Objectives:
Small subunit ribosomal RNA (rRNA) gene is one of the most widely used phylogenetic markers or DNA barcodes. A similarity between two DNA sequences is an important measure in many biology disciplines including taxonomy.
Answer the following questions:
1. Explain the terms: small subunit, 16S and 18S rRNA?
Obtain the small subunit rRNA gene sequences of the following organisms.
- Human nuclear small subunit rRNA gene
- Human mitochondrial small subunit rRNA gene
- Mouse nuclear small subunit rRNA gene
- Mouse mitochondrial small subunit rRNA gene
- Select two plant species of your choice
- Nuclear small subunit rRNA gene
- Mitochondrial small subunit rRNA
- Chloroplast small subunit rRNA
- Select two bacterial species in the same genus
- Small subunit rRNA gene
- Select two archaeal species in the same genus
- Small subunit rRNA gene
Calculate the sequence similarities to answer the following questions
2. Compare pairwise similarities between 2 animals (nuclear/mitochondria), 2 plants (nuclear/mitochondria/chloroplast), 2 bacteria, and 2 archaea. Discuss the results in the light of the general public view of the evolution of organisms.
3. Generate single FASTA files to include all sequences. Generate multiple sequence alignment and maximum likelihood phylogenetic trees using the MEGA tool. Also, draw the tree using only nuclear 18S sequences and bacterial/archaeal 16S. Discuss the results.
[Tips]
- You can obtain sequences at https://www.ncbi.nlm.nih.gov/nuccore/. Only the (nearly) full length of the gene should be obtained.
- For bacterial and archaeal 16S genes, use www.ezbiocloud.net.
- For pairwise similarity calculation, use this web service.
- Small subunit (=SSU) rRNA is called differently for the different domains. For Bacteria and Archaea (Prokaryotes), it is called 16S rRNA. For Eukarya (Eukaryotes), it is called 18S rRNA. Please note that SSU rRNA of mitochondrion and plastid (chloroplast) is also called 16S rRNA. All genes coding SSU (16S or 18S) are homologous, meaning that they share a common ancestor. Therefore, you should be able to (roughly) see how they are phylogenetically related by calculating sequence similarities.
By Jon Jongsik Chun
