Sexagem de embriões bovinos fecundados in vitro pela técnica de PCR multiplex

Neste trabalho, a técnica de PCR (polymerase chain reaction) foi utilizada para a sexagem de 92 embriões bovinos fertilizados in vitro. Os embriões originaram-se de fertilização in vitro de oócitos aspirados de ovários de fêmeas bovinas, provenientes de abatedouros comerciais. Os oócitos foram maturados, fertilizados e cultivados até o estádio de blastocisto. Os embriões foram lavados em solução de PBS, transferidos para tubos de polipropileno contendo água ultrapura, e imediatamente congelados a -196ºC. Os embriões foram descongelados sobre isopor contendo gelo picado e tratados com proteinase K. Para a reação de PCR, utilizaram-se alíquotas de 34 µl de cada tudo, onde foram acrescidos dois pares de primers, seqüência BC1.2 e seqüência satélite 1.715, desoxinucleotídeos, MgCl2, tampão PCR 10X, TaqDNA polimerase e água, em um volume final de 50 µl. As amostras foram amplificadas e a eletroforese realizada em gel de poliacrilamida a 8%. Os géis foram corados com solução de brometo de etídio e analisados em transiluminador de luz ultravioleta. Um índice de 93,47% de amplificação foi atingido, com 41 embriões (47,67%) machos e 45 (52,32%) embriões fêmeas. O uso de gel de poliacrilamida a 8% foi eficaz na separação de fragmentos de DNA muito próximos.

Mitochondrial translation factors of Trypanosoma brucei: elongation factor-Tu has a unique subdomain that is essential for its function

Mitochondrial translation in the parasitic protozoan Trypanosoma brucei relies on imported eukaryotic-type tRNAs as well as on bacterial-type ribosomes that have the shortest known rRNAs. Here we have identified the mitochondrial translation elongation factors EF-Tu, EF-Ts, EF-G1 and release factor RF1 of trypanosomatids and show that their ablation impairs growth and oxidative phosphorylation. In vivo labelling experiments and a SILAC-based analysis of the global proteomic changes induced by EF-Tu RNAi directly link EF-Tu to mitochondrial translation. Moreover, EF-Tu RNAi reveals downregulation of many nuclear encoded subunits of cytochrome oxidase as well as of components of the bc1-complex, whereas most cytosolic ribosomal proteins were upregulated. Interestingly, T. brucei EF-Tu has a 30-amino-acid-long, highly charged subdomain, which is unique to trypanosomatids. A combination of RNAi and complementation experiments shows that this subdomain is essential for EF-Tu function, but that it can be replaced by a similar sequence found in eukaryotic EF-1a, the cytosolic counterpart of EF-Tu. A recent cryo-electron microscopy study revealed that trypanosomatid mitochondrial ribosomes have a unique intersubunit space that likely harbours the EF-Tu binding site. These findings suggest that the trypanosomatid-specific EF-Tu subdomain serves as an adaption for binding to these unusual mitochondrial ribosomes.

Function of cardiolipin in mitochondria of Saccharomyces cerevisiae

Cardiolipin and its precursor phosphatidylglycerol, phospholipids found uniquely in membranes engaged in oxidative phosphorylation, play important roles in multimeric complexes of the energy transducing system (ETS) associated with the inner mitochondrial membrane. A combined molecular genetic and biochemical approach was used to more precisely define the role of cardiolipin in cell processes. ^ Strains of yeast Saccharomyces cerevisiae unable to synthesize cardiolipin because of the crd1Δ allele (encodes cardiolipin synthase) with different phenotypes were analyzed to determine which phenotypes are due to lack of cardiolipin. We concluded that many of the severe phenotypes ascribed to cells lacking cardiolipin, particularly when grown at 37°C, are because of the synergistic interaction of the crd1Δ mutation with the reduced expression of the PET56 gene which encodes a component essential for the formation of functional mitochondrial ribosomes. We also demonstrate that much of the reduced mitochondrial function in crd1Δ is because of reduced expression of ETS components at elevated temperature. ^ A crd1Δ mutant of S. cerevisiae has less severe physiological changes than strains lacking both phosphatidylglycerol and cardiolipin due to an increased level of phosphatidylglycerol, which might partially substitute for the cardiolipin-requiring functions. By varying the level of cardiolipin, we were able to correlate phenotypes in a dose-dependent manner with the level of cardiolipin to support more strongly an involvement of cardiolipin in a particular cellular process. There is almost complete lack of a supercomplex composed of cytochrome bc1 complex (complex III) and cytochrome c oxidase (complex IV) in extracts of cardiolipin-lacking mitochondria when compared to wild type cells and the level of supercomplex varies in proportion to the cardiolipin levels. Reduced cardiolipin levels also compromise the growth properties of yeast in a dose-dependent manner suggesting that the loss in growth efficiency is related to a role of cardiolipin that cannot be replaced by phosphatidylglycerol. An independent kinetic approach was performed to compare organization of the respiratory chain in wild-type and cardiolipin-lacking mitochondria. Cardiolipin-lacking mitochondria display kinetic properties for electron transfer between complexes III and IV via cytochrome c consistent with cytochrome c being a freely diffusible carrier, confirming complexes III and IV exist as individual complexes and not associated into a supercomplex in cardiolipin-lacking mitochondria. ^

Estudio genético y molecular de la respuesta a la vernalización en zanahoria (Daucus carota L.)

La zanahoria es una planta bienal de estación fría que requiere de un período de vernalización para florecer. Sin embargo, algunos cultivares adaptados a zonas más cálidas requieren de menor vernalización y son clasificados como anuales o de floración temprana. El objetivo de esta tesis fue determinar la base genética e identificar los genes y/o regiones cromosómicas involucradas en los requerimientos de vernalización en la zanahoria. Para ello fueron evaluadas a campo familias segregantes F1, F2, F3, RC1 y RC2 obtenidas a partir de un cruzamiento entre una planta anual y una bienal. En base a los patrones de segregación observados se concluyó que la anualidad, o bajos requerimientos de vernalización, estaría determinada por un gen simple dominante. Al evaluar introducciones de zanahoria anuales y bienales de diversos orígenes geográficos y sus cruzamientos, se volvió a observar la total dominancia de la anualidad y se encontró variabilidad en el ciclo entre materiales anuales y entre materiales bienales. Utilizando un método molecular que se basa en similitud completa (BLAST), no se encontró en el genoma de la zanahoria secuencias homólogas al gen FLC, el cual juega un rol central en la respuesta a la vernalización en Arabidopsis y otras especies como las Brassicas. Mediante la técnica de mapeo se encontró una región cromosómica ligada a la respuesta a la vernalización en zanahoria. La misma se localizó en un grupo de ligamiento con 78 marcadores moleculares a una distancia de 0,69 cM y de 0,79 cM de los marcadores más cercanos. Este mapa servirá como base para el desarrollo de marcadores moleculares ligados al carácter y en un futuro para el mapeo físico y secuenciación de la región de interés utilizando una librería génica de BACs de zanahoria.

Silicate content in sediment ofthe Santa Monica Basin, California

Seafloor recycling of organic materials in Santa Monica Basin, California was examined through in situ benthic chamber experiments, shipboard whole-core incubations and pore water studies. Mass balance calculations indicate that the data are internally consistent and that the estimated benthic exchange rates compare well with those derived from deep, moored conical sediment traps and hydrographic modeling. Pore water and benthic flux observations indicate that the metabolizable organic matter at the seafloor must be composed of at least two fractions of very different reactivities. While the majority of reactive organic compounds degrade quickly, with a half-life of <=6.5 years, 1/4 of the total metabolizable organic matter appears to react more slowly, with a half-life on the order of 1700 years. Down-core changes in pore water sulfate and titration alkalinity are not explained by stoichiometric models of organic matter diagenesis and suggest that reactions not considered previously must be influencing the pore water concentrations. Measured recycling and burial rates indicate that 43% of the organic carbon reaching the basin seafloor is permanently buried. The results for Santa Monica Basin are compared to those reported for other California Borderland Basins that differ in sedimentation rate and bottom water oxygen content. Organic carbon burial rates for the Borderland Basins are strongly correlated with total organic carbon deposition rate and bulk sedimentation rate. No significant correlation is observed between carbon burial and bottom water oxygen, extent of oxic mineralization and sediment mixing. Thus, for the California Borderlands, it appears that carbon burial rates are primarily controlled by input rates and not by variations in preservation.

Endosperm balance number manipulation for direct in vivo germplasm introgression to potato from a sexually isolated relative (Solanum commersonii Dun.)

Diploid (2n = 2x = 24) Solanum species with endosperm balance number (EBN) = 1 are sexually isolated from diploid 2EBN species and both tetraploid (2n = 4x = 48, 4EBN) and haploid (2n = 2x = 24, 2EBN) S. tuberosum Group Tuberosum. To sexually overcome these crossing barriers in the diploid species S. commersonii (1EBN), the manipulation of the EBN was accomplished by scaling up and down ploidy levels. Triploid F1 hybrids between an in vitro-doubled clone of S. commersonii (2n = 4x = 48, 2EBN) and diploid 2EBN clones were successfully used in 3x × 4x crosses with S. tuberosum Group Tuberosum, resulting in pentaploid/near pentaploid BC1 progenies. This provided evidence of 2n (3x) egg formation in the triploid female parents. Two selected BC1 pentaploid hybrids were successfully backcrossed both as male and as female parents with S. tuberosum Group Tuberosum. The somatic chromosome number varied greatly among the resulting BC2 progenies, which included hyperaneuploids, but also a number (4.8%) of 48-chromosome plants. The introgression of S. commersonii genomes was confirmed by the presence of S. commersonii-specific randomly amplified polymorphic DNA markers in the BC2 population analyzed. The results clearly demonstrate the feasibility of germplasm introgression from sexually isolated diploid 1EBN species into the 4x (4EBN) gene pool of the cultivated potato using sexual hybridization. Based on the amount and type of genetic variation generated, cumbersomeness, general applicability, costs, and other factors, it would be interesting to compare the approach reported here with other in vitro or in vivo, direct or indirect, approaches previously reported.

Expression of galectin-3 modulates T-cell growth and apoptosis.

Galectin-3 is a member (if a large family of beta-galactoside-binding animal lectins. It has been shown that the expression of galectin-3 is upregulated in proliferating cells, suggesting a possible role for this lectin in regulation of cell growth. Previously, we have shown that T cells infected with human T-cell leukemia virus type I express high levels of galectin-3, in contrast to uninfected cells, which do not express detectable amounts of this protein. In this study, we examined growth properties of human leukemia T cells transfected with galectin-3 cDNA, and thus constitutively overexpressing this lectin. Transfectants expressing galectin-3 displayed higher growth rates than control transfectants, which do not express this lectin. Furthermore, galectin-3 expression in these cells confers resistance to apoptosis induced by anti-Fas antibody and staurosporine. Galectin-3 was found to have significant sequence similarity with Bcl-2, a well-characterized suppressor of apoptosis. In particular, the lectin contains the NWGR motif that is highly conserved among members of the Bcl-2 family and shown to be critical for the apoptosis-suppressing activity. We further demonstrated that galectin-3 interacts with Bc1-2 in a lactose-inhibitable manner. We conclude that galectin-3 is a regulator of cell growth and apoptosis and it may function through a cell death inhibition pathway that involves Bcl-2.

Platelet-derived growth factor induces apoptosis in growth-arrested murine fibroblasts.

The platelet-derived growth factor (PDGF) is a potent mitogen for murine fibroblasts. PDGF-stimulated cells express a set of immediate-early-response genes but require additional (progression) factors in serum to progress through the cell cycle. Serum-deprived cells are reversibly arrested in G0 phase and fail to fully traverse the G1 phase of the cell cycle when stimulated by PDGF alone. We now report that serum-deprived normal rat kidney fibroblast (NRK) cells stimulated by either PDGF AA or PDGF BB homodimers undergo apoptotic cell death. Furthermore, we show that epidermal growth factor also induces apoptotic cell death in serum-deprived NRK cells, epidermal growth factor enhances the rate of apoptosis in PDGF-treated cells, and a progression factor (insulin) but not endogenously expressed Bc1-2 fully protects NRK cells from PDGF-stimulated apoptosis. The results indicate that PDGF induces apoptosis in growth-arrested NRK cells and that the inability of NRK cells to transit the G1/S checkpoint is the critical determinant in establishing the genetic program(s) to direct the PDGF signal to apoptosis. The results suggest that polypeptide growth factors in vivo may signal cell fate positively or negatively in settings that limit the potential of cells to completely transit the cell cycle.

Construction of a 2.8-megabase yeast artificial chromosome contig and cloning of the human methylthioadenosine phosphorylase gene from the tumor suppressor region on 9p21.

Many human malignant cells lack methylthioadenosine phosphorylase (MTAP) enzyme activity. The gene (MTAP) encoding this enzyme was previously mapped to the short arm of chromosome 9, band p21-22, a region that is frequently deleted in multiple tumor types. To clone candidate tumor suppressor genes from the deleted region on 9p21-22, we have constructed a long-range physical map of 2.8 megabases for 9p21 by using overlapping yeast artificial chromosome and cosmid clones. This map includes the type IIFN gene cluster, the recently identified candidate tumor suppressor genes CDKN2 (p16INK4A) and CDKN2B (p15INK4B), and several CpG islands. In addition, we have identified other transcription units within the yeast artificial chromosome contig. Sequence analysis of a 2.5-kb cDNA clone isolated from a CpG island that maps between the IFN genes and CDKN2 reveals a predicted open reading frame of 283 amino acids followed by 1302 nucleotides of 3' untranslated sequence. This gene is evolutionarily conserved and shows significant amino acid homologies to mouse and human purine nucleoside phosphorylases and to a hypothetical 25.8-kDa protein in the pet gene (coding for cytochrome bc1 complex) region of Rhodospirillum rubrum. The location, expression pattern, and nucleotide sequence of this gene suggest that it codes for the MTAP enzyme.

Moving molecules: mRNA trafficking in mammalian oligodendrocytes and neurons

Numerous mRNA molecules are localized in regions of the dendrites of neurons, some moving along dendrites in response to synaptic activity. The proteins encoded by these RNAs have diverse functions, including participation in memory formation and long-term potentiation. Recent experiments have shown that a cytoplasmic RNA trafficking pathway described for oligodendrocytes also operates in neurons. Transported RNAs possess a cis-acting element that directs them to granules, which are transported along microtubules by the motor proteins kinesin and dynein. These RNA molecules are recruited to the cytoplasmic transport granules by cooperative interaction with a cognate trans-acting factor. mRNAs containing the 11-nucleotide A2RE11 or 21-nucleotide A2RE sequences bind heterogeneous nuclear ribonucleoproteins A2 and A3, which are abundant in the brain. Mutations in this cis-acting element that weaken its interaction with hnRNP A2 also interfere with RNA trafficking. Several dendritically localized mRNAs, including those encoding calcium-calmodulin-dependent protein kinase 11 a subunit and neurogranin, possess A2RE-like sequences, suggesting that they may be localized by interaction with these heterogeneous nuclear ribonucleoproteins. Calcium-calmodulin-dependent protein kinase 11 a subunit is of particular interest: Its RNA is transported in depolarized neurons, and the protein it encodes is essential for establishing long-term memory. Several other cis-acting sequences and trans-acting factors that participate in neuronal RNA localization have been discovered.

Non-coding RNAs in the nervous system

Increasing evidence suggests that the development and function of the nervous system is heavily dependent on RNA editing and the intricate spatiotemporal expression of a wide repertoire of non-coding RNAs, including micro RNAs, small nucleolar RNAs and longer non-coding RNAs. Non-coding RNAs may provide the key to understanding the multi-tiered links between neural development, nervous system function, and neurological diseases.

GENETIC EVALUATION OF SEED TRAITS FROM INTRASPECIFIC CROSSING OF GENETICALLY DISTINCT WATERMELON VARIETIES

Citrullus lanatus (Thunb.) Matsumura and Nakai (Cucurbitaceae) is an important cucurbit crop worldwide. Global production of watermelon is about 90 million metric tonnes per annum, making it among the top five most consumed fresh fruits. The objective of this study was to evaluate seed variability in different segregating populations, and determine heritability of traits of watermelon. Interspecific crosses were made between two cultivars of C. lanatus (Bebu and Wlêwlê Small Seeds (WSS) were performed at Research Station of Nangui Abrogoua University in Abidjan, Côte d’Ivoire. There was wide variability between parental, F1, BC1 (first generation of back-crossing) and F2 seeds. Seeds of all hybrid populations were intermediate versus those of the parents. Also, crossing did not affect F1 and F2 seed characters, but affected those of BC1 because of maternal effects. Thus, back-crossing on Bebu cultivar produced seeds which looked like those of Bebu; while back-crossing on WSS cultivar produced seeds similar to those of WSS. Principal Component Analysis (PCA) and individuals repartitioning revealed that Bebu and WSS cultivars were genetically distinct and showed three main groups: two groups from each parental line and one from a recombinant line (hybrids). F2 population had a wide individual’s dispersion, and contained seeds of all other populations. High heritability was observed for all evaluated characters.