No description available.

No description available.

No description available.

No description available.

No description available.

The differentiation of melanocytes from multipotential neural crest cells is an ideal system for studying the processes underlying lineage determination in development. Tyrosinase is a key enzyme in melanin biosynthesis and the activation of the tyrosinase gene is characteristic of differentiated melanocytes. In order to study the mechanisms underlying activation of melanocyte-specific genes during differentiation in chick embryos, a chicken genomic DNA library was screened for tyrosinase-encoding sequences using a mouse tyrosinase cDNA probe. Two identical hybridising clones were identified. Restriction mapping and sequencing revealed that both clones contained a 4.3 kb genomic DNA fragment, CTYR4.3, that included 2125 nt of the 5' flanking region, the first exon and part of the first intron of the chicken tyrosinase gene. The 5' flanking sequence of CTYR4.3, which is the most extensive to be reported for a lower vertebrate tyrosinase gene to date, was analysed further using computer-aided homology searches and primer extension. Alignment of the promoter sequences of CTYR4.3 with those of the human, mouse, quail and turtle tyrosinase genes revealed two evolutionary conserved regions. These regions may be functionally significant as they contain regulatory elements previously reported to play a role in melanocyte-specific expression of the tyrosinase gene in mammals. These include an initiator region and an associated SP1-binding site, the M-box and an upstream enhancer element, TDE. In addition, other potential transcription factor binding motifs were identified, including an AP-1-binding site, a UV-responsive element and glucocorticoid-responsive elements. Although several TATA box motifs were identified, they were situated more than 200 bp upstream of the transcription start sites mapped by primer extension analysis and therefore are unlikely to function as TFIID-binding sites. Transcription initiation appears to occur at heterogeneous start sites, and given the absence of a functional TATA box, may be mediated via the conserved initiator region and SP1-binding site. To test the ability of the 5' flanking sequence of CTYR4.3 to drive transcription and to begin to assess the functional significance of the various conserved elements, transient transfection assays were carried out. Constructs were generated in which 2.1 kb, 1.1 kb, 0.5 kb and 0.2 kb fragments of the 5' flanking sequence were linked to a luciferase reporter gene. These constructs were introduced into cultures of chicken retinal pigment epithelial cells (RPE), immortalised quail neural crest cells (MQTNC), and human liver cells (Hep G2) by calcium phosphate-mediated transfection. Transfections with all constructs resulted in luciferase activities significantly greater than those that were observed with the promoterless luciferase construct, thus confirming that the 5' flanking sequence of CTYR4.3 does possess promoter activity. However, the level of expression from the various constructs differed markedly in the different cell types. In the tyrosinase-negative Hep G2 cells, low levels of expression were observed with all constructs. In the tyrosinase-positive RPE cells, a high level of luciferase activity was obtained specifically with the smallest (0.2 kb) promoter construct. Since the 0.2 kb promoter fragment does not include the conserved initiator region, SP1-binding site, or M-box, the role of these elements in tissue-specific transcription initiation of the chicken tyrosinase gene is now questionable. These results suggest the existence of transcription regulatory mechanisms that are unique to avians and possibly other lower vertebrates. In contrast to the results obtained for RPE cells, the highest luciferase activity was obtained with the full length 2.1 kb promoter construct in the immortalised quail neural crest-derived cells. These results may have developmental significance since they suggest that the chicken tyrosinase gene promoter is regulated differently in RPE cells and neural crest-derived cells.

The objective of this study was to identify peptide ligands, using phage display techniques, which bind sites on mouse embryos, ovaries, cytoplasmic membranes and/or intracytoplasmic components. Specifically, M13 coliphage 7-mer, 12-mer and 15-mer random peptide libraries were used separately for biopanning. Peptides derived from the amplified pools were sequenced and studied. The phage display for in vivo ovary experiments yielded no pool of peptides after two cycles of biopanning and re-amplification. With the same initial concentration of a random 7-mer or 12-mer library, there were repeating sequences derived after three and four biopanning cycles on mouse embryos and unfertilized ova. The sequences were not distinguishable from a control group. Subsequent experimentation using a random 15-mer library to select for internalized phage-peptides yielded two apparent consensus sequences, RNVPPIFNDVYWIAF (9/32 or 28%) and HGRFILPWWYAFSPS (11/32 or 34%). The 15-mer control group yielded no clones. The deduced peptide sequences were compared to known sequences to ascertain their uniqueness. No significant similarities were found, yielding two possible novel motifs. Through this adapted process of phage display and further research, the phage display technology may be used as a tool in the recognition of specific mouse gamete sites. By identifying binding sites of mouse gametes, the peptides might be exploited as a means of studying the embryo cell surface or cytoplasmic components and mouse sperm-egg interactions. Such peptides may also be used for macromolecule delivery in transfection or transgenesis. / Master of Science

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No description available.

O ácido retinóico (AR) é essencial para a embriogênese. A principal enzima sintetizadora de AR durante o desenvolvimento é a ALDH1A2 (RALDH2), uma retinaldeído desidrogenase que converte retinaldeído a AR. Para entendermos como o gene da aldh1a2 é regulado identificamos regiões evolutivamente conservadas (ECRs) em vertebrados e testamos seu potencial regulatório. Identificamos uma ECR localizada no intron1 da aldh1a2, conservada em anfíbios, aves e mamíferos que atua como um enhancer em estruturas derivadas de ectoderme, endoderme e mesoderme. Animais transgênicos transientes e permanentes mostram a ativação desse enhancer na região da placa do teto do tubo neural e epicárdio, local onde esse enhancer é ativado em células derivadas do órgão pro-epicárdico após o contato e/ou proximidade com células do miocárdio. A identificação de um enhancer conservado no gene da aldh1a2 suporta a idéia de que esse gene possui uma regulação modular e mostra que a abordagem evolutiva é uma eficiente ferramenta para a identificação de mecanismos de controle desse gene. / Retinoic acid (RA) is essential for embryogenesis. The key RA synthetic enzyme during early development is ALDH1A2 (RALDH2), a retinaldehyde dehydrogenase that converts retinaldehyde into RA. To understand how aldh1a2 is regulated we screened the gene for evolutionary conserved regions (ECRs) among vertebrates and assayed their regulatory potential. We describe an aldh1a2 intron 1 ECR (identified as RALDH2.2) that is conserved in amphibians, avians and humans and acts as an enhancer in derivatives of ectoderm, endoderm and mesoderm. Transient and stable transgenesis in mice reveal strong activity of the raldh2 intron 1 enhancer at the roof plate of the neural tube and at the growing epicardium. Transgenic mice indicate that the enhancer is activated in proepicardium-derived cells by contact and/or close proximity to the myocardium. The identification of an aldh1a2 conserved enhancer supports the idea of a modular regulation and shows that the evolutionary approach is an efficient tool to identify control mechanisms of the aldh1a2 gene.