Problem 7 - Campbell Biology

Question

Level \(2 :\) Application/Analysis Expression of a cloned eukaryotic gene in a bacterial cell involves many challenges. The use of mRNA and reverse transcriptase is part of a strategy to solve the problem of (A) post-transcriptional processing. (B) post-translational processing. (C) nucleic acid hybridization. (D) restriction fragment ligation.

Step-by-Step Solution

Answer

The correct answer is (A) post-transcriptional processing.

Step 1: Understand the Problem

To express a eukaryotic gene in a bacterial cell, one must overcome differences in gene expression mechanisms between eukaryotes and bacteria. This involves processing the eukaryotic gene in a way that allows the bacteria to read and express it correctly.

Step 2: Identify Key Terms

mRNA and reverse transcriptase are mentioned as part of the strategy. mRNA is a mature transcript that has undergone post-transcriptional processing, including splicing and addition of a poly-A tail.

Step 3: Role of mRNA and Reverse Transcriptase

Using mRNA means we are starting with a transcript that has already been processed, which contains only exons. Reverse transcriptase converts this mRNA back into DNA (cDNA), which can then be inserted into a bacterial plasmid.

Step 4: Link to Solution

Given that we use mRNA (a mature transcript) and reverse transcriptase to create cDNA, the purpose is to bypass the need for eukaryotic post-transcriptional modifications in the bacterial system.

Step 5: Choose the Correct Answer

The use of mRNA and reverse transcriptase is intended to handle post-transcriptional processing. Hence, the correct answer is (A) post-transcriptional processing.

Key Concepts

Eukaryotic Gene Expression

Expressing eukaryotic genes within bacterial cells can be quite challenging. This difficulty arises due to the fundamental differences in gene expression mechanisms between eukaryotes and bacteria. We must process eukaryotic genes in such a way that bacteria can read and express them properly.

In eukaryotic cells, genes have introns (non-coding regions) and exons (coding regions). Before the mRNA can be translated into proteins, these introns must be removed through a process known as splicing. Additionally, more modifications such as the addition of a 5' cap and a 3' poly-A tail are performed on the mRNA.

Bacteria, on the other hand, do not naturally carry out these modifications. When expressing a eukaryotic gene in bacteria, scientists must find methods to ensure that the bacterial cells can synthesize the correct protein. One effective method involves using processed mRNA and reverse transcriptase to generate complementary DNA (cDNA), which can then be introduced into bacterial cells.

Post-Transcriptional Processing

Post-transcriptional processing is a crucial step in the conversion of a primary RNA transcript into mature mRNA, ready for translation. This process includes several modifications:

• Splicing: The removal of introns and joining of exons.
• Addition of a 5' Cap: A modified guanine nucleotide that protects mRNA from degradation.
• Addition of a 3' Poly-A Tail: A series of adenine nucleotides that stabilize mRNA and assist in its export out of the nucleus.

Bacteria lack the machinery to perform such modifications. Therefore, when expressing eukaryotic genes in bacteria, we make use of mRNA that has already undergone these modifications. By using mRNA and reverse transcriptase, we can bypass these steps in the bacterial system. The mRNA is first converted into cDNA, which can then be inserted into bacterial plasmids for further expression.

This strategy circumvents the requirement of post-transcriptional processing within the bacterial host, making it an essential component when working with eukaryotic genes.

Reverse Transcriptase

Reverse transcriptase is an enzyme that catalyzes the formation of DNA from an RNA template. This process is referred to as reverse transcription. Here’s how it works:

• The mRNA, which contains only exons, is used as a template.
• Reverse transcriptase synthesizes complementary DNA (cDNA) from this mRNA template.

This cDNA can be cloned into a bacterial plasmid, bypassing the need for the bacterial cell to perform post-transcriptional modifications.

In the context of gene expression, the use of reverse transcriptase is vital for converting already processed mRNA into a DNA form that bacteria can replicate and express. This approach helps solve the problem of post-transcriptional processing by providing bacteria with a version of the gene that doesn't require further modification.

Thus, reverse transcriptase plays a key role in enabling the expression of eukaryotic genes within a bacterial system efficiently.