Na +] was found at [Na +> 10 mM. From the difference spectrum ratio, dA 280 dA 260 , the G-C content in the local structures was calculated to be 91 and 56%. The temperature jump to 70–85°C in aqueous 30 mM Na + of the RNA solution induced first-order kinetics, from which the kinetically determined melting curve was calculated. The curve could be approximately described in a Gaussian form with a T m which agrees well with the high T m in the static melting curve at 30 mM Na +. The kinetic properties of the reaction indicated a double helix-coil transition. However, the temperature jump to 20–60°C did not induce monophasic kinetics. The kinetic amplitude of the slow component showed a T m which corresponded to the low T m in the static melting curve at 30 mM Na +. The slow relaxation had the characteristics of a double helix-to-coil transition. However, contributions from very fast processes including single strand unstacking, were most noticeable in the low temperature melting region of the static curve. The thermodynamic parameters of both transitions from double helix to coil were analysed in detail. Both activation energies for helix formation were negative, and the nucleation is thought to follow a process similar to that in oligonucleotides. Values of T m and enthalpy change of both helix-coil transitions indicated the cloverleaf model as the most plausible one for some limited regions of yeast 5.8S RNA among the previously proposed models: burp gun, cloverleaf and Rubin's models.

Sequence organization of the origins of DNA replication in lambdoid coliphages

We have determined the sequences of the ori region DNA of several phage λ mutants and hybrids, which shed light on the mechanism of DNA replication in the lambdoid phages. These include the heterologous substitution hybrids λrep82:λ and λrep80:λ, a pseudorevertant of the ori−r93 mutant λr93 mutant λr93hot5, and the insertion mutant λpk35. The ori regions of the three lambdoid phages, λ, φ80 and 82, all have repeated sequences, termed iterons, and A·T-rich zones. We note that a similar arrangement of DNA is also found in several other prokaryotic origins of replication. λ and φ80 have four iterons, and 82 has five. The origin of λr93hot5 is unusual in that contains only three iterons, yet the phage grows normally. Analysis of this mutant indicates that the spacing of iterons is crucial to ori function, whereas their number is not. This argues against the cloverleaf model for λ ori structure (Hobom et al., 1979). In λpk35 the drug resistance element Tn903 is inserted into the “inceptor” (ice) site, proposed to be crucial for λ replication initiation (Hobom et al., 1979); yet this phage grows normally.

Analysis of the secondary and tertiary structures of Rous sarcoma virus RNA.

The secondary and tertiary structures of the 35S RNA of Rous Sarcoma virus have been investigated. T1 RNase partial digests have been first resolved into their components by gel electrophoresis under non denaturing conditions and then each component analyzed further by various techniques. More than one hundred structured fragments have thus been isolated and shown to consist of several individual nucleotide sequences located far apart on the genome. On the basis of the results, a cloverleaf model for the structure of RSV 35S RNA is proposed that has implications for the various biological functions of this RNA.

The primary structure of cytoplasmic initiator transfer ribonucleic acid from Torulopsis utilis.

Cytoplasmic initiator transfer ribonucleic acid (tRNAinit) was purified from bulk Torulopsis (Candida) utilis tRNA by a series of column chromatography procedures. Sequence analysis of the products of complete and partial digestion of this tRNA with ribonuclease A [EC 3.1.4.22] and ribonuclease T1 [EC 3.1.4.8] enabled us to determine the complete primary structure of the molecule. The chain length of this tRNA was 76, including 11 modified nucleotides. The structure of the tRNA was arranged into a cloverleaf model and compared with those of other initiator tRNA species. As in the cytoplasmic initiator tRNA's of most other eukaryotic cells, the sequence -A-U-C-G- is contained in this tRNA in place of the usual -T-psi-C-G (or A)- found in other tRNA's.

Laser Raman evidence for new cloverleaf secondary structures for eukaryotic 5.8S RNA and prokaryotic 5S RNA.

Neither of the two previously proposed secondary structures for eukaryotic 5.8S RNA is consistent with the present laser Raman results. A new, highly stable "cloverleaf" secondary structure not only fits the Raman data but also accounts for previously determined enzymatic partial cleavage patterns, base sequence and pairing homologies, and G-C and A-U base pair numbers and ratios. The new cloverleaf model also conserves several structural features (constant loops, bulges, and stems) consistent with known 5.8S RNA functions. Finally, we propose a similar new cloverleaf secondary structure for Escherichia coli 5S RNA, consonant with many known properties of prokaryotic 5S RNA.

Determination of the base-pairing content of four specific tRNAs by infrared spectroscopy.

AbstractInfrared spectroscopy was used for the determination of the base‐pairing content of four specific tRNAs in deuterium oxide solution. Infrared spectra were obtained in the 1750–1550 cm−1 region at various temperatures ranging from about 15 to 90°C. Melting curves were constructed by plotting the molar extinction coefficient at ν = 1657 cm−1 versus temperature. These transition curves enabled us to determine the ranges of temperature which correspond to the ordered (partially double‐stranded) or randomly coiled structure of the tRNA. For a set of wavenumbers the extinction coefficients at these temperatures were used for the calculation of the base‐pairing content. The procedure employed here is based on a method described earlier by Thomas [(1969) Biopolymers 7, 325–334]. For the conditions selected for this investigation (Mg2+‐free D2O‐buffer; 0.01M tris‐DCl, 0.015M NaCl, pD 7.5) the results of this determination agree within the limits of errors with the number of base pairs predicted by the cloverleaf model.

Bisulfite-induced C changed to U transitions in yeast alanine tRNA.

The reaction of yeast tRNAAla1ab with NaHSO3 at 25 degrees and pH 5.8 has been studied. Five reactive residues have been located. Four of these (C-17 in Loop I, C-36 in the anticodon, C-74 and C-75 near the acceptor end) react to the same extent (42%) under the conditions of the experiment. The other (C-72 in the first base pair of the acceptor stem) reacts much more slowly (8%). No other changes were detected, but kinetic data suggest two or more additional residues may react very slowly. The C changed to U change in the anticodon (igc changed to igu) is a missense change (Ala changed to Thr). Both mechanistic considerations and experimental data from the literature show that HSO3--induced deamination of cytosine residues occurs only at unstacked residues. The quantitative changes for tRNAAla indicate that the stacking lifetimes of C-17, C-36, C-74, and C-75 are about equal. All other cytidine residues are much more tightly stacked. These results are consistent with the folded cloverleaf models that have been proposed from x-ray diffraction studies of yeast tRNAPhe. Residues 48 and 56, which are in single-stranded regions in the unfolded cloverleaf structure, do not react suggesting that they are tightly stacked in solution under the conditions of this experiment. The data also indicate that the anticodon loop is flexible in solution.

Bisulfite-induced C changed to U transitions in yeast valine tRNA.

The reaction of yeast tRNAVallab with NaHSO3 at 25 degrees and pH 5.8 has been studied. Six reactive residues have been located. C-17 in loop I is the most reactive (51% conversion) and C-73 in the first base pair of the acceptor stem the least reactive (8%). Three of the remaining reactive residues (C-39 in loop II, C-75 and C-76 near the acceptor stem) react to the same extent (36 to 38%) under the conditions of the experiment. C-37 in the anticodon reacted to a lesser extent (28%) than C-39 (36%), located just 2 residues away in the anticodon loop. No other changes were detected, but kinetic data suggest one or more additional residues may react very slowly. The C changed to U change in the anticodon (iac changed to iau) is a missense change (Val changed to Ile). Both mechanistic considerations and experimental data from the literature show that HSO3--induced deamination of cytosine residues occurs only at unstacked residues. We interpret the quantitative changes in tRNAVal to indicate that C-17 spends a large portion of its lifetime in an unstacked conformation. The stacking lifetimes of C-37, C-39, C-75, and C-76 seem to be similar but not identical. All other cytidine residues are much more tightly stacked. These results are consistent with the folded cloverleaf models that have been proposed from x-ray diffraction studies of yeast tRNAPhe. Residues C-46, C-49, C-57, and C-61, which are present in the single-stranded regions of the unfolded cloverleaf structure, do not react, suggesting that they are tightly stacked in solution under the conditions of this experiment. The data also suggest that anticodon-loop conformations other than the extremes with five bases stacked on either the 3' or 5' portion of the anticodon stem exist in solution and that the anticodon loop is flexible.

Does 5S RNA function by a switch between two secondary structures?

THE structure of 5S RNA has been a lively field for experiment and speculation, with no definitive resolution of the problem in sight (see refs 1 and 2 for reviews). Fox and Woese3 suggested comparative sequence analysis of prokaryotic 5S RNAs as a method for locating base paired regions, an appealing procedure because of its similarity to the method used to substantiate the cloverleaf model for tRNA. Its logic states that if all known 5S RNA sequences contain complementary bases at a site, a double helix probably exists there in the functional form. We propose that comparative sequence analysis suggests two different functional structures for prokaryotic 5S RNA. One of them has been introduced by Fox and Woese3, and its set condary structure is based on four conserved helices: the ‘molecular stalk’ (Escherichia coli residues 1–10 and 119–110), the weak ‘tuned helix’ (18–23/65–60), the ‘common arm base’ (31–34/51–48) and the ‘prokaryotic loop’ (82-86/94-90). The base paired structure that results is consistent with enzymatic digestion14 and chemical modification1,5 results on native 5S RNA, if allowance is made for protection of residues by tertiary bonding as well as secondary structure. This ‘model’, however, does not explain the apparent conservation of a double helix (33–39/88–82 in E. coli) (Table 1) which is sterically inconsistent with two of the helices listed (the common arm base and the prokaryotic loop). Fox and Woese3 noted this conservation but considered it unimportant because of the variable helix length and the conflict with the other two helices.

Nuclear magnetic resonance of the base-pairing structure of the native and denatured conformers of Escherichia coli transfer RNA Trp

The native and denatured forms of transfer RNA Trp from Escherichia coli have been studied by high-resolution (300 MHz) proton nuclear magnetic resonance spectroscopy. The low-field nuclear magnetic resonance spectrum indicates that there are 22 ± 2 base-pairs present in the native form, and the observed resonance positions can be accounted for in terms of 19 base-pairs from the cloverleaf secondary structure, one protected U, and two tertiary structure base-pairs including A 14-s 4U 8. The denatured conformer contains one or two less base-pairs than the native conformer, and the low-field spectrum of the denatured conformer can be accounted for in terms of a model which retains the amino acid acceptor stem, T ΨC stem, and the anticodon stem of the cloverleaf model, but which pairs some bases of the dihydrouridine stem with bases from the minor loop. The proposed model is consistent with the ease of interconversion of the native and denatured conformers, the pattern of kethoxalation of the native and denatured conformera, and the retention of a tertiary structure A 14-s 4U 8 base-pair in the denatured conformer. Alternative models of the denatured form are considered, but they do not adequately explain the observed nuclear magnetic resonance spectrum. The binding of chloroquine to the native conformer has also been studied. A comparison of the binding of ethidium bromide and chloroquine to E. coli tRNA Glu indicates that the two drugs have very similar binding properties and this suggests a possible mechanism for the chloroquine-induced renaturation of tRNA Trp.