In the microscopic world, different species of bacteria and fungi are engaged in a never-ending turf war, fighting to defend their territory and to advance and overtake competing organisms.

This battle is conducted through chemical warfare. Microbes use small molecules that we commonly refer to as antibiotics (or antifungals) to kill off enemy cells while they are protected from that antibiotic by harboring a resistance gene.

Some antibiotics don’t just kill bacteria; they can also kill eukaryotic cells such as fungi and mammalian cells. These molecules are called cell selection agents and are useful tools in biotechnology applications and life sciences research.

Puromycin, blasticidin, hygromycin, and G418 are popular cell selection agents that kill cells by inhibiting protein translation. These selection agents are used to establish stable cell lines, execute genetic screens, and more.

In this article, we’ll discuss how these cell selection agents kill cells and briefly cover which kinds of experiments that they’re used for

 

Article Table of Contents

What is a cell selection agent?

Puromycin

Blasticidin

Hygromycin

G418

References

 

What is a cell selection agent?

Cell selection agents are reagents and tools that are used to isolate or distinguish specific cells from a mixed population.

For example, common uses for cell selection agents include mammalian cell culture and genetic engineering. When establishing a stable cell line or conducting a CRISPR screen, you would use a cell selection agent to kill off cells that have not taken up the DNA of interest. This selection works because the DNA, in addition to its intended use, carries a resistance gene that neutralizes the cell selection agent, allowing the cells to grow.

You may recognize that puromycin, blasticidin, hygromycin, and G418 are antibiotics. Since they also kill eukaryotic cells such as fungi, plant, and mammalian cells they are also considered cell selection agents. For any of these types of cells to grow in the presence of the selection agent they need to express a resistance gene.

Each of the selection agents we’re discussing in this article functions by inhibiting protein translation, so let’s cover some basic background about how that process works.

The ribosome “translates” messenger RNA (mRNA) into a protein. Transfer RNAs (tRNA) enter the ribosome, build the nascent polypeptide chain one amino acid at a time. Once the tRNA has “transferred” its amino acid to the growing peptide, then that spent tRNA exits the ribosome to make room for the next tRNA to add an amino acid. This process is called translational elongation. Eventually, when the ribosome reaches the stop codon, it will release the mRNA and the newly synthesized protein which is called translational termination (Figure 1).

Figure 1. Protein translation. An aminoacyl-tRNA enters the ribosome and binds to the translating mRNA. The growing polypeptide chain is transferred to the newest aminoacyl-tRNA, and then the deacylated tRNA exits the ribosome. Different colored circles represent different amino acids on the aminoacyl-tRNAs that are transferred to the growing protein. The growing polypeptide chain shifts to the central spot, and then the cycle of protein elongation repeats over and over until protein translation is finished.

 

The ribosome is conserved through all kingdoms of life, so it’s no surprise that selection agents targeting protein translation kill prokaryotic and eukaryotic cells. While each selection agent binds to the ribosome and inhibits translation, the exact mechanism by which they do so is different. We’ll cover these differences in more detail in the following sections.

The most common use of these compounds is as cell selection agents. However, since they each inhibit or perturb translation in slightly different ways, these molecules are also important tools for detailed studies of protein translation.

In the cases of these selection agents, the resistance genes encode for enzymes that modify the selection agent. The specific resistance gene and type of modification vary by selection agent, but what they all have in common is that each modification prevents the selection agent from binding to the ribosome and inhibiting protein translation (Figure 2). 

Figure 2. Cell selection agents are inactivated by resistance genes encoding enzymes that modify the selection agent.

 

Puromycin

Puromycin is a structural analog of aminoacyl-tRNA, which is the molecule that adds a single amino acid onto a growing polypeptide chain in the ribosome.

Since puromycin is a good mimic for the tRNA, puromycin can sneak into the ribosome and is added onto the growing polypeptide chain. While puromycin can be added to the translating protein, other amino acids can’t be added onto puromycin. This results in the ribosome prematurely releasing the partial protein that it was not done translating yet (Figure 3).

Figure 3. Puromycin enters the tRNA binding site and is conjugated to the growing polypeptide chain. No more amino acids can be added onto puromycin, causing premature translational termination.

 

In these experiments, cells that have a plasmid with the pac gene survive the selection. pac encodes puromycin N-acetyl-transferase, which is an enzyme that modifies puromycin in a way that prevents puromycin from inhibiting translation (Vara et al, 1986).

Think about pac like a muzzle that prevents a dog from biting. Puromycin is still present in cells, but in its acetylated form puromycin cannot inhibit translation. In this way, cells that express the pac gene are immune to selection by puromycin.

 

Blasticidin

Blasticidin S is a potent peptidyl nucleoside antibiotic that was originally isolated from the fungus Streptomyces griseochromogenes. The term “peptidyl nucleoside” simply means there are two parts to blasticidin: one side that looks like a peptide or small protein (peptidyl), and another side that looks like a nucleic acid such as DNA or RNA (nucleoside).

Blasticidin essentially sticks tRNA to the ribosome in a way that the tRNA can’t be released and causes a traffic jam blocking the next aminoacyl-tRNA from binding to the ribosome (Svidritskiy et al, 2013).

Blasticidin can be neutralized through a few different resistance genes. The BSD and bsr genes encode deaminase enzymes that convert blasticidin into a slightly different molecule that cannot inhibit translation (Izumi et al, 1991; Kimura et al, 1994).

The bls gene encodes for an acetyltransferase that inactivates blasticidin by acetylating it, similar to puromycin’s inactivation (Pérez-González et al, 1990).

 

Hygromycin

Hygromycin B is an aminoglycoside antibiotic produced by the bacterium Streptomyces hygroscopicus. Hygromycin binds to the small subunit (30S) of the ribosome and perturbs tRNA-mRNA interactions that are essential for faithful translation. This perturbation can prevent translational elongation, and it can also result in mistranslated and non-functional proteins (Borovinskaya et al, 2008). Mistranslation is when the wrong amino acid (a different one than what the mRNA codes for) is put into the translated protein.

The enzyme hygromycin phosphotransferase modifies hygromycin by phosphorylating it, which disrupts its ability to inhibit translation (Blochlinger and Diggelmann, 1984). So, hygromycin is used to select cells that express the gene for hygromycin phosphotransferase. 

 

G418

G418 is an aminoglycoside antibiotic that binds to the ribosome in the same spot where a protein called the ribosome recycling factor normally resides. When a ribosome has finished translating one mRNA into a protein, this recycling factor helps finish that round of translation, and then “recycles” the ribosome to start translating a new mRNA (Janosi et al, 1994). When G418 blocks the ribosome recycling factor from binding to the ribosome, that leads to the ribosome getting trapped on a single mRNA, and it can’t proceed to translate additional mRNA molecules (Borovinskaya et al, 2007).

Part of the impact of G418 preventing normal translational termination is that it enhances stop codon readthrough. The stop codon in the mRNA is the signal that tells the ribosome to stop translating and release the newly synthesized protein. When G418 is present, ribosomes more frequently misread the stop codon and incorporate an amino acid and keep translating. For this reason, G418 is used as a tool to promote stop codon readthrough in the lab.

For example, one cause of proximal spinal muscular atrophy is the premature termination of the SMN1 protein when it’s being translated. Spinal muscular atrophy is a group of neuromuscular disorders characterized by the death of neurons that control motor function. Adding G418 counteracts the premature termination of SMN1, which functionally improved motor function in mice (Heier and DiDonato, 2009).

While stop codon readthrough is a good thing for SMN1 in spinal muscular atrophy, and in other specific cases, G418 kills cells because it indiscriminately reads through thousands of different mRNAs. While some of these read through events generate functional proteins, most of them yield aggregation-prone proteins that cause proteotoxic stress that overwhelm and kill the cells (Kramarski and Arbely, 2020).  

Cells that carry the neo gene will be able to survive and grow in the presence of G418. neo encodes the enzyme aminoglycosidase 3’-phosphotransferase which phosphorylates neomycin, thereby making it ineffective at inhibiting translation (Kucherlapati et al, 1984).

 

That’s an overview of how the cell selection agents puromycin, blasticidin, hygromycin, and G418 kill cells by inhibiting translation in slightly different ways. If you already know which selection agent is right for your experiments, then GoldBio is your go-to source for high quality cell selection agents, and other antibiotics, at an affordable price. See the related products below and the links throughout the link for more details on size, pricing, and more.  

 

 

References

Aviner R. (2020). The science of puromycin: From studies of ribosome function to applications in biotechnology. Computational and structural biotechnology journal, 18, 1074–1083. https://doi.org/10.1016/j.csbj.2020.04.014

Blochlinger, K., & Diggelmann, H. (1984). Hygromycin B phosphotransferase as a selectable marker for DNA transfer experiments with higher eucaryotic cells. Molecular and cellular biology, 4(12), 2929–2931. https://doi.org/10.1128/mcb.4.12.2929-2931.1984

Borovinskaya, M. A., Pai, R. D., Zhang, W., Schuwirth, B. S., Holton, J. M., Hirokawa, G., Kaji, H., Kaji, A., & Cate, J. H. (2007). Structural basis for aminoglycoside inhibition of bacterial ribosome recycling. Nature structural & molecular biology, 14(8), 727–732. https://doi.org/10.1038/nsmb1271

Borovinskaya, M. A., Shoji, S., Fredrick, K., & Cate, J. H. (2008). Structural basis for hygromycin B inhibition of protein biosynthesis. RNA (New York, N.Y.), 14(8), 1590–1599. https://doi.org/10.1261/rna.1076908

Heier, C. R., & DiDonato, C. J. (2009). Translational readthrough by the aminoglycoside geneticin (G418) modulates SMN stability in vitro and improves motor function in SMA mice in vivo. Human molecular genetics, 18(7), 1310–1322. https://doi.org/10.1093/hmg/ddp030

Izumi, M., Miyazawa, H., Kamakura, T., Yamaguchi, I., Endo, T., & Hanaoka, F. (1991). Blasticidin S-resistance gene (bsr): a novel selectable marker for mammalian cells. Experimental cell research, 197(2), 229–233. https://doi.org/10.1016/0014-4827(91)90427-v

Janosi, L., Shimizu, I., & Kaji, A. (1994). Ribosome recycling factor (ribosome releasing factor) is essential for bacterial growth. Proceedings of the National Academy of Sciences of the United States of America, 91(10), 4249–4253. https://doi.org/10.1073/pnas.91.10.4249

Kimura, M., Takatsuki, A., & Yamaguchi, I. (1994). Blasticidin S deaminase gene from Aspergillus terreus (BSD): a new drug resistance gene for transfection of mammalian cells. Biochimica et biophysica acta, 1219(3), 653–659. https://doi.org/10.1016/0167-4781(94)90224-0

Ko, T., Oliveira, M. M., Alapin, J. M., Morstein, J., Klann, E., & Trauner, D. (2022). Optical Control of Translation with a Puromycin Photoswitch. Journal of the American Chemical Society, 144(47), 21494–21501. https://doi.org/10.1021/jacs.2c07374

Kramarski, L., & Arbely, E. (2020). Translational read-through promotes aggregation and shapes stop codon identity. Nucleic acids research, 48(7), 3747–3760. https://doi.org/10.1093/nar/gkaa136

Kucherlapati, R. S., Eves, E. M., Song, K. Y., Morse, B. S., & Smithies, O. (1984). Homologous recombination between plasmids in mammalian cells can be enhanced by treatment of input DNA. Proceedings of the National Academy of Sciences of the United States of America, 81(10), 3153–3157. https://doi.org/10.1073/pnas.81.10.3153

Pérez-González, J. A., Ruiz, D., Esteban, J. A., & Jiménez, A. (1990). Cloning and characterization of the gene encoding a blasticidin S acetyltransferase from Streptoverticillum sp. Gene, 86(1), 129–134. https://doi.org/10.1016/0378-1119(90)90125-b

Rao, R. N., Allen, N. E., Hobbs, J. N., Jr, Alborn, W. E., Jr, Kirst, H. A., & Paschal, J. W. (1983). Genetic and enzymatic basis of hygromycin B resistance in Escherichia coli. Antimicrobial agents and chemotherapy, 24(5), 689–695. https://doi.org/10.1128/AAC.24.5.689

Svidritskiy, E., Ling, C., Ermolenko, D. N., & Korostelev, A. A. (2013). Blasticidin S inhibits translation by trapping deformed tRNA on the ribosome. Proceedings of the National Academy of Sciences of the United States of America, 110(30), 12283–12288. https://doi.org/10.1073/pnas.1304922110

 

 

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antibiotic resistance antibiotics Simon Currie

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