Evaluating potential of multi-parametric MRI using co-registered histology: Application to a mouse model of glioblastoma.

BACKGROUND: Conventional MRI fails to detect regions of glioblastoma cell infiltration beyond the contrast-enhanced T1 solid tumor region, with infiltrating tumor cells often migrating along host blood vessels. PURPOSE: MRI is capable of generating a range of image contrasts which are commonly assessed individually by qualitative visual inspection. It has long been hypothesized that better diagnoses could be achieved by combining these multiple images, so called multi-parametric or multi-spectral MRI. However, the lack of clinical histology and the difficulties of co-registration, has meant this hypothesis has never been rigorously tested. Here we test this hypothesis, using a previously published multi-dimensional dataset consisting of registered MR images and histology. STUDY TYPE: Animal Model. SUBJECTS: Mice bearing orthotopic glioblastoma xenografts generated from a patient-derived glioblastoma cell line. FIELD STRENGTH/SEQUENCES: 7 Tesla, T1/T2 weighted, T2 mapping, contrast enhance T1, diffusion-weighted, diffusion tensor imaging. ASSESSMENT: Immunohistochemistry sections were stained for Human Leukocyte Antigen (probing human-derived tumor cells). To achieve quantitative MRI-tissue comparison, multiple histological slices cut in the MRI plane were stacked to produce tumor cell density maps acting as 'ground truth'. STATISTICAL TESTS: Sensitivity, specificity, accuracy and Dice similarity indices were calculated. ANOVA, t-test, Bonferroni correction and Pearson coefficients were used for statistical analysis. RESULTS: Correlation coefficient analysis with co-registered 'ground truth' histology showed interactive regression maps had higher correlation coefficients and sensitivity values than T2W, ADC, FA, and T2map. Further, the interaction regression maps showed statistical improved detection of tumor volume. DATA CONCLUSION: Voxel-by-voxel analysis provided quantitative evidence confirming the hypothesis that mpMRI can, potentially, better distinguish between the tumor region and normal tissue.

Stacked in-plane histology for quantitative validation of non-invasive imaging biomarkers: Application to an infiltrative brain tumour model.

BACKGROUND: While it is generally agreed that histopathology is the gold standard for assessing non-invasive imaging biomarkers, most validation has been by qualitative visual comparison. To date, the difficulties involved in accurately co-registering histology sections with imaging slices have prevented a voxel-by-voxel assessment of imaging modalities. By contrast with previous studies, which focus on improving the registration algorithms, we have taken the approach of improving the quality of the histological processing and analysis. NEW METHOD: To account for imaging slice orientation and thickness, multiple histology sections were cut in the MR imaging plane and averaged to produce stacked in-plane histology (SIH) maps. When combined with intensity sensitive staining this approach gives histopathology maps, which can be used as the gold standard to validate imaging biomarkers. RESULTS: We applied this pipeline to a patient-derived mouse model of glioblastoma multiforme (GBM). Increasing the number of stacked histology sections significantly increased SIH measured tumour volume. The SIH technique proposed here resulted in reduced variability of volume measurements and this allowed significant improvements in the quantitative volumetric assessment of multiple MRI modalities. Further, high quality registration enabled a voxel-wise comparison between MRI and histopathology maps. Previous approaches to the validation of imaging biomarkers with histology, have been either qualitative or of limited accuracy. Here we propose a pipeline that allows for a more accurate validation via co-registration with SIH maps, potentially allowing validation in a voxel-wise mode. CONCLUSION: This work demonstrates that methodically produced SIH maps facilitate the quantitative histopathologic assessment of imaging biomarkers.

Assessing the effects of Ang-(1-7) therapy following transient middle cerebral artery occlusion.

The counter-regulatory axis, Angiotensin Converting Enzyme 2, Angiotensin-(1-7), Mas receptor (ACE2/Ang-1-7/MasR), of the renin angiotensin system (RAS) is a potential therapeutic target in stroke, with Ang-(1-7) reported to have neuroprotective effects in pre-clinical stroke models. Here, an extensive investigation of the functional and mechanistic effects of Ang-(1-7) was performed in a rodent model of stroke. Using longitudinal magnetic resonance imaging (MRI) it was observed that central administration of Ang-(1-7) following transient middle cerebral artery occlusion (MCAO) increased the amount of tissue salvage compared to reperfusion alone. This protective effect was not due to early changes in blood brain barrier (BBB) permeability, microglia activation or inflammatory gene expression. However, increases in NADPH oxidase 1 (Nox1) mRNA expression were observed in the treatment group compared to control. In order to determine whether Ang-(1-7) has direct cerebrovascular effects, laser speckle contrast imaging (LSCI) was performed to measure dynamic changes in cortical perfusion following reperfusion. Delivery of Ang-(1-7) did not have any effect on cortical perfusion following reperfusion however; it showed an indication to prevent the 'steal phenomenon' within the contralateral hemisphere. The comprehensive series of studies have demonstrated a moderate protective effect of Ang-(1-7) when given alongside reperfusion to increase tissue salvage.

Quantitative histopathologic assessment of perfusion MRI as a marker of glioblastoma cell infiltration in and beyond the peritumoral edema region.

BACKGROUND: Conventional MRI fails to detect regions of glioblastoma cell infiltration beyond the contrast-enhanced T1 solid tumor region, with infiltrating tumor cells often migrating along host blood vessels. PURPOSE: To quantitatively and qualitatively analyze the correlation between perfusion MRI signal and tumor cell density in order to assess whether local perfusion perturbation could provide a useful biomarker of glioblastoma cell infiltration. STUDY TYPE: Animal model. SUBJECTS: Mice bearing orthotopic glioblastoma xenografts generated from a patient-derived glioblastoma cell line. FIELD STRENGTH/SEQUENCES: 7T perfusion images acquired using a high signal-to-noise ratio (SNR) multiple boli arterial spin labeling sequence were compared with conventional MRI (T1 /T2 weighted, contrast-enhanced T1 , diffusion-weighted, and apparent diffusion coefficient). ASSESSMENT: Immunohistochemistry sections were stained for human leukocyte antigen (probing human-derived tumor cells). To achieve quantitative MRI-tissue comparison, multiple histological slices cut in the MRI plane were stacked to produce tumor cell density maps acting as a "ground truth." STATISTICAL TESTS: Sensitivity, specificity, accuracy, and Dice similarity indices were calculated and a two-tailed, paired t-test used for statistical analysis. RESULTS: High comparison test results (Dice 0.62-0.72, Accuracy 0.86-0.88, Sensitivity 0.51-0.7, and Specificity 0.92-0.97) indicate a good segmentation for all imaging modalities and highlight the quality of the MRI tissue assessment protocol. Perfusion imaging exhibits higher sensitivity (0.7) than conventional MRI (0.51-0.61). MRI/histology voxel-to-voxel comparison revealed a negative correlation between tumor cell infiltration and perfusion at the tumor margins (P = 0.0004). DATA CONCLUSION: These results demonstrate the ability of perfusion imaging to probe regions of low tumor cell infiltration while confirming the sensitivity limitations of conventional imaging modalities. The quantitative relationship between tumor cell density and perfusion identified in and beyond the edematous T2 hyperintensity region surrounding macroscopic tumor could be used to detect marginal tumor cell infiltration with greater accuracy. LEVEL OF EVIDENCE: 1 Technical stage: 2 J. Magn. Reson. Imaging 2019;50:529-540.

Atherosclerotic Carotid Plaque Composition: A 3T and 7T MRI-Histology Correlation Study.

BACKGROUND AND PURPOSE: Carotid artery atherosclerotic plaque composition may influence plaque stability and risk of thromboembolic events, and noninvasive plaque imaging may therefore permit risk stratification for clinical management. Plaque composition was compared using noninvasive in vivo (3T) and ex vivo (7T) MRI and histopathological examination. METHODS: Thirty-three endarterectomy cross-sections, from 13 patients, were studied. The data sets consisted of in vivo 3T MRI, ex vivo 7T MRI, and histopathology. Semiautomated segmentation methods were used to measure areas of different plaque components. Bland-Altman plots and mean difference with 95% confidence interval were carried out. RESULTS: There was general quantitative agreement between areas derived from semiautomated segmentation of MRI data and histology measurements. The mean differences and 95% confidence bounds in the relative to total plaque area between 3T versus Histology were: fibrous tissue 4.99%(-4.56 to 14.56), lipid-rich/necrotic core (LR/NC) with hemorrhage -1.81%(-14.11 to 10.48), LR/NC without hemorrhage -2.43%(-13.04 to 8.17), and calcification -3.18%(-11.55 to 5.18). The mean differences and 95% confidence bounds in the relative to total plaque area between 7T and histology were: fibrous tissue 3.17%(-3.17 to 9.52), LR/NC with hemorrhage -0.55%(-9.06 to 7.95), LR/NC without hemorrhage -12.62%(-19.8 to -5.45), and calcification -2.43%(-9.97 to 4.73). CONCLUSIONS: This study provides evidence that semiautomated segmentation of 3T/7T MRI techniques can help to determine atherosclerotic plaque composition. In particular, the high resolution of ex vivo 7T data was able to highlight greater detail in the atherosclerotic plaque composition. High-field MRI may therefore have advantages for in vivo carotid plaque MRI.

Investigation of nanoparticle transport inside coarse-grained geological media using magnetic resonance imaging.

Quantifying nanoparticle (NP) transport inside saturated porous geological media is imperative for understanding their fate in a range of natural and engineered water systems. While most studies focus upon finer grained systems representative of soils and aquifers, very few examine coarse-grained systems representative of riverbeds and gravel based sustainable urban drainage systems. In this study, we investigated the potential of magnetic resonance imaging (MRI) to image transport behaviors of nanoparticles (NPs) through a saturated coarse-grained system. MRI successfully imaged the transport of superparamagnetic NPs, inside a porous column composed of quartz gravel using T(2)-weighted images. A calibration protocol was then used to convert T(2)-weighted images into spatially resolved quantitative concentration maps of NPs at different time intervals. Averaged concentration profiles of NPs clearly illustrates that transport of a positively charged amine-functionalized NP within the column was slower compared to that of a negatively charged carboxyl-functionalized NP, due to electrostatic attraction between positively charged NP and negatively charged quartz grains. Concentration profiles of NPs were then compared with those of a convection-dispersion model to estimate coefficients of dispersivity and retardation. For the amine functionalized NPs (which exhibited inhibited transport), a better model fit was obtained when permanent attachment (deposition) was incorporated into the model as opposed to nonpermanent attachment (retardation). This technology can be used to further explore transport processes of NPs inside coarse-grained porous media, either by using the wide range of commercially available (super)paramagnetically tagged NPs or by using custom-made tagged NPs.

Application of paramagnetically tagged molecules for magnetic resonance imaging of biofilm mass transport processes.

Molecules become readily visible by magnetic resonance imaging (MRI) when labeled with a paramagnetic tag. Consequently, MRI can be used to image their transport through porous media. In this study, we demonstrated that this method could be applied to image mass transport processes in biofilms. The transport of a complex of gadolinium and diethylenetriamine pentaacetic acid (Gd-DTPA), a commercially available paramagnetic molecule, was imaged both in agar (as a homogeneous test system) and in a phototrophic biofilm. The images collected were T(1) weighted, where T(1) is an MRI property of the biofilm and is dependent on Gd-DTPA concentration. A calibration protocol was applied to convert T(1) parameter maps into concentration maps, thus revealing the spatially resolved concentrations of this tracer at different time intervals. Comparing the data obtained from the agar experiment with data from a one-dimensional diffusion model revealed that transport of Gd-DTPA in agar was purely via diffusion, with a diffusion coefficient of 7.2 x 10(-10) m(2) s(-1). In contrast, comparison of data from the phototrophic biofilm experiment with data from a two-dimensional diffusion model revealed that transport of Gd-DTPA inside the biofilm was by both diffusion and advection, equivalent to a diffusion coefficient of 1.04 x 10(-9) m(2) s(-1). This technology can be used to further explore mass transport processes in biofilms, either by using the wide range of commercially available paramagnetically tagged molecules and nanoparticles or by using bespoke tagged molecules.

Magnetic resonance imaging of structure, diffusivity, and copper immobilization in a phototrophic biofilm.

Magnetic resonance imaging (MRI) was used to spatially resolve structure, water diffusion, and copper transport and fate in a phototrophic biofilm [corrected]. MRI was able to resolve considerable structural heterogeneity, ranging from classical laminations approximately 500 mum thick to structures with no apparent ordering. Pulsed-field gradient (PFG) analysis spatially resolved water diffusion coefficients which exhibited relatively little or no attenuation (diffusion coefficients ranged from 1.7 x 10(-9) m(2) s(-1) to 2.2 x 10(-9) m(2) s(-1)). The biofilm was then reacted with a 10-mg liter(-1) Cu(2+) solution, and transverse relaxation time parameter maps [corrected].were used to spatially and temporally map copper immobilization within the biofilm. Significantly, a calibration protocol similar to that used in biomedical research successfully quantified copper concentrations throughout the biofilm. Variations in Cu concentrations were controlled by the biofilm structure. Copper immobilization was most rapid (approximately 5 mg Cu liter(-1) h(-1)) over the first 20 to 30 h and then much slower for the remaining 60 h of the experiment. The transport of metal within the biofilm is controlled by both diffusion and immobilization. This was explored using a Bartlett and Gardner model which examined both diffusion and adsorption through a hypothetical film exhibiting properties similar to those of the phototrophic biofilm. Higher adsorption constants (K) resulted in longer lag times until the onset of immobilization at depth but higher actual adsorption rates. MRI and reaction transport models are versatile tools which can significantly improve our understanding of heavy metal immobilization in naturally occurring biofilms.

High-resolution 3D isotropic MR imaging of mouse flank tumours obtained in vivo with solenoid RF micro-coil.

The investigation of mouse flank tumours by magnetic resonance imaging (MRI) is limited by the achievable spatial resolution, which is generally limited by the critical problem of signal-to-noise ratio. Sensitivity was improved by using an optimized solenoid RF micro-coil, built into the animal cradle. This simple design did not require extensive RF engineering expertise to construct, yet allowed high-resolution 3D isotropic imaging at 60 x 60 x 60 microm(3) for a flank tumour in vivo, revealing the heterogeneous internal structure of the tumour. It also allowed dynamic contrast enhanced (DCE) experiments and angiography (MRA) to be performed at 100 x 100 x 100 microm(3) resolution. The DCE experiments provided an excellent example of the diffusive spreading of contrast agent into less vascularized tumour tissue. This work is the first step in using high-resolution 3D isotropic MR to study transport in mouse flank tumours.

Rheo-NMR phenomena of wormlike micelles.

Using a combination of rheology and nuclear magnetic resonance (NMR) spectroscopy/velocimetry we demonstrate the existence of shear banding fluctuations under Couette flow of the micellar system 10% w/v cetylpyridinium chloride and sodium salicylate (CPyCl-NaSal) molar ratio 2 : 1 in 0.5 M NaCl in either HO or HO, using both time-averaged and real-time measurements. These shear banding fluctuations are consistent not only with the shear stress fluctuations observed in rheological measurements but also with fluctuations in the change of the constrained fraction of the amphiphile chain (Δ) observed in H-NMR spectroscopy experiments. Using H-NMR spectroscopy on a deuterated probe molecule (-decane) located in the wormlike micellar interior, direct measurement of the shear-induced nematic phase transition is reported.

Shear banding fluctuations and nematic order in wormlike micelles.

Using rapid NMR velocimetry we demonstrate the existence of shear band fluctuations in the Couette flow of the wormlike micelle system, 10% w/v cetylpyridinium chloride and sodium salicylate (molar ratio 2:1) in 0.5 M 2H2O NaCl brine. We show that the fluctuations may be either quasirandom or periodic, the fluctuation spectrum being similar to that observed in the stress. Despite the equilibrium fluid being far from an isotropic-nematic transition, deuterium NMR shows that the onset of shear banding is associated with a nematic micellar state whose order parameter depends on shear rate.

Diffusion in thin films on the surface of a porous solid.

A water-wet mono-dispersed glass bead system is saturated with two phases, a wetting phase of water and a non-wetting phase of tetrachloroethylene (no 1H signal). Pulsed field gradient NMR measurements of the one-dimensional probability density distribution P(Delta) (X) for the diffusive displacements of water molecules in times, Delta, are presented for the whole accessible water saturation range. At lower water contents the distributions show a distinctive shape, which is attributed to the distribution of the aqueous phase in thin surface wetting films connecting pendular rings where the beads are in contact. The data are reproduced well by a computer simulation of a random walk model based on diffusion of molecules within such a structure, allowing determination of the surface film thickness.

The feedback response of Escherichia coli rRNA synthesis is not identical to the mechanism of growth rate-dependent control.

Growth rate-independent rrn P1 promoter mutants were tested for their ability to respond to changes in rrn gene dosage. Most were found to be normal for the feedback response. In addition, cellular levels of the initiating nucleoside triphosphates remained unchanged when the rrn gene dosage was altered. These results suggest that the feedback response cannot be the mechanism for growth rate-dependent control of rRNA synthesis and that the relationship between these two processes may be more complicated than is currently understood.

The Candida albicans CUG-decoding ser-tRNA has an atypical anticodon stem-loop structure.

In many Candida species, the leucine CUG codon is decoded by a tRNA with two unusual properties: it is a ser-tRNA and, uniquely, has guanosine at position 33 (G33). Using a combination of enzymatic (V1 RNase, RnI nuclease) and chemical (Pb(2+), imidazole) probing of the native Candida albicans ser-tRNACAG, we demonstrate that the overall tertiary structure of this tRNA resembles that of a ser-tRNA rather than a leu-tRNA, except within the anticodon arm where there is considerable disruption of the anticodon stem. Using non-modified in vitro transcripts of the C. albicans ser-tRNACAG carrying G, C, U or A at position 33, we demonstrate that it is specifically a G residue at this position that induces the atypical anticodon stem structure. Further quantitative evidence for an unusual structure in the anticodon arm of the G33-tRNA is provided by the observed change in kinetics of methylation of the G at position 37, by purified Escherichia coli m(1)G37 methyltransferase. We conclude that the anticodon arm distortion, induced by a guanosine base at position 33 in the anticodon loop of this novel tRNA, results in reduced decoding ability which has facilitated the evolution of this tRNA without extinction of the species encoding it.

Multiple mechanisms are used for growth rate and stringent control of leuV transcriptional initiation in Escherichia coli.

Expression of the Escherichia coli leuV operon, which contains three tRNA(1)(Leu) genes, is regulated by several mechanisms including growth-rate-dependent control (GRDC) and stringent control (SC). Structural variants of the leuV promoter which differentially affect these regulatory responses have been identified, suggesting that promoter targets for GRDC and SC may be different and that GRDC of the leuV promoter occurs in the absence of guanosine 3', 5'-bisdiphosphate. To determine the mechanisms of the leuV promoter regulation, we have examined the stability of promoter open complexes and the effects of nucleotide triphosphate (NTP) concentration on the efficiency of the leuV promoter and its structural variants in vitro and in vivo. The leuV promoter open complexes were an order of magnitude more stable to heparin challenge than those of rrnBp(1). The major initiating nucleotide GTP as well as other NTPs increased the stability of the leuV promoter open complexes. When the cellular level of purine triphosphates was increased at slower growth rates by pyrimidine limitation, a 10% reduction in leuV promoter activity was seen. It therefore appears that transcription initiation from the leuV promoter is less sensitive to changes in intracellular NTP concentration than that from rrnBp(1). Comparative analysis of regulation of the leuV promoter with and without upstream activating sequences (UAS) demonstrated that the binding site for factor of inversion stimulation (FIS) located in UAS is essential for maximal GRDC. Moreover, the presence of UAS overcame the effects of leuV promoter mutations, which abolished GRDC of the leuV core promoter. However, although the presence of putative FIS binding site was essential for optimal GRDC, both mutant and wild-type leuV promoters containing UAS showed improved GRDC in a fis mutant background, suggesting that FIS protein is an important but not unique participant in the regulation of the leuV promoter.

Activation of Escherichia coli leuV transcription by FIS.

The transcription factor FIS has been implicated in the regulation of several stable RNA promoters, including that for the major tRNALeu species in Escherichia coli, tRNA1Leu. However, no evidence for direct involvement of FIS in tRNA1Leu expression has been reported. We show here that FIS binds to a site upstream of the leuV promoter (centered at -71) and that it directly stimulates leuV transcription in vitro. A mutation in the FIS binding site reduces transcription from a leuV promoter in strains containing FIS but has no effect on transcription in strains lacking FIS, indicating that FIS contributes to leuV expression in vivo. We also find that RNA polymerase forms an unusual heparin-sensitive complex with the leuV promoter, having a downstream protection boundary of approximately -7, and that the first two nucleotides of the transcript, GTP and UTP, are required for formation of a heparin-stable complex that extends downstream of the transcription start site. These studies have implications for the regulation of leuV transcription.

Effect of a mutation in the anticodon of human mitochondrial tRNAPro on its post-transcriptional modification pattern.

Although the gene sequences of all 22 tRNAs encoded in the human mitochondrial genome are known, little information exists about their sequences at the RNA level. This becomes a crucial limitation when searching for a molecular understanding of the growing number of maternally inherited human diseases correlated with point mutations in tRNA genes. Here we describe the sequence of human mt-tRNAPropurified from placenta. It shows absence of editing events in this tRNA and highlights the presence of eight post-transcriptional modifications. These include T54, never found so far in an animal mt-tRNA, and m1G37, a modification known to have fundamental functional properties in a number of canonical tRNAs. Occurrence of m1G37 was further investigated in an analysis of the substrate properties of in vitro transcripts of human mt-tRNAProtowards pure Escherichia coli methylguanosine transferase. This enzyme properly methylates G37 in mt-tRNA and is sensitive to the presence of a second G at position 36, neighboring the target nucleotide for methylation. Since mutation of nt 36 was shown to be correlated with myopathy, the potential consequences of non-modification or under-modification of mt-tRNA nucleotides in expression of the particular myopathy and of mitochondrial diseases in general are discussed.

tRNA recognition for modification: solution probing of tRNA complexed with Escherichia coli tRNA (guanosine-1) methyltransferase.

The interaction of Escherichia coli tRNA (guanosine-1) methyltransferase and tRNA(1Leu) transcripts has been probed using cleavage with iodine of phosphorothioate-substituted transcripts, lead acetate, and enzymes specific for single- and double-stranded RNA. All lytic agents protect the anticodon stem-loop and variable loop regions against cleavage, and some protection is also seen in core structures of the tRNA. Residues from both strands of the anticodon stem are protected against cleavage with iodine and lead by enzyme, yet positions G37 and G36, which are crucial for catalysis and binding, are not. This suggests that these residues may undergo structural perturbation in the presence of S-adenosyl methionine. Occupancy of the AdoMet site by the product S-adenosyl-homocysteine, a potent inhibitor of the enzyme, has little or no effect on tRNA binding or protection. Enhanced reactivity with lead is seen at residues located in the anticodon stem-loop, extra-loop, and core (C34, U47c, and G49), which suggests some perturbations in RNA structure might accompany binding.

RNA-protein interactions within the 3 ' untranslated region of vimentin mRNA.

Several functions have been attributed to protein binding within the 3'untranslated region (3'UTR) of mRNA, including mRNA localization, stability, and translational repression. Vimentin is an intermediate filament protein whose 3'untranslated sequence is highly conserved between species. In order to identify sequences that might play a role in vimentin mRNA function, we synthesized32P-labeled RNA from different regions of vimentin's 3'UTR and assayed for protein binding with HeLa extracts using band shift assays. Sequences required for binding are contained within a region 61-114 nucleotides downstream of the stop codon, a region which is highly conserved from Xenopus to man. As judged by competition assays, binding is specific. Solution probing studies of 32P-labeled RNA with various nucleases and lead support a complex stem and loop structure for this region. Finally, UV cross-linking of the RNA-protein complex identifies an RNA binding protein of 46 kDa. Fractionation of a HeLa extract on a sizing column suggests that in addition to the 46 kDa protein, larger complexes containing additional protein(s) can be identified. Vimentin mRNA has been shown to be localized to the perinuclear region of the cytoplasm, possibly at sites of intermediate filament assembly. To date, all sequences required for localization of various mRNAs have been confined to the 3'UTR. Therefore, we hypothesize that this region and associated protein(s) might be important for vimentin mRNA function such as in localization.

Interaction of tRNA with tRNA (guanosine-1)methyltransferase: binding specificity determinants involve the dinucleotide G36pG37 and tertiary structure.

The sequence G37pG36 is present in all tRNA species recognized and methylated by the Escherichia coli modification enzyme tRNA (guanosine-1)methyltransferase. We have examined whether this dinucleotide sequence provides the base specific recognition signal for this enzyme and have assessed the role of the remaining tRNA in recognition. E. coli tRNAHis and yeast tRNAAsp were substituted with G at positions 36 and 37 and were found to be excellent substrates for methylation. This suggested that the general tRNA structure can be specifically bound by the enzyme. In addition, heterologous tRNA species including fully modified tRNA1Leu are excellent inhibitors of tRNA1Leu transcript methylation. Analyses of structural variants of yeast tRNAAsp and E. coli tRNA1Leu demonstrate clearly that the core tertiary structures of tRNA are required for recognition and that G37 must be in the correct position in space relative to important contacts elsewhere in the molecule. This latter conclusion was reached because the addition of one to three stacked base pairs in the anticodon stem of tRNA1Leu dramatically alters activity. In this case, the G37 base is rotated away from the correct position in space relative to other tRNA contact sites. The acceptor stem structure is required for optimal activity since deletion of three or five base pairs is detrimental to activity; however, specific base sequence may not be important because (i) the addition of three stacked base pairs of different sequence had little effect on activity and (ii) heterologous tRNAs with little or no sequence homology in the acceptor stem are excellent substrates. Both poly G and GpG are potent and specific inhibitors of enzyme activity and are minimal substrates which can be methylated, forming m1G. Taken together, these studies suggest that 1MGT can bind the general tRNA structure and that the crucial base-pair contacts are G37 and G36.