Question

The genes for both the \(\alpha\) - and \(\beta\) -globin 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 \(\alpha\) -globin into a bacterial plasmid and have the bacteria produce \(\alpha\) -globin?

Short answer

Convert the \( \alpha \)-globin mRNA to cDNA (without introns) and then insert this cDNA into the bacterial plasmid.

Step-by-step solution

- Understand the Problem

The \( \alpha \)-globin gene in eukaryotes contains introns, which are non-coding regions. Bacteria do not have the machinery to process introns. Therefore, directly inserting the eukaryotic \( \alpha \)-globin gene into a bacterial plasmid will not result in proper \( \alpha \)-globin production. The gene needs to be modified.

- Extract Exons Only

First, obtain the mRNA transcript for \( \alpha \)-globin from a eukaryotic cell. mRNA processing in eukaryotes removes introns, so this transcript will only contain the exons (coding regions). The mRNA cannot be directly inserted into plasmids.

- Convert mRNA to cDNA

Use reverse transcriptase to convert the \( \alpha \)-globin mRNA into complementary DNA (cDNA). This cDNA will be free of introns and can now be utilized for cloning into a plasmid.

- Insert cDNA into Plasmid

Incorporate the \( \alpha \)-globin cDNA into a bacterial plasmid. This can be done using restriction enzymes and DNA ligase to ensure the cDNA is correctly inserted into the plasmid vector.

- Transform Bacteria

Transform the bacteria with the recombinant plasmid containing the \( \alpha \)-globin cDNA. The bacteria will then use this cDNA to produce the \( \alpha \)-globin protein since it is free from the introns that bacteria cannot process.

Key concepts

Introns and Exons

Understanding introns and exons is crucial when it comes to gene cloning. Introns are non-coding regions within a gene that do not encode protein sequences. They are often removed during mRNA processing. Exons, on the other hand, are coding regions that actually get translated into proteins. In eukaryotes, genes are often 'split' by these introns. Bacteria, however, do not possess the machinery to process introns. This means that simply inserting a eukaryotic gene into a bacterial plasmid won't work for protein production. The gene needs to be free of introns for the bacteria to effectively produce the protein.

mRNA Processing

mRNA processing is a key step in gene expression in eukaryotes. After a gene is transcribed into pre-mRNA, several processing steps occur:

• Splicing: Introns are removed, and exons are joined together.
• Capping: A 5' cap is added to the start of the mRNA.
• Polyadenylation: A poly-A tail is added to the end of the mRNA.

These steps ensure that the mRNA is mature and can be translated into proteins. For cloning purposes, we focus on splicing where the introns are removed, resulting in an mRNA that only contains the exons. This is highly significant, as bacteria can't remove introns themselves.

Reverse Transcription

Reverse transcription is a method used to convert RNA into DNA. This process is essential in cloning intron-free genes from eukaryotes.

• First, the mature mRNA (exon-only) is extracted from the eukaryotic cells.
• Then, reverse transcriptase enzyme transcribes this mRNA into complementary DNA (cDNA).

The cDNA is an exact DNA copy of the mRNA and is free of introns. This cDNA can then be used for further cloning processes, like insertion into bacterial plasmids.

Plasmid Transformation

Plasmid transformation is the process of introducing foreign DNA into bacteria. This is essential for cloning genes and producing proteins in bacterial systems.

• The cDNA (intron-free) of the \(\alpha\)-globin gene is first inserted into a bacterial plasmid vector. This involves using restriction enzymes to cut the plasmid and DNA ligase to join the cDNA to the plasmid.
• The recombinant plasmid is then introduced into bacteria, usually via chemical transformation or electroporation.
• Bacteria that successfully take up the plasmid can then express the \(\alpha\)-globin protein, as the cDNA does not contain introns the bacteria can't handle.

This method allows the production of eukaryotic proteins in bacterial systems for research and biotechnology applications.