Question

In this exercise you will examine the sequences for both Pax6 and eyeless and consider the differences and sims. larities between them, First, download the sequences for \(\operatorname{Pax} 6(82069480)\) and eyeless (12643549) from the \(\mathrm{NCB}\) Entrez Protein site using their accession numbers, given in parentheses. Go to the BLAST homepage (http://www.ncbi.nim.nih. gov/blast) and choose Align two sequences using BLAST (bl2seq) under the Specialized BLAST heading. Instead of searching a large database as is typical with BLAST, we will only be aligning two sequences with one another. Paste your sequences for Pax6 and eyeless into boxes for Sequence 1 and Sequence 2 and make sure that you choose blastp as the program. When all of this is done, push the Align button. In your BLAST alignments there is a line between "Query" and "Sbjct" that helps guide the eye with the alignment: if "Query' and "Sbjct" agree identically, the matching letter is repeated in the middle; if they do not match exactly but the amino acids are compatible in some sense (a favorable mismatch), then a " \(+\) " is displayed on the middle line to indicate a positive score. Where there is no letter on the middle line indicates an unfavorable mismatch or a gap. The numbers at the beginning and end of the "Query" and "Sbjct" lines tell you the position in the sequence. (a) Choose one of the alignments returned by BLAST and give a tally of the number of (i) identical amino acids: (ii) favorable mismatches: (iii) unfavorable mismatches: (iv) gaps. (b) Give two examples of unfavorable mismatches and two examples of favorable mismatches in your chosen alignment. Based on what you know of the chemistry and structure of the amino acids why might these amino acid pairs give rise to negative and positive scores, respectively? (c) Choose one unfavorable mismatch pair and one favor able mismatch pair from your chosen alignment. What codons may give rise to each of these amino acids? What is the minimum number of mutations necessary in the DNA to produce this particular unfavorable mismatch? How many DNA mutations would be required to produce the particular favorable mismatch you chose?

Short answer

Depending on the personalized data retrieved from the BLAST tool, the answer will vary. Therefore, refer to the step-by-step solution for instructions on how to systematically process and analyze the data in order to answer the given exercise.

Step-by-step solution

Download the Sequences

First, go to the NCBI Entrez Protein site. Enter the accession numbers '82069480' and '12643549' for Pax6 and eyeless, respectively, to download the sequences.

Using the BLAST Tool

Go to the BLAST homepage and choose the 'Align two sequences using BLAST (bl2seq)' option under the 'Specialized BLAST' heading. In the 'Sequence 1' and 'Sequence 2' boxes, paste the downloaded sequences for Pax6 and eyeless. Ensure to select 'blastp' as the program, then click 'Align'.

Interpretation of the BLAST Result

In the BLAST results, find the alignment between the 'Query' and 'Sbjct'. If the amino acids match exactly, the matching letter is repeated. A '+' indicates a favorable mismatch, and an absence of a letter indicates an unfavorable mismatch or a gap. Tally up the identical amino acids, favorable mismatches, unfavorable mismatches, and gaps. The numbers at the beginning and end of the 'Query' and 'Sbjct' lines signify the position in the sequence.

Analyzing Mismatches

Identify two examples of unfavorable mismatches and two of favorable mismatches. Consider why these specific amino acid pairs result in positive or negative scores by examining the chemistry and structure of the amino acids involved.

Examining Codons

Choose one unfavorable mismatch pair and one favorable mismatch pair from the selected alignment. Determine what codons may give rise to each of these amino acids. Calculate the minimum number of DNA mutations needed for the unfavorable match to occur, and determine the number of DNA mutations needed for a favorable mismatch to occur.

Key concepts

Pax6 gene

The Pax6 gene plays a crucial role in the development of the eyes and brain in animals. While it is best known for its involvement in eye formation, Pax6 also influences the development of other sensory organs and neural structures. This gene is highly conserved across species, meaning the gene's structure and function remain similar from flies to humans.

Such conservation underscores the gene's importance in fundamental biological processes, and analyzing its sequence through tools like BLAST can reveal significant insights into protein homology and function across different organisms.

eyeless gene

The eyeless gene is the fruit fly version of the Pax6 gene in humans. It holds the instructions for a protein that triggers the development of eyes in Drosophila. Mutations in this gene can lead to the absence of eyes, hence the name 'eyeless'.

By comparing the sequences of eyeless and Pax6, researchers can explore the evolutionary relationship between these genes. Despite their divergence, they often exhibit a high degree of similarity, which is detectable with sequence alignment tools like BLAST. This similarity also extends to the resulting proteins, which share common functions.

amino acids

Amino acids are the building blocks of proteins, where a chain of amino acids fold into complex shapes to form a functional protein. There are 20 different amino acids that combine in countless ways to create the diversity of proteins found in living organisms.

Differences in the properties of amino acids can affect protein structure and function, which is why understanding the sequence of amino acids within a protein can be critical. Sequence alignment tools, such as BLAST, help identify these sequences and predict how amino acid changes might impact protein activity.

DNA mutations

DNA mutations are changes in the nucleotide sequence of DNA. These mutations can occur naturally or be induced by external factors. They can have a range of consequences, from benign to causing significant structural and functional changes in proteins.

In the context of sequence alignment, mutations are reflected in mismatches between aligned sequences. These can be favorable if they don't disrupt or even enhance function, or unfavorable if they result in a loss of function or detrimental effect.

codons

Codons are triplets of nucleotides in DNA that specify which amino acid will be added to a growing protein chain. Each set of three nucleotides corresponds to a specific amino acid, and the full set of instructions for making a protein is determined by the sequence of codons in a gene.

When analyzing sequence alignments, it's useful to consider not only amino acid mismatches but also the codons that lead to them. This helps to determine how many DNA mutations might be necessary to produce a particular amino acid substitution.

sequence analysis

Sequence analysis involves examining the arrangement of amino acids in proteins or nucleotides in DNA to discern patterns, identify similarities, and predict functions. This analysis is essential in molecular biology as it helps in understanding evolutionary relationships, identifying mutations, and comparing gene sequences between different species.

Tools like BLAST are specifically designed to perform sequence comparisons quickly and accurately, making them indispensable for researchers and students who are analyzing the genetic and protein similarities.

protein homology

Protein homology refers to the similarity between proteins across different species, which is usually an indication of shared ancestry and common functions. By utilizing sequence alignment, scientists can identify homologous proteins and infer evolutionary relationships.

These relationships are often visualized through similarity scores, which tell us how closely related different proteins are in terms of their amino acid sequences. This concept is fundamental when comparing the Pax6 gene and eyeless gene, as it provides insights into how proteins have conserved functions across diverse life forms.

molecular biology

Molecular biology is the branch of biology that focuses on the molecular basis of biological activity, including the interactions between the various systems of a cell and the structure and function of the cell's genetic material. It combines aspects of biochemistry, genetics, and biophysics.

Molecular biology techniques, such as sequence alignment with BLAST, are critical for advancing our understanding of genes and proteins, how they evolve, and how they contribute to health and disease. In educational settings, introducing these techniques helps to bridge the gap between theoretical concepts and their practical applications in research.