Production of N‐Acetyl‐Phosphinothricin: A Substance used for Inducing Male Sterility in Transgenic Plants

The non‐toxic compound N‐acetyl‐L‐phosphinothricin (N‐Ac‐L‐PPT) is used in a so‐called deacetylation system to induce male sterility in transgenic plants by tapetum specific deacetylation to the herbicide L‐phosphinothricin (L‐PPT). A procedure was developed to produce pure racemic and L‐isomeric N‐Ac‐PPT containing less than 30 ppm residual PPT. Experiments applied to wild type tobacco and PPT‐resistant tobacco showed that the maximal tolerated N‐Ac‐PPT concentration would be less than 45 mM of the L‐isomer. Otherwise unspecific deacetylation by several acylases, as well as by environmental conditions like higher temperatures or pHs beyond neutrality, increased the residual L‐PPT content to toxic concentrations. In contrast, N‐acetyl‐L‐phosphinothricyl‐alanyl‐alanine (N‐Ac‐L‐PPTT), a substance also occurring during the biosynthesis of phosphinothricyl‐alanyl‐alanine (PPTT) by some Streptomyces species, was tolerated up to 274 mM by wild type tobacco plants. However, the ArgE deacatylase from Escherichia coli originally used in the deacetylation system, as well as some other acylases, showed no activity towards N‐Ac‐L‐PPTT.     

Formation of a Tree having a Low Lignin Content

Received 30 September 2001/ Accepted in revised form 26 October 2001     

GABAB Receptor Constituents Revealed by Tandem Affinity Purification from Transgenic Mice

GABA_B receptors function as heterodimeric G-protein-coupled receptors for the neurotransmitter γ-aminobutyric acid (GABA). Receptor subtypes, based on isoforms of the ligand-binding subunit GABA_B1, are thought to involve a differential set of associated proteins. Here, we describe two mouse lines that allow a straightforward biochemical isolation of GABA_B receptors. The transgenic mice express GABA_B1 isoforms that contain sequences for a two-step affinity purification, in addition to their endogenous subunit repertoire. Comparative analyses of purified samples from the transgenic mice and wild-type control animals revealed two novel components of the GABA_B1 complex. One of the identified proteins, potassium channel tetramerization domain-containing protein 12, associates with heterodimeric GABA_B receptors via the GABA_B2 subunit. In transfected hippocampal neurons, potassium channel tetramerization domain-containing protein 12 augmented axonal surface targeting of GABA_B2. The mice equipped with tags on GABA_B1 facilitate validation and identification of native binding partners of GABA_B receptors, providing insight into the molecular mechanisms of synaptic modulation.     

Mice carrying a conditional Serca2 allele for the generation of Ca handling-deficient mouse models

Sarco(endo)plasmic reticulum calcium ATPases (SERCA) are cellular pumps that transport Ca²⁺ into the sarcoplasmic reticulum (SR). Serca2 is the most widely expressed gene family member. The very early embryonic lethality of Serca2^null mouse embryos has precluded further evaluation of loss of Serca2 function in the context of organ physiology. We have generated mice carrying a conditional Serca2^flox allele which allows disruption of the Serca2 gene in an organ-specific and/or inducible manner. The model was tested by mating Serca2^flox mice with MLC-2v^wt/Cre mice and with αMHC-Cre transgenic mice. In heterozygous Serca2^wt/floxMLC-2v^wt/Cre mice, the expression of SERCA2a and SERCA2b proteins were reduced in the heart and slow skeletal muscle, in accordance with the expression pattern of the MLC-2v gene. In Serca2^flox/flox Tg(αMHC-Cre) embryos with early homozygous cardiac Serca2 disruption, normal embryonic development and yolk sac circulation was maintained up to at least embryonic stage E10.5. The Serca2^flox mouse is the first murine conditional gene disruption model for the SERCA family of Ca²⁺ ATPases, and should be a powerful tool for investigating specific physiological roles of SERCA2 function in a range of tissues and organs in vivo both in adult and embryonic stages.     

The structure and function of phytochrome A: the roles of the entire molecule and of its various parts

Phytochrome A is readily cleavable by proteolytic agents to yield an amino-terminal fragment of 66 kilodalton (kDa), which consists of residues 1 to approximately 600, and a dimer of the carboxy-terminal 55-kDa fragment, from residue 600 or so to the carboxyl terminus. The former domain, carrying the tetrapyrrole chromophore, has been studied extensively because of its photoactivity, while less attention has been paid to the non-chromophoric portion until quite recently. However, the evidence gathered to date suggests that this domain is also of great improtance. We present here a review of the structure and the biochemical and physiological functions of the two domains, of parts of these domains, and of the cooperation between them.     

Environmental assessment of maize production alternatives: Traditional,intensive and GMO-based cropping patterns

The evolution of maize production patterns in Argentina is evaluated over the last 25 years to compare costs, benefits, environmental performance and sustainability as well as to identify the main driving sources and improvement potential. Results from Argentina cropping systems are compared to other systems worldwide in order to put the Argentina results in a broader context. The study focuses on three farming categories: (1) traditional, low-intensity systems, (2) conventional, high-intensity systems, and (3) GMO-based cropping systems. Low input intensity systems include traditional cropping patterns with seed selection by farmers and conventional hybrid seed coupled to plowing and crop-animal rotation techniques; high input intensity systems use conventional hybrid seeds and recommended chemicals, irrigation and machinery with important soil erosion consequences; and GMO-based cropping systems use herbicide resistant transgenic hybrids, pesticides, higher fertilizer rates, and no-till practices. In each of the three cases, input flows are compared to the achieved yield (in mass and income terms) to better understand relative efficiencies and options for improvement. The study of GMO systems required a preliminary investigation of GMO seed production by seed companies, where a large investment in terms of prior knowledge and high-tech laboratory research is required. The assessments used the Emergy Accounting (EMA) approach. EMA includes material, energy, labor, money, and knowledge flows into the assessment and expands its focus over larger time and spatial scales than conventional economic and cumulative energy demand methods. Emergy-based environmental indicators of grain production for high-intensity hybrid and GMO systems both show a lower performance than low-intensity, traditional patterns in terms of resource return, renewability and sustainability. The fraction of renewability in low-intensity systems is between 28% and 63%, while it is between 8% and 26% for high-intensity hybrid and GMO systems. Calculated indicators also show that GMO-based maize production patterns do not guarantee the expected improvement over conventional high-intensity cropping systems or low-intensity systems in terms of performance and sustainability. Strong reliance on nonrenewable resources and technology, as well as role of direct and indirect labor costs are important factors in determining long-term sustainability and environmental stability of maize production systems.     

Site-specific modification of the bovine genome using Cre recombinase-mediated gene targeting.

Cre recombinase (Cre)-mediated targeted insertion of a transgene is a powerful technique that can be used to tailor genomes. When combined with somatic cell nuclear transfer it could offer an efficient way to generate transgenic livestock with site-specific genetic modifications that are free of antibiotic selection markers. We have engineered primary bovine fibroblasts to contain a chromosomal acceptor site with incompatible loxP/lox2272 sites for Cre-mediated cassette exchange and show for the first time that Cre-mediated targeting can be applied in these acceptor cells. Molecular characterization of the resulting cell clones revealed Cre-mediated transgene insertion efficiencies of up to 98% when antibiotic selection was used to identify transgene containing cell clones. Most clonal lines also contained random insertions of the targeting and Cre expression plasmids with only about 10% of the clones being exclusively modified by the intended targeted insertion. This targeting efficiency was sufficient to enable the isolation of correctly targeted clones without the help of antibiotic selection. Therefore, this recombinase-mediated insertion strategy has the potential to produce transgenic cattle from antibiotic selection marker-free somatic cells with transgenes inserted into proven genomic loci ensuring reliable expression levels.     

High somatic mutation frequencies in a LacZ transgene integrated on the mouse X-chromosome

To study spontaneous and induced mutagenesis in vivo we recently constructed a series of transgenic mice harboring different numbers of bacteriophage lambda shuttle vectors, provided with a LacZ mutational target gene, integrated in their genome. The transgenic mice enabled analysis of spontaneous and induced mutation frequencies in postmitotic tissues like liver and brain. The obtained data indicated spontaneous mutation frequencies in the order of ⁻⁵ - 10⁻⁶. Here we report a 25–100 times higher spontaneous mutation frequency in liver and brain DNA of mice from strain 35.5, with the lambda-gt10LacZ concatemer integrated on the X-chromosome. These results indicate the presence of a mutational ‘hot spot’ in the mammalian somatic genome in vivo.     

人源抗体制备及临床应用研究进展

与常规治疗药物相比,抗体药物具有靶向性强、特异性好等优点,其作为一类重要的治疗性药物,近年来在临床中的应用逐渐增多,为疾病的治疗提供了新的选择,应用范围逐渐从肿瘤、自身免疫性疾病及慢性炎症扩展到心血管和感染性等疾病中。人源抗体的全部结构是由人类抗体基因所编码的,因此避免了异种蛋白长期应用引起的不良反应,加之人源抗体制备技术的不断发展完善,使其逐渐成为治疗性抗体研发的首要选择。综述了近年来治疗性人源抗体的主要制备技术及其在临床中应用的最新进展,同时探讨了人源抗体制备技术的不足之处,以期为人源抗体的发展提供借鉴和思路。     

米修链霉菌TF78对香蕉枯萎病的田间防效及根际土壤微生物的影响

【背景】香蕉枯萎病是香蕉生产上的毁灭性病害,生物防治是遏制该病害发生的有效手段。在前期的研究中,从健康香蕉根际土壤中分离获得一株对香蕉枯萎病具有良好盆栽防治效果的生防菌——米修链霉菌(Streptomyces misionensis) TF78,但其对香蕉枯萎病的田间生防潜力和对土壤微生物环境的影响尚不清楚。【目的】评价米修链霉菌TF78对香蕉枯萎病的田间防治效果,明确其对香蕉根际土壤微生物群落的影响。【方法】选取两块发病香蕉园,测定该生防菌株对香蕉枯萎病的防治效果,并利用扩增子测序技术分析施用菌剂组和空白对照组共12份香蕉根际土壤的微生物多样性和丰度。【结果】米修链霉菌TF78对两块香蕉园的田间防效分别达55.30%和45.32%。该生防菌株处理组的物种稀释曲线坡度大于空白对照组,并显著富集了优势种群梳霉门(Kickxellomycota),消减了绿弯菌门(Chloroflexi)、酸杆菌门(Acidobacteria)和苔藓杆菌(Bryobacter)的丰度,对土壤中优势种群变形菌门(Proteobacteria)、厚壁菌门(Firmicutes)、放线菌门(Actinobacteria)、芽单胞菌门(Gemmatimonadetes)及木霉属(Trichoderma)、鞘氨醇单胞菌属(Sphingomonas)、寡养单胞菌属(Stenotrophomonas)和芽孢杆菌属(Bacillus)的相对丰度影响不显著。【结论】米修链霉菌TF78塑造了不利于香蕉枯萎病菌Fusarium oxysporum f.sp.cubense存活的土壤环境,有效降低了田间香蕉枯萎病的发生,同时对土壤中大部分具有重要生态功能和抑菌功能的优势微生物种群影响不显著。该研究结果为米修链霉菌TF78的进一步开发应用奠定了基础。