Pràctiques de laboratori de Genètica: Grau en Biotecnologia

Es realitzaran dues pràctiques, una sobre la "Segregació de caràcters", en què es farà ús de la Drosophila melanogaster com a organisme experimental, i una altra sobre "Cromosomes politènics", en què es farà ús de la Drosophila subobscura. Ambdues pràctiques estaran imbricades en el temps per optimitzar les sessions de laboratori

Prácticas de laboratorio de Genética: Grado en Biotecnología

Se realizarán dos prácticas, una sobre la "Segregación de Caracteres" utilizando Drosophila melanogaster como organismo experimental, y otra sobre "Cromosomas Politénicos" utilizando Drosophila subobscura. Ambas prácticas estarán imbricadas en el tiempo para optimizar las sesiones de laboratorio

Bacillus thuringiensis Toxins: Functional Characterization and Mechanism of Action

Bacillus thuringiensis (Bt)-based products are the most successful microbial insecticides to date. This entomopathogenic bacterium produces different kinds of proteins whose specific toxicity has been shown against a wide range of insect orders, nematodes, mites, protozoa, and human cancer cells. Some of these proteins are accumulated in parasporal crystals during the sporulation phase (Cry and Cyt proteins), whereas other proteins are secreted in the vegetative phase of growth (Vip and Sip toxins). Currently, insecticidal proteins belonging to different groups (Cry and Vip3 proteins) are widely used to control insect pests and vectors both in formulated sprays and in transgenic crops (the so-called Bt crops). Despite the extensive use of these proteins in insect pest control, especially Cry and Vip3, their mode of action is not completely understood

A Genomic and Proteomic Approach to Identify and Quantify the Expressed Bacillus thuringiensis Proteins in the Supernatant and Parasporal Crystal

The combined analysis of genomic and proteomic data allowed us to determine which cry and vip genes are present in a Bacillus thuringiensis (Bt) isolate and which ones are being expressed. Nine Bt isolates were selected from Spanish collections of Bt based on their vip1 and vip2 gene content. As a first step, nine isolates were analyzed by PCR to select those Bt isolates that contained genes with the lowest similarity to already described vip1 and vip2 genes (isolates E-SE10.2 and O-V84.2). Two selected isolates were subjected to a combined genomic and proteomic analysis. The results showed that the Bt isolate E-SE10.2 codifies for two new vegetative proteins, Vip2Ac-like_1 and Sip1Aa-like_1, that do not show expression differences at 24 h vs. 48 h and are expressed in a low amount. The Bt isolate O-V84.2 codifies for three new vegetative proteins, Vip4Aa-like_1, Vip4Aa-like_2, and Vip2Ac-like_2, that are marginally expressed. The Vip4Aa-like_1 protein was two-fold more abundant at 24 h vs. 48 h, while the Vip4Aa-like_2 was detected only at 24 h. For Vip2Ac-like_2, no differences in expression were found at 24 h vs. 48 h. Moreover, the parasporal crystal of the E-SE10.2 isolate contains a single type of crystal protein, Cry23Aa-like, while the parasporal crystal from O-V84.2 contains three kinds of crystal proteins: 7.0-9.8% weight of Cry45Aa-like proteins, 35-37% weight of Cry32-like proteins and 2.8-4.3% weight of Cry73-like protein

Specific binding of Bacillus thuringiensis Cry2A insecticidal proteins to a common site in the midgut of Helicoverpa species

For a long time, it has been assumed that the mode of action of Cry2A toxins was unique and different from that of other three-domain Cry toxins due to their apparent nonspecific and unsaturable binding to an unlimited number of receptors. However, based on the homology of the tertiary structure among three-domain Cry toxins, similar modes of action for all of them are expected. To confirm this hypothesis, binding assays were carried out with 125 I-labeled Cry2Ab. Saturation assays showed that Cry2Ab binds in a specific and saturable manner to brush border membrane vesicles (BBMVs) of Helicoverpa armigera. Homologous-competition assays with 125 I-Cry2Ab demonstrated that this toxin binds with high affinity to binding sites in H. armigera and Helicoverpa zea midgut. Heterologous-competition assays showed a common binding site for three toxins belonging to the Cry2A family (Cry2Aa, Cry2Ab, and Cry2Ae), which is not shared by Cry1Ac. Estimation of Kd (dissociation constant) values revealed that Cry2Ab had around 35-fold less affinity than Cry1Ac for BBMV binding sites in both insect species. Only minor differences were found regarding Rt (concentration of binding sites) values. This study questions previous interpretations from other authors performing binding assays with Cry2A toxins and establishes the basis for the mode of action of Cry2A toxins

Mutations in the Bacillus thuringiensis Cry1Ca toxin demonstrate the role of domains II and III in specificity towards Spodoptera exigua larvae

Several mutants of the Bacillus thuringiensis Cry1Ca toxin affected with regard to specific activity towards Spodoptera exigua were studied. Alanine was used to replace single residues in loops 2 and 3 of domain II (mutant pPB19) and to replace residues 541– 544 in domain III (mutant pPB20). Additionally, a Cry1Ca mutant combining all mutations was constructed (mutant pPB21). Toxicity assays showed a marked decrease in toxicity against S. exigua for all mutants, while they retained their activity against Manduca sexta, confirming the importance of these residues in determining insect specificity. Parameters for binding to the specific receptors in BBMV (brush border membrane vesicles) of S. exigua were determined for all toxins. Compared with Cry1Ca, the affinity of mutant pPB19 was slightly affected (2-fold lower), whereas the affinity of the mutants with an altered domain III (pPB20 and pPB21) was approx. 8-fold lower. Activation of Cry1Ca protoxin by incubation with S. exigua or M. sexta BBMV revealed the transient formation of an oligomeric form of Cry1Ca. The presence of this oligomeric form was tested in the activation of the different Cry1Ca mutants, and we found that those mutated in domain II (pPB19 and pPB21) could not generate the oligomeric form when activated by S. exigua BBMV. In contrast, when oligomerization was tested using BBMV prepared from M. sexta, all of the Cry1Ca mutants showed the formation of a similar oligomeric form as did the wild-type toxin. Our results show how modification of insect specificity can be achieved by manipulation of different parts of the toxin structure involved in different steps of the mode of action of B. thuringiensis [email protected]; [email protected]

Reduced membrane-bound alkaline phosphatase does not affect binding of Vip3Aa in a Heliothis virescens resistant colony

The Vip3Aa insecticidal protein from Bacillus thuringiensis (Bt) is produced by specific transgenic corn and cotton varieties for efficient control of target lepidopteran pests. The main threat to this technology is the evolution of resistance in targeted insect pests and understanding the mechanistic basis of resistance is crucial to deploy the most appropriate strategies for resistance management. In this work, we tested whether alteration of membrane receptors in the insect midgut might explain the >2000-fold Vip3Aa resistance phenotype in a laboratory-selected colony of Heliothis virescens (Vip-Sel). Binding of 125I-labeled Vip3Aa to brush border membrane vesicles (BBMV) from 3rd instar larvae from Vip-Sel was not significantly different from binding in the reference susceptible colony. Interestingly, BBMV from Vip-Sel larvae showed dramatically reduced levels of membrane-bound alkaline phosphatase (mALP) activity, which was further confirmed by a strong downregulation of the membrane-bound alkaline phosphatase 1 (HvmALP1) gene. However, the involvement of HvmALP1 as a receptor for the Vip3Aa protein was not supported by results from ligand blotting and viability assays with insect cells expressing HvmALP1

Tècniques d'anàlisi genètica: Tema 7

El document forma part dels materials docents programats mitjançant l'ajut del Servei de Política Lingüística de la Universitat de València.Tema 7 de 8, de què consta l'assignatura. Es presenta les metodologies per detectar lligament en pedigrís humans, l'utilització de la puntuació LOD i els experiments en híbrids de cèl·lules somàtiques d'home-ratol

Selecció de problemes resolts, pas a pas, de l'assignatura Tècniques d'Anàlisi Genètica

El document s'ha enviat per concursar en els Premis Fernando Sapiña.Selecció de problemes resolts, pas a pas, de l'assignatura Tècniques d'Anàlisi Genètic

Tècniques d'anàlisi genètica: Tema 1

El document forma part dels materials docents programats mitjançant l'ajut del Servei de Política Lingüística de la Universitat de València.Tema 1 dels 8 temes de què consta l'assignatura, que tracta de marcadors molecular i el lligament al locus d'interé