Insight into de-regulation of amino acid feedback inhibition: a focus on structure analysis method.

Regulation of amino acid's biosynthetic pathway is of significant importance to maintain homeostasis and cell functions. Amino acids regulate their biosynthetic pathway by end-product feedback inhibition of enzymes catalyzing committed steps of a pathway. Discovery of new feedback resistant enzyme variants to enhance industrial production of amino acids is a key objective in industrial biotechnology. Deregulation of feedback inhibition has been achieved for various enzymes using in vitro and in silico mutagenesis techniques. As enzyme's function, its substrate binding capacity, catalysis activity, regulation and stability are dependent on its structural characteristics, here, we provide detailed structural analysis of all feedback sensitive enzyme targets in amino acid biosynthetic pathways. Current review summarizes information regarding structural characteristics of various enzyme targets and effect of mutations on their structures and functions especially in terms of deregulation of feedback inhibition. Furthermore, applicability of various experimental as well as computational mutagenesis techniques to accomplish feedback resistance has also been discussed in detail to have an insight into various aspects of research work reported in this particular field of study.

Identification of the pathogenic effects of missense variants causing PRKAG2 cardiomyopathy.

BACKGROUND: Pathogenic missense variants in PRKAG2, the gene for the gamma 2 regulatory subunit of adenosine monophosphate-activated protein kinase (AMPK), cause severe progressive cardiac disease and sudden cardiac death, named PRKAG2 cardiomyopathy. In our previous study, we reported a E506K variant in the PRKAG2 gene that was associated with this disease. This study aimed to functionally characterize the three missense variants (E506K, E506Q, and R531G) of PRKAG2 and determine the possible effects on AMPK activity. METHODS: The proband was clinically monitored for eight years. To investigate the functional effects of three missense variants of PRKAG2, in vitro mutagenesis experiments using HEK293 cells with wild and mutant transcripts and proteins were comparatively analyzed using quantitative RT-PCR, immunofluorescence staining, and enzyme-linked immunosorbent assay. RESULTS: In the long-term follow-up, the proband was deceased due to progressive heart failure. In the in vitro experimental studies, PRKAG2 was overexpressed after 48 h of transfection in three mutated cells, after which the expression levels of PRKAG2 were regressed to the level of wild-type cells in 3-weeks stably transformed cells, except for the cells with E506K variant. E506K, E506Q, and R531G variants had caused a reduction in the AMPK activity and resulted in the formation of cytoplasmic glycogen deposits. CONCLUSION: Three missense variants that alter AMPK activity affect a residue in the CBS4 domain associated with ATP/AMP-binding. Detailed information on the influence of PRKAG2 pathogenic variants on AMPK activity would be helpful to improve the treatment and management of patients with metabolic cardiomyopathy.

Prediction of improved therapeutics for fabry disease patients generated by mutagenesis of the α-galactosidase A active site, dimer interface, and glycosylation region.

Fabry disease is an X-linked lysosomal storage disorder caused by the deficiency of the enzyme, α-galactosidase A that induces the accumulation of the substrate globotriaosylceramide. Currently approved enzyme replacement therapy using recombinant human α-galactosidase A improves patient symptoms but a majority of patients experience adverse events due to the multiple infusions required for full therapeutic efficacy. Our approach is to use medicinal chemistry and phylogenic comparisons to introduce mutations into the human enzyme to increase catalytic activity and/or stability to generate an improved therapeutic enzyme that may require fewer infusions. We designed mutations at three regions of the human α-galactosidase A: the active site, the dimer interface, and a site for glycosylation. The M208E mutation, adjacent to the Y207 active site residue, increased enzyme activity 3.01-fold. This mutation introduced a charged Glu residue that is adjacent to the Y207 active site residue and close to a site of N-glycosylation. The W277C mutation, designed to promote dimer stability, introduced a strong thiol-aromatic interaction (Cys-Phe) at the dimer interface and increased activity 2.31-fold. The W277C and M208E mutations modify the structure of the enzyme into forms with enhanced thermal stability 3.7- and 3.9-fold, respectively and positive cooperativity resulting in increased Hill coefficient from 1.0 to 4.60 and 3.47, respectively. Enhanced thermal stability and positive cooperativity predict improved in vivo activity and superior therapeutic properties. Our results demonstrate the value of in vitro mutagenesis for α-galactosidase A and support future perspectives to validate these results in Fabry disease patients.

Reengineering the ligand sensitivity of the broadly tuned human bitter taste receptor TAS2R14.

BACKGROUND: In humans, bitterness perception is mediated by ~25 bitter taste receptors present in the oral cavity. Among these receptors three, TAS2R10, TAS2R14 and TAS2R46, exhibit extraordinary wide agonist profiles and hence contribute disproportionally high to the perception of bitterness. Perhaps the most broadly tuned receptor is the TAS2R14, which may represent, because of its prominent expression in extraoral tissues, a receptor of particular importance for the physiological actions of bitter compounds beyond taste. METHODS: To investigate how the architecture and composition of the TAS2R14 binding pocket enables specific interactions with a complex array of chemically diverse bitter agonists, we carried out homology modeling and ligand docking experiments, subjected the receptor to point-mutagenesis of binding site residues and performed functional calcium mobilization assays. RESULTS: In total, 40 point-mutated receptor constructs were generated to investigate the contribution of 19 positions presumably located in the receptor's binding pocket to activation by 7 different TAS2R14 agonists. All investigated positions exhibited moderate to pronounced agonist selectivity. CONCLUSIONS: Since numerous modifications of the TAS2R14 binding pocket resulted in improved responses to individual agonists, we conclude that this bitter taste receptor might represent a suitable template for the engineering of the agonist profile of a chemoreceptive receptor. GENERAL SIGNIFICANCE: The detailed structure-function analysis of the highly promiscuous and widely expressed TAS2R14 suggests that this receptor must be considered as potentially frequent target for known and novel drugs including undesired off-effects.

Site-directed mutagenesis switching a dimethylallyl tryptophan synthase to a specific tyrosine C3-prenylating enzyme.

The tryptophan prenyltransferases FgaPT2 and 7-DMATS (7-dimethylallyl tryptophan synthase) from Aspergillus fumigatus catalyze C(4)- and C(7)-prenylation of the indole ring, respectively. 7-DMATS was found to accept l-tyrosine as substrate as well and converted it to an O-prenylated derivative. An acceptance of l-tyrosine by FgaPT2 was also observed in this study. Interestingly, isolation and structure elucidation revealed the identification of a C(3)-prenylated l-tyrosine as enzyme product. Molecular modeling and site-directed mutagenesis led to creation of a mutant FgaPT2_K174F, which showed much higher specificity toward l-tyrosine than l-tryptophan. Its catalytic efficiency toward l-tyrosine was found to be 4.9-fold in comparison with that of non-mutated FgaPT2, whereas the activity toward l-tryptophan was less than 0.4% of that of the wild-type. To the best of our knowledge, this is the first report on an enzymatic C-prenylation of l-tyrosine as free amino acid and altering the substrate preference of a prenyltransferase by mutagenesis.

Enhanced heterotetrameric assembly of potato ADP-glucose pyrophosphorylase using reverse genetics.

ADP-glucose pyrophosphorylase (AGPase) is a key allosteric enzyme in plant starch biosynthesis. Plant AGPase is a heterotetrameric enzyme that consists of large (LS) and small subunits (SS), which are encoded by two different genes. Computational and experimental studies have revealed that the heterotetrameric assembly of AGPase is thermodynamically weak. Modeling studies followed by the mutagenesis of the LS of the potato AGPase identified a heterotetramer-deficient mutant, LS(R88A). To enhance heterotetrameric assembly, LS(R88A) cDNA was subjected to error-prone PCR, and second-site revertants were identified according to their ability to restore glycogen accumulation, as assessed with iodine staining. Selected mutations were introduced into the wild-type (WT) LS and co-expressed with the WT SS in Escherichia coli glgC(-). The biochemical characterization of revertants revealed that LS(I90V)SS(WT), LS(Y378C)SS(WT) and LS(D410G)SS(WT) mutants displayed enhanced heterotetrameric assembly with the WT SS. Among these mutants, LS(Y378C)SS(WT) AGPase displayed increased heat stability compared with the WT enzyme. Kinetic characterization of the mutants indicated that the LS(I90V)SS(WT) and LS(Y378C)SS(WT) AGPases have comparable allosteric and kinetic properties. However, the LS(D410G)SS(WT) mutant exhibited altered allosteric properties of being less responsive and more sensitive to 3-phosphoglyceric acid activation and inorganic phosphate inhibition. This study not only enhances our understanding of the interaction between the SS and the LS of AGPase but also enables protein engineering to obtain enhanced assembled heat-stable variants of AGPase, which can be used for the improvement of plant yields.

Inducing mutation and ascertaining lethal dosage of in vitro cultures of banana cv. Ney Poovan to ethyl methane sulfonate.

In vitro mutation breeding in vegetatively propagated crops like banana offers a benefit in screening for beneficial variants in plant cells or cultured tissues. An attempt was made to induce mutants and determine the lethal dose, as it is the prerequisite to optimize the concentration and duration of the mutagen used to recover a larger population in mutation research. Shoot tip cultures were treated for 2 and 4 h at six different EMS concentrations ranging from 80 mM to 160 mM, whereas proliferating multiple shoots were exposed for 30 and 60 min at six different EMS concentrations ranging from 8 mM to 40 mM. Survival percentage, shoot length, and number of shoots reduced linearly and significantly as concentration and duration increased in both shoot tips and proliferating multiple buds. The probit curve-based analysis of mortality of treated explants revealed that the LD50 was 155.83 mM for 2 h and 113.72 mM for 4 h, respectively for shoot tip cultures, whereas for proliferating multiple buds, the LD50 value was adjusted to 39.11 mM for 30 min and 30.41 mM for 60 min. 160 mM EMS for 4 h resulted in a shorter shoot, a longer rooting duration, a lesser number of roots, and decreased root development. In proliferating multiple shoots, the smallest shoot, longest rooting duration, least number of roots, and shortest root were observed in 40 mM EMS for 60 min. Similar reductions in growth parameters were observed in proliferating multiple shoots at higher exposure to EMS for a longer duration.

Biochemical analysis of H2O2-induced mutation spectra revealed that multiple damages were involved in the mutational process.

Reactive oxygen species (ROS) are a major threat to genomic integrity and believed to be one of the etiologies of cancers. Here we developed a cell-free system to analyze ROS-induced mutagenesis, in which DNA was exposed to H2O2 and then subjected to translesion DNA synthesis by various DNA polymerases. Then, frequencies of mutations on the DNA products were determined by using next-generation sequencing technology. The majority of observed mutations were either C>A or G>A, caused by dAMP insertion at G and C residues, respectively. These mutations showed similar spectra to COSMIC cancer mutational signature 18 and 36, which are proposed to be caused by ROS. The in vitro mutations can be produced by replicative DNA polymerases (yeast DNA polymerase δ and ε), suggesting that ordinary DNA replication is sufficient to produce them. Very little G>A mutation was observed immediately after exposure to H2O2, but the frequency was increased during the 24 h after the ROS was removed, indicating that the initial oxidation product of cytosine needs to be maturated into a mutagenic lesion. Glycosylase-sensitivities of these mutations suggest that the C>A were made on 8-oxoguanine or Fapy-guanine, and that G>A were most likely made on 5-hydroxycytosine modification.

Radiation induced mutagenesis, physio-biochemical profiling and field evaluation of mutants in sugarcane cv. CoM 0265.

PURPOSE: Sugarcane is an important cash crop and is affected by soil salinity. CoM 0265, a moderately salt-tolerant variety grown in the Maharashtra region (India), has low sugar content. The present study was aimed to employ gamma ray induced in vitro mutagenesis with repeated and step-wise selection in sugarcane for the isolation and physio-biochemical profiling of the selected salt-tolerant mutants for improved agronomic performance and sugar content. MATERIALS AND METHODS: Embryogenic callus culture of CoM 0265 variety was subjected to different doses of gamma radiation (10, 20, 30, 40, 50, and 60 Gy) followed by selection on NaCl containing media (50, 100, 150, 200, and 250 mM NaCl). The regenerated plantlets were hardened and selected based on ground nursery field trial on normal soil and saline field trial, in augmented block design for the selected mutant clones. Different physio-biochemical changes and activity of antioxidant enzymes were analyzed in the salt selected in vitro cultures and field-grown mutant clones. RESULTS: Dose optimization showed 40 Gy as the LD50 for gamma radiation and 150 mM NaCl as the dose for in vitro selection experiments. The selected mutant clones showed higher tissue water content (TWC), chlorophyll, and lower sodium content indicative of tolerance to salt stress. Catalase and peroxidase enzyme activities in the top visible dewlap (TVD) of the putative mutant clones were significantly higher than the control. The average yield and sucrose percent of the selected mutant clones were significantly higher than control checks in the saline field trial. Mutant clones M8457 and M8721 exhibited improved yield and commercial cane sugar over the parent control check varieties under saline field conditions. Catalase activity was strongly associated with TWC (r = 0.34) and chlorophyll content (r = 0.41) while it was negatively correlated with sodium ion content (r = -0.38). Peroxidase activity in TVD also showed a significant positive correlation with chlorophyll content (r = 0.42) and a negative correlation with sodium ion content (r=-0.39). The improvement in yield and CCS (t/ha) was strongly associated with the lower sodium ion content of the mutant clones (r=-0.54 and -0.53, respectively). CONCLUSIONS: Gamma ray induced mutants were isolated for improved sucrose and high yield in sugarcane var. CoM 0265. The results suggest that gamma radiation induced mutations result in physiological and metabolomic alterations for better growth and adaptation under in vitro and field stress conditions in sugarcane. The improved mutants can be further useful for commercial cultivation in saline areas.

[Directional breeding of high oil content peanut variety Yuhua 9 by in vitro mutagenesis and screening].

Leaf water potential of peanut subjected to drought stress is positively related to the oil content of peanut kernels. The aim of this study was to directly screen the high oil mutants of peanut and create the new peanut varieties using hydroxyproline as water potential regulator. In vitro mutagenesis was carried out with the embryonic leaflets of peanut variety Huayu 20 as explants and pingyangmycin as a mutagen added into the somatic embryo formation medium. The formed somatic embryos were successively transferred to somatic embryo germination and selection medium containing 6 mmol/L hydroxyproline (at -2.079 MPa water potential ) to induce regeneration and directionally screen high oil content mutants. After that, these plantlets were grafted and transplanted to the experimental field and 132 high oil mutants with oil content over 55% were obtained from the offspring of regenerated plants. Finally, among them, the oil contents of 27 lines were higher than 58% and of 2 lines were higher than 60%. A new peanut variety Yuhua 9 with high yield and oil content was bred from the regenerated plant progenies combining the pedigree breeding method. The yield was 14.0% higher than that of the control cultivar in the testing new peanut varieties of Liaoning province, and also it has passed the national registration of non-major crop varieties. Yuhua 9 with an oil content of 61.05%, which was 11.55 percentage points higher than that of the parent Huayu 20, was the peanut cultivar with the highest oil content in the world. The result showed that it was an effective way for directional breeding of high oil peanut varieties by means of the three-step technique including in vitro mutagenesis, directional screening by reducing water potential in medium and pedigree selection of regenerated plant progenies.

Gamma irradiation induced effects on in vitro shoot cultures and influence of monochromatic light regimes on irradiated shoot cultures of Dendrobium sonia orchid.

The effects of gamma radiation and monochromatic lights on growth of in vitro shoot cultures of orchid, Dendrobium sonia, were investigated. The gamma irradiated shoot cultures grown under white, blue, yellow and red monochromatic lights exhibited differential growth pattern. Shoot cultures gamma irradiated at 15-45 Gy showed reduced shoot length, fresh weight and leaf area. The monochromatic light significantly influenced survival rate and growth of irradiated shoots. The yellow and red light treatments positively influenced survival of gamma irradiated shoots with significant increase in fresh weight, shoot length and chlorophyll content. Yellow light was found to be most effective as leaf area was increased across the radiation dose range (15-100 Gy) compared to red light. The results demonstrated that the method of post-irradiation exposure could be useful to improve growth of gamma irradiated in vitro shoots, and help to recover orchid mutants with novel modifications.

[Breeding of peanut variety Yuhua 4 by in vitro mutagenesis].

The embryonic leaflets of peanut (Arachis hypogaea) variety Huayu 20 were used as explants and pingyangmycin as a mutagen to induce somatic embryos. Four weeks after the inoculation, the survived explants were transferred to somatic embryo germination medium containing screening reagent hydroxyproline, and finally 15 regenerated plants were obtained. Pedigree breeding method was used during the following selection breeding, and three lines with significantly increased yield and 23 lines with high oil content were obtained from these mutant offsprings. The line with both high yield and high oil content has passed peanut variety multi-location in Anhui province and was named "Yuhua 4". Its yield was 16.63% higher than that of the control variety Baisha 1016, ranking the first in all the testing varieties. Yuhua 4 showed the characteristics of early maturity, small pod and high oil content. The oil content of kernels was 56.10%, higher than that of original parent Huayu 20 with 49.50% oil content, tested by the Ministry of Agriculture of Oil and Products Quality Supervision, Inspection and Test Center (Wuhan), and the yield was 15% higher than that of Huayu 20. It was concluded that in vitro mutagenesis and target screening was an effective way on creating new germplasm and breeding new variety in peanut.

Metachromatic leukodystrophy: Biochemical characterization of two (p.307Glu→Lys, p.318Trp→Cys) arylsulfatase A mutations.

Metachromatic leukodystrophy (MLD) is a lysosomal storage disease caused by Arylsulfatase A (ASA) deficiency. The hallmark of the disease is central and peripheral neurodegeneration. More than 200 mutations have been identified in ARSA gene so far. Some of these mutations were characterized. The aim of this study is to reinforce genotype-phenotype correlation and to understand the effect of mutations on the enzyme by biochemical characterization. Two missense mutations (c.919G→A, p.307Glu→Lys and c.954G→T, p.318Trp→Cys in exon 5) were constructed on WT-ASA cDNA and were confirmed by DNA sequence analysis. Plasmid DNA carrying mutant or normal ASA cDNA was transferred to Chinese Hamster Ovary (CHO) cells through transient transfection. ASA protein was produced by CHO cells. Hexosaminidase beta-subunit gene was cotransfected into the CHO cells as a control gene of transfection efficiency. 48 hours after transfection, cells were collected and homogenized. ASA and hexosaminidase activities were measured in supernatant. ASA enzyme activity is decreased 100% according to the control by the effect of both mutations. The mutations are located in the higly conserved region of the protein. In this study, we showed that both mutations result in null ASA activity in CHO cells making the protein nonfunctional. We confirmed that p.307Glu→Lys and p.318Trp→Cys mutations cause late infantile form of MLD disease.

Development of broadly reactive H5N1 vaccine against different Egyptian H5N1 viruses.

The H5N1 highly pathogenic avian influenza (HPAI) virus was isolated for the first time in Egypt in 2006, since then, the virus has become endemic causing a significant threat to the poultry industry and humans. H5N1 HPAI outbreaks continue to occur despite extensive vaccination programs that have been implemented nationwide in different poultry species. Several studies showed that the co-circulating H5N1 viruses in Egypt are genetically and antigenically distant raising a question on the cross protective efficacy of commercial vaccines. In this study, we introduced mutations at the antigenic sites of the hemagglutinin (HA) to broaden reactivity of the Egyptian H5N1 virus. A reverse genetically created variant H5N1 virus (A/chicken/Egypt/1063/2010) with five amino acid mutations (G140R, Y144F, I190L, K192Q, D43N) in the HA gene showed enhanced cross reactivity. This virus showed up to 16 fold increase in reactivity to the classic-lineageH5N1viruses measured by hemagglutination inhibition (HI) assay while maintaining similar level of reactivity with the variant-lineage viruses compared to wild-type virus. In addition, a single amino acid substitution (N165H), which removes potential glycosylation site at the HA globular head of two classic strains (A/chicken/Egypt/527/2012 and A/chicken/Egypt/102d/2010) broadened the reactivity to antisera generated against H5N1 viruses from different clusters. The broadened reactivity of the mutant viruses were also confirmed by testing reactivity of antisera prepared from the mutant viruses against reference viruses from both classic and variant clades. The virus neutralization test using selected antisera and viruses further confirmed the cross HI results. This study highlights that targeted mutation in the HA may be effectively used as a tool to develop broadly reactive influenza vaccines to cope with the continuous antigenic evolution of viruses.

Directional breeding of high oil content peanut variety Yuhua 9 by in vitro mutagenesis and screening / 生物工程学报

Leaf water potential of peanut subjected to drought stress is positively related to the oil content of peanut kernels. The aim of this study was to directly screen the high oil mutants of peanut and create the new peanut varieties using hydroxyproline as water potential regulator. In vitro mutagenesis was carried out with the embryonic leaflets of peanut variety Huayu 20 as explants and pingyangmycin as a mutagen added into the somatic embryo formation medium. The formed somatic embryos were successively transferred to somatic embryo germination and selection medium containing 6 mmol/L hydroxyproline (at -2.079 MPa water potential ) to induce regeneration and directionally screen high oil content mutants. After that, these plantlets were grafted and transplanted to the experimental field and 132 high oil mutants with oil content over 55% were obtained from the offspring of regenerated plants. Finally, among them, the oil contents of 27 lines were higher than 58% and of 2 lines were higher than 60%. A new peanut variety Yuhua 9 with high yield and oil content was bred from the regenerated plant progenies combining the pedigree breeding method. The yield was 14.0% higher than that of the control cultivar in the testing new peanut varieties of Liaoning province, and also it has passed the national registration of non-major crop varieties. Yuhua 9 with an oil content of 61.05%, which was 11.55 percentage points higher than that of the parent Huayu 20, was the peanut cultivar with the highest oil content in the world. The result showed that it was an effective way for directional breeding of high oil peanut varieties by means of the three-step technique including in vitro mutagenesis, directional screening by reducing water potential in medium and pedigree selection of regenerated plant progenies.

EFECTO DE DIFERENTES EXPLANTES IRRADIADOS EN LA REGENERACIÓN in vitro DE FRIJOL COMÚN (Phaseolus vulgaris L.) CULTIVAR "ICA Pijao" / Effect of Different Irradiated Explants in the in vitro Regeneration of Common Bean (Phaseolus vulgaris L.) Cultivar "ICA Pijao"

RESUMEN El mejoramiento genético convencional en frijol común resulta difícil debido a que presenta una base genética estrecha y muy estable. En este sentido, la combinación de la mutagénesis y el cultivo de tejidos, es una alternativa para inducir variabilidad genética en la búsqueda de tolerancia a factores bióticos y abióticos. Es por ello, que el presente trabajo tuvo como objetivo determinar el efecto de diferentes explantes irradiados en la regeneración in vitro de frijol común (Phaseolus vulgaris L.) cultivar "ICA Pijao". Se aplicaron radiaciones gamma en callos, en el nudo cotiledonal con un cotiledón (NC-1) con dosis de 0, 10, 20, 30, 40, 50 y 60 Gy y semillas con 0, 100, 200, 300 y 400 Gy. Se evaluó el porcentaje de germinación, longitud de las raíces, porcentaje de explantes que formaron callos, masa fresca (g) de los callos, número de brotes por callo y el número de brotes con raíces. La radiación gamma provocó una disminución en la masa fresca del callo y NC-1. Los callos y el NC-1 solamente formaron brotes con las dosis de 10 y 20 Gy, pero estos fueron hiperhíricos. Los resultados demostraron que la semilla irradiada fue el explante con el que se logró la regeneración in vitro de plantas con hojas definidas, por lo que se recomienda como explante inicial para el uso combinado de mutagénesis y regeneración in vitro de plantas para el cultivar P. vulgaris "ICA Pijao" a través de la organogénesis indirecta.

ABSTRACT Conventional breeding in common bean is difficult because they have a close and very stable genetic base. In this connection the combination of mutagenesis and tissue culture is an alternative to induce genetic variability in the search for tolerance to biotic and abiotic factors. For this reason, the present study aimed to determine the effect of different irradiated explants in the in vitro regeneration of common bean (Phaseolus vulgaris L.) cultivar "ICA Pijao". To do this, were applied doses gamma radiation in callus, cotiledonary node with one cotyledon (NC-1) with doses of radiation 0, 10, 20, 30, 40, 50 and 60 Gy and seeds with 0, 100, 200, 300 and 400 Gy. The length of roots in the germinated seeds, fresh mass (g) of the callus and the number of shoots per callus were determined. The gamma radiation caused a decrease in the fresh weight of callus and NC-1. The callus and NC-1, irradiated with doses of 10 and 20 Gy they formed buds but these were hyperhydric. Results demonstrated that the irradiated seed was the explant with which it was achieved regeneration of shoots with leaves defined, so it is recommended as initial explant for combined use of mutagenesis and in vitro regeneration of plants for P. vulgaris cultivar "ICA Pijao" via organogenesis indirect.

Breeding of peanut variety Yuhua 4 by in vitro mutagenesis / 生物工程学报

The embryonic leaflets of peanut (Arachis hypogaea) variety Huayu 20 were used as explants and pingyangmycin as a mutagen to induce somatic embryos. Four weeks after the inoculation, the survived explants were transferred to somatic embryo germination medium containing screening reagent hydroxyproline, and finally 15 regenerated plants were obtained. Pedigree breeding method was used during the following selection breeding, and three lines with significantly increased yield and 23 lines with high oil content were obtained from these mutant offsprings. The line with both high yield and high oil content has passed peanut variety multi-location in Anhui province and was named "Yuhua 4". Its yield was 16.63% higher than that of the control variety Baisha 1016, ranking the first in all the testing varieties. Yuhua 4 showed the characteristics of early maturity, small pod and high oil content. The oil content of kernels was 56.10%, higher than that of original parent Huayu 20 with 49.50% oil content, tested by the Ministry of Agriculture of Oil and Products Quality Supervision, Inspection and Test Center (Wuhan), and the yield was 15% higher than that of Huayu 20. It was concluded that in vitro mutagenesis and target screening was an effective way on creating new germplasm and breeding new variety in peanut.

Embryonic transcriptional activation of aXenopus cytoskeletal actin gene does not require a serum response element.

The Serum Response Element (SRE) is a sequence motif which activates transcription of certain genes in response to factors that stimulate cell proliferation. This motif is found in the promoter of aXenopus laevis cytoskeletal actin gene, which is transcriptionally activated very early in embryonic development. We tested whether the SRE plays a role in the developmentally-timed transcriptional activation of this gene by constructing an SRE replacement mutant and studying its transcription after microinjection intoXenopus embryos. Normal amounts of actin mRNA are transcribed at the normal time in development from this mutant, suggesting that the SRE is not the sole determinant of temporal specificity of actin gene transcription in the embryo.

Nuclear protein binding patterns in the 5'-upstream regulatory elements of HLA class I genes

The expression of MHC class I genes has been thought to be regulated by two major cis-acting regulatory elements. The first region, enhancer A (Enh A) spanning from positions -210 to -165 contains perfect palindrome (PP), TGGGGATTCCCCA. The PP is well-conserved both in mouse and human MHC class I genes, even though the PP is disrupted by 2 bp substitutions (TGAGGATTCTCCA) in HLA-C genes. Three proteins binding to the Enh A of HLA-A and -B locus genes, but very weakly or nearly not to the Enh A of HLA-C locus gene have been identified. To determine functional importance of the PP for binding of trans-acting protein, mutant DNA probes were made by site-directed in vitro mutagenesis and then electrophoretic mobility shift assay was performed. HLA-A mutant DNA probe, in which the PP is disrupted, shows the same nuclear protein binding pattern as that of the HLA-C gene, and HLA-C mutant DNA probe, in which the PP is introduced, shows the same nuclear protein binding pattern as that of the wild type HLA-A gene. These data suggest that the perfect palindrome and its cognate DNA binding nuclear protein play an important role in the HLA class I gene regulation, and thus the lower expression of HLA-C antigen may be ascribed to no or very weak factor binding to the nonpalindromic sequences of HLA-C upstream DNA.