A number of yeast-derived elements were added to thecircular bacterial plasmid pBR322. Yeast that requireuracil for growth (Ura− cells) were transformed withthese modified plasmids and Ura+ colonies were selected by growth in media lacking uracil. For plasmidscontaining each of the elements listed in parts (a) to(c), indicate whether you expect the plasmid to integrate into a chromosome by recombination, or insteadwhether it is maintained separately as a plasmid. If theplasmid is maintained autonomously, is it stably inherited by all of the daughter cells of subsequent generations when you no longer select for Ura+ cells (that is,when you grow the yeast in media containing uracil)?a. URA+ geneb. URA+ gene, ARS c. URA+ gene, ARS, CEN (centromere)d. What would need to be added in order for these sequences to be maintained stably in yeast cells as alinear artificial chromosome?

A number of yeast-derived elements were added to thecircular bacterial plasmid pBR322. Yeast that requireuracil for growth (Ura− cells) were transformed withthese modified plasmids and Ura+ colonies were selected by growth in media lacking uracil. For plasmidscontaining each of the elements listed in parts (a) to(c), indicate whether you expect the plasmid to integrate into a chromosome by recombination, or insteadwhether it is maintained separately as a plasmid. If theplasmid is maintained autonomously, is it stably inherited by all of the daughter cells of subsequent generations when you no longer select for Ura+ cells (that is,when you grow the yeast in media containing uracil)?a. URA+ geneb. URA+ gene, ARS c. URA+ gene, ARS, CEN (centromere)d. What would need to be added in order for these sequences to be maintained stably in yeast cells as alinear artificial chromosome?

Biochemistry

6th Edition

ISBN:9781305577206

Author:Reginald H. Garrett, Charles M. Grisham

Publisher:Reginald H. Garrett, Charles M. Grisham

Chapter28: Dna Metabolism: Replication, Recombination, And Repair

Section: Chapter Questions

Problem 10P: Homologous Recombination, Heteroduplex DNA, and Mismatch Repair Homologous recombination in E. coli...

See similar textbooks

Similar questions

A plasmid vector pBS281 is cleaved by the enzymeBamHI (5′ G^GATCC 3′), which recognizes only onesite in the DNA molecule. Human DNA is digestedwith the enzyme MboI (5′ ^GATC 3′), which recognizes many sites in human DNA. These two digestedDNAs are now ligated together. Consider only thosemolecules in which the pBS281 DNA has been joinedwith a fragment of human DNA. Answer the following questions concerning the junction between thetwo different kinds of DNA. a. What proportion of the junctions between pBS281and all possible human DNA fragments can becleaved with MboI?b. What proportion of the junctions between pBS281and all possible human DNA fragments can becleaved with BamHI?c. What proportion of the junctions between pBS281and all possible human DNA fragments can becleaved with XorII (5′ C^GATCG 3′)?d. What proportion of the junctions between pBS281and all possible human DNA fragments can becleaved with BstYI (5′ R^GATCY 3′)? (R and Ystand for purine and pyrimidine, respectively.)e.…
For each of the pLM160 plasmid elements listed below: define the component AND briefly discuss the functional significance of this component for recombineering in Chlamydomonas reinhardtii. a. mNeonGreen-3xFLAG b. KanR2 c. 5’ cloning primer annealing site
Consider the following plasmid (size 8000 bp), with restriction sites at the positions indicated: (see image) a) This plasmid is digested with the enzymes listed below. Indicate how many fragments will begenerated in each case, and give the sizes of the fragments.PstIXhoICombination of PstI + XhoI + EcoRI (triple digest) b) Draw the banding pattern you would expect to observe if each of these digestions is loaded into a separate well of an agarose gel, and the fragments separated by electrophoresis. In the first well you load a DNA marker (M) containing fragments with sizes of 1000 bp, 2000 bp, 4000 bp and 8000 bp. c) This gel is transferred to a membrane in a Southern blot experiment, and hybridised to a radioactively labelled 200 bp probe, which anneals to the plasmid DNA at the position indicated on the diagram above. Draw the autoradiographic profile you would expect to observe for the membrane.
You are attempting to clone a 3 kb gene from the bacterial sp Microbacterium foliarum into the EcoRI site of the 6.0 kb plasmid shown. If you restrict the plasmid with EcoR1, how many bands will you obtain on agarose gel electrophoresis? Compare it with a plasmid in which no gene has been inserted. Draw a representative agarose gel showing the bands, and the direction of current flow as well point out the positive and negative electrodes
RNA polymeraze binds 100x quicker to the promotor when the promotor is in a 8kb plasmid than to the same promotor on a 200bp linear segment. Assuming that the incubation is performed with equal concentrations of the two types of DNA, explain this difference.
If the luciferase open reading frame is 1,650 bp in the control plasmid, and the Rel open reading frame is 1,764 bp, what are the predicted masses of the proteins encoded in these two open reading frames? Show all work below and give your answers in kilodaltons (kD
You 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 contain
Do virtual restriction digests of 300ng pGLO plasmid using HinDIII, EcoRI, and XhoI separately, and a double digest of pGLO with HinDIII plus EcoRI. How many nanograms of DNA should we expect to be contained within the slowest bandfrom the HinDIII-digest of pGLO?
How many fragments will be formed after digestion in sample C, if the plasmid is circular and why? How was RAD52 expression observed?
Genes encoding toxins are often located on plasmids.A recent outbreak has just occurred in which a bacterium that is usually nonpathogenic is producing atoxin. Plasmid DNA can be isolated from this newlypathogenic bacterial strain and separated from thechromosomal DNA. To establish whether the plasmidDNA contains a gene encoding the toxin, you coulddetermine the sequence of the entire plasmid andsearch for a sequence that looks like other toxin genespreviously identified. An easier way exists to determine whether the plasmid DNA carries the gene(s)for the toxin; this strategy does not involve DNAsequence analysis. Describe this easier method.
You digest a 5 kb plasmid with two enzymes that yield fragments of 4 kb and 1 kb. After running the digest on a gel, what would you predict to see in terms of the intensities of the bands?
U have the plasmid pUC18/19, which is a circular plasmid that consists of 2686 bp. What would the number of and length of the fragments be if you cut the plasmid with the following restriction enzymes or combination of enzymes? Give a schematic representation of the digestions. PscI & GsuI ______________________________________________________________ ScaI, PdmI & BsaXI ______________________________________________________________ ScaI, SspI & EheI ______________________________________________________________