Concept explainers
A researcher has a Trp auxotrophic strain of E. coli with a mutation in a single gene. To identify that mutant
gene, she uses a genomic library made from a wild-type version of that same strain to find plasmids that rescue the mutant
of the plasmids contain gene X, while the other four contain gene Y. Our scientist has encountered a
phenomenon called multicopy suppression, related to the fact that plasmids are usually present in several copies per bacterium. Because the genes in the plasmids are present in more than their usual single copy in the
bacterial chromosome, more than the usual amount of Protein X or Protein Y is being produced from the plasmids. Sometimes, overexpression of one protein can rescue the mutant phenotype caused by loss of a
different protein. Suggest at least two ways that our scientist could determine which of the two genes, gene X or gene Y, actually corresponds to the mutant gene causing the Trp phenotype.
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Genetics: From Genes to Genomes
- A shuttle vector is a vector (usually a plasmid) constructed so that it can propagate in two different host species. One of the most common types of shuttle vectors is the yeast shuttle vector. Examples of such vectors derived from yeast are Yeast Episomal Plasmid (YEP), Yeast Integrating Plasmid (YIP) and Yeast Replicating Plasmid (YRP). Why is YEP preferred over YIP and YRP? Give your thoughts on this.arrow_forwardThe Molecular Cell Biology Unit of University X is working on a particular gene segment called ABC20. For a specific kind of experiment, they need to quantify the gene and require a lot of copies of the gene. The initial gel run showed a very faint band of the gene which made the quantification process difficult to proceed. Now propose a method with the help of which they can generate many copies of the gene. Here you need to know that they have the plasmid from where they can get the gene. You do not need to explain the process in detail rather answer in no more than 10 sentences on what can be done about this problem!arrow_forwardScientists modified the tumor-inducing (Ti) plasmid, a naturally occurring plant plasmid found in Agrobacterium tumefaciens, to create a tool used to introduce any gene of interest into plant cells. They created a binary system because a single Ti plasmid is too large to be easily manipulated. One part of the system is a disarmed plasmid, and the second part is a transformation vector. The following sentences describe the function of key DNA elements in the system. Virulence region Genes for conjugative transfer Disarmed Ti plasmid (T-region removed) ori Kan selectable marker plant selectable marker constitutive promoter T-region conjugative transfer virulence Kan (bacterial selectable marker) Amp selectable marker Plant selectable marker (e.g., herbicide resistance) Constitutive promoter T-DNA left border Place the terms in the appropriate blanks to complete the sentences. Not all terms will be used. 3' transcriptional terminator MCS (inserted gene of interest) T-region Transformation…arrow_forward
- When a 5-kb circular plasmid is digested with a restriction enzyme that has three recognition sites on the plasmid, how many bands can be visualized on an agarose gel? a)1 b)2 c)5 d)3arrow_forwarda) A plasmid DNA in bacteria has a length of 14,000 bp and an Lk of 1300. Calculate the superhelical density o for this plasmid. Show your work for partial credit, round to one digit after the decimal point. b) You use a Type II topoisomerase to change the linking number of this plasmid to 1310. How many turnovers must the topoisomerase perform? Is this resulting plasmid underwound or overwound?arrow_forwardA shuttle vector is a vector (usually a plasmid) constructed so that it can propagate in two different host species. One of the most common types of shuttle vectors is the yeast shuttle vector. Examples of such vectors derived from yeast are Yeast Episomal Plasmid (YEp), Yeast Integrating Plasmid (YIp) and Yeast Replicating Plasmid (YRp). Among these three vectors, YIp has the lowest transformation frequency and copy number per cell. Explain why Ylp is still popularly used despite its limitations.arrow_forward
- The genes for both the α- and βglobin chains of hemoglobin contain introns (i.e., they are split genes). How would this fact affect your plans if you wanted to introduce the gene for α-globin into a bacterial plasmid and have the bacteria produce α-globin?arrow_forwardThis plasmid was digested using different restriction enzymes whose sites have been mapped. The plasmid is 7896 base pairs long. This is a long question so u can count this as two or even three but please answer the question? Determine the size (base pairs) and number of fragments that would be produced if the plasmid was digested with the following enzymes: a) EcoRI b) BamHI c) HindIII d) EcoRI and HindIII e)EcoRI, HindIII, and BamHI *Hint- this is actually an EASY question, since the restriction map is already drawn for you!arrow_forwardYou transform bacteria with a plasmid carrying the ampicillin-resistance gene ampR. How would you determine which bacteria took up the plasmid? O Bacteria containing the plasmid would be able to grow in the absence of ampicillin. O Bacteria containing the plasmid would be able to grow in the presence of ampicillin. O Bacteria containing the plasmid would be able to produce ampicillin. O Ampicillin-resistance is necessary for the plasmid DNA get into the bacteria.arrow_forward
- Bacterial plasmids often serve as cloning vectors. Describe the essential features of a plasmid vector.arrow_forwardYou are about to isolate a 3000 bp large plasmid from an E.coli culture. You know that the plasmid is present in 100 copies per E. coli cell. You aim to have a final plasmid concentration of 100 ng/µl in a total volume of 50 µl. Assuming the yield is 100 %, how many E. coli cells should the culture from which the plasmid is to be isolated at least containarrow_forwardWhen cloning a foreign DNA fragment into a plasmid, it is often useful to insert the fragment at a site that interrupts a selectable marker(such as the tetracycline-resistance gene of pBR322). The loss of function of the interrupted gene can be used to identify clones containing recombinant plasmids with foreign DNA. With a yeast artificial chromosome (YAC) vector, it is not necessary to do this; the researcher can still distinguish vectors that incorporate large foreign DNA fragments from those that do not. How are these recombinant vectors identified?arrow_forward
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