Biochemistry Chapter 9

Which general procedure is NOT used in the cloning of DNA?

• determining the function of the protein coded for by the DNA

• joining two DNA fragments covalently

• selecting a DNA molecule that is capable of autonomous replication

• obtaining the DNA segment to be cloned

Answer explanation

The procedure of determining the function of the protein coded for by the DNA is not part of the cloning process. Cloning focuses on obtaining DNA segments, joining them, and ensuring they can replicate autonomously.

Restriction endonucleases:

• recognize and cleave DNA at specific sequences.

• restrict the choice of host organisms for cloned DNA sequences.

• always produce sticky ends.

• include the subclass known as DNA ligases.

Answer explanation

Restriction endonucleases are enzymes that recognize specific DNA sequences and cleave them at those sites, making the first answer choice correct. The other options are inaccurate regarding their functions.

How can a target DNA fragment that has been prepared and digested with EcoRI and PvuII restriction endonucleases be inserted into a plasmid?

• A linker sequence must be used to insert the DNA fragment.

• Carefully designed primers can be used to insert the DNA fragment into the plasmid during PCR.

• DNA ligase can be used to join it to a vector digested by the same restriction endonucleases.

• The target DNA fragment cannot be inserted because the blunt ends of DNA formed by the restriction endonucleases cannot be ligated.

Answer explanation

The correct choice is that DNA ligase can be used to join the target DNA fragment to a plasmid that has been digested with the same restriction endonucleases, allowing for compatible ends to be ligated together.

For a standard plasmid to be useful for molecular biology, what feature must it NOT have?

• A. origin of replication

• B. unique restriction enzyme sites

• C. antibiotic resistance genes

• D. DNA methylation sites

Answer explanation

A standard plasmid should not have DNA methylation sites, as these can interfere with cloning and expression. The other features, like an origin of replication, unique restriction sites, and antibiotic resistance genes, are essential for plasmid function.

Why would a lab technician choose a bacterial artificial chromosome, rather than a plasmid, for cloning a gene?

• because the DNA insert is from genomic DNA and not plasmid DNA

• because the insert to be cloned is very large

• because bacterial DNA will be used to make many copies of the gene (i.e. cloning it)

• because work on the gene will only be performed in a cell-free system, one without the genomic DNA required for cloning

Answer explanation

A bacterial artificial chromosome (BAC) is preferred for cloning large DNA inserts, as plasmids can only accommodate smaller fragments. This makes BACs suitable for handling large genomic DNA sequences.

Which element is NOT shared by both bacterial artificial chromosomes (BACs) and yeast artificial chromosomes (YACs)?

• origin of replication (ori)

• selectable markers

• specialized sequences derived from telomeres

• restriction endonuclease cleavage sites

Answer explanation

Specialized sequences derived from telomeres are unique to yeast artificial chromosomes (YACs) and are not found in bacterial artificial chromosomes (BACs), making this the correct choice.

Which statement regarding cloning vectors is false?

• They often contain polylinker sequences.

• They usually have selectable markers, screenable markers, or both.

• They can sometimes be artificial chromosomes, both yeast and bacterial.

• They are sometimes plasmids, small circular DNA molecules lacking an ori sequence.

Answer explanation

The false statement is that cloning vectors are sometimes plasmids lacking an ori sequence. Plasmids must have an origin of replication (ori) to replicate within a host cell, making this statement incorrect.

The operator of a typical E. coli expression vector:

• allows efficient transcription of the inserted gene.

• improves the amount and stability of the mRNA produced.

• permits regulation by a repressor that binds to it.

• provides sequence signals for the efficient translation of the mRNA derived from the gene.

Answer explanation

The correct choice, 'permits regulation by a repressor that binds to it,' highlights that E. coli expression vectors often include operators that allow for controlled gene expression through repressor binding.

Which statement is NOT an advantage of using bacterial hosts?

• Bacterial regulatory sequences are well understood.

• Bacteria are easy to store and grow in the laboratory.

• Efficient methods exist to get DNA into bacteria and extract DNA from them.

• Bacteria contain protein chaperones and modification systems to properly fold and modify protein products.

Answer explanation

The statement about bacteria containing protein chaperones and modification systems is NOT an advantage of using bacterial hosts, as these systems are often less effective in bacteria compared to eukaryotic systems.

Why is a system involving transcription from the bacteriophage T7 promoter and polymerase sometimes preferred over a system involving transcription from the Lac promoter?

• The system allows for cloning of very large genes (>100,000 bp).

• The system affords tight regulation and directs high levels of expression of genes fused to the T7 promoter.

• The system permits proper folding of the protein products.

• The system can be easily turned off by removing lactose from the growth medium.

Answer explanation

The T7 promoter system allows for tight regulation and high expression levels, making it preferable for producing proteins efficiently compared to the Lac promoter system.