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

Use cDNA for \(\alpha\) -globin gene, remove introns, and insert into bacterial plasmid for expression in bacteria.

Step-by-step solution

- Understanding Introns and Exons

In eukaryotic genes, introns are non-coding sequences while exons are coding sequences. Bacteria do not have the cellular machinery to remove introns since their own genes do not contain them.

- Gene Splicing Requirement

Before inserting the \(\alpha\) -globin gene into a bacterial plasmid, the introns within the gene need to be removed. This ensures that the bacteria can correctly process and produce the \(\alpha\) -globin protein.

- Use of cDNA

Extract mRNA corresponding to the \(\alpha\) -globin gene, which has introns already removed during RNA processing. Then, use reverse transcriptase to create complementary DNA (cDNA) from the mRNA. This cDNA will only contain exons.

- Insert cDNA into the Plasmid

Insert the generated cDNA into the bacterial plasmid. Since the cDNA lacks introns, the bacteria will be able to transcribe and translate the gene to produce \(\alpha\) -globin.

- Transformation and Expression

Transform bacteria with the plasmid containing the \(\alpha\) -globin cDNA. Select transformed colonies and induce the expression of the \(\alpha\) -globin protein.

- Verification

Verify the production of \(\alpha\) -globin by isolating proteins from the bacteria and conducting analysis such as SDS-PAGE or Western blotting.

Key concepts

Introns and Exons

In eukaryotic genes, sequences called introns and exons play crucial roles. Introns are non-coding sections of DNA, meaning they do not code for proteins and must be removed from the RNA transcript during processing. This process, known as splicing, ensures that only the coding sequences, or exons, are expressed in the final mRNA.
Bacteria, however, do not have introns. Their genes are continuous sequences without interruptions. Because bacteria lack the cellular machinery for splicing, they cannot process eukaryotic genes containing introns. Thus, for successful gene expression in bacteria, any introns must be removed from the gene you wish to insert.

cDNA Synthesis

To express a eukaryotic gene containing introns in bacteria, scientists use complementary DNA (cDNA). Here's how it works.
First, mRNA is extracted from the eukaryotic cells since it reflects the mature, intron-free gene. This mRNA is produced through natural splicing in the eukaryotic cell.
Next, the enzyme reverse transcriptase is used to create cDNA from this mRNA template. Reverse transcriptase converts the single-stranded mRNA into a complementary double-stranded DNA molecule. This resulting cDNA contains only exons, making it suitable for bacterial expression.

Bacterial Plasmid Transformation

Next is integrating the cDNA into a bacterial plasmid—a small, circular DNA molecule separate from the bacterial chromosome.
Scientists first cut open the plasmid using restriction enzymes, creating a location to insert the cDNA.
Once the cDNA is inserted into the plasmid, DNA ligase enzyme is used to seal the gaps, forming a continuous DNA molecule.
This recombinant plasmid can then be introduced into bacterial cells in a process called transformation. The bacteria take up the plasmid from their environment.

Protein Expression Verification

After transforming the bacteria with the plasmid containing the cDNA, it’s essential to verify that the \(\alpha\)-globin protein is being produced. Steps for verification include:
1. Isolating the proteins from the bacterial cells.
2. Using techniques like SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis), which separates proteins based on their size.
3. Western blotting to detect the specific \(\alpha\)-globin protein using antibodies.
These methods confirm the presence and size of the expressed protein, ensuring that the transformation and expression were successful.

Gene Splicing

Gene splicing in the context of bacteria involves creating a continuous gene sequence free of introns. This splicing happens naturally during eukaryotic mRNA processing.
When scientists synthesize cDNA for bacterial expression, they essentially capitalize on this natural splicing. The cDNA is a spliced version of the original gene, ready for bacterial machinery to use without further modifications.
This process ensures that the inserted gene can be efficiently transcribed and translated into a functional protein by the bacteria, completing the process from DNA to a usable protein product.