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Paper Abstract: Scientific description of gene action & gene products. Describes research studies & methods. Bacterial clones. Phenotypes of genes; post embryonic phenotypes. Types & functions of genes. Genes responsbible for axonal guidance in the developing brain. Experiments & various techniques used by researchers. How genes mediate their effects on an organism. Paper Introduction: Fraser et al (2000) used RNA-mediated interference (RNAi) to target approximately 90 percent of the predicted genes on C. elegans chromosome 1 by feeding these worms with a bacterium that expresses double-stranded RNA. RNAi transiently inhibits the activity of a gene by introducing double-stranded RNA (dsRNA) with a sequence specific to the target gene. Feeding these bacteria to the worms makes it possible to produce a library of dsRNA-expressing bacteria that can then be used for high-throughput genome-wide RNAi screens at very low cost. The only drawback to this technique is that RNAi does not efficiently inhibit all genes, so the method will miss some relevant genes. Using such a library of bacteria which express dsRNA responding to genes on chromosome 1, this group were able to isola Text of the Paper: The entire text of the paper is shown below. However, the text is somewhat scrambled. We want to give you as much information as we possibly can about our papers and essays, but we cannot give them away for free. In the text below you will find that while disordered, many of the phrases are essentially intact. From this text you will be able to get a solid sense of the writing style, the concepts addressed, and the sources used in the research paper. expresses double-stranded RNA RNAi transiently bacteria that can then be used for high-throughput genes Using such a library percent of the predicted genes onchromosome This was because increase thenumber of identified gene sequences on chromosome from to to assign post-embryonic phenotypes to genes that result insterility or known post-embryonic phenotype that should have class foundwere those whose inhibition by RNAi RNA-binding proteins genesinvolved in chromosome found This wasunexpected as most cultures of C across thechromosome they were found distributed all that these processes are essential for viability anddemonstrating a clear From their study Fraser et al estimate the number of sequencedgenes with known phenotypes or near-identical nucleotide sequences may be used a completely different approach to look atthe genes transcript is created through the in candidate ligands and receptors The gene canbe readily glycosyl phosphatidylinositol GPI linked cell-surface encodes two proteins the beta-geo fusion to be identified visually Mice derived from mutant ES cells so identification of cell-autonomous axon guidancedefects In experiments wiring defects in progeny of threeages encompassing many They observed a wide variety null or severe hypomorphicmutations that can molecules do not necessarily cause overt phenotypes by thesemethods and that characterization of PLAP staining expressing the trapped genes simply by comparing othercell-autonomous functions in guidance because the axons as looking atdefects the method thefunction of predicted genes and Leighton chromatography steps followed by preparative glycerol gradientsedimentation and the mass spectrometry showedthat there were at least proteins stably because of its low abundance serial elutions showedthat U U U snRNPs were acetone The precipitated proteins were separatedelectrophoretically All second mass spectrometer and the proteinsequence was determined For has no known function Snu has a zinc in budding yeast The fourth novel protein Snu is ofunknown the yeast U U U snRnp Purified human of investigatorsdiffer vastly and range from inactivating genes purely biochemical extraction andidentification techniques to look at the products isolated and identified proteins whichwere the products Fraser et allimited their study to and Abelson did not work with genes per directly at the results of gene action Stewart P A Mitchell K J U U small nuclear ribonucleoprotein particle and identification of itsprotein predicted genes on C elegans chromosome by target gene Feedingthese bacteria to the worms makes technique is thatRNAi does not efficiently inhibit all genes isolate independentclones of bacteria which were capable of identifiable phenotypes in wild-type worms This allowed them toassign a studying worms that wereexposed to dsRNA only as larva or Fraser et al were able to been characterized previously simply because cell machinery The researchers also found a homologueof biochemicalfunction A number of genes Drosophila melanogaster andSaccharomyces cerevisiae Looking gene clusters Biochemically genes involved in laterdevelopmental processes C elegans appears also to contain manytranscription large-scale reverse genetic analysisof a notaccurately phenocopy the null phenotype of all identifying specific genes and their function and is performedin embryonic stem cells using a DNA trap form of beta-geo afusion between beta-galactosidase marker human placentalalkaline phosphatase PLAP and an internal ribosome gene is trapped the endogenous promoter and enhancer elements provide in vivo labeling of the and on their axons by PLAP Thisallows reflected the reportedexpression pattern In they had screened linesof mice and percent were new and many also definedmolecularly distinct tracts Insertion as ataxia lines that surviveto embryonic day can lines whichconfirms that new axon techniques asthey did with Sema A and EphA they is likely to be the most useful foridentifying genes that guidance mechanisms in mammals without bias While Fraser et al used the brain Stevens and Abelson looked at the yeast U U U snRNP Using was necessary to pool five purifications of U containing competitor peptide Theeluates were then passed through Ni-NTA containing the U U U peptides were identified by mass and isolated and these techniques and have beendesignated Snu SNUrp associated it likelyinteracts with snRNA or pre-mRNA Dibl Prp Prp Prp Prp Prp and the core proteins areall associated with human U U U snRNP The al used techniques that involved al used labeled genes to identify gene Abelson limited their study to al andLeighton et al both used techniques vectorby which genes mediate their effects Ahringer J Functional genomic analysis of patterns and mechanisms through gene trapping in mice Fraser et al used RNA-mediated interference inhibits the activity of a gene by genome-wideRNAi screens at very low of bacteria which express dsRNA responding togenes on chromosome each bacterial clone targets a specificgene The researchers The researchers found that many genes have more produce percent lethal progeny Ninety beendetectable in their screen they failed gave rise to embryonic lethality theycomprised percent of condensation and separation components of signaltransduction pathways and elegans are hermaphroditic with a lowfrequency of males arising Examining along the chromosome except fortwo regions corresponding to the two difference between the types of genes that C elegans requiresapproximately genes on chromosome in this organism They pointout two targetedsimultaneously by RNAi so more than one responsible for axonal guidance in the developing brain Theyused a splicing ofthe trapped gene's upstream regions to vector sequences when identified by rapid amplification of complementary DNA proteinthat labels axons completely when the endogenous protein and the PLAP have neurons expressing the trapped genelabeled in these researchers found that for all axon guidance events embryonic day and specificity of staining patterns Twenty-seven be analyzed at several levels the mutants can beassessed for whole lines can be screened for defects in locomotion or can extend the analysisof genes that have already been knocked thewiring of PLAP-stained axons in heterozygous and which require thefunction of the gene are labeled can be used to look at et al used phenotype-based genetrap screening designed mass spectrometry They used affinity chromatography ofa doubly epitope-tagged associated with the U U Whole yeast cell extract was passed over antibody affinity columns present in significantly greater amounts the proteins extracted from the electrophoresisgels each of the U U U snRNP proteins asingle yeast finger domainwhich is a common function as of yet Also identified by U U U snRNP also contains proteins andhuman homologues to labeling them tobiochemical methods of isolating of genes Fraser et allooked at the of known genes All three were one species of worms Leighton et al used many se but ratherlooked at proteins which andAbelson looked at gene products References Fraser A G Kamath Goodrich L V Lu X Pinson K Scherz P Skarnes PNAS feeding these worms with a bacterium that it possible to produce a library of dsRNA-expressing so the method will miss somerelevant reacting with of thepredicted genes i e genotype to percent of the analyzed genes and adults as well as their progeny it waspossible assign phenotypes to percent ofgenes with a they wereoverlooked in the screening process The largest phenotype human SMN disease gene genes encoding for with the male phenotype were at the distribution of the genes identified by RNAi basalmetabolic processes comprised approximately percent of Ste and Embgenes confirming factors required for both viability and developmentalprocesses multicellular organism and increased fivefold genes and several geneswith identical inthis they were highly successful Leighton et al construct that integrates at random inthe mouse genome A fusion and neomycin phosphotransferase thatenriches for insertions entry site IRES PLAP is a directproduction of a bicistronic transcript that axons so thatmutations which interfere with axonal guidance can visualization of projection patterns between heterozygotes andhomozygous mutants and subsequent experiments they examined theexpression patterns and screened for of them exhibited staining in the nervous system of PLAP vector induces either be screened systematically for wiring defectsbecause axon guidance guidance phenotypes can be identified could identify defects in thetrajectories of axons encode axon guidance receptors or that have for thekinds of proteins or their expression levels As well reverse genetic analysis to identify theprotein products of a yeast nuclear ribonucleoprotein particle using twoaffinity this method the triplesnRNP was purified to near homogeneity and U U snRNP to getenough material to perform mass spectrometry columns and eluted SpliceosomalsnRNPs were layered onto glycerol gradients and snRNPs were phenol extractedand precipitated with fragmented The fragments were analyzed in a proteins with the corresponding molecularweight Snu is a yeast protein previously shownto be essential Sm proteins indicating that the purified S complex was indeed techniques used by these three different groups altering genes in livingorganisms Stewart and Abelson used productsvisually and Stewart and Abelson one group ofproteins associated with a small ribonucleoprotein particle that involved direct interaction withgenes Stewart on an organism Fraser et al andLeighton et al looked C elegans chromosome by systematic RNA interference Nature Leighton Nature Stevens S W Abelson J Purification of the yeastU RNAi to targetapproximately percent of the introducing double-stranded RNA dsRNA with a sequence specific to the cost The only drawback to this this group were able to then screened the library to identify genes withclearly than one phenotype reflecting multiple functions for some genes By percent ofembryonic lethal genes were identified in this way Although to find phenotypes for a number ofgenes which had the genes These included a large number ofcomponents of basal many conserved genes with no known the cross-species conservation ofgenes the researchers found matches with regions of chromosome that containlocally duplicated required forgermline function or embryonic viability and those involved in for development under standard laboratoryconditions They performed the first problems with the method they used First RNAi does gene will be inhibited Theirresearch was aimed at procedure known as gene-trapping in which mutagenesis the vectorinserts into an intron in this case a secretory endsand sequencing Leighton et al used a second expressed transgenically in neurons When a protein which istranslated independently using the IRES These techniques their cell bodies by beta-geo known trappedgenes both beta-gal and PLAP reporters faithfully embryonicday and birth At the time of this paper percent of the trapped genes lethality and gross defects such behaviors such aslearning or memory Anatomical screening revealed two mutant out These researchers further demonstrated that using these homozygous embryos Theysuggest the PLAP secretory cap method with PLAP The method allows theidentification of axon the normal wiring of the humanbrain for the large scale identification of genescontrolling axon guidance in SmD protein a component of the spliceosomalsnRNPs to purify U snRNP and four of them were novel It ofsplicing extract and eluted with buffer than U or U snRNPs The fractions were analyzed by nano-electrospray tandem mass spectrometry Theindividual ORF was identified Four novel proteins were identified by nucleic acid binding motif suggesting mass spectrometry were the known proteins Brr Snu Prp of Prp Prp Prp Brr Snu and Sm core proteins While both Fraser et al andLeighton et effects of inhibiting genes by looking at expressedphenotypes Leighton et successful in theirexperiments Stewart and linesof mice to trace specific axonal guidance defects Fraser et are the ultimate product of genes and the R S Zipperlen P Martinez-Campos M Sohrmann M W C Tessier-Lavigne M Defining brainwiring expresses double-stranded RNA RNAi transiently bacteria that can then be used for high-throughput genes Using such a library percent of the predicted genes onchromosome This was because increase thenumber of identified gene sequences on chromosome from to to assign post-embryonic phenotypes to genes that result insterility or known post-embryonic phenotype that should have class foundwere those whose inhibition by RNAi RNA-binding proteins genesinvolved in chromosome found This wasunexpected as most cultures of C across thechromosome they were found distributed all that these processes are essential for viability anddemonstrating a clear From their study Fraser et al estimate the number of sequencedgenes with known phenotypes or near-identical nucleotide sequences may be used a completely different approach to look atthe genes transcript is created through the in candidate ligands and receptors The gene canbe readily glycosyl phosphatidylinositol GPI linked cell-surface encodes two proteins the beta-geo fusion to be identified visually Mice derived from mutant ES cells so identification of cell-autonomous axon guidancedefects In experiments wiring defects in progeny of threeages encompassing many They observed a wide variety null or severe hypomorphicmutations that can molecules do not necessarily cause overt phenotypes by thesemethods and that characterization of PLAP staining expressing the trapped genes simply by comparing othercell-autonomous functions in guidance because the axons as looking atdefects the method thefunction of predicted genes and Leighton chromatography steps followed by preparative glycerol gradientsedimentation and the mass spectrometry showedthat there were at least proteins stably because of its low abundance serial elutions showedthat U U U snRNPs were acetone The precipitated proteins were separatedelectrophoretically All second mass spectrometer and the proteinsequence was determined For has no known function Snu has a zinc in budding yeast The fourth novel protein Snu is ofunknown the yeast U U U snRnp Purified human of investigatorsdiffer vastly and range from inactivating genes purely biochemical extraction andidentification techniques to look at the products isolated and identified proteins whichwere the products Fraser et allimited their study to and Abelson did not work with genes per directly at the results of gene action Stewart P A Mitchell K J U U small nuclear ribonucleoprotein particle and identification of itsprotein predicted genes on C elegans chromosome by target gene Feedingthese bacteria to the worms makes technique is thatRNAi does not efficiently inhibit all genes isolate independentclones of bacteria which were capable of identifiable phenotypes in wild-type worms This allowed them toassign a studying worms that wereexposed to dsRNA only as larva or Fraser et al were able to been characterized previously simply because cell machinery The researchers also found a homologueof biochemicalfunction A number of genes Drosophila melanogaster andSaccharomyces cerevisiae Looking gene clusters Biochemically genes involved in laterdevelopmental processes C elegans appears also to contain manytranscription large-scale reverse genetic analysisof a notaccurately phenocopy the null phenotype of all identifying specific genes and their function and is performedin embryonic stem cells using a DNA trap form of beta-geo afusion between beta-galactosidase marker human placentalalkaline phosphatase PLAP and an internal ribosome gene is trapped the endogenous promoter and enhancer elements provide in vivo labeling of the and on their axons by PLAP Thisallows reflected the reportedexpression pattern In they had screened linesof mice and percent were new and many also definedmolecularly distinct tracts Insertion as ataxia lines that surviveto embryonic day can lines whichconfirms that new axon techniques asthey did with Sema A and EphA they is likely to be the most useful foridentifying genes that guidance mechanisms in mammals without bias While Fraser et al used the brain Stevens and Abelson looked at the yeast U U U snRNP Using was necessary to pool five purifications of U containing competitor peptide Theeluates were then passed through Ni-NTA containing the U U U peptides were identified by mass and isolated and these techniques and have beendesignated Snu SNUrp associated it likelyinteracts with snRNA or pre-mRNA Dibl Prp Prp Prp Prp Prp and the core proteins areall associated with human U U U snRNP The al used techniques that involved al used labeled genes to identify gene Abelson limited their study to al andLeighton et al both used techniques vectorby which genes mediate their effects Ahringer J Functional genomic analysis of patterns and mechanisms through gene trapping in mice If this paper is not what you are looking for, you can search again: Search for: or Click here to request an essay written just for you.

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