Concatenation of Transgenic DNA: Random or Orchestrated?

Generation of transgenic organisms by pronuclear microinjection has become a routine procedure. However, while the process of DNA integration in the genome is well understood, we still do not know much about the recombination between transgene molecules that happens in the first moments after DNA injection. Most of the time, injected molecules are joined together in head-to-tail tandem repeats—the so-called concatemers. In this review, we focused on the possible concatenation mechanisms and how they could be studied with genetic reporters tracking individual copies in concatemers. We also discuss various features of concatemers, including palindromic junctions and repeat-induced gene silencing (RIGS). Finally, we speculate how cooperation of DNA repair pathways creates a multicopy concatenated insert.

Response of physiological parameters in Dionaea muscipula J. Ellis teratomas transformed with rolB oncogene

Background Plant transformation with rol oncogenes derived from wild strains of Rhizobium rhizogenes is a popular biotechnology tool. Transformation effects depend on the type of rol gene, expression level, and the number of gene copies incorporated into the plant’s genomic DNA. Although rol oncogenes are known as inducers of plant secondary metabolism, little is known about the physiological response of plants subjected to transformation. Results In this study, the physiological consequences of rolB oncogene incorporation into the DNA of Dionaea muscipula J. Ellis was evaluated at the level of primary and secondary metabolism. Examination of the teratoma (transformed shoots) cultures of two different clones (K and L) showed two different strategies for dealing with the presence of the rolB gene. Clone K showed an increased ratio of free fatty acids to lipids, superoxide dismutase activity, synthesis of the oxidised form of glutathione, and total pool of glutathione and carotenoids, in comparison to non-transformed plants (control). Clone L was characterised by increased accumulation of malondialdehyde, proline, activity of superoxide dismutase and catalase, total pool of glutathione, ratio of reduced form of glutathione to oxidised form, and accumulation of selected phenolic acids. Moreover, clone L had an enhanced ratio of total triglycerides to lipids and accumulated saccharose, fructose, glucose, and tyrosine. Conclusions This study showed that plant transformation with the rolB oncogene derived from R. rhizogenes induces a pleiotropic effect in plant tissue after transformation. Examination of D. muscipula plant in the context of transformation with wild strains of R. rhizogenes can be a new source of knowledge about primary and secondary metabolites in transgenic organisms.

QUALITY OF HELIANTHUS ANNUUS HONEY OBTAINED IN THE CONDITIONS OF RADIOACTIVE CONTAMINATION

Natural honey is a source of vital amino acids, easily digestible carbohydrates, macro, microelements, biologically active substances that determine nutritional, antibacterial and antioxidant properties. In the conditions of man-caused pollution of Polissya of Ukraine due to the accident at the Chernobyl nuclear power plant, systematic control of the quality and safety of beekeeping products is important. To conduct such research, we created a group of twelve bee families - analogs of the Ukrainian breed, medium strength. Families were kept in unified multifunctional hives. At the beginning of the honey harvest, the bee families were transported to the sunflower fields, where they stayed during the blossoming of the plants. The density of radioactive contamination of 137Cs soils where sunflower was grown was 47.0 kBq / m2. We used organoleptic, physicochemical, microscopic, microbiological, and radiological methods in the study. According to standard methods, we studied the species composition of pollen grains, physicochemical parameters of centrifugal, honeycomb, and «zabrus» sunflower honey.(zabrus honey was obtained from wax caps, which we cut with an apiary knife from honeycombs filled with nectar and sealed by bees). The content of lead (Pb) in honey from sunflower obtained in the conditions of Polissya is 1.8 - 2.1 times higher than the State sanitary norms. The largest amount of it is in the centrifugal honey. In acceptable amounts, the heavy metals cadmium (Cd), arsenic (As), and 137Cs were present in honey. Pesticides, dichlorodiphenyltrichloromethylmethane, and hexachlorane were not detected in the samples. We investigated the bactericidal action against bacterial growth of typical cultures of Proteus vulgaris, Escherichia coli, Klebsiella pneumonia, Salmonella Typhimurium, and Staphylococcus aureus. Zubrus sunflower honey showed the highest antimicrobial and antioxidant properties. We found that the value of antioxidant activity (AOA) of sunflower honey depends on the method of its production, duration of storage, and solutions of extracts (alcohol, aqueous) used in research. Laboratory control of transgenic organisms in flowers and sunflower pollen did not reveal the target sequences of the cauliflower mosaic virus (CaMV) 35S promoter and the NOS terminator (nopaline synthase) of the plasmid Agrobacterium tumefaciens.

The effect of mating complexity on gene drive dynamics

Gene drive technology is being presented as a means to deliver on some of the global challenges humanity faces today in healthcare, agriculture and conservation. However, there is a limited understanding of the consequences of releasing self-perpetuating transgenic organisms into the wild populations under complex ecological conditions. In this study, we analyze the impact of three factors, mate-choice, mating systems and spatial mating network, on the population dynamics for two distinct classes of modification gene drive systems; distortion and viability-based ones. All three factors had a high impact on the modelling outcome. First, we demonstrate that distortion based gene drives appear to be more robust against the mate-choice than viability-based gene drives. Second, we find that gene drive spread is much faster for higher degrees of polygamy. With fitness cost, speed is the highest for intermediate levels of polygamy. Finally, the spread of gene drive is faster and more effective when the individuals have fewer connections in a spatial mating network. Our results highlight the need to include mating complexities while modelling the population-level spread of gene drives. This will enable a more confident prediction of release thresholds, timescales and consequences of gene drive in populations.

Difundindo a Biotecnologia na sociedade: Relato de experiência extensionista no contexto da pandemia da COVID-19

A Biotecnologia se refere a qualquer aplicação tecnológica que utilize sistemas biológicos, organismos vivos ou seus derivados, para fabricar ou modificar produtos e processos, visando utilização humana. O presente relato de experiência retrata o projeto de extensão “Difundindo a Biotecnologia na sociedade”, o qual visou trabalhar com alunos do 2º ano do Ensino Médio do Centro Educacional Colares Moreira (Codó-MA), abrangendo conteúdos amplamente divulgados pela mídia e minimamente compreendidos pela sociedade, tais como: clonagem, terapia gênica, testes de paternidade, organismos transgênicos e uso da informação genética para diagnóstico de doenças. O projeto foi dividido em 4 etapas: Etapa 1: Realização de uma avaliação diagnóstica com os estudantes da escola; Etapa 2: Ciclo de palestras; Etapa 3: Desenvolvimento de duas oficinas abordando organismos transgênicos e clonagem e Etapa 4: Discussão acerca da Bioética relacionada à Biotecnologia. A maior parte dos estudantes participantes desconheciam a importância da Biotecnologia, bem como sua aplicação na sociedade. Na avaliação diagnóstica, dos 103 alunos, apenas 34% souberam informar pelo menos uma importância da Biotecnologia; 44% se mostraram favoráveis à comercialização dos transgênicos e somente 6% já ouviram falar da terapia genética e conheciam exames de DNA. Com a pandemia do novo coronavírus, a execução do projeto continuou de forma remota, porém com o passar dos meses, houve grande evasão e diminuição de participação. Contudo, ressalta-se que, para a minoria que conseguiu acompanhar o projeto remotamente, notou-se grande interesse e uma melhor compreensão acerca da temática. Com o desenvolvimento das atividades, observou-se a importância da extensão universitária enquanto prática social formadora e transformadora. Palavras-chave: Educação; Ciência; Informação; Divulgação científica Diffusing Biotechnology in society: Report of extensionist experience in the context of the pandemic OF COVID-19 Abstract: Biotechnology refers to any technological application that uses biological systems, living organisms, or their derivatives to manufacture or modify products and processes for human use. The present experience report portrays the extension project “Diffusing Biotechnology in society”, which aimed to work with High School students at Centro Educacional Colares Moreira (Codó-MA, Brazil), covering contents widely disseminated by the media and minimally understood by society, such as cloning, gene therapy, paternity tests, transgenic organisms and use of genetic information for the prevention of genetic diseases. The project was divided into four stages: Stage 1: Conducting a diagnostic evaluation with High School students; Stage 2: Cycle of lectures; Stage 3: Developing two workshops addressing transgenic organisms and cloning. Step 4: Discussion about Bioethics related to Biotechnology. Most of the participating students were unaware of the importance of Biotechnology and its application in society. In the diagnostic evaluation, of the 103 students, only 34% could inform at least one importance of Biotechnology; 44% were in favor of transgenics, and only 6% had heard of gene therapy and knew about DNA testing. With the new coronavirus pandemic, the project execution continued remotely, but there was a significant dropout over the months. However, it is noteworthy that, for the minority that managed to monitor the project remotely, there was great interest and a better understanding of the theme. With the development of activities, the importance of university extension was observed as a formative action and transforming social practice. Keywords: Education; Science; Information; Scientific divulgation

The fushi tarazu zebra element is not required for Drosophila viability or fertility

Expression of genes in precisely controlled spatiotemporal patterns is essential for embryonic development. Much of our understanding of mechanisms regulating gene expression comes from the study of cis-regulatory elements (CREs) that direct expression of reporter genes in transgenic organisms. This reporter-transgene approach identifies genomic regions sufficient to drive expression but fails to provide information about quantitative and qualitative contributions to endogenous expression, although such conclusions are often inferred. Here we evaluated the endogenous function of a classic Drosophila CRE, the fushi tarazu (ftz) zebra element. ftz is a pair-rule segmentation gene expressed in seven stripes during embryogenesis, necessary for formation of alternate body segments. Reporter transgenes identified the promoter-proximal zebra element as a major driver of the seven ftz stripes. We generated a precise genomic deletion of the zebra element (ftzΔZ) to assess its role in the context of native chromatin and neighboring CREs, expecting large decreases in ftz seven-stripe expression. However, significant reduction in expression was found for only one stripe, ftz stripe 4, expressed at ∼25% of wild type levels in ftzΔZ homozygotes. Defects in corresponding regions of ftzΔZ mutants suggest this level of expression borders the threshold required to promote morphological segmentation. Further, we established true-breeding lines of homozygous ftzΔZ flies, demonstrating that the body segments missing in the mutants are not required for viability or fertility. These results highlight the different types of conclusions drawn from different experimental designs and emphasize the importance of examining transcriptional regulatory mechanisms in the context of the native genomic environment.

Using Fluorescence in Biotechnology Instruction to Visualize Antibiotic Resistance & DNA

Fluorescence technology has many useful applications for both research and teaching, among them the detection of fluorescence in live transgenic organisms and of DNA in agarose gels. However, dedicated fluorescence imaging systems can be expensive and complex. We describe a simple apparatus for non-microscopic fluorescence imaging using affordable and readily available parts. We describe three activities of increasing complexity that utilize fluorescence illumination and teach principles of fluorescence. At the high school level, our more advanced activities can be used for lessons addressing NGSS performance expectations in HS-LS3 and HS-LS4.

Advances in Agricultural Biotechnology

Agricultural biotechnology is becoming the major sector in crop improvement through the use of scientific techniques for the modification of genes conferring resistance to biotic, abiotic stress and improving the quality of crops. With the evolvement from Mendelian genetics to molecular biotechnology, there have been several developments in the field of crop improvement. Recent biotechnological advances have aimed towards removing the physiological constraints of the crops and increasing crop yield potential. With the use of different tools of agricultural biotechnologies like genetic engineering, tissue culture, embryo rescue, somatic hybridization, molecular marker-assisted selection, genome doubling, and omics technologies, various transgenic crops have been developed over the decades and have been approved for commercialization. This development and adoption of transgenic technology have been shown to increase crop yields, reduce CO2 emission, reduce pesticide and insecticide use and decrease the costs of crop production. Even though the biotechnological approach and transgenic organisms have immense potential to contribute to the world’s food security, several concerns of genetically modified crops being a threat to the environment and human health have developed. This review will address applications and concerns of biotechnology in crop improvement considering health hazards and ecological risks.

Challenges for the characterization of genetically modified animals by the qPCR technique in the era of genomic editing

Characterization of genetically modified organisms through determination of zygosity and transgene integration concerning both copy number and genome site is important for breeding a transgenic line and the use of these organisms in the purpose for which it was obtained. Southern-blot, fluorescence in situ hybridization or mating are demanding and time-consuming techniques traditionally used in the characterization of transgenic organisms and, with the exception of mating, give ambiguous results. With the emergence of the real-time quantitative PCR technology, different applications have been described for the analysis of transgenic organisms by determination of several parameters to transgenic analysis. However, the accuracy in quantitation by this method can be influenced in all steps of quantification. This review focuses on the aspects that influence pre-analytical steps (DNA extraction and DNA quantification methods), quantification strategies and data analysis in quantification of copy number and zygosity in transgenic animals.

A Phosphite Dehydrogenase Variant with Promiscuous Access to Nicotinamide Cofactor Pools Sustains Fast Phosphite-Dependent Growth of Transplastomic Chlamydomonas reinhardtii

Heterologous expression of the NAD+-dependent phosphite dehydrogenase (PTXD) bacterial enzyme from Pseudomonas stutzerii enables selective growth of transgenic organisms by using phosphite as sole phosphorous source. Combining phosphite fertilization with nuclear expression of the ptxD transgene was shown to be an alternative to herbicides in controlling weeds and contamination of algal cultures. Chloroplast expression of ptxD in Chlamydomonas reinhardtii was proposed as an environmentally friendly alternative to antibiotic resistance genes for plastid transformation. However, PTXD activity in the chloroplast is low, possibly due to the low NAD+/NADP+ ratio, limiting the efficiency of phosphite assimilation. We addressed the intrinsic constraints of the PTXD activity in the chloroplast and improved its catalytic efficiency in vivo via rational mutagenesis of key residues involved in cofactor binding. Transplastomic lines carrying a mutagenized PTXD version promiscuously used NADP+ and NAD+ for converting phosphite into phosphate and grew faster compared to those expressing the wild type protein. The modified PTXD enzyme also enabled faster and reproducible selection of transplastomic colonies by directly plating on phosphite-containing medium. These results allow using phosphite as selective agent for chloroplast transformation and for controlling biological contaminants when expressing heterologous proteins in algal chloroplasts, without compromising on culture performance.