Whole body screening using high-temperature superconducting MR volume resonators: mice studies

High temperature superconducting (HTS) surface resonators have been used as a low loss RF receiver resonator for improving magnetic resonance imaging image quality. However, the application of HTS surface resonators is significantly limited by their filling factor. To maximize the filling factor, it is desirable to have the RF resonator wrapped around the sample so that more nuclear magnetic dipoles can contribute to the signal. In this study, a whole new Bi(2)Sr(2)Ca(2)Cu(2)O(3) (Bi-2223) superconducting saddle resonator (width of 5 cm and length of 8 cm) was designed for the magnetic resonance image of a mouse's whole body in Bruker 3 T MRI system. The experiment was conducted with a professionally-made copper saddle resonator and a Bi-2223 saddle resonator to show the difference. Signal-to-noise ratio (SNR) with the HTS saddle resonator at 77 K was 2.1 and 2 folds higher than that of the copper saddle resonator at 300 K for a phantom and an in-vivo mice whole body imaging. Testing results were in accordance with predicted ones, and the difference between the predicted SNR gains and measured SNR gains were 2.4%～2.7%. In summary, with this HTS saddle system, a mouse's whole body can be imaged in one scan and could reach a high SNR due to a 2 folds SNR gain over the professionally-made prototype of copper saddle resonator at 300 K. The use of HTS saddle resonator not only improves SNR but also enables a mouse's whole body screen in one scan.     

Comparisons between the 35 mm Quadrature Surface Resonator at 300 K and the 40 mm High-Temperature Superconducting Surface Resonator at 77 K in a 3T MRI Imager

This study attempts to compare the signal-to-noise ratio (SNR) of the 40 mm High-Temperature Superconducting (HTS) surface resonator at 77 K and the 35 mm commercial quadrature (QD) surface resonator at 300 K in a 3 Tesla (T) MRI imager. To aquire images for the comparison, we implemented a phantom experiment using the 40 mm diameter Bi₂Sr₂Ca₂Cu₃O_x (Bi-2223) HTS surface resonator, the 35 mm commercial QD surface resonator and the 40 mm professionally-made copper surface resonator. The HTS surface resonator at 77 K provided a 1.43-fold SNR gain over the QD surface resonator at 300 K and provided a 3.84-fold SNR gain over the professionally-made copper surface resonator at 300 K on phantom images. The results agree with the predictions, and the difference between the predicted SNR gains and measured SNR gains is 1%. Although the geometry of the HTS surface resonator is different from the QD surface resonator, its SNR is still higher. The results demonstrate that a higher image quality can be obtained with the HTS surface resonator at 77 K. With the HTS surface resonator, the SNR can be improved, suggesting that the HTS surface resonator is a potentially helpful diagnostic tool for MRI imaging in various applications.     

A Temperature-Stable Cryo-System for High-Temperature Superconducting MR In-Vivo Imaging

To perform a rat experiment using a high-temperature superconducting (HTS) surface resonator, a cryostat is essential to maintain the rat''s temperature. In this work, a compact temperature-stable HTS cryo-system, keeping animal rectal temperature at 37.4°C for more than 3 hours, was successfully developed. With this HTS cryo-system, a 40-mm-diameter Bi₂Sr₂Ca₂Cu₃O_x (Bi-2223) surface resonator at 77 K was demonstrated in a 3-Tesla MRI system. The proton resonant frequency (PRF) method was employed to monitor the rat''s temperature. Moreover, the capacity of MR thermometry in the HTS experiments was evaluated by correlating with data from independent fiber-optic sensor temperature measurements. The PRF thermal coefficient was derived as 0.03 rad/°C and the temperature-monitoring architecture can be implemented to upgrade the quality and safety in HTS experiments. The signal-to-noise ratio (SNR) of the HTS surface resonator at 77 K was higher than that of a professionally made copper surface resonator at 300 K, which has the same geometry, by a 3.79-fold SNR gain. Furthermore, the temperature-stable HTS cryo-system we developed can obtain stable SNR gain in every scan. A temperature-stable HTS cryo-system with an external air-blowing circulation system is demonstrated.     

Planar Quadrature RF Transceiver Design Using Common-Mode Differential-Mode (CMDM) Transmission Line Method for 7T MR Imaging

The use of quadrature RF magnetic fields has been demonstrated to be an efficient method to reduce transmit power and to increase the signal-to-noise (SNR) in magnetic resonance (MR) imaging. The goal of this project was to develop a new method using the common-mode and differential-mode (CMDM) technique for compact, planar, distributed-element quadrature transmit/receive resonators for MR signal excitation and detection and to investigate its performance for MR imaging, particularly, at ultrahigh magnetic fields. A prototype resonator based on CMDM method implemented by using microstrip transmission line was designed and fabricated for 7T imaging. Both the common mode (CM) and the differential mode (DM) of the resonator were tuned and matched at 298MHz independently. Numerical electromagnetic simulation was performed to verify the orthogonal B₁ field direction of the two modes of the CMDM resonator. Both workbench tests and MR imaging experiments were carried out to evaluate the performance. The intrinsic decoupling between the two modes of the CMDM resonator was demonstrated by the bench test, showing a better than -36 dB transmission coefficient between the two modes at resonance frequency. The MR images acquired by using each mode and the images combined in quadrature showed that the CM and DM of the proposed resonator provided similar B₁ coverage and achieved SNR improvement in the entire region of interest. The simulation and experimental results demonstrate that the proposed CMDM method with distributed-element transmission line technique is a feasible and efficient technique for planar quadrature RF coil design at ultrahigh fields, providing intrinsic decoupling between two quadrature channels and high frequency capability. Due to its simple and compact geometry and easy implementation of decoupling methods, the CMDM quadrature resonator can possibly be a good candidate for design blocks in multichannel RF coil arrays.     

High resolution MR eye protocol optimization: Comparison between 3D-CISS, 3D-PSIF and 3D-VIBE sequences

PurposeThe purpose of this study was to compare selected MRI pulse sequences and to evaluate their utility for depicting specific anatomic regions in the eye.MethodsA High-Resolution (HR) 0.08 × 0.08 × 0.60 mm³ MRI protocol was developed on a 1.5-T clinical system and applied in the left eye of an albino rabbit, utilizing a small field of view surface coil. The comprehensive MRI protocol consisted of two 3D (T2/T1)w sequences (3D-PSIF and 3D-CISS), and one 3D T1w sequence (3D-VIBE). The T1w 3D-VIBE sequence was acquired, before and after intravenous injection of 0.2 mmol/kgr gadolinium-DTPA. Signal-to-Noise Ratios (SNR) and Contrast-to-Noise Ratios (CNR) amongst specific eye anatomical areas were calculated for each sequence. The presence of artifacts was rated subjectively utilizing a 5 point scale.Results3D-PSIF and 3D-CISS provide better delineation and visualization of the eye as compared with 3D-VIBE sequences. 3D-CISS images present the highest SNR and revealed better discrimination of the ocular surrounding tissues; its basic drawback though is related to ghost artifacts appearing in the anterior chamber and resulting in the lowest image quality. In post-contrast imaging, the T1w 3D-VIBE sequence provided the best overall image quality. Moreover, 3D (T2/T1)w sequences can provide good anatomic depiction of the eye segments. Agreement between the two independent readers was good.ConclusionsOptimization of a comprehensive MR eye imaging protocol is achieved. A higher SNR, a better spatial resolution and a reduction of the total scan time were obtained, thus making clinical MRI systems more reliable in eye imaging.     

Proton/Deuterium Magnetic Resonance Imaging of Rodents at 9.4T Using Birdcage Coils

The purpose of the present study was to fabricate a volume coil for proton/deuterium magnetic resonance imaging (MRI) in rodents at 9.4 T. Two birdcage radiofrequency (RF) coils have been designed for proton/deuterium MRI: the rungs of two concentric birdcages were azimuthally interleaved with each other for better decoupling, and the two coils were tuned to 400.3 and 61.4 MHz for ¹H/²H resonance at 9.4 T. Compared to a commercially available coil, the proposed ¹H/²H RF coil provides reasonable transmission efficiency and imaging signal-to-noise ratio (SNR); the relationships among imaging parameters such as SNR, voxel size, and deuterium oxide concentrations have been quantitatively studied, and the linear correlation results together with the spectroscopic data in vivo indicate its feasibility in deuterium metabolic imaging (DMI) in vivo. Our study indicates that using the birdcage design for MRI signal excitation combined with surface coil array for signal reception can facilitate DMI investigations more effectively towards future pre-clinical and clinical applications. As a noninvasive method by measuring nonhydrogen nuclear deuterium signals to reflect metabolite information, DMI will feature prominently in future precision medicine through the whole process of diagnosis, treatment, and prognosis. © 2021 Bioelectromagnetics Society.     

Cell adhesion monitoring using a quartz crystal microbalance: comparative analysis of different mammalian cell lines

The quartz crystal microbalance (QCM) has been widely accepted as a sensitive technique to follow adsorption processes in gas as well as in liquid environments. However, there are only a few reports about the use of this technique to monitor the attachment and spreading of mammalian cells onto a solid support in culture. Using a QCM-setup we investigated the time course of cell attachment and spreading as a function of seeding density for three widespread and frequently used cell lines (MDCK strains I and II and Swiss 3T3-fibroblasts). Results were found to be in good agreement with the geometrical properties of the individual cell types. The shifts of the resonance frequency associated with confluent cell layers on top of the quartz resonators were found to be dependent on the cell species [MDCK-I: (320±20) Hz; MDCK-II: (530±25) Hz; 3T3: (240±15) Hz] reflecting their individual influence on the shear oscillation of the resonator. These findings are discussed with respect to the basic models of materials in contact with an oscillating quartz resonator. We furthermore showed by inhibition-assays using soluble RGD-related peptides, that only specific, integrin mediated cell adhesion is detected using this QCM approach, whereas the sole presence of the cellular body in close vicinity to the resonator surface is barely detectable.     

Tunable Multiple Fano Resonances and Stable Plasmonic Band-Stop Filter Based on a Metal-Insulator-Metal Waveguide

Plasmonics - A metal-insulator-metal (MIM) waveguide consisting of two stub resonators and a ring resonator is proposed, which can be used as refractive index sensor and stop-band filter at the...     

Novel bimodal bifunctional ligands for radioimmunotherapy and targeted MRI

The structurally novel bifunctional ligands C-NETA and C-NE3TA, each possessing both acyclic and macrocyclic moieties, were prepared and evaluated as potential chelates for radioimmunotherapy (RIT) and targeted magnetic resonance imaging (MRI). Heptadentate C-NE3TA was fortuitously discovered during the preparation of C-NETA. An optimized synthetic method to C-NETA and C-NE3TA including purification of the polar and tailing reaction intermediates, tert-butyl C-NETA (2) and tert-butyl C-NE3TA (3) using semiprep HPLC was developed. The new Gd(III) complexes of C-NETA and C-NE3TA were prepared as contrast enhancement agents for use in targeted MRI. The T 1 relaxivity data indicate that Gd(C-NETA) and Gd(C-NE3TA) possess higher relaxivity than Gd(C-DOTA), a bifunctional version of a commercially available MRI contrast agent; Gd(DOTA). C-NETA and C-NE3TA were radiolabeled with (177)Lu, (90)Y, (203)Pb, (205/6)Bi, and (153)Gd; and in vitro stability of the radiolabeled corresponding complexes was assessed in human serum. The in vitro studies indicate that the evaluated radiolabeled complexes were stable in serum for 11 days with the exception being the (203)Pb complexes of C-NETA and C-NE3TA, which dissociated in serum. C-NETA and C-NE3TA radiolabeled (177)Lu, (90)Y, or (153)Gd complexes were further evaluated for in vivo stability in athymic mice and possess excellent or acceptable in vivo biodistribution profile. (205/6)Bi- C-NE3TA exhibited extremely rapid blood clearance and low radioactivity level at the normal organs, while (205/6)Bi- C-NETA displayed low radioactivity level in the blood and all of the organs except for the kidney where relatively high renal uptake of radioactivity is observed. C-NETA and C-NE3TA were further modified for conjugation to the monoclonal antibody Trastuzumab.     

Modular Coils with Low Hydrogen Content Especially for MRI of Dry Solids

IntroductionRecent advances have enabled fast magnetic resonance imaging (MRI) of solid materials. This development has opened up new applications for MRI, but, at the same time, uncovered new challenges. Previously, MRI-invisible materials like the housing of MRI detection coils are now readily depicted and either cause artifacts or lead to a decreased image resolution. In this contribution, we present versatile, multi-nuclear single and dual-tune MRI coils that stand out by (1) a low hydrogen content for high-resolution MRI of dry solids without artifacts; (2) a modular approach with exchangeable inductors of variable volumes to optimally enclose the given object; (3) low cost and low manufacturing effort that is associated with the modular approach; (4) accurate sample placement in the coil outside of the bore, and (5) a wide, single- or dual-tune frequency range that covers several nuclei and enables multinuclear MRI without moving the sample.

Materials and Methods

The inductors of the coils were constructed from self-supporting copper sheets to avoid all plastic materials within or around the resonator. The components that were mounted at a distance from the inductor, including the circuit board, coaxial cable and holder were manufactured from polytetrafluoroethylene.

Results and Conclusion

Residual hydrogen signal was sufficiently well suppressed to allow ¹H-MRI of dry solids with a minimum field of view that was smaller than the sensitive volume of the coil. The SNR was found to be comparable but somewhat lower with respect to commercial, proton-rich quadrature coils, and higher with respect to a linearly-polarized commercial coil. The potential of the setup presented was exemplified by ¹H / ²³Na high-resolution zero echo time (ZTE) MRI of a model solution and a dried human molar at 9.4 T. A full 3D image dataset of the tooth was obtained, rich in contrast and similar to the resolution of standard cone-beam computed tomography.     

High Resolution T1ρ Mapping of In Vivo Human Knee Cartilage at 7T

Purpose

Spin lattice relaxation time in rotating frame (T1ρ) mapping of human knee cartilage has shown promise in detecting biochemical changes during osteoarthritis. Due to higher field strength, MRI at 7T has advantages in term of SNR compared to clinical MR scanners and this can be used to increase in image resolution. Objective of current study was to evaluate the feasibility of high resolution T1ρ mapping of in vivo human knee cartilage at 7T MR scanner.

Materials and Methods

In this study we have used a T1ρ prepared GRE pulse sequence for obtaining high resolution (in plan resolution  = 0.2 mm²) T1ρ MRI of human knee cartilage at 7T. The effect of a global and localized reference frequency and reference voltage setting on B₀, B₁ and T1ρ maps in cartilage was evaluated. Test-retest reliability results of T1ρ values from asymptomatic subjects as well as T1ρ maps from abnormal cartilage of two human subjects are presented. These results are compared with T1ρ MRI data obtained from 3T.

Results

Our approach enabled acquisition of 3D-T1ρ data within allowed SAR limits at 7T. SNR of cartilage on T1ρ weighted images was greater than 90. Off-resonance effects present in the cartilage B₀, B₁ and T1ρ maps obtained using global shim and reference frequency and voltage setting, were reduced by the proposed localized reference frequency and voltage setting. T1ρ values of cartilage obtained with the localized approach were reproducible. Abnormal knee cartilage showed elevated T1ρ values in affected regions. T1ρ values at 7T were significantly lower (p<0.05) compared to those obtained at 3T.

Conclusion

In summary, by using proposed localized frequency and voltage setting approach, high-resolution 3D-T1ρ maps of in vivo human knee cartilage can be obtained in clinically acceptable scan times (<30 min) and SAR constraints, which provides the ability to characterize cartilage molecular integrity.     

Serological characterization of a pluripotent mouse embryonal stem cell line, two transformed derivatives, and an endoderm-like cell line

The pluripotent mouse embryonal stem cell line BLC 1 and two transformants derived from it by DNA transformation (T1 and T2/K26) as well as the blastocyst-derived cell line BLC 3 were tested for the presence of cell surface antigens recognized by the monoclonal antibodies ECMA-7, anti-SSEA-1 and M 1/22.25, and intermediate filament proteins labeled by the monoclonal antibodies TROMA 1 and TROMA 2 using a three-step indirect immunofluorescence technique. According to present concepts and in agreement with previous data (Wobus et al., 1984a), the results obtained indicate that BLC 1, T1 and T2/K26 are undifferentiated embryonal stem cells, and BLC 3 is an endoderm-like cell line.     

Wide Bandpass and Narrow Bandstop Microstrip Filters Based on Hilbert Fractal Geometry: Design and Simulation Results

This paper presents new Wide Bandpass Filter (WBPF) and Narrow Bandstop Filter (NBSF) incorporating two microstrip resonators, each resonator is based on 2^nd iteration of Hilbert fractal geometry. The type of filter as pass or reject band has been adjusted by coupling gap parameter (d) between Hilbert resonators using a substrate with a dielectric constant of 10.8 and a thickness of 1.27 mm. Numerical simulation results as well as a parametric study of d parameter on filter type and frequency responses are presented and studied. WBPF has designed at resonant frequencies of 2 and 2.2 GHz with a bandwidth of 0.52 GHz, −28 dB return loss and −0.125 dB insertion loss while NBSF has designed for electrical specifications of 2.37 GHz center frequency, 20 MHz rejection bandwidth, −0.1873 dB return loss and 13.746 dB insertion loss. The proposed technique offers a new alternative to construct low-cost high-performance filter devices, suitable for a wide range of wireless communication systems.     

Reference 3 T MRI parameters of the normal human eye

The purpose of this study is to establish magnetic resonance imaging (MRI) standard normative reference quantitative markers for future possible diagnosis in Ophthalmology based on relaxation times (T1 and T2) and retina/choroid complex (RCC) layer thickness values measured in vivo in normal human eyes.This research followed the tenets of the Declaration of Helsinki and was approved by the local Ethical Committee. 15 healthy subjects volunteered to undergo MRI of both eyes. 3 T MRI was performed using a circular surface detector coil with a 15 min acquisition protocol for each eye. The most important normal human eye structures were visualized and characterized geometrico-physicochemically by the 35 MRI standard normative reference markers (20 RCC thicknesses, 8 T1 and 7 T2) calculated. Future possible pathology management could be based on the relative-to-normal differences between the standard normative reference MRI markers calculated in this study and the corresponding MRI markers calculated in the future in disease-suspected eyes.In conclusion, this research demonstrates that ocular MRI at 3 T, performed without contrast agents, brings useful additional multiparametric quantitative information for future possible automated medical diagnosis, staging and evaluation of ocular disease mechanisms.     

Interleukin 2 acts directly on a Tac-positive cloned B cell line established from a patient with adult T cell leukemia

A Tac-positive B cell line termed K3B was established from a patient with adult T cell leukemia (ATL). This cell line had EBNA antigen and human T cell leukemic virus (HTLV) provirus besides B1 antigen and surface immunoglobulin. A cloned Tac-positive B cell line termed K3B01 was obtained from K3B by the limiting dilution method. The K3B01 cells were shown to absorb IL 2 activity in a tonsillar IL 2 preparation. By using this cloned cell line and a purified recombinant IL 2 preparation, it was shown that the proliferation of K3B01 cells was enhanced by the addition of recombinant IL 2. Moreover, this response was inhibited by anti-Tac antibody. These results demonstrate definitively that IL 2 acts directly on B cells through IL 2 receptors on them.     

Birdcage volume coils and magnetic resonance imaging: a simple experiment for students

Background

This article explains some simple experiments that can be used in undergraduate or graduate physics or biomedical engineering laboratory classes to learn how birdcage volume radiofrequency (RF) coils and magnetic resonance imaging (MRI) work. For a clear picture, and to do any quantitative MRI analysis, acquiring images with a high signal-to-noise ratio (SNR) is required. With a given MRI system at a given field strength, the only means to change the SNR using hardware is to change the RF coil used to collect the image. RF coils can be designed in many different ways including birdcage volume RF coil designs. The choice of RF coil to give the best SNR for any MRI study is based on the sample being imaged.

Results

The data collected in the simple experiments show that the SNR varies as inverse diameter for the birdcage volume RF coils used in these experiments. The experiments were easily performed by a high school student, an undergraduate student, and a graduate student, in less than 3 h, the time typically allotted for a university laboratory course.

Conclusions

The article describes experiments that students in undergraduate or graduate laboratories can perform to observe how birdcage volume RF coils influence MRI measurements. It is designed for students interested in pursuing careers in the imaging field.

     

Evaluation of microbubbles as contrast agents for ultrasonography and magnetic resonance imaging

Background

Microbubbles (MBs) can serve as an ultrasound contrast agent, and has the potential for magnetic resonance imaging (MRI). Due to the relatively low effect of MBs on MRI, it is necessary to develop new MBs that are more suitable for MRI. In this study, we evaluate the properties of SonoVue® and custom-made Fe₃O₄-nanoparticle-embedded microbubbles (Fe₃O₄-MBs) in terms of contrast agents for ultrsonography (US) and MRI.

Methodology/Principal Findings

A total of 20 HepG2 subcutaneous-tumor-bearing nude mice were randomly assigned to 2 groups (i.e., n = 10 mice each group), one for US test and the other for MRI test. Within each group, two tests were performed for each mouse. The contrast agent for the first test is SonoVue®, and the second is Fe₃O₄-MBs. US was performed using a Technos^MPX US system (Esaote, Italy) with a contrast-tuned imaging (CnTI™) mode. MRI was performed using a 7.0T Micro-MRI (PharmaScan, Bruker Biospin GmbH, Germany) with an EPI-T₂* sequence. The data of signal-to-noise ratio (SNR) from the region-of-interest of each US and MR image was calculated by ImageJ (National Institute of Health, USA). In group 1, enhancement of SonoVue® was significantly higher than Fe₃O₄-MBs on US (P<0.001). In group 2, negative enhancement of Fe₃O₄-MBs was significantly higher than SonoVue® on MRI (P<0.001). The time to peak showed no significant differences between US and MRI, both of which used the same MBs (P>0.05). The SNR analysis of the enhancement process reveals a strong negative correlation in both cases (i.e., SonoVue® r = −0.733, Fe₃O₄-MBs r = −0.903, with P<0.05).

Conclusions

It might be important to change the Fe₃O₄-MBs'' shell structure and/or the imagining strategy of US to improve the imaging quality of Fe₃O₄-MBs on US. As an intriguing prospect that can be detected by US and MRI, MBs are worthy of further study.     

Spectrally Tunable Optical Transmission of Titanium Nitride Split Ring Resonators

In this paper, the optical properties of titanium nitride split ring resonators as an intermetallic metamaterial nanostructure were studied. Our simulation shows the presence of plasmon and LC resonances in the transmission spectrum of a cell consists of four u-shape split ring resonators. The effect of different parameters of resonator such as size, periodic constant, and the material between arms in addition to the polarization of incident beam was examined on the resonance behavior of the system. Also, the optical properties of a cell consist of four complementary split ring resonators within titanium nitride thin film were investigated. An excited mode was observed at λ = 840 nm that was attributed to the plasmon resonance. Changing the arrangement and configuration of the system from C _1v to C _2v symmetry led to the presence of the LC mode beside the plasmon mode in the transmission spectrum. Also, we explored a connection between the complementary split ring resonators and orderly perforated surface plasmon systems. It was determined that a transition occurred from resonator-type to surface plasmon behavior by increasing the size of resonator above 170 nm.     

The distance between cytochromes a and a3 in the azide compound of bovine-heart cytochrome oxidase

The electron-spin relaxation rates of the two species of cytochrome a3(3+)-azide found in the azide compound of bovine-heart cytochrome oxidase were measured by progressive microwave saturation at T = 10 K. It has been shown previously that Cyt a3(3+)-azide gives rise to two distinct EPR resonances, depending upon the oxidation state of Cyt a. When Cyt a is ferrous, Cyt a3(3+)-azide has g = 2.88, 2.19 and 1.64; upon oxidation of Cyt a, the a3(3+)-azide g-values become g = 2.77, 2.18, and 1.74 (Goodman, G. (1984) J Biol. Chem. 259, 15094-15099). The relaxation effect of Cyt a on Cyt a3 could be measured as the difference in microwave field saturation parameter H1/2 between the g = 2.77 and g = 2.88 species. For each signal the spin-lattice relaxation time T1 was determined from H1/2 using the transverse relaxation time T2. The value of T2 at 10 K was extrapolated from a plot of line-width vs. temperature at higher temperature. The dipolar contribution to T1 was related to the Cyt a-Cyt a3 spin-spin distance utilizing available information on the relative orientation of Cyt a3-azide and Cyt a (Erecińska, M., Wilson, D.F. and Blasie, J.K. (1979) Biochim. Biophys. Acta 545, 352-364). By taking into account the relaxation parameters for both gx and gz components of the Cyt a3-azide g-tensor, the angle between the gz components of the Cyt a and Cyt a3 g-tensors was determined to be between 0 and 18 degrees, and the Cyt a-Cyt a3 spin-spin distance was found to be 19 +/- 8 A.     

Molecular analysis of the acetolactate synthase gene of Chlamydomonas reinhardtii and development of a genetically engineered gene as a dominant selectable marker for genetic transformation

Genomic and cDNA clones of the acetolactate synthase (ALS) gene of Chlamydomonas reinhardtii have been isolated from a mutant, c85-20 (Hartnett et al., 1987), that is resistant to high concentrations of sulfometuron methyl (SMM) and related sulfonylurea herbicides. Comparison of the ALS gene sequences from the wild-type and the SMM resistant (SMMr) strains revealed two amino acid differences in the mature enzyme, a lysine to threonine change at position 257 (K257T) and a leucine to valine change at position 294 (L294V). Transformation of wild-type C. reinhardtii with the mutant ALS gene produced no transformants with ability to grow in the presence of a minimum toxic concentration of SMM (3 microm). Substitution of the ALS promoter with the promoter of the C. reinhardtii Rubisco small subunit gene (RbcS2) permitted recovery of SMMr colonies. In vitro mutagenesis of the wild-type ALS gene to produce various combinations of mutations (K257T, L294V and W580L) indicated that the K257T mutation was necessary and sufficient to confer the SMMr phenotype. Optimum transformation rates were obtained with two constructs (pJK7 and pRP-ALS) in which all introns in the coding region were present. Rates of transformation with construct pJK7 were approximately 2.5 x 10-4 transformants/cell (i.e. one transformant for each of 4000 initial cells) using electroporation and 8.5 x 10-6 transformants/cell using the glass bead vortexing method. These results suggest that pJK7 and pRP-ALS can serve as important additional dominant selectable markers for the genetic transformation of C. reinhardtii.