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GENE RESEARCH.
Term Paper ID:30116
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Essay Subject:
Scientific description of gene action & gene products.... More...
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9 Pages / 2025 Words
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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
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Snu 23 has a zinc finger domainwhich is a common nucleic acid binding motif, suggesting it likelyinteracts with snRNA or pre-mRNA. From their study, Fraser et al estimate that C. A fusion transcript is created through the splicing ofthe "trapped" gene's upstream regions to vector sequences when the vectorinserts into an intron: in this case, a secretory trap form of beta-geo (afusion between beta-galactosidase and neomycin phosphotransferase) thatenriches for insertions in candidate ligands and receptors. This was because each bacterial clone targets a specificgene. In experiments, these researchers found that for all known trappedgenes, both beta-gal and PLAP reporters faithfully reflected the reportedexpression pattern. J., Goodrich, L. W., & Abelson, J. (2 1). References Fraser, A. Fraser et al andLeighton et al both used techniques that involved direct interaction withgenes. The fractions containing the U4/U6.U5/ snRNPs were phenol extractedand precipitated with acetone. Examining the cross-species conservation ofgenes, the researchers found matches with Drosophila melanogaster andSaccharomyces cerevisiae. elegans are hermaphroditic, with a lowfrequency of males arising. Although Fraser et al were able to assign phenotypes to 45 percent ofgenes with a known post-embryonic phenotype that should have beendetectable in their screen, they failed to find phenotypes for a number ofgenes which had been characterized previously simply because they wereoverlooked in the screening process. Purification of the yeastU4/U6.U5 small nuclear ribonucleoprotein particle and identification of itsprotein. Stevens, S. First, RNAi does notaccurately phenocopy the null phenotype of all genes, and several geneswith identical or near-identical nucleotide sequences may be targetedsimultaneously by RNAi, so more than one gene will be inhibited. The precipitated proteins were separatedelectrophoretically. By studying worms that wereexposed to dsRNA only as larva or adults as well as their progeny, it waspossible to assign post-embryonic phenotypes to genes that result insterility or produce 1 percent lethal progeny. As well as looking atdefects, the method can be used to look at the normal wiring of the humanbrain. Stewart and Abelson did not work with genes per se, but ratherlooked at proteins, which are the ultimate product of genes, and the vectorby which genes mediate their effects on an organism. A number of genes with the male phenotype were found. At the time of this paper, they had screened 24 linesof mice and 88 percent of them exhibited staining in the nervous system.They observed a wide variety and specificity of staining patterns. The method allows theidentification of axon guidance mechanisms in mammals without bias for thekinds of proteins or their expression levels. It was necessary to pool five purifications of U4/U6.U5 snRNP to getenough material to perform mass spectrometry because of its low abundance.Whole yeast cell extract was passed over antibody affinity columns ofsplicing extract and eluted with buffer containing competitor peptide. This wasunexpected as most cultures of C. Theindividual peptides were identified by mass and isolated and fragmented.The fragments were analyzed in a second mass spectrometer and the proteinsequence was determined. This allowed them toassign a genotype to 13.9 percent of the analyzed genes, and increase thenumber of identified gene sequences on chromosome 1 from 7 to 378. All three were successful in theirexperiments. Theeluates were then passed through Ni-NTA columns and eluted. While both Fraser et al andLeighton et al used techniques that involved altering genes in livingorganisms, Stewart and Abelson used purely biochemical extraction andidentification techniques to look at the products of genes. Insertion of PLAP vector induces either null or severe hypomorphicmutations that can be analyzed at several levels: the mutants can beassessed for lethality and gross defects such as ataxia; lines that surviveto embryonic day 11.5 can be screened systematically for wiring defectsbecause axon guidance molecules do not necessarily cause overt phenotypes;whole lines can be screened for defects in locomotion or behaviors such aslearning or memory. G., Kamath, R. Biochemically, genes involved in basalmetabolic processes comprised approximately 5 percent of Ste and Embgenes, confirming that these processes are essential for viability, anddemonstrating a clear difference between the types of genes required forgermline function or embryonic viability and those involved in laterdevelopmental processes. 87.9 percent of the 2,769 predicted genes onchromosome 1. Using this method, the triplesnRNP was purified to near homogeneity and the mass spectrometry showedthat there were at least 24 proteins stably associated with the U4/U6.U5snRNP, and four of them were novel. The researchers found that many genes have more than one phenotype,reflecting multiple functions for some genes. All the proteins extracted from the electrophoresisgels were analyzed by nano-electrospray tandem mass spectrometry. Feedingthese bacteria to the worms makes it possible to produce a library of dsRNA-expressing bacteria that can then be used for high-throughput genome-wideRNAi screens at very low cost. The researchers also found a homologueof human SMN disease gene, genes encoding for RNA-binding proteins, genesinvolved in chromosome condensation and separation, components of signaltransduction pathways and many conserved genes with no known biochemicalfunction. The only drawback to this technique is thatRNAi does not efficiently inhibit all genes, so the method will miss somerelevant genes. Twenty-seven percent of the trapped genes were new, and many also definedmolecularly distinct tracts. The largest phenotype class foundwere those whose inhibition by RNAi gave rise to embryonic lethality: theycomprised 6 percent of the genes. Dibl is a yeast protein previously shownto be essential in budding yeast. RNAi transiently inhibits the activity of a gene by introducing double-stranded RNA (dsRNA) with a sequence specific to the target gene. The fourth novel protein, Snu13, is ofunknown function as of yet. Leighton et al used a second marker, human placentalalkaline phosphatase (PLAP) and an internal ribosome entry site (IRES).PLAP is a glycosyl phosphatidylinositol (GPI)-linked cell-surface proteinthat labels axons completely when expressed transgenically in neurons.When a gene is trapped the endogenous promoter and enhancer elements directproduction of a bicistronic transcript that encodes two proteins: the beta-geo fusion to the endogenous protein and the PLAP protein, which istranslated independently using the IRES. Ninety percent ofembryonic lethal genes were identified in this way. SpliceosomalsnRNPs were layered onto glycerol gradients, and serial elutions showedthat U4/U6.U5 snRNPs were present in significantly greater amounts than U1or U2 snRNPs. In subsequent experiments, they examined theexpression patterns and screened for wiring defects in progeny of threeages encompassing many axon guidance events: embryonic day 11.5, embryonicday 15.5 and birth. Looking at the distribution of the genes identified by RNAi across thechromosome, they were found distributed all along the chromosome except fortwo regions corresponding to the two regions of chromosome 1 that containlocally duplicated gene clusters. Nature, 41 ,174-179. Leighton et al (2 1) used a completely different approach to look atthe genes responsible for axonal guidance in the developing brain. V., Lu, X., Pinson, K.,Scherz, P., Skarnes, W. These techniques provide in vivo labeling of the axons so thatmutations which interfere with axonal guidance can be identified visually.Mice derived from mutant ES cells have neurons expressing the trapped genelabeled in their cell bodies by beta-geo and on their axons by PLAP. They used affinity chromatography ofa doubly epitope-tagged SmD3 protein, a component of the spliceosomalsnRNPs, to purify the yeast U4/U6.U5 snRNP. Theirresearch was aimed at identifying specific genes and their function, and inthis they were highly successful. Anatomical screening revealed two mutant lines, whichconfirms that new axon guidance phenotypes can be identified by thesemethods and that characterization of PLAP staining can extend the analysisof genes that have already been knocked out. Purified human U4/U6.U5 snRNP also contains 2 -25 proteins andhuman homologues of Prp3, Prp4, Prp8, Brr2, Snu114 and Sm core proteins areall associated with human U4/U6.U5 snRNP. These researchers further demonstrated that using these techniques, asthey did with Sema6A and EphA4, they could identify defects in thetrajectories of axons expressing the trapped genes simply by comparing thewiring of PLAP-stained axons in heterozygous and homozygous embryos. They performed the first large-scale reverse genetic analysisof a multicellular organism, and increased fivefold the number of sequencedgenes with known phenotypes on chromosome 1 in this organism. PNAS, 96, 7226-7231. Nature, 4 8, 325-33 . (2 1). C. elegans appears also to contain manytranscription factors required for both viability and developmentalprocesses. Theyused a procedure known as gene-trapping in which mutagenesis is performedin embryonic stem cells using a DNA construct that integrates at random inthe mouse genome. For each of the U4/U6.U5. While Fraser et al used reverse genetic analysis to identify thefunction of predicted genes, and Leighton et al used phenotype-based genetrap screening designed for the large scale identification of genescontrolling axon guidance in the brain, Stevens and Abelson looked at theprotein products of a yeast nuclear ribonucleoprotein particle using twoaffinity chromatography steps followed by preparative glycerol gradientsedimentation and mass spectrometry. The researchers then screened the library to identify genes withclearly identifiable phenotypes in wild-type worms. Using such a library of bacteria which express dsRNA responding togenes on chromosome 1, this group were able to isolate 2,445 independentclones of bacteria which were capable of reacting with 2,416 of thepredicted genes, i.e. The gene canbe readily identified by 5' rapid amplification of complementary DNA endsand sequencing. S., Zipperlen, P., Martinez-Campos, M.,Sohrmann, M., & Ahringer, J. Snu66 has no known function. The techniques used by these three different groups of investigatorsdiffer vastly, and range from inactivating genes to labeling them tobiochemical methods of isolating proteins. Four novel proteins were identified by these techniques and have beendesignated Snu (SNUrp associated) proteins with the corresponding molecularweight. elegans chromosome 1by feeding these worms with a bacterium that expresses double-stranded RNA. Fraser et al andLeighton et al looked directly at the results of gene action: Stewart andAbelson looked at gene products. Also identified by mass spectrometry were the known proteins Brr2,Snu114, Prp3, Prp6, Prp4, Prp8, Prp31, Prp38 and the core Sm proteins,indicating that the purified 25S complex was indeed the yeast U4/U6.U5snRnp. Leighton, P. These included a large number ofcomponents of basal cell machinery. C., & Tessier-Lavigne, M. Defining brainwiring patterns and mechanisms through gene trapping in mice. snRNP proteins, asingle yeast ORF was identified. (2 ). Functional genomic analysis of C.elegans chromosome 1 by systematic RNA interference. They pointout two problems with the method they used. Fraser et allooked at the effects of inhibiting genes by looking at expressedphenotypes; Leighton et al used labeled genes to identify gene productsvisually; and Stewart and Abelson isolated and identified proteins whichwere the products of known genes. elegans requiresapproximately 5, genes for development under standard laboratoryconditions. Thisallows visualization of projection patterns between heterozygotes andhomozygous mutants, and so identification of cell-autonomous axon guidancedefects. Theysuggest the PLAP secretory cap method is likely to be the most useful foridentifying genes that encode axon guidance receptors or that have othercell-autonomous functions in guidance because the axons which require thefunction of the gene are labeled with PLAP. Fraser et al (2 ) used RNA-mediated interference (RNAi) to targetapproximately 9 percent of the predicted genes on C. Stewart and Abelson limited their study to one group ofproteins associated with a small ribonucleoprotein particle; Fraser et allimited their study to one species of worms; Leighton et al used many linesof mice to trace specific axonal guidance defects. A., Mitchell, K.
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