The progress of studies on genes concerning the development and differentiation of early swine embryos have been delayed by limited paucity material. In order to identify the porcine ESTs associates with promoting its breeding efficiency, a cDNA library and ESTs database from oocytes of high reproductive swine is established. Oocytes were obtained from Duroc pig by superovulation which was performed by Taiwan Livestock Research Institute, Council of Agriculture. Total RNA was isolated from 50 mature oocytes, reverse transcription is then performed, followed by PCR based amplification of the cDNA. The amplified cDNA size ranges from 0.4 to 5 kb. The derived cDNA were ligated to a pCR2.1 vector, and the library has complexities of about 5.26¡Ñ104 independent clones. A total of 320 clones was picked and sequenced. By BLASTx analysis, among the 123 sequences, more than 43.07%¡]53/123¡^ mitochondrial proteins are found, 56.91¢H¡]70/123¡^ of the sequence were homologous to known transcripts from human, mouse, Drosophila. In nucleotide level analysis, 82.11¢H¡]101/123¡^ matched with the mitochondrial, ribosome genes and 17.89¢H¡]22/123¡^matched with other homologous genes by BLASTn. PCR analysis of the oocyte library for specific genes revealed transcripts for genes including homologous genes¡]2 pairs highly abundance and 2 pairs low abundance genes¡^, housekeeping genes¡]ACT£] and G3PDH¡^ and developmental genes¡]NEK2 and ZP1¡^. However, novel genes of swine are supposed to be the candidates for high productive phenotypes of swine. The library is a valuable resource for the isolation of clones representing genes active at the early stage. The ability to construct cDNA expression library from a few cells will allow gene expression analysis from oocyte biopsies and derived by nuclear transfer procedures.

An infectious cDNA clone of Vero cell adapted Beaudette strain of IBV was constructed using in vitro assembly of cDNA fragments. The entire genome of IBV was RT-PCR amplified into seven fragments, with each piece overlapping about 10 nucleotides. The fragments were ligated and transcribed to synthesize RNA, which was transfected into BHK-21 cells. These cells were then overlaid onto IBV susceptible Vero cells. After five days transfection, the virus was successfully rescued from the transfected cells. The cDNA clone from our laboratory strain has a five nucleotide insertion not present in the originally sequenced virus, resulting in total genome size of 27,613 nucleotides. The infectious cDNA clone was further manipulated to demonstrate its potential as a gene transfer vector, by replacing the ORF5a open reading frame with enhanced green fluorescent protein. The recombinant infectious cDNA clone was also successfully rescued after three days transfection of BHK-21 cells followed by co-culturing with Vero cells. This study showed that the 5a protein, whose function is not known, is not necessary for in vitro IBV replication. This study also showed that the 5a ORF is a good candidate for an insertion site of recombinant genes for the development of IBV infectious cDNA clone as a gene transfer vector.

Made available in DSpace on 2014-06-12T15:51:40Z (GMT). No. of bitstreams: 2 arquivo1575_1.pdf: 998437 bytes, checksum: bbe05532417b4b624c68c59d53e3fa89 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2009 / O objetivo deste estudo foi otimizar um protocolo de extração do RNA de fragmentos de tecidos tumorais de próstatas, contendo hiperplasia e adenocarcinoma, fixados em formol e conservados em blocos de parafina. Em seguida, a qualidade do RNA extraído foi avaliada mediante a amplificação por PCR do exon 4 do gene TP53, onde se observa um polimorfismo no códon 72 muito relacionado com processos cancerígenos em vários órgãos. Os resultados obtidos demonstraram que foi possível aperfeiçoar um protocolo para detecção do RNA a partir de tecidos tumorais de próstatas parafinizados, sem utilizar inibidores de RNAse usualmente empregados em semelhantes protocolos

No abstract available. / Dissertation (MSc)--University of Pretoria, 2011. / Genetics / unrestricted

As achieving higher frost resistance is one of the apricot breeding goals in the Czech Republic, it is important to obtain a deeper understanding of the genetic background of the exit from endogenous dormancy for apricot flower buds. The reason is that frost resistance of flower buds is significantly reduced after exit from endogenous dormancy. The aim of this work was to contribute to the understanding of the process of exit of various plant tissues from dormancy, as the problem is very complex and containing a number of physiological processes that are controlled by the function and regulation of many various substances and corresponding genes. The time of exit of flower buds from endogenous dormancy was established by counting of flowering buds on sampled twigs after their transport to laboratory conditions. Furthermore, the production of ethylene, ethane, and CO2 was monitored for sampled twigs with flower and leaf buds. Other physiological parameters monitored during the course of this study were the level of abscisic acid in flower buds and their weigh during the sampling close to the period of exit from endogenous dormancy. An analysis of transcriptome using the cDNA-AFLP method was carried out on four apricot variants ('Sundrop', SEO, 'Vestra' a 'Betinka') to achieve a deeper understanding of the genetic background of exit of flower buds from endogenous dormancy. The transcription profiles obtained from this experiment were evaluated for changes in the expression profiles and fragments of genes with modified expression during the monitored period were sequenced identified. Obtained sequences were compared with sequences in electronic databases (NCBI and TIGR). The results of this comparison led to the identification of a number of the sequenced genes. The results of this study confirmed the suitability of use of the cDNA-AFLP method for the identification of gene candidates and getting a preliminary picture of the main molecular mechanisms taking place during the time of exit of flower buds from endogenous dormancy. The suitability of the used procedure was confirmed by the match between the genes identified from the sequencing and genes already described in the literature as being linked to the exit of plants from endogenous dormancy (e.g. gene coding for acquaporin, GTP-binding proteins, elongation factor 1 alpha, ubiquitin, and xyloglucan endotransglycosylase hydrolase). Measurements of concentration of selected substances during exit from endogenous dormancy do not allow definite conclusions about their influence on this process. On the other hand were identified several genes that might be candidates for markers identifying the output timing of endogenous dormancy of buds and provide a good starting point for further scientific research in this field.

No description available.

A cDNA clone encoding the entire amino acid sequence of rabbit brown adipose tissue uncoupling protein has been isolated and sequenced. The coding region of this cDNA is 80.6% identical to that of the rat uncoupling protein cDNA. In contrast to rat uncoupling protein for which there are two mRNAs of 1500 and 2000 nucleotides there is only one rabbit uncoupling protein mRNA of 2000 nucleotides. Whereas the rat cDNA hybridizes more strongly to the shorter rat uncoupling protein mRNA the rabbit cDNA hybridizes more strongly to the longer rat uncoupling protein mRNA. Primer extension and Northern blot analysis were performed to try to account for the difference of 430 ± 75 nucleotides between the two rat uncoupling protein mRNAs. Northern blot analysis indicated the presence of 355 more nucleotides in the 3'-untranslated region of the 2000 nucleotide long rat uncoupling protein mRNA than in the 1500 nucleotide long rat uncoupling protein mRNA. The two rat uncoupling protein mRNAs could therefore arise by differential processing. Primer extension revealed that the two rat uncoupling protein mRNAs have a 5'-untranslated region of approximately 186 nucleotides. The deduced amino acid sequence of rabbit UCP is 86% identical with both the rat and hamster proteins. Several regions are conserved in all three uncoupling proteins. The two longest regions of conservation are residues 52 to 69 and 82 to 100 of the mature proteins and correspond to two of several basic regions of the protein that have been suggested as possible targeting sequences. These conserved regions fall within amino acids 52 to 104 of the mature rat protein, which has been shown by others to target a passenger protein to mitochondria. Helical wheel diagrams that correspond to residues 52 to 68 and residues 72 to 92 reveal possible amphiphilic α-helical formations that may be involved in targeting. Regions corresponding to those conserved in the three UCPs are also conserved in three mammalian ADP/ATP carriers and may indicate a common role for these regions, perhaps including targeting. There is almost complete conservation of lysine, arginine, and cysteine residues that are thought to be involved in nucleotide binding and proton transport in the three UCPs. There is a threonine to alanine change at the carboxyl-terminus of the rabbit protein compared to the rat protein. This amino acid difference may explain the differential reactivities of rabbit and rat UCP with an antibody preparation against rat UCP. / Thesis / Master of Science (MSc)

The DNA microarray is a powerful tool to study expression levels of thousands of genes simultaneously. Often, cDNA libraries representing expressed genes of an organism are available, along with expressed sequence tags (ESTs). ESTs are widely used as the probes for microarrays. Designing custom microarrays, rich in genes relevant to the experimental objectives, requires selection of probes based on their sequence. We have designed a probe selection method, called GeneSieve, to select EST probes for custom microarrays. To assign annotations to the ESTs, we cluster them into contigs using PHRAP. The larger contig sequences are then used for similarity search against known proteins in model organism such as Arabidopsis thaliana. We have designed three different methods to assign annotations to the contigs: bidirectional hits (BH), bidirectional best hits (BBH), and unidirectional best hits (UBH). We apply these methods to pine and potato EST sets. Results show that the UBH method assigns unambiguous annotations to a large fraction of contigs in an organism. Hence, we use UBH to assign annotations to ESTs in GeneSieve. To select a single EST from a contig, GeneSieve assigns a quality score to each EST based on its protein homology (PH), cross hybridization (CH), and relative length (RL). We use this quality score to rank ESTs according to seven different measures: length, 3' proximity, 5' proximity, protein homology, cross hybridization, relative length, and overall quality score. Results for pine and potato EST sets indicate that EST probes selected by quality score are relatively long and give better values for protein homology and cross hybridization. Results of the GeneSieve protocol are stored in a database and linked with sequence databases and known functional category schemes such as MIPS and GO. The database is made available via a web interface. A biologist is able to select large number of EST probes based on annotations or functional categories in a quick and easy way. / Master of Science

The goal of the thesis was to characterize transcriptional profiles of in vivo and in vitro derived embryos during bovine minor and major embryonic genome activation and to identify mRNA transcripts newly synthesized during these stages. In our first work we have concentrated on the study of minor genome activation at the 4-cell stage of embryo. Using SSH, we have identified 31 amplicons homologous with already identified genes. We have selected 5 of these for detailed study of their expression during the whole period of preimplantation development: centromere protein, 350/400 kDa (CENPF, mitosin), splicing factor arginine/serine-rich 3 (SRFS3), high mobility group nucleosomal binding domain 2 (HMGN2) protein and eukaryotic translation initiation factors EIF4A2 a EIF4E. All these genes play an important role in the early embryo development. SRFS3 is the first described gene with an important function in preimplantation development, which is expressed already during bovine minor genome activation, and its transcription is α-amanitin sensitive during this period. We have selected CENPF gene for a more thorough study. By silencing its expression by the injection of CENPF dsRNA into the zygote, we have studied its function throughout the whole preimplantation development of bovine embryo....

Neste trabalho realizamos a análise das variações na expressão gênica global do fungo aquático Blastocladiella emersonii submetido ao estresse de carência de oxigênio (hipóxia), utilizando a técnica de microarranjos de cDNA em lâminas contendo 3773 genes distintos. Nos experimentos de hipóxia gradual (diminuição gradual da concentração de oxigênio dissolvido, seguido de reoxigenação) e hipóxia direta (diminuição direta da concentração de oxigênio dissolvido, seguido de reoxigenação) observamos que 650 genes foram diferencialmente expressos em pelo menos uma das condições de estresse e que 534 deles mostraram-se afetados (direta ou indiretamente) pela disponibilidade de oxigênio, uma vez que apresentaram recuperação (ou tendência à recuperação) da sua expressão aos níveis normais, quando as células foram reoxigenadas. Além de modular a expressão de diversos genes sem função conhecida, B. emersonii responde à hipóxia reajustando a expressão de genes responsáveis pela produção e consumo de energia. Pelo menos transcricionalmente, este fungo favorece o metabolismo anaeróbico, através da indução de genes que codificam enzimas da via glicolítica e lactato desidrogenase, ao passo que no ciclo do ácido cítrico, a maioria dos genes encontram-se reprimidos ou não sofrem alteração na expressão. Processos dispendiosos em energia como síntese protéica, metabolismo de aminoácidos, enovelamento de proteínas e transporte por membrana apresentaram perfis predominantemente de repressão gênica quando em carência de oxigênio. Ainda utilizando a técnica de microarranjos, mostramos semelhanças entre os perfis transcricionais nos experimentos hipóxia e de carência de Fe2+ (tratamento com quelante de Fe2+ 2,2´-dipyridyl) sugerem que estes estresses estão de alguma forma relacionados, fornecendo bons indícios de que o íon Fe2+ possa ter um papel importante no mecanismo sensor de oxigênio e/ou de resposta a hipóxia em B. emersonii. Além disso, o tratamento prévio de células submetidas à hipóxia com o antibiótico geldanamicina, um conhecido inibidor da proteína de choque térmico HSP90, levou à diminuição da indução de certos genes de hipóxia, indicando que este fungo pode possuir algum mecanismo semelhante ao do fator de transcrição de hipóxia HIF1-α de mamíferos, uma vez que este fator também é afetado por geldanamicina. Adicionalmente, desenvolvemos um protocolo para transformação de B. emersonii mediada por Agrobacterium tumefasciens que se mostrou promissor. A transferência do T-DNA contendo um gene de resistência a higromicina B, presente no vetor binário pBINPLUS-Hph, foi evidenciada pelo crescimento normal e esporulação das células transformadas, na presença do antibiótico e pela amplificação do gene de resistência no DNA genômico de células transformadas. / In this work we analyzed global gene expression changes in the aquatic fungus Blastocladiella emersonii submitted to oxygen deprivation (hypoxia), using cDNA microarrays containing 3,773 distinct genes. In gradual hypoxia (gradual decrease in dissolved oxygen concentration, followed by reoxygenation) and direct hypoxia (direct decrease of dissolved oxygen concentration, followed by reoxygenation) we observed 650 differentially expressed genes in at least one of the stress conditions tested, 534 of them being affected (directly or indirectly) by oxygen availability, since they showed recovery of normal expression levels or a tendency to recover, when cells were reoxygenated. Besides modulating many genes with no previously assigned function, B. emersonii responds to hypoxia by readjusting the expression levels of genes responsible for energy production and consumption. At least transcriptionally, this fungus seems to favour anaerobic metabolism through the induction of genes encoding glycolytic enzymes and lactate dehydrogenase, while in the TCA-cycle, most genes were repressed or unchanged. Energy-costly processes like protein synthesis, amino acid metabolism, protein folding and transport had their gene expression profiles predominantly repressed during oxygen deprivation. Microarray experiments also showed similarities between the transcriptional profile of genes in hypoxia and iron (II) deprivation (treatment with the iron (II) chelator 2,2\'-dipyridyl), suggesting that these stresses are somehow related, giving good evidence that Fe2+ ion could have a role in the mechanism of oxygen sensing and/or response to hypoxia in B. emersonii. Furthermore, pretreatment of cells subjected to hypoxia with the antibiotic geldanamycin, a known inhibitor of the heat shock protein HSP90, caused a significant decrease in the induction of certain hypoxic genes, indicating that this fungus could have a mechanism similar to that of the mammalian hypoxia transcription factor HIF-1α, which is also affected by geldanamycin. Additionally, we developed an Agrobacterium tumefasciens-mediated protocol for transformation of B. emersonii that has shown to be promising. The capacity to transfer the T-DNA containing a hygromycin B resistance gene, present in the pBINPLUSHph binary vector, was evidenced by the normal growth and sporulation of the transformed cells in the presence of antibiotic and by amplification of the resistance gene from the genomic DNA of transformed cells