No description available.

No description available.

The control of gene expression by rearrangement of DNA sequences, in prokaryotes and eukaryotes, is recorded in several instances. These accompany the differentiation of cells, yielding a new phenotype. The possibility of such a means of gene control operating in Physarum was considered; this organism undergoes marked changes in cell morphology and function during the amoebal-plasmodial transition. Genes activated or inactivated in this transition were examined for possible changes in structure. This was done by using amoebal- and plasmodial-specific cDNAs to probe Southern blots of amoebal and plasmodial DNA, digested with restriction endonucleases. This procedure should have revealed any restriction enzyme polymorphisms that might have existed between amoebae and plasmodia as a result of DNA rearrangements. However, no changes in DNA structure were observed between amoebae and plasmodia. The scope of this investigation is critically assessed. The methylation of cytosine residues has also been proposed as a means of controlling gene expression in eukaryotes. The available amoebal- and plasmodial-specific cDNAs were used therefore to probe Southern blots of amoebal and plasmodial DNA digested with methylation sensitive and insensitive restriction enzymes, in order to examine the methylation patterns of DNA from the two forms. For all phase-specific genes tested, the patterns in amoebae and plasmodia were identical, suggesting that no changes had occurred. Again, the scope of this investigation is assessed, and the possibility of a more extensive search for putative DNA rearrangements or changes in methylation pattern is mooted. To study closely the structure of three plasmodial-specific genes, attempts were made to clone regions of Physarum genomic DNA containing these sequences. It was not possible to isolate positive clones in any useful quantity. The probable reasons for the difficulties encountered are discussed.

Abstract Lysyl hydroxylase (E.C. 1.14.11.4, protocollagen-lysine 2-oxoglutarate 5-dioxygenase, PLOD) catalyses the formation of hydroxylysine in collagens and other proteins with collagen-like sequences. The hydroxylysine residues participate in the formation of collagen crosslinks and serve as attachment sites for carbohydrate units. The importance of lysine hydroxylation is demonstrated by the critical manifestations found in patients with the type VI variant of the Ehlers-Danlos syndrome, which is caused by a deficiency in lysyl hydroxylase activity. Lysyl hydroxylase requires Fe2+, 2-oxoglutarate, O2 and ascorbate. The binding site for the C-5 carboxyl group of 2-oxoglutarate is characterized here by site-directed mutagenesis. Two conserved and one non-conserved amino acid residues at the possible binding site in human lysyl hydroxylase 1 were converted individually to alanine or lysine and the mutant polypeptides were expressed in insect cells. Mutation of arginine-700 to alanine inactivated the enzyme completely, whereas mutation of the other two residues had only a minor effect. In addition, the Km of the arginine-700 to lysine mutant polypeptide for 2-oxoglutarate was increased 10-fold. The results thus indicate that this conserved arginine is the residue that binds the C-5 carboxyl group of 2-oxoglutarate in lysyl hydroxylases. A novel human lysyl hydroxylase isoenzyme, termed lysyl hydroxylase 3, was identified, cloned and characterized here. The overall amino acid sequence identity between the novel human lysyl hydroxylase isoenzyme and the other human lysyl hydroxylase isoenzymes is about 60%. The highest expression levels of the mRNA for lysyl hydroxylase 3 among the tissues studied were found in the placenta, pancreas and heart. The novel isoenzyme was expressed as a recombinant protein in insect cells, and the protein was shown to function as a lysyl hydroxylase in vitro hydroxylation experiments using short synthetic peptides as substrates. No differences in catalytic properties were found between the recombinant lysyl hydroxylases 3 and 1. The structure of the human gene for lysyl hydroxylase 3 was determined in the last part of this work. The gene is shown to be only 11.6 kb in size and to contain 19 exons. Transcription was found to be initiated at multiple sites, and the introns contained 15 full-length Alu retroposons or partial Alu fragments of more than 100 bp. The present characterization of the exon-intron organization of the gene will provide a basis for further studies to determine whether there is any genetic disease that is attributable to mutations in this gene.

The goals of gene prediction using computational approaches are to determine gene location and the corresponding functionality of the coding region. A subset of gene prediction is the gene structure prediction problem, which is to define the exon-intron boundaries of a gene. Gene prediction follows two general approaches: statistical patterns identification and sequence similarity comparison. Similarity based approaches have gained increasing popularity with the recent vast increase in genomic data in GenBank. The proposed gene prediction algorithm is a similarity based algorithm which capitalizes on the fact that similar sequences bear similar functions. The proposed algorithm, like most other similarity based algorithms, is based on dynamic programming. Given a genomic DNA, X = x1 xn and a closely related cDNA, Y = y1 yn, these sequences are aligned with matching pairs stored in a data set. These indexes of matching sets contain a large jumble of all matching pairs, with a lot of cross over indexes. Dynamic programming alignment is again used to retrieve the longest common non-crossing subsequence from the collection of matching fragments in the data set. This algorithm was implemented in Java on the Unix platform. Statistical comparisons were made against other software programs in the field. Statistical evaluation at both the DNA and exonic level were made against Est2genome, Sim4, Spidey, and Fgenesh-C. The proposed gene structure prediction algorithm, by far, has the best performance in the specificity category. The resulting specificity was greater than 98%. The proposed algorithm also has on par results in terms of sensitivity and correlation coeffcient. The goal of developing an algorithm to predict exonic regions with a very high level of correctness was achieved.

The goals of gene prediction using computational approaches are to determine gene location and the corresponding functionality of the coding region. A subset of gene prediction is the gene structure prediction problem, which is to define the exon-intron boundaries of a gene. Gene prediction follows two general approaches: statistical patterns identification and sequence similarity comparison. Similarity based approaches have gained increasing popularity with the recent vast increase in genomic data in GenBank. The proposed gene prediction algorithm is a similarity based algorithm which capitalizes on the fact that similar sequences bear similar functions. The proposed algorithm, like most other similarity based algorithms, is based on dynamic programming. Given a genomic DNA, X = x1 xn and a closely related cDNA, Y = y1 yn, these sequences are aligned with matching pairs stored in a data set. These indexes of matching sets contain a large jumble of all matching pairs, with a lot of cross over indexes. Dynamic programming alignment is again used to retrieve the longest common non-crossing subsequence from the collection of matching fragments in the data set. This algorithm was implemented in Java on the Unix platform. Statistical comparisons were made against other software programs in the field. Statistical evaluation at both the DNA and exonic level were made against Est2genome, Sim4, Spidey, and Fgenesh-C. The proposed gene structure prediction algorithm, by far, has the best performance in the specificity category. The resulting specificity was greater than 98%. The proposed algorithm also has on par results in terms of sensitivity and correlation coeffcient. The goal of developing an algorithm to predict exonic regions with a very high level of correctness was achieved.

We previously reported that the CNGB1 locus encoding the rod photoreceptor cGMP-gated channel β-subunit is complex, comprising non- overlapping transcription units that give rise to at least six transcripts (Ardell, M.D., Aragon, I., Oliveira, L., Porche, G.E., Burke, E., Pittler, S.J., 1996. The beta subunit of human rod photoreceptor cGMP-gated cation channel is generated from a complex transcription unit. FEBS Lett. 389, 213- 218). To further understand the transcriptional regulation of this extraordinarily complex locus, and to develop a screen for defects in the gene in patients with hereditary disease, we determined its genomic organization and DNA sequence. The CNGB1 locus consists of 33 exons, which span approximately 100 kb of genomic DNA on chromosome 16. The β-subunit comprises two domains, an N-terminal glutamic acid-rich segment (GARP), and a C-terminal channel-like portion. Two additional exons encoding a short GARP transcript and a truncated channel-like transcript have been identified. A major transcription start point was identified 79 bp upstream of the initiator ATG. To begin analysis of the basis for the generation of multiple transcripts, and to identify promoters driving expression in retina, approximately 2.5 kb of the upstream region were sequenced. Putative cis- elements, which can bind the retina-specific transcription factors Crx and Erx, were found immediately upstream of the transcription start point, and may be important for gene expression in this tissue. From our analysis, a model is reported to account for at least four of the retinal transcripts.

No description available.

No description available.

Submitted by Mariana de Lara Campos Arcuri null (mariana.arcuri@aluno.ibb.unesp.br) on 2017-07-24T23:48:37Z No. of bitstreams: 1 Dissertação Final Ficha Catalográfica.pdf: 1462289 bytes, checksum: 6374b4edc59dd6c0ef4dab57c0176073 (MD5) / Approved for entry into archive by Luiz Galeffi (luizgaleffi@gmail.com) on 2017-07-26T16:41:02Z (GMT) No. of bitstreams: 1 arcuri_mlc_me_bot.pdf: 1462289 bytes, checksum: 6374b4edc59dd6c0ef4dab57c0176073 (MD5) / Made available in DSpace on 2017-07-26T16:41:02Z (GMT). No. of bitstreams: 1 arcuri_mlc_me_bot.pdf: 1462289 bytes, checksum: 6374b4edc59dd6c0ef4dab57c0176073 (MD5) Previous issue date: 2017-05-24 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / As lacases, p-diphenol-O2-oxidoredutases, são enzimas que desempenham papel fundamental na oxidação de monolignóis durante a biossíntese de lignina, estando, portanto, associadas a processos de crescimento e tolerância a alguns tipos de estresses abióticos. As lacases podem ser encontradas em bactérias, fungos, plantas e insetos. Estudos apontam que as lacases vegetais apresentam comportamento similar às de origem fúngica, atuando de formar complementar à rota de lignificação, em resposta ao estresse oxidativo, promovendo a detoxificação celular. As lacases são geralmente codificadas por famílias multigênicas. Através de análises in silico no genoma de eucalipto, o presente estudo identificou 54 genes codificadores de lacases (denominados EgLAC) que, filogeneticamente, se distribuem em seis diferentes subgrupos. Com base em dados de RNA-Seq, padrões distintos de expressão das lacases identificadas foram observados, sendo algumas enriquecidas em um dado órgão/tecido e outras com expressão não detectável pelo método. Análises de RT–qPCR de alguns genes selecionados com base em um banco de sequências expressas confirmaram, por exemplo, a expressão raiz-específica do gene EgLAC52 bem como as expressões preferenciais em raiz e folha dos genes EgLAC4 e EgLAC32, respetivamente. Em paralelo, a expressão de alguns destes genes em reposta a estresses foi investigado, e alterações na expressão relativa em resposta aos estresses oxidativo e osmótico foram constatadas, sugerindo a participação destas lacases em respostas a estresses abióticos. / Laccases are p-diphenol-O2-oxidoreductases encoded by multigene families widely distributed throughout the plant kingdom. They exhibit important roles in the oxidation of monolignols during lignin biosynthesis and are reported to be functionally involved in plant development, tolerance and response to stress. Apart from plants, laccases can be also found in bacteria, fungi and insects. Here, a genome-wide survey of the eucalyptus genome revealed the presence of 88 putative laccases genes. However, after meticulous analyzes using different approaches, the redundant sequences were discarded and 54 laccases genes (referred as EgLAC) were retrieved. These genes were phylogenetically distributed in six different subgroups. Based on RNA-Seq data, distinct organ/tissue expression patterns of the identified EgLAC genes were ascertained. The vast majority showed organ/tissue-enriched expression, while certain genes exhibited no detectable expression. RT-qPCR analyzes confirmed the organ/tissue expression patterns of a representative set of genes such as, for example, the rootspecific expression of EgLAC52 and the root and leaf preferential expressions of genes EgLAC4 and EgLAC32. Further expression profiling of selected EgLAC genes in response to oxidative and osmotic stresses revealed differences in their relative expression, with some genes being stress-induced. These results suggest that certain laccases might be implicated in abiotic stress responses.