TextText BSCI410 Final Review Hey guys this is a review for the final We can post questions and answers and just pretty much explanation for terms on the review sheet It ll be quicker if we all contribute rather than just going through and doing them individually Hey guys I did the the left side of the list of terms you need to know as contribution Could 1 do the right side then 2 do the topics and concepts Knockdown vs Knockout mutants Okay the differences are subtle gene knockout is where a gene is completely erased from the genome this usually occurs through of process of homologous recombination or CRE Lox excision you can look this up in wikipedia where a sequence of gene X say the ATG transcription start site is exchanged for a neutral sequence As a consequence after recombination there is 0 of the gene product and this is called a knockout Now knockdown occurs through a different process where you introduce short RNA sequences homologous to your target gene RNA sequence in a cell and through specific binding the RNA sequences coding for your gene will be depleted however the original gene sequence in the genome is still there and because this method is not 100 effective but at the most 90 of you target RNA will be depleted this is referred to as knockDOWN instead of knockOUT Because doing a knockdown can give you an approximation of what your phenotype after knockout may look like is the pathway on questions like this and because one can usually do it in a week where a knockout in a cell line will take you al least a couple of months researchers usually do a knockdown first and if necessary to prove their point or have a continuous supply of cells to work with they ll create a knockout Gene knockdown refers to techniques by which the expression of one or more of an organism s genes is reduced either through genetic modification a change in the DNA of one of the organism s chromosomes or by treatment with a reagent such as a short DNA or RNA oligonucleotide with a sequence complementary to either an mRNA transcript or a gene If genetic modification of DNA is done the result is a knockdown organism If the change in gene expression is caused by an oligonucleotide binding to an mRNA or temporarily binding to a gene this results in a temporary change in gene expression without modification of the chromosomal DNA and is referred to as a transient knockdown A gene knockout abbreviation KO is a genetic technique in which an organism is engineered to carry genes that have been made inoperative have been knocked out of the organism Also known as knockout organisms or simply knockouts they are used in learning about a gene that has been sequenced but which has an unknown or incompletely known function Researchers draw inferences from the difference between the knockout organism and normal individuals The term also refers to the process of creating such an organism as in knocking out a gene The technique is essentially the opposite of a Gene Knock in Knockout is often abbreviated as KO Knocking out two genes simultaneously in an organism is known as a double knockout DKO Similarly the terms triple knockout TKO and quadruple knockouts QKO are used to describe 3 or 4 knocked out genes respectively RNA interference RNAi RNA interference RNAi also called post transcriptional gene silencing PTGS is a biological process in which RNA molecules inhibit gene expression typically by causing the destruction of specific mRNA molecules Two types of small ribonucleic acid RNA molecules microRNA miRNA and small interfering RNA siRNA are central to RNA interference RNAs are the direct products of genes and these small RNAs can bind to other specific messenger RNA mRNA molecules and either increase or decrease their activity for example by preventing an mRNA from producing a protein RNA interference has an important role in defending cells against parasitic nucleotide sequences viruses and transposons but also in directing development as well as gene expression in general The RNAi pathway is found in many eukaryotes including animals and is initiated by the enzyme Dicer which cleaves long double stranded RNA dsRNA molecules into short double stranded fragments of 20 nucleotides that are called siRNAs Each siRNA is unwound into two single stranded ss ssRNAs namely the passenger strand and the guide strand The passenger strand is degraded and the guide strand is incorporated into the RNA induced silencing complex RISC The most well studied outcome is post transcriptional gene silencing which occurs when the guide strand base pairs with a complementary sequence in a messenger RNA molecule and induces cleavage by Argonaute the catalytic component of the RISC complex The enzyme dicer trims double stranded RNA to form small interfering RNA or microRNA These processed RNAs are incorporated into the RNA induced silencing complex RISC which targets messenger RNA to prevent translation 37 Genome wide mutant collections The sequencing of entire genomes has led to the identification of many genes A future challenge will be to determine the function of all of the genes of an organism One of the best ways to ascertain function is to disrupt genes and determine the phenotype of the resulting organism Novel large scale approaches for generating gene disruptions and analyzing the resulting phenotype are underway in the budding yeast Saccharomyces cerevisiae and other organisms including flies Mycoplasma worms plants and mice T DNA The transfer DNA abbreviated T DNA is the transferred DNA of the tumor inducing Ti plasmid of some species of bacteria such as Agrobacterium tumefaciens and Agrobacterium rhizogenes It derives its name from the fact that the bacterium transfers this DNA fragment into the host plant s nuclear DNA genome The T DNA is bordered by 25 base pair repeats on each end Transfer is initiated at the right border and terminated at the left border and requires the vir genes of the Ti plasmid Agrobacterium mediated T DNA transfer is widely used as a tool in biotechnology In genetic engineering the tumor promoting and opine synthesis genes are removed from the T DNA and replaced with a gene of interest and or a selection marker this is required so that it is possible to establish which plants have been successfully transformed Examples of selection markers include neomycin phosphotransferase hygromycin B phosphotransferase which both phosphorylate antibiotics and phosphinothricin acetyltransferase which