Slide 1Spotted MicroarraysSlide 3Spotted DNA MicroarraySlide 510_15_DNA.microarrays .jpgSlide 7Slide 8Slide 9Slide 1010_02_cell_sorter.jpgSlide 12Slide 13Slide 14Slide 15Slide 16Slide 17Slide 18Slide 19Slide 20Slide 21FertilizationSlide 23Homologous chromosomes pair before lining up on the spindle during meiosisSlide 25Maternal and paternal chromatids recombine in paired chromosomes to form chiasmataChiasmata ensure proper pairing and segregation of chromosomes in meiosis IKinetochores on sister chromatids function independently in meiosis II (as in mitosis)Slide 29Meiosis generates new combinations of genes in two waysErrors during chromosome segregation result in gametes with extra or missing chromosomesSlide 32Slide 33Slide 34Slide 35Slide 36Slide 37Slide 38Figure 7.1 The Human Infant Preformed in the Sperm, as Depicted by Nicolas Hartsoeker (1694)Slide 40Development of a human embryo from fertilization to implantationSlide 42Slide 43Slide 44Slide 45Slide 46Slide 47Slide 48Phospholipase C activates two signaling pathways How can fertilization activate Phospholipase C?Phospholipase C activates two signaling pathwaysSlide 51Slide 52Slide 53Slide 54Slide 55Slide 56Slide 57Experiments to test importance of Ca++Slide 59Microarrays (gene chips) for assaying global gene expression patterns(some slides courtesy of J. Lieb)Spotted Microarrays•PCR products (direct from genome, or from cDNA clones), or oligonucleotides are spotted by capillary action onto a glass microscope slide.•Up to ~44,000 features per slide •Typically hybridized with 2 differentially labeled samples simultaneously.The Fordham Hall DNA MicroarrayerSpotted DNA Microarray18,000 PCR-amplified cDNA clone insertsPrinted on poly-lysine coated 1 X 3 inch glass slide Each spot corresponds to a different genecirca 1998TumorPool of Cell LinesA Typical DNA Microarray ExperimentReference or ControlExperimentalSampleLower in TumorHigher in Tumor(tests thousands of genes at a time)10_15_DNA.microarrays .jpgMicroarray technologyincreased expressiondecreased expressionUse computers to keep track of expression levels of all of the genes in large datasets. Use bioinformatics software to identify genes with interesting expression characteristics.these genes have higher expression in normal tissue than breast cancersthese genes have lower expression in basal-like breast cancers than in normal tissue or luminal breast cancersExpression profiling:overall patterns of gene expression can be used in diagnosisEach column is from a different tumor.Each row represents one gene. Rows are clustered by similar expression pattern.Chu et al., Science 282: 699-705 (1998)Transcription response during yeast sporulation (1116 out of ~5000 genes changed)Iyer et al., Science 283: 83-87 (1999) Transcriptional response of human fibroblasts to serum(~6% of genes on microarray changed)10_02_cell_sorter.jpgUsing a fluorescence-activated cell sorter (FACS) to separate cellsBirnbaum et al., Nature Methods 2, 615 - 619 (2005)Brady et al., Science 318: 801-806 (2007) Global expression map depicting major patterns of gene activity in Arabidopsis root cell types(Each row represents a group of genes with similar expression pattern)Sorted cell typeThe latest technology: RNA-SeqUses “next-generation” sequencing methodsRNA-Seq summary(tests virtually all genes at once)Sequence millions of cDNA molecules.Determine which gene each sequence came from.Compile data in computers.Reverse transcription to make cDNA copies of many RNA molecules in a sampleZhang G et al. Genome Res. 2010;20:646-654©2010 by Cold Spring Harbor Laboratory PressAligning short sequence reads to genome sequence to identify and count transcriptsSequence readsZhang G et al. Genome Res. 2010;20:646-654©2010 by Cold Spring Harbor Laboratory PressAligning short sequence reads to genome sequence to identify and count transcriptsVenn diagram showing the genes expressed in each of the three stages of grape berry developmentZenoni S. et.al. Plant Physiol. 2011:152:1787-1795Copyright © 2010. © 2010 American Society of Plant Biologists. All rights reserved.Suppose that in a particular developmental state, 5000 genes are expressed.Which genes are important for specifying that developmental state?Suppose that in a particular developmental state, 5000 genes are expressed.Which genes are important for specifying that developmental state?Suppose that 500 of those genes change in expression level after a signaling event.Which genes are important for carrying out the change induced by the signal?Coordinated regulation of multiple genesDevelopmental functionsFertilizationHow are gametes produced?How does fertilization occur?What events does fertilization initiate?Homologous chromosomes pair before lining up on the spindle during meiosisMeiosismeiosis II resembles mitosismeiosis IMaternal and paternal chromatids recombine in paired chromosomes to form chiasmataSister chromatids are held together by cohesinsChiasmata ensure proper pairing and segregation of chromosomes in meiosis IKinetochores on sister chromatids function independently in meiosis II (as in mitosis)Meiosis generates new combinations of genes in two ways(Some colors are wrong in this diagram.)Errors during chromosome segregation result in gametes with extra or missing chromosomesWould these be viable?Meiosismeiosis II resembles mitosismeiosis IOogenesis occurs before birth in humans.(from Gilbert, Developmental Biology)Oocyte formation in ovarian follicle(from Gilbert, Developmental Biology)Ovulation in rabbit ovaryCell cycle progression in maturing frog oocyteThe immature oocyte is arrested at G2 of meiosis I until stimulated by hormone.Cell cycle progression in maturing frog oocyteProgesterone stimulates meiosis I (oocyte maturation). The mature oocyte arrests again at metaphase of meiosis II.It stays arrested until fertilized by a sperm.SpermatogenesisFigure 7.1 The Human Infant Preformed in the Sperm, as Depicted byNicolas Hartsoeker (1694)The sperm: a stripped downmachine for delivering DNADevelopment of a human embryo from fertilization to implantationFertilization: a multistepprocess(ECM)chemotaxis of spermContact between the sperm and the egg Zona pellucidaSperm galactosyltransferase binds ZP3protein(from Gilbert, Developmental Biology)Releases ZP-degrading enzymesSperm entry into hamster egg(from Gilbert, Developmental Biology)(from Gilbert, Developmental Biology)Fusion of sperm and egg plasma membranesFertilization
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