Molecular Characterization of a Gene Encoding a Mucin-Like Protein From Caenorhabditis elegans

A mutator strain containing a Tc 1 transposon disrupting the dpy-6 gene activity was used to isolate the gene. This strain was backcrossed to the N2 wild-type strain in order to reduce and stabilize the Tc 1 copy number. After six rounds of backcrosses, a single Tc1 insertion associated with the dumpy phenotype could be identified. This Tc 1 insertion was located in a 9.5 kb EcoRI fragment, which was physically mapped, by hybridization to YACs and cosmids, to a region in the X chromosome of C. elegans were the dpy-6 gene is located. The Tc 1 tagged fragment was cloned and the sequence adjacent to the insertion site was determined. An open reading frame containing 1857 by was found, capable of encoding a protein with 619 amino acids. The putative protein was similar to mucin-like sequences, with the presence of thirty four copies of a six amino acid repeat arranged in tandem near the carboxy-terminus. Some of the repeats were also found dispersed in other parts of the putative protein. The amino acid repeats, like in other mucin-like proteins, were rich in threonine, serine and proline. Another aspect which has been investigated was the identification of a collagenolytic activity in C. elegans. Using substrate gels containing collagen, this activity was found associated exclusively with the cuticle material. Attempts were made to isolate the corresponding gene by Polymerase Chain Reaction using oligonucleotides specific for conserved regions within metalloproteases

Introdução à bioquímica clínica veterinária

3. ed. rev. ampl

Classical scrapie diagnosis in ARR/ARR sheep in Brazil

Background: Scrapie is a transmissible spongiform encephalopathy (TSE) that affects sheep flocks and goat herds. The transfer of animals or groups of these between sheep farms is associated with increased numbers of infected animals and with the susceptibility or the resistance to natural or classical scrapie form. Although several aspects linked to the etiology of the natural form of this infection remain unclarified, the role of an important genetic control in scrapie incidence has been proposed. Polymorphisms of the PrP gene (prion protein, or simply prion), mainly in codons 136, 154, and 171, have been associated with the risk of scrapie. Case: One animal from a group of 292 sheep was diagnosed positive for scrapie in the municipality of Valparaíso, state of São Paulo, Brazil. The group was part of a flock of 811 free-range, mixed-breed Suffolk sheep of the two genders and ages between 2 and 7 years from different Brazilian regions. Blood was collected for genotyping (for codons 136, 141, 154 and 171), and the third lid and rectal mucosa were sampled for immunohistochemistry (IHC) for scrapie, from all 292 animals of the group. IHC revealed that seven (2.4%) animals were positive for the disease. Collection of samples was repeated for 90 animals, among which the seven individuals diagnosed positive and 83 other animals that had some degree of kinship with those. These 90 sheep were sacrificed and necropsied, when samples of brain (obex), cerebellum, third eyelid, rectal mucosa, mesenteric lymph node, palatine tonsil, and spleen were collected for IHC. The results of IHC analyses carried out after necropsy of the seven positive animals submitted to the second collection of lymphoreticular tissue and of the 83 animals with some degree of kinship with them confirmed the positive diagnosis obtained in the first analysis, and revealed that three other sheep were also positive for scrapie. Samples of 80 animals (89%) were negative for the disease in all organs and tissues analyzed. In turn, 10 sheep (11%) were positive, presenting immunoreactivity in one or more tissues. Genotyping revealed the presence of four of the five alleles of the PrP gene commonly detected in sheep: ARR, ARQ, VRQ and ARH. These allele combinations formed six haplotypes: ARR/ARR, ARR/ARQ, ARH/ARH, ARQ/ARH, ARQ/ARQ and ARQ/VRQ. Animals were classified according to susceptibility to scrapie, when 8.9% of the genotyped sheep were classified into risk group R1 (more resistant, with no restriction to breeding). In turn, 40% of the animals tested ranked in groups R4 and R5 (genetically very susceptible, cannot be used for breeding purposes). Discussion: The susceptibility of sheep flocks depends on the genetic pattern of animals and is determined by the sequence of the gene that codifies protein PrP. Additionally, numerous prion strains are differentiated based on pathological and biochemical characteristics, and may affect animals differently, depending on each individual’s genotype. Most epidemiologic data published to date indicate that animals that carry the ARR/ARR genotype are less susceptible to classical scrapie. However, in the present study, the fact that two scrapie-positive sheep presented the haplotype ARR/ARR indicates that this genotype cannot always be considered an indicator of resistance to the causal agent of the classical manifestation of the disease. The coexistence in the same environment of several crossbred animals from different flocks and farms, which characterizes a new heterogeneous flock, may have promoted a favorable scenario to spread the disease, infecting animals in the most resistant group

Introdução à bioquímica clínica veterinária

ePub rev., atual. e ampl

Caracterização funcional de promotores de Mycoplasma hyopneumoniae

43

Caracterização funcional de promotores de Mycoplasma hyopneumoniae

43

Atividade in vivo de promotores de Mycoplasma hyopneumoniae : avaliação de um sistema reporter

40