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Genetic Analysis: An Integrated Approach (3rd Edition)
- Some S. aureus strains can produce an enzyme called beta-lactamase, which breaks down beta-lactam drugs like methicillin. These strains that make beta-lactamase have a gene called mecA. Draw a diagram of the bacterial DNA encoding this gene, including the following terms: promoter, terminator, coding region.arrow_forwardYou are attempting to prepare a single gene knockout library using the pRL27 transposon system. You grow the donor E. coli in Luria broth containing both kanamycin and diaminopimelic acid (DAP) and your recipient Serratia rubidaea in plain Luria broth. You combine an equal ratio of donor and recipient cultures and plate the mixture onto Luria agar supplemented with DAP. After 24 hours incubation at 37°C, you create a cell slurry and plate the cells onto Luria agar aupplemented with kanamycin. After 24 hours incubation at 37°C, you find that no colonies grow. What best explains this outcome? A. Failure to supplement media with DAP B. Failure to remove antiobiotic containing media C. Failure to incubate for a sufficient length of time D. Failure to incubate at the appropiate temperature E. Failure to use the proper mating mix ratioarrow_forwardIn the bacteriophage T7 system used to express recombinant proteins, the gene of interest is fused to T7 promoter and T7 RNA polymerase is separately cloned into the same cell. What is the main reason this system uses T7 RNA polymerase instead of relying on the bacterial RNA polymerase? To restrict the expression of bacterial protein expression To enhance the amount of recombinant protein expression To enhance the expression of bacterial protein expression To restrict the amount of recombinant protein expression To enable the expression of T7 viral protein expressionarrow_forward
- You are using Drosophila as a model system to investigate eye development. You do positional cloning to isolate a mutant gene called eyeless (eyl), which causes flies to develop with reduced sized eyes when homozygous. Using several DNA markers from the candidate interval as probes for colony hybridization to a CDNA library, you obtain a 10 Kb cDNA insert cloned into the unique EcoRI site of a 3.5 Kb plasmid vector. a. Briefly describe how would you use the Drosophila eyl cDNA clone to identify a clone from a mouse genomic DNA library that is homologous to the Drosophila eyl gene? b. You isolate a clone of mouse genomic DNA that appears to be homologous to the Drosophila eyl gene. After DNA sequencing of a small piece of the mouse genomic clone, you use the DNA sequence to search the mouse genome database. You find that there is a SNP locus (n1234) that does NOT cause an RFLP in this genomic clone. You have identified several breeding lines of mice, each having been bred for several…arrow_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_forwardConsider three genes in E. coli: thr+, ara+, and leu+ (which give the cell the ability to synthesize threonine, arabinose, and leucine, respectively). All three of these genes are close together on the E. coli chromosome. Phages are grown in a thr+ ara+ leu+ strain of bacteria (the donor strain). The phage lysate is collected and used to infect a strain of bacteria that is thr− ara− leu −. The recipient bacteria are then tested on selective medium lacking leucine. Bacteria that grow and form colonies on this medium (leu+ transductants) are then replica-plated on medium lacking threonine and on medium lacking arabinose to see which are thr+ and which are ara+. Another group of the recipient bacteria are tested on medium lackingthreonine. Bacteria that grow and form colonies on this medium (thr+ transductants) are then replica-plated on medium lacking leucine and onto medium lacking arabinose to see which are ara+ and which are leu+. Results from these experiments are as follows:…arrow_forward
- You want to clone a eukaryotic gene and express the protein in yeast. The protein typically localizes in the mitochondria. What does your clone need so that the protein is localized in the ER rather than the mitochondria?arrow_forwardThe technique of fluorescence in situ hybridization (FISH) is described. This is another method for examining sequence complexity within a genome. In this method, a DNA sequence, such as a particular gene sequence, can be detected within an intact chromosome by using a DNA probe that is complementary to the sequence.For example, let’s consider the β-globin gene, which isfound on human chromosome 11. A probe complementary to theβ-globin gene binds to that gene and shows up as a brightly colored spot on human chromosome 11. In this way, researchers can detectwhere the β-globin gene is located within a set of chromosomes. Becausethe β-globin gene is unique and because human cells are diploid(i.e., have two copies of each chromosome), a FISH experimentshows two bright spots per cell; the probe binds to each copy ofchromosome 11. What would you expect to see if you used thefollowing types of probes?A. A probe complementary to the Alu sequenceB. A probe complementary to a tandem array near…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
- The mRNA used for the SARS-CoV-2 vaccine from Pfizer/BioNTech is generated in a cell-free system by transcribing a DNA template. The T7 promoter is used as a signal to start transcription. What is the T7 promoter, and give one reason why it is used.arrow_forwardAs part of a project investigating potential new drug targets in the fight against malaria, you are seeking to clone the gene for a protein from the malaria parasite Plasmodium falciparum. You wish to express this protein in BL21 (DE3) cells, a standard laboratory strain of Escherichia coli. After purification of your protein, you run an SDS-PAGE gel and notice that the major band has lower molecular weight than expected, so you fear you are getting a truncated version. 1. What technique could you use to confirm that you are obtaining a shortened version of your intended protein? explainarrow_forwardAs part of a project investigating potential new drug targets in the fight against malaria, you are seeking to clone the gene for a protein from the malaria parasite Plasmodium falciparum. You wish to express this protein in BL21 (DE3) cells, a standard laboratory strain of Escherichia coli. After purification of your protein, you run an SDS-PAGE gel and notice that the major band has lower molecular weight than expected, so you fear you are getting a truncated version. (a) Give TWO possible causes of your protein becoming truncated. explainarrow_forward
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