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Combination of Genes

-unique in each cell type within organism
-makes cells different from each other
-ex: photoreceptor neurons express opsin genes but skin cells don't
-determine which genes expressed in cell and at what level can help determine character of cell


DNA Microarrays-What

-used to determine character of cell
-tens of thousands of small DNA fragments spotted onto glass slide
-usually a fragment of each gene or cDNA corresponding to each gene is spotted onto the slide
-ex: human genome ~35,000 so you have ~35,000 genomic or cDNAs spotted onto slides
-fragments of DNA will serve as target for hybridization reaction with mRNA collected from tissue samples


DNA Microarrays-How

-mRNA collected and converted into cDNAs
-fluorescently labeled nucleotides added to reactions such that one sample will emit red or green light when excited with laser beam
-two samples compared
-labeled cDNAs hybridize with genomic fragments affixed to glass slide
-cDNAs from each sample able to simultaneously hybridize with target sequences
-after incubation period, glass slide washed and prepared for detection step
-laser focused on each spot on microarray and computer records hybridization leves


Recordings by Computer

-at most basic level there are four types
-red spot
-green spot
-yellow spot
-black spot
-colors correspond to level of hybridization from two samples


Microarray Expression Profiling Example in notes

-assume green labeled samples correspond to wild type tissue and red corresponds to mutant tissue
-shades of red, green, and yellow in picture corresponds to level of hybridization from each sample
-if green indicates corresponding gene is expressed at higher levels in wild type tissue
-if red indicates corresponding gene expressed at higher levels in mutant tissue
-if yellow indicates corresponding gene expressed at equal levels in both tissues
-if black indicates corresponding gene not expressed in either tissue


cDNA Microarray Exons
vs. Introns

-will involves spotting of fragments that only correspond to exons of coding genes
-intronic and intergenic regions ignored so mRNA transcribed from intronic or intergenic DNA would not have target to hybridize to on array slide
-small genes and ncRNAs often missed in this assay
-computer program that predicts existence of genes is not perfect and some bona fide genes not placed on array slide


Tiling Arrays

-overcome shortcomings of cDNA microarrays
-ideal tiling array uses overlapping DNA fragments
-human tiling array requires several hundred thousand genomic fragments to be spotted onto glass slide
-other arrays will make use of fragments separated by gaps
-size of fragments and gaps can vary
-in all versions RNA samples are still hybridized to slide like in cDNA array


Transcriptional Profile of Genomic Region Containing Two Genes

-picture on slide
-in addition to two genes, see lots of transcription being initiated in intergenic regions
-could correspond to new genes


Review of D/V Patterning

-during D/V patterning there is gradient of Dorsal nuclear localization
-cells along ventral surface have high levels, lateral surface have moderate to low levels, and dorsal surface has no nuclear Dorsal
-asymmetry results in differential gene expression and adoption of distinct cell fates across D/V axis


DNA Microarrays and D/V Patterning

-initially only a handful of dorsal genes were known (twi, sog, rho, sna)
-had some bioinformatic evidence that additional Dorsal targets were encoded in genome
-to identify new targets DNA microarrays were used to identify genes expressed along ventral surface (gives rise to mesoderm), lateral surface (gives rise to neuroectoderm), and dorsal surface (gives rise to ectoderm)


Identification of D/V Genes using DNA Microarrays-Ideal Method

-requires isolation of RNA from tissue samples
-isolate RNA from cells along ventral, lateral, and dorsal surface
-expression profile of each population of cells could then be determined
-not feasible because of technical limitations


Identification of D/V Genes using DNA Microarrays-Actual Method

-manipulation of Toll pathway using mutants to generate whole embryos that mimicked high, low, and absent levels of nuclear localized Dorsal
-accomplished by use of gain-of-function Toll allele, combination of Toll loss-of-function alleles and a pipe loss-of-function allele
-RNA from populations of these embryos were isolated and hybridized to DNA microarray


Identification of D/V Genes using DNA Microarrays-Results

-led to identification of dozens of new genes
-89 genes expressed at higher levels in Toll10B mutant than in other two mutants
-Toll10B mutant has higher nuclear Dorsal levels and thus mimics ventral cells of wild type embryos
-the 89 genes could be new genes that are required for formation of mesoderm
-these genes would be predicted to be expressed along ventral surface like twi and sna


Expression Patterns of New D/V Genes

-determined and compared to genes that were already known to be expressed in mesoderm, neuroectoderm, and ectoderm



-expressed within cells of ventral surface
-known target of Dorsal in part because of embryonic enhancer contains cluster of Dorsal binding sites
-in Toll10B mutant, sna expressed in nearly all cells of embryo
-because Toll10B mutant is gain-of-fucntion allele so all Dorsal is localized to the nucleus in all embryonic cells



-one of 89 new genes expressed at higher levels in Toll10B mutant
-in wild type embryos it's expressed within cells just along ventral surface like snail
-in Toll10B mutant it's also expanded throughout the entire embryo


dpp Gene

-normally expressed with cells that are found along dorsal surface
-in pipe mutants Dorsal protein is prevented from entering nucleus of all cells thus entire embryo mimics dorsal surface
-dpp expressed throughout entire embryo as expected



-found in microarray scan to be expressed at higher levels in pipe mutants than in two other genotypes
-in wild type embryos Ect1 gene transcribed only in cells on dorsal embryo like dpp
-in pipe mutants Ect1 expression is expanded throughout entire embryo, like dpp