Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The Quaternary period was marked by considerable changes on climate and vegetation. In the Southern Hemisphere, glaciations were milder than in the Northern Hemisphere, however, there were temperature reductions, and the climate became drier. Thus, climate-related modifications occurred in the Atlantic forest vegetation, which contracted its distribution being substituted towards other types, such as Cerrado and Caatinga . The original Atlantic forest vegetation became restricted to moist locations with milder temperatures, called refuges, which sheltered most part of the biodiversity at this time. It is known that such paleoclimatic changes affected the population dynamics of many species, especially in the Northern Hemisphere. However, it is still not clear how much these impacts influenced the species of the Neotropical region. The two species of this study, Drosophila maculifrons and Drosophila griseolineata, belong to the guaramunu group of Drosophila, and are considered sister-species that diverged about four million years ago. However, disregarding their close phylogenetic relationships, they present some distinct ecological patterns, the first species being more generalist, and the second more restricted to forest environments. Due to this ecological heterogeneity, these two species are potential indicators of the genetic consequences caused by the climatic fluctuations of the Quaternary, especially in face of a comparative perspective. The aims of this study were to evaluate the intraspecific diversity of different D. maculifrons and D. griseolineata populations, analyze the structure of individuals and populations in these two species of the guaramunu group and identify the ecological and evolutionary forces that modeled their distribution in Southern/Southeastern Brazil. In order to do so, 114 individuals were collected along the South, Southeast and Center-west regions of Brazil and Medellin, Colombia. Modeling analysis was performed, together with phylogenetic and phylogeographic analyses, with the last based in sequences of COI (Cytochrome Oxidase Subunit I) and COII (Cytochrome Oxidase Subunit II) genes. In general, the results that inferred the distribution of the most suitable habitat for each species indicate that the two species occur in sympatry at several points, although D. maculifrons is more widely distributed than D. griseolineata, at least in the brazilian territory. According to our phylogeographic analysis, D. maculifrons presents low levels of diversity and structure for mtDNA, which could be explained by a recent populational expansion event, dated for about 20 to 30 thousand years ago. On the other hand, D. griseolineata shows moderate levels of diversity and population structure, and its populations seem to have remained stables along time, showing a pattern of isolation by distance. So, it is interesting to evaluate the ecological and/or evolutionary factors which are responsible for all this difference, and this work represents a first step towards this understanding. / O período do Quaternário foi marcado por alterações consideráveis no clima e vegetação. No Hemisfério Sul, as glaciações foram mais amenas, entretanto, houve a redução da temperatura e o clima tornou-se mais seco. Dessa forma, ocorreram contrações na distribuição da Mata Atlântica e sua substituição por outros tipos, condizentes ao clima, como o Cerrado e a Caatinga. Assim, a vegetação de Mata Atlântica ficou restrita a locais úmidos e com temperaturas mais amenas, conhecidos como refúgios, os quais abrigaram grande parte da biodiversidade neste período. Sabe-se que essas alterações paleoclimáticas influenciaram a dinâmica populacional de muitas espécies, especialmente no Hemisfério Norte, porém ainda não está claro o quanto esses impactos influenciaram as espécies de distribuição Neotropical. As duas espécies em questão, Drosophila maculifrons e D. griseolineata, pertencem ao grupo guaramunu de Drosophila e são consideradas espécies irmãs, que divergiram aproximadamente há 4 milhões de anos. Porém, apesar do grau de parentesco, elas apresentam alguns padrões ecológicos distintos, sendo a primeira mais generalista e a segunda mais restrita a ambientes florestais. Devido a essa heterogeneidade ecológica, essas duas espécies são potenciais indicadoras das conseqüências genéticas ocasionadas pelas flutuações climáticas do Quaternário, principalmente em face de uma perspectiva comparativa. Os objetivos deste trabalho foram avaliar a diversidade intra-específica de diferentes populações de D. maculifrons e D. griseolineata, analisar a estruturação de indivíduos e populações nestas duas espécies do grupo guaramunu e inferir as forças ecológicas e evolutivas que modelaram sua distribuição ao longo do sul e sudeste brasileiros. Para isso, nossa amostragem conta com 114 indivíduos distribuídos ao longo das regiões Sul, Sudeste, Centro-Oeste do Brasil e Medellin na Colômbia. Foram realizadas análises de modelagem, bem como análises filogenéticas e filogeográficas, sendo que essas duas últimas basearam-se nas sequências dos genes COI (Citocromo Oxidase Subunidade I) e COII (Citocromo Oxidase Subunidade II). De uma forma geral, os resultados que inferiram a distribuição dos habitats mais adequados para cada espécie indicam que as duas espécies apresentam diversos pontos de simpatria, embora D. maculifrons seja mais amplamente distribuída em território brasileiro. De acordo com as análises filogeográficas, D. maculifrons apresenta baixos níveis de diversidade e estruturação em nível de DNA mitocondrial, o que pode ser explicado por um evento de expansão populacional recente, datado para aproximadamente 20 a 30 mil anos. Por outro lado, D. griseolineata apresenta níveis moderados de diversidade e estruturação populacional e suas populações parecem ter se mantido estáveis ao longo do tempo, apresentando um padrão de isolamento por distância. É, pois, interessante avaliar os fatores ecológicos e/ou evolutivos responsáveis por toda essa diferença, e esta dissertação representa um primeiro passo rumo a esse entendimento.

Na levedura Saccharomyces cerevisiae, o gene KIN3 codifica uma serina-treonina cinase cuja função ainda é desconhecida. A proteína Kin3 é homóloga estrutural da proteína NIMA, de Aspergillus nidulans, a qual é necessária para a transição da fase G2/M do ciclo celular, e ainda possui papel na resposta a danos no DNA. Cinases relacionadas à NIMA (Nek´s) foram identificadas em diferentes espécies, e mamíferos contêm 11 Nek´s. Algumas destas cinases apresentam propriedades semelhantes a NIMA, estando envolvidas na progressão do ciclo celular e na resposta a danos no DNA. A proteína Kin3 possui propriedades semelhantes a Nek1 e Nek2. Nek1 interage com proteínas envolvidas na sinalização e reparação de danos no DNA. Linhagens mutantes kin3Δ são defectivas na parada da fase G2/M do ciclo celular em resposta a danos no DNA e apresentam uma pronunciada sensibilidade a diversos agentes mutagênicos. O objetivo deste trabalho foi avaliar a interação do gene KIN3 com os genes do complexo MRX (MRE11/RAD50/XRS2), envolvido na sinalização e reparação de danos no DNA, bem como na manutenção da integridade dos cromossomos. O perfil de sensibilidade dos simples e duplos mutantes sugerem uma interação epistática entre o gene KIN3 e os genes do complexo MRX, na resposta a danos causados por 8-metoxipsoraleno fotoativado, cisplatina e mustarda nitrogenada. A expressão do gene KIN3 durante estresse mutagênico não apresentou diferenças significativas entre a linhagem selvagem e as linhagens mutantes mre11Δ, rad50Δ e xrs2Δ. Os resultados obtidos ainda indicam que a proteína Kin3 possa estar agindo junto com o complexo MRX na via Mec1/Tel1 de sinalização de danos no DNA. / In the yeast Saccharomyces cerevisiae, the KIN3 gene codifies a serine-threonine cinase whose function is still unknown. The Kin3 protein is a structural homolog of NIMA protein of Aspergillus nidulans, which is necessary to G2/M transition, and has a role in DNA damage response. NIMA-related kinases (Nrk´s) were identified in different species, and mammals contain eleven Nrk´s. Some of these kinases display similar properties with NIMA, being involved in cell cycle progression and DNA damage response. The Kin3p have similar properties with Nek1 and Nek2. Nek1 interacts with proteins involved in signaling and repair of DNA damage. Mutant strains in KIN3 are defective in the G2/M-phase checkpoint in response to DNA damage and display a pronounced sensitivity to several mutagens. The aim of this work was to evaluate the interaction between KIN3 gene and the genes of MRX complex (MRE11/RAD50/XRS2), involved in signaling and repair of DNA damage, as well in the maintenance of chromosome integrity. The sensitivity profile of single and double mutants suggests an epistatic interaction between the KIN3 gene and the genes of MRX complex, in damage response induced by photo-activated methoxypsoralen, cisplatin and nitrogen mustard. The KIN3 gene expression during mutagenic stress does not show significant differences between the wild type and mutant strains mre11Δ, rad50Δ and xrs2Δ. The obtained results indicate that the Kin3 protein can act together with the MRX complex in the Mec1/Tel1 pathway of DNA damage signaling.

Um dos maiores objetivos na ciência atual é poder relacionar alterações nos genes a mudanças nas morfologias de organismos multicelulares. Além disso, é importante poder determinar se estas modificações foram obras do acaso ou se a seleção natural teve um papel central moldando a forma e o tamanho das criaturas vivas. A busca por nutrientes e sua utilização é uma das questões mais básicas para a sobrevivência de uma espécie. Em mamíferos a diversificação e especialização dos dentes para triturar, rasgar e mastigar diferentes tipos de comida possibilitou um incremento na geração de energia e representou uma inovação chave para a sobrevivência deste grupo. No presente estudo foi investigado o gene PAX9, que codifica um fator de transcrição chave no desenvolvimento da dentição de mamíferos. Para 125 indivíduos de origem ameríndia foi seqüenciado o exon 3 (138 pb), conjuntamente com as regiões intrônicas flanqueadoras 5´e 3´(232 pb e 220 pb, respectivamente) e os dados integrados com a informação disponível para a mesma região de 115 indivíduos de diferentes origens geográficas. Além disso, os exons 2, 3 e 4 (627 pb, 138 pb e 240 pb respectivamente) foram seqüenciados do DNA de 25 espécies de mamíferos e agrupados com os dados disponíveis de mais 29 espécies. Em humanos, o polimorfismo Ala240Pro possui uma distribuição variada entre os Ameríndios da América do Sul e está presente em Esquimós, Asiáticos e Europeus, mas não está presente entre os Afro-Americanos. O panorama que surge é que provavelmente a partir da saída da África a freqüência da mutação Ala240Pro aumenta, sendo possivelmente selecionada positivamente, o que pode estar relacionado à agenesia do terceiro molar, ou ainda, estar associado a genes conectados a adaptações ao frio. Com a chegada dos Ameríndios a regiões equatoriais, outro cenário parece dominar a evolução do exon 3, seja pela perda inicial da possível vantagem adaptativa, ou por causa da ação da deriva genética amplamente documentada nestas populações. Provavelmente em algum momento na história destas populações, fatores casuais poderiam ter sido preponderantes às restrições funcionais da proteína. Em geral, há uma maior variabilidade em nível de aminoácidos no exon 3 considerando todos os mamíferos estudados quando comparada com a do exon 2, ficando o exon 4 numa posição intermediária. O exon 3 seria um exemplo notável de uma situação que conferiria “evolvabilidade” ao PAX 9. / One of the main goals in science today is to establish the relation between gene changes and variations in the morphologies of multicellular organisms. In addition, it is important to determine if these changes were randomic or whether natural selection had a central role shaping the form and size of living beings. Searching for nutrients and its use is one of the most basic issues for a species survival. In mammals teeth diversification and specialization to grind, tear and chew different types of food were a key innovation that allowed an increase in the generation of energy and in the survival of this group. In this study an investigation was performed in the Paired box gene 9 (PAX9), which codes for a transcription factor that was a key factor in the development of mammal dentition. A total of 125 persons from Amerindian populations were studied by sequencing the PAX9 gene exon 3 (138 base pairs) as well as its 5´and 3´flanking intronic segments (232 bp and 220 bp, respectively) and the data integrated with the information available for the same genetic region from 115 individuals of different geographical origins. Moreover, exons 2, 3 and 4 (627 bp, 138 bp and 240 bp, respectively) were sequenced from DNA of 25 mammalian species and the results combined with the data available from other 29 additional species. In humans, the Ala240Pro polymorphism has a varied distribution among the South American Amerindians and is present in Eskimos, Asians and Europeans, but it is not present among Afro-Americans. The pattern that emerges is that probably starting with the out-Africa migration, and possibly as a result of positive selection, the Ala240Pro mutation frequency increased, that could be related to third molar agenesis or an associated gene connected to cold adaptation. With the Amerindian arrival again in equatorial regions, another pattern seems prevalent in exon 3 evolution, either due to the loss of a possible initial adaptive advantage, or to the action of genetic drift, widely documented in these populations. Probably at some point in the history of these populations, random factors could have been dominant despite the protein functional restrictions. In a general way, there is a greater variability at the amino acid level in exon 3 of all mammals considered when compared to exon 2, exon 4 occupying an intermediary position. Exon 3 would be a remarkable example of a condition that would confer PAX9 evolvability.

A espécie humana sempre foi fascinada pela variação de cor exibida pelos animais. Atualmente, investigações genéticas têm permitido que os pesquisadores caracterizem os mecanismos moleculares envolvidos na produção de pigmentos que afetam fenótipos de cor bem como na percepção de tal variação de cor entre diferentes espécies. Essa tese apresenta três investigações sobre esses assuntos. Na primeira, foi estudada a base molecular para a evolução da cor de plumagem em Ramphastidae. Essa família de aves inclui tucanos, araçaris e saripocas, possuindo uma ampla variação em plumagem. Foi encontrada seleção positiva no gene do receptor da melanocortina-1 (MC1R) no ramo que leva ao gênero Ramphastos, sugerindo que a plumagem mais escura nessas espécies foi favorecida pela seleção natural. Interessantemente, três de cinco substituições de aminoácido encontradas nesse gênero foram associadas ao melanismo em outras espécies de aves. No segundo estudo, uma análise do gene MC1R foi geia em perus domésticos (Meleagris gallopavo) brancos e pigmentados. Esse gene é um dos pelo menos cinco locos genéticos conhecidos que determinam a cor da plumagem nessa espécie, e três alelos funcionais já foram caracterizados (B, b+, b1). Os resultados mostraram que as aves brancas têm alguma diversidade genética em nível de DNA, mas quase todos os alelos foram b+, com um único alelo b1 encontrado, sugerindo que essas aves têm um valor limitado para o cruzamento e desenvolvimento de novas variedades coloridas. Entre as aves coloridas, os resultados mostraram que a classificação fenotípica da plumagem feita pelos criadores brasileiros não é preditiva para os alelos que podem ser encontrados no gene MC1R. Finalmente, no terceiro estudo a base molecular da visão colorida em uacari (Cacajao calvus) foi explorada. Essa é uma espécie de primata do Novo Mundo que exibe uma distintiva face avermelhada intensa, um sistema social complexo, e uma dieta especializada. O loco da opsina ligado ao cromossomo X foi caracterizado, e os resultados revelaram a ocorrência de extensivo polimorfismo molecular e o maior número de alelos funcionais (seis) já encontrados em uma espécie de primata. Esse achado provavelmente deriva de seleção para visão tricromática, talvez mediada por seleção sexual. Em conjunto, esses resultados mostram a importância de estudos moleculares que permitam uma melhor compreensão da evolução e manutenção da variação biológica associada à produção e percepção de cores em diferentes grupos de vertebrados. / Humans have been always fascinated by body color variation in animals. Current genetic investigations have allowed researchers to characterize the molecular mechanisms involved in the production of pigments affecting color phenotypes, as well as in the perception of such color variation among different species. This thesis presents three investigations concerning these subjects. In the first, the molecular basis for the evolution of plumage coloration in Ramphastidae was studied. This bird family includes toucans and toucanets, showing wide plumage variation. Positive selection in the melanocortin-1 receptor (MC1R) gene was found in the branch leading to the genus Ramphastos, suggesting that the darker plumage in these species was favored by natural selection. Interestingly, three out of five aminoacid substitutions found in this genus have been associated to melanism in other bird species. In the second study, an analysis of the MC1R gene was performed in white and pigmented domestic turkeys (Meleagris gallopavo). This gene is one of the at least five genetic loci known to determine plumage color in this species, and three functional alleles have been identified (B, b+, b1). The results showed that white birds have some genetic diversity at the DNA level, but almost all alleles were b+, with a single b1 allele found, suggesting that these birds have a limited value for breeding new color varieties. Among colored birds, the results showed that the plumage phenotypic classification used by Brazilian breeders is not predictive of the alleles that can be found in the MC1R gene. Finally, in the third study, the basis of color vision in uakari (Cacajao calvus) was explored. This is a species of New World primate exhibiting a distinctive intense red face, a complex social system, and a specialized diet. The X-lined Opsin locus was characterized, and the results revealed the occurrence of extensive molecular polymorphism and the largest number of functional alleles (six) ever found for a primate. This finding possibly derives from selection for trichromatic vision, perhaps mediated by sexual selection. Taken together, these results show the importance of molecular studies allowing a better understanding of the evolution and maintenance of biological variation associated to color production and perception in different vertebrate groups.

A coloração dos animais é uma característica que apresenta uma grande diversidade de fenótipos nas diferentes espécies. Diferentes abordagens podem ser utilizadas para o entendimento da diversidade na coloração existente nas espécies animais. Através da análise de genes candidatos as mutações responsáveis pela variação na coloração têm sido descritas em diferentes espécies, demonstrando o envolvimento de mecanismos moleculares variados na sua regulação. Este trabalho tem por objetivo a utilização de duas abordagens genéticas para o estudo da variação na coloração, a análise de genes candidatos e a predição computacional do efeito de polimorfismos não sinônimos. Em ovinos a coloração da lã é uma característica com importância na produção e para a identificação das raças. Polimorfismos em diferentes genes foram associados com a coloração da lã, entretanto, estes não foram estudados em muitas raças que apresentam variação fenotípica. A ovelha crioula é uma raça local existente no sul do Brasil que apresenta uma ampla diversidade de cores na lã, incluindo branco, preto e diversos tons intermediários. O gene receptor de melanocortina 1 (MC1R) foi previamente associado com a coloração na raça crioula, entretanto, outros genes também devem estar envolvidos na regulação da coloração na raça. Este trabalho avaliou a influência dos genes MC1R, ASIP (proteína sinalizadora agouti), TYRP1 (proteína relacionada à tirosinase 1) e KIT (homólogo do oncogene de sarcoma felino viral v-kit Hardy-Zuckerman 4) na coloração da lã na raça ovina crioula. Amostras de 410 animais de diferentes cores foram analisadas, sendo a variação na coloração da lã determinada por colorimetria. O padrão de herança dos fenótipos foi avaliado através de cruzamentos dirigidos entre indivíduos de diferentes cores. Os polimorfismos nos genes foram avaliados através da realização do sequenciamento e da análise de fragmentos e a quantificação da expressão do gene ASIP foi realizada por PCR em Tempo Real. Foi observada a associação significativa entre polimorfismos nos genes MC1R e ASIP e a cor da lã na raça crioula. O alelo dominante do gene MC1R, provocado pelas mutações p.M73K e p.D121N, foi encontrado apenas em indivíduos pigmentados. Este alelo resulta na ativação constitutiva do receptor, e consequentemente na produção constante de eumelanina, sendo epistático sobre o gene ASIP. Nos animais homozigotos para o alelo selvagem do MC1R a manifestação do fenótipo branco ocorreu somente nos portadores de um alelo contendo a duplicação do gene ASIP. Os portadores da duplicação do ASIP apresentaram níveis elevados de expressão do gene enquanto os homozigotos para a cópia simples do ASIP não expressaram o gene e apresentaram fenótipos pigmentados. Os resultados obtidos permitiram identificar a influência da interação epistática dos genes MC1R e ASIP na coloração da lã nos ovinos crioulos. O estudo de genes candidatos envolvidos na rota da pigmentação mostrou-se uma abordagem adequada para a análise da variação na coloração nestes animais. Espera-se que o conhecimento adquirido neste trabalho auxilie na criação e na preservação da raça através da manutenção da diversidade fenotípica existente. A avaliação computacional dos polimorfismos não sinônimos vem sendo utilizada recentemente a fim de determinar os SNPs que potencialmente afetam o funcionamento dos genes e identificar os mecanismos responsáveis por doenças complexas e pela variação nos fenótipos. A predição do efeito de polimorfismos nos genes utilizando ferramentas computacionais apresenta-se como uma abordagem alternativa para o estudo da genética da coloração. O gene MC1R humano apresenta uma grande quantidade de polimorfismos alguns dos quais foram associados com a variação na pigmentação e com suscetibilidade a tumores de pele. Entretanto, muitas das variações existentes no gene não foram avaliadas quanto às suas consequências funcionais e o seu papel na variação da pigmentação. Foi realizada a predição computacional dos polimorfismos não sinônimos no gene MC1R humano com o objetivo de identificar os nsSNPs mais provavelmente danosos, e estabelecer aqueles com potencial efeito na função do MC1R. Foram utilizadas 11 ferramentas de predição individuais (SIFT, MutPred, Polyphen-2, PROVEAN, I-Mutant 3.0, PANTHER, SNPs3D, Mutation Assessor, PhD-SNP, SNPs&GO e SNAP) e dois programas consenso (PON-P e PredictSNP 1.0) para a análise de 92 nsSNPs localizados no gene. Os programas utilizados baseiam-se em métodos evolutivos, estruturais e computacionais, resultando na identificação dos 14 nsSNPs mais danosos (L48P, R67W, H70Y, P72L, S83P, R151H, S172I, L206P, T242I, G255R, P256S, C273Y, C289R e R306H). Apesar das diferenças nos resultados de cada programa a combinação dos diferentes métodos permitiu diferenciar os polimorfismos neutros dos danosos, mostrando concordância com os programas consenso. A predição computacional demonstrou ser uma abordagem eficiente para a identificação dos alelos danosos no gene MC1R e para a priorização de mutações para posteriores estudos funcionais e populacionais. / Animal color is a characteristic that presents a large diversity of phenotypes. Different approaches can be used to understand the color diversity existing among and within species. Through analysis of candidate genes the mutations responsible for the color variation have been described in different species, showing the involvement of various molecular mechanisms of regulation. The objective of this work is the use of two genetic approaches to the study of color variation, the analysis of candidate genes and the computational prediction of non-synonym polymorphism effects (nsSNPs). In sheep the wool color is a feature with commercial importance and in identifying breeds. Polymorphisms in different genes have been associated with wool color, but they have not been studied in many breeds that show phenotypic variation regarding such a charactere. The Creole is a local breed from southern most Brazil that presents a wide range of wool color, varying from white to black, and including several intermediate hues. The melanocortin 1 receptor (MC1R) was previously associated with the wool color in the Creole, however, other genes might also be involved in the regulation of color in the breed. This study evaluated the influence of the genes MC1R, ASIP (agouti signaling protein), TYRP1 (tyrosinase related protein 1) and KIT (v-kit Hardy- Zuckerman 4 feline sarcoma viral oncogene homolog) in the Creole breed wool color. Samples from 410 specimens of different colors were analyzed. The variation in the color of the wool was performed by colorimetry. The inheritance pattern of the phenotypes was assessed by crossbreeding individuals of different colors. Polymorphisms in the genes were evaluated by performing sequencing and fragment analysis, and the quantification of the ASIP gene expression was performed by Real Time-PCR. It was observed a significant association between polymorphisms in MC1R and ASIP gene and the wool color in Creole breed. The dominant allele of the MC1R gene, caused by p.M73K and p.D121N mutations was found only in pigmented individuals. This allele leads to the constitutive activation of the receptor and therefore in constant production of eumelanin and is epistatic on the ASIP gene. In the homozygous to the wild-type allele of MC1R the manifestation of white phenotype occurred only in individuals with one allele containing a duplication of the ASIP gene. The carriers of the duplicated copy of ASIP showed high levels of gene expression while homozygous for the simple copy of the ASIP did not expressed the gene, and showed pigmented phenotypes. The results allowed the identification of the influence of epistatic interaction of MC1R and ASIP gene in the wool color in Creole breed. The study of candidate genes involved in the pigmentation pathway proved to be a suitable approach for the analysis of variation in pigmentation in these animals. It is expected that the knowledge acquired in this work will assist on stablishment of commercial breeding and preservation policies of this sheep breed. The computational evaluation of non-synonymous polymorphism has been used to determine SNPs that potentially affect the function of the genes and identify the mechanisms responsible for complex diseases and by the variation in phenotypes. The prediction of the effect of polymorphisms in genes using computational tools presents an alternative approach to the study of the genetic of coloration. The human MC1R gene has a large number of know polymorphisms, some of which were associated with changes in pigmentation and susceptibility to skin tumors. However, many existing variations in the gene have not been evaluated regarding the functional consequences and its role in the variation of pigmentation. Computational prediction of nonsynonymous polymorphisms was performed in the human MC1R gene in order to identify the most likely harmful nsSNPs and to establish those with potential effect on the function of MC1R. Eleven individual tools (SIFT, MutPred, Polyphen-2, PROVEAN, I-Mutant 3.0, PANTHER, SNPs3D, Mutation Assessor, PhD-SNP, SNPs&GO and SNAP) and two consensus programs (PON-P and PredictSNP 1.0) were used to the analysis of 92 nsSNPs located in the gene. The programs used are based in evolutionary, structural and computational methods, resulting in the identification of the 14 most damaging nsSNPs (L48P, R67W, H70Y, P72L, S83P, R151H, S172I, L206P, T242I, G255R, P256S, C273Y, C289R and R306H). Despite the differences in the results of the each program the combination of different methods allowed the differentiation of the neutral polymorphisms from the most damaging, showing agreement with the consensus programs. The computational prediction has proved to be an efficient approach for the identification of harmful alleles in the MC1R gene and for the prioritization of mutations for further functional and population studies.

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.

A cluster of QUT genes within A. nidulans, encoding the enzymes required for the catabolism of quinate to protocatechuic acid, has previously been isolated within the recombinant phage Q1 by hybridization to certain genes in the equivalent N. crassa qa cluster. The location and functional integrity of the QUTE, QUTD and QUTA genes within the QUT gene cluster has been confirmed by the transformation of appropriate A. nidulans qut mutant strains. A.nidulans DNA homologous to the N. crassa qa-2 gene, encoding catabolic dehydroquinase, is able to transform a qutE mutant strain. Biochemical analysis of QUTE transformants containing multiple copies of the QUTE gene has shown that upon induction by quinate there is no increase in the level of catabolic dehydroquinase over that observed in a wild-type strain and that the transformants are subject to normal regulatory control. A. nidulans DNA homologous to the N. crassa qa-y gene is able to transform a qutD mutant strain. Biochemical studies of a number of qutD mutants suggests that in A.nidulans the QUTD gene encodes an essential component of a permease system required for the uptake of quinate. A.nidulans DNA homologous to the N. crassa qa-1F gene is able to transform a qutA mutant strain showing that the QUTA gene is equivalent to the N. crassa qa-1F gene and encodes a positively-acting regulatory protein. A small number of QUTA transformants exhibited constitutive expression of the QUT genes but these strains were subsequently found to be phenotypically unstable and therefore unsuitable for further analysis. DNA sequence analysis of the genes described above by the research group has confirmed their location within Q1 and their physical organisation in chromosome VIII of Aspergillus nidulans.

The aim of this study was to assess the feasibility of using the budding yeast Saccharomyces cerevisiae as a system in which to analyse plant promoters. The promoters chosen for study were the 19S and 35S promoters of cauliflower mosaic virus (CaMV) which, like cellular plant promoters, are transcribed in the plant nucleus by host cell RNA polymerase II. A complete CaMV genome was introduced into yeast on a 2 micron plasmid-based vector and using Northern blot analysis, several CaMV-hybridising transcripts were detected. More precise information on the activity of the promoters was obtained by constructing gene fusions in which the 19S and 35S promoters were linked to the bacterial lacZ gene. Biochemical assays for β-galactosidase showed that cells harbouring the 19S-lacZ gene expressed β-galactosidase but those harbouring the 35S-lacZ gene did not. The insertion of a yeast transcription termination signal upstream of the 19S promoter did not abolish or diminish expression of the 19S-lacZ gene. β-galactosidase was present at low levels in cells expressing 19S-lacZ, constituting less than 0.01% of total cell protein. The 5'ends of 19S-lacZ transcripts present in yeast were mapped by primer extension. The major RNA species initiated approximately 250bp upstream of the 19S-lacZ coding region, indicating the existence of a fortuitous promoter in this region of the CaMV DNA. Two less abundant RNA species initiated within the 19S-lacZ open reading frame at positions +9 and +25bp and may be produced from the genuine 19S promoter. There is evidence to suggest that one or both of these shorter transcripts is the functional mRNA for β-galactosidase. All three classes of RNA were polyadenylated. Coupling of the 19S-lacZ gene to a yeast enhancer (the GAL UAS) produced a 5-fold increase in β-galactosidase activity. At the transcriptional level, activation of the enhancer resulted in a massive increase in the level of the RNA initiating at -250bp but had a minor influence of the levels of the two RNA species initiating at +9 and +25. A series of deletion mutations within the 19S promoter was constructed using Ba131 nuclease. Analysis of these mutations in yeast revealed that sequences from -500 to -193bp and from -137 to -62bp were not required for 19S promoter function, but a deletion from -62 to -21bp (which removes the putative TATA box) severely reduced 19S-1acZ gene expression. Transgenic tobacco plants containing the 19S promoter deletions fused to a CAT gene were produced by A.tumefaciens-mediated gene transfer but the analysis of these plants was not completed.

The homoprotocatechuate degradative pathway of Escherichia coli C contains two isomerisation reactions with chemically similar intermediates, which were thought to be catalysed by distinct but genetically linked enzymes. The possibility that these two isomerases may have arisen from the same ancestral precursor, given their similar substrate structures and close physical location of their genes, was investigated by nucleotide sequencing and purification of the individual enzymes. The purified proteins are of very different subunit molecular weight and have been shown by kinetic measurements to be specific for their respective substrates. The two isomerisation events are separated by an enzyme catalysed decarboxylation and it has been shown that the second isomerisation and the decarboxylation reactions are distinct activities of the same protein subunit. Comparison of the amino acid sequence of the latter with that of the first isomerase of the pathway, reveals a very low level of similarity, suggesting that the two enzymes are unlikely to be derived from a common ancestor. Subcloning of the rest of hpc gene cluster has revealed that the gene order and direction of transcription are not as previously reported. All the genes for the pathway enzymes are transcribed in the same direction and are subject to negative regulation by a protein which appears to be transcribed in the opposite direction. A putative operator site to which the regulatory protein could bind has been located in the same region as a mapped promoter for the pathway genes, which also contains a binding site for the catabolite activator protein. Pairwise comparison of the amino acid sequences of the rest of the Hpc enzymes have shown only low levels of similarity. However, the single dehydrogenase enzyme of the pathway is similar to isoforms of human aldehyde dehydrogenase. The subcloning procedures used in this study have enabled high level expression of several of the pathway enzymes. This has enabled preliminary crystallographic analysis of the isomerase and decarboxylase/isomerase enzymes.