Concept explainers
Researchers at the University of California at San Diego have designed a strategy, alternatively called the mutagenic chain reaction (MCR) or gene drive, that can introduce rapidly a designed mutation into almost all of the chromosomes within an entire interbreeding population. Their idea was surprisingly simple, and it depends
on plasmids such as that in the diagram that follows. In these MCR constructs, genes that can express high levels of Cas9 protein (gray) and an sgRNA (green) for a particular target in the genome are flanked by sequences that surround the target site in the genome (blue).
Suppose you make a recombinant MCR plasmid in which the plasmid contains sequences for the X-linked yellow body color gene in Drosophila. The Cas9 gene and sgRNA genes in the plasmid replace a protein-coding exon of the yellow gene that is needed for yellow gene function. The sgRNA is specific for a site within the wild-type yellow gene.
a. | The researchers injected this plasmid into a wild-type male embryo, where it became incorporated into some germ-line cells by homologous recombination. The sperm that developed from these germ-line cells fertilized a wild-type egg. The females that developed from these fertilized eggs were yellow-bodied, which was surprising because loss-of-function alleles of yellow are recessive to wild-type alleles. Explain (includediagrams) the genesis of these yellow-bodied females. (Hint: Think about the name mutagenic chain reaction.) |
b. | When a single such yellow-bodied female was introduced into a population of 100 wild-type flies, within a couple of generations almost every fly had yellow bodies. Explain this result. |
c. | Researchers are now trying to use the gene drive system to prevent Anopheles stephensi mosquitoes from spreading malaria, a disease caused by a protozoan called Plasmodium that |
d. | In 2016, an expert panel convened by the National Academies of Science, Engineering, and Medicine released a report that cautioned against the release to the environment of Anopheles mosquitoes engineered as in part (c). Why was this panel so concerned about using MCR to control malaria? |
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Genetics: From Genes to Genomes
- Now that you understand how the CRISPR-Cas9 system works, think back to the experiments discussed in the introduction to this chapter, in which researchers used CRISPR-Cas9 genome editing to treat mice with Duchenne muscular dystrophy. Why did the researchers choose to cut out the entire exon 23 in the mice with the disorder? Why not replace the specific mutation using a donor piece of DNA and homologous recombination? Propose some possible explanations.arrow_forwardAfter Drosophila DNA has been treated with a restriction enzyme, the fragments are inserted into plasmids and selected as clones in E. coli. With the use of this “shotgun” technique, every DNA sequence of Drosophila in a library can be recovered.a. How would you identify a clone that contains DNA encoding the protein actin, whose amino acid sequence is known?b. How would you identify a clone encoding a specific tRNA?arrow_forwardThe human hexokinase enzyme has the same function as the bacterial hexokinase enzyme but is somewhat different in its amino acid sequence. You have obtained a mutant bacterial strain in which the gene for hexokinase is missing. If you introduce into your mutant strain a DNA plasmid engineered to contain the DNA coding sequence of the human hexokinase gene, what must you also include? a)The human hexokinase promoter b)The bacterial hexokinase promoter c)Both the human and bacterial promoters d)You cannot engineer a bacteria to produce a human enzymearrow_forward
- Below is a portion of an exon from a gene that encodes protein X in the genome of the plant Arabidopsis. Wildtype DNA3’ TTC AAT GCT CCG AAT ACC 5’ template strand5’ AAG TTA CGA GGC TTA TGG 3’ non-template strand A new strain (Strain B) of Arabidopsis is identified with the same region of the gene coding for protein X: 3’ TTC AAT GCT CCC AAT ACC 5’ template strand5’ AAG TTA CGA GGG TTA TGG 3’ non-template strand Compare the two DNA sequences and look for any differences. Based on what you find a. There is no mutation in Strain B compared to Strain A. b. After the point of the mutation, all the amino acids encoded by the Strain B template will be different than the Strain A protein X. c. Protein X made from the Strain B template will be much shorter than protein X made from the Strain A template d. Protein X from Strain B will have one amino acid difference that would not affect protein function. e. There is a mutation but there will not be any difference in the…arrow_forwardAfter Drosophila DNA has been treated with a restrictionenzyme, the fragments are inserted into plasmids and selected as clones in E. coli. With the use of this “shotgun”technique, every DNA sequence of Drosophila in a librarycan be recovered.a. How would you identify a clone that contains DNAencoding the protein actin, whose amino acid sequenceis known?b. How would you identify a clone encoding a specifictRNAarrow_forwardThere is a hypothetical gene related to the nervous system of Drosophila. Describe all the methods, steps, and key substances you need to obtain to use the following techniques in experimental design to study the gene: - In situ hybridization (to find the mRNA) - Immunohistochemistry (to find the protein) - CRISPR-Cas9 (for loss of function) - Expression vector (for gain of function)arrow_forward
- Suppose a researcher previously cloned gene Y into M13 bacteriophage vector. Gene Y encodes a product called peptide Y. A region of gene Y contains the DNA sequence ATG-CGC-GAA-CTG-GTG-AAC-TAA. The researcher wishes to change a Val residue to an Ala residue in this region of peptide Y using site-directed mutagenesis. What should be the sequence of the mutant oligonucleotide primer in this region? You may use a codon table. mutant oligonucleotide primer sequence: GGC-GGC-GAA-CTG-GTG-AAC-TAA Incorrectarrow_forwardThe figure below shows RNA-Seq data (RED) for the D. melanogaster transformer (tra) gene obtained from both adult female and male fruit flies. The blue lines indicate the tra gene structure, with thicker lines indicating exons, and thin lines introns. The 5' end of the gene is on the left, and the 3' end of the gene is on the right. Based on these data, the most likely conclusion is: Males and females express identical isoforms of tra Males express more tra RNA than females The female isoform has fewer amino acids The female isoform has more amino acids The male and female isoforms have different 3'UTRsarrow_forwardIn the practical you have been analysing a human genomic library. You know from your calculations that only a small proportion of the human genome is represented, even when the entire class results are considered. Therefore, the chance of finding a particular single-copy gene in your library is very small. Outline a strategy for constructing a genomic DNA library more representative of the entire human genome. You will need to consider alternative vectors and the efficiency of transformation of the bacterial cells.arrow_forward
- Gerald Rubin and Allan Spradling devised a method of introducinga transposon into Drosophila. This approach has been importantfor the transposon tagging of many Drosophila genes. Theresearchers began with a P element that had been cloned on a plasmid.Using cloning methods, they inserted the wild-type allele for the rosygene into the P element in this plasmid. The recessive allele, rosy,results in a rosy eye color, while the wild-type allele, rosy+, producesred eyes. The plasmid also had an intact transposase gene.The cloned DNA is shown to the right.Rubin and Spradling used a micropipette to inject this DNAinto regions of embryos that would later become reproductivecells. These embryos were originally homozygous for the recessiverosy allele. However, the P element carrying the rosy+ allele could “hop” out of the plasmid and into a chromosome of the cells that were destined to become germ cells (i.e., sperm or egg cells). After the embryos had matured to adults, the flies were then…arrow_forwardResearchers are manipulating the gene cxx2 for their experiments, and they have inserted a smallnumber of base pairs randomly somewhere into the gene. They isolate several versions (with differentinsertions) of this modified gene and carry out an RT-PCR using a primer that recognizes thetranscriptional start site area (eg. from +1 to +20), and a primer that binds to the polyA tail.For the second modified gene sample, they observe that the mature mRNA is now several basepairs longer, however splicing has not been affected. They examine the protein and identify thatit has significant changes to its amino acid sequence, altering its length. Which of the letteredarrowheads indicates the location where this insertion could be? (There could be one answer orseveral. Give all answers that apply)arrow_forwardYou receive purified mRNA that codes for the spike protein of SARS-CoV-2 as well as plasmid pET32(a+). Explain how you will prepare SARS-CoV-2 spike cDNA and name the enzymes that you will need how you will clone the cDNA into pET32(a+) and select the recombinant plasmid how you will then produce a recombinant SARS-CoV-2 spike and purify itarrow_forward
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