Download A golden fish reveals pigmentation loss in Europeans Data Activity

GENETIC ORIGIN OF PIGMENTATION LOSS IN ZEBRAFISH AND HUMANS
Lamason et al. 2014—Accompanying Student Worksheet
Genetic origin of golden mutant zebrafish
DNA or RNA sequences can be changed in many different ways. Some common types of mutations are
single base pair changes (for example from A to T or C to G), insertions of additional nucleotides, or
deletions (removal) of existing nucleotides. Parts of a gene can also be rearranged and end up in a
different location on the chromosome (translocation). All these mutations can have different effects on
the organism, dependent on what gene is affected and how the mutation changes the encoded protein
structure or the expression of a gene.
Lamason and colleagues mapped the golden allele responsible for the loss of pigmentation in zebrafish
to a gene called slc24a5. They described two golden mutant zebrafish lines, golb1 and golb13, that both
had reduced pigmentation but differed in the type of mutation causing it.
Below is a map showing a region of chromosome 18 that includes the slc24a5 gene and various
polymorphisms with known location (called gene markers) that are indicated with a “z” followed by a
number. Polymorphisms are like fingerprints and can serve as mile-markers along the chromosome.
They were used here to locate the golden mutation.
The middle row labeled “golb13 mutation” indicates with either a plus or minus sign whether or not a
particular section of chromosome around each marker could be copied (or “amplified”) from the mutant
golb13 fish DNA sample using PCR (polymerase chain reaction). PCR fails when a stretch of chromosome is
completely missing.
Figure 1. Gene map of a portion on chromosome 18 that includes the golden gene (top row). The map shows
the approximate position and range of different gene markers. Markers from z928 to z13836 are deleted in
the golb13 variant (red box). The bottom row zooms in on a chromosome region that contains gene marker
z9484, which is present in the golb13 variant (+), and z13836, which is absent () in golb13. The gene sequence
of this region includes slc24a5, the golden gene. (Modified after Fig. S2, Lamason et al. 2014, Supplementary
Materials)
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1. The middle row labeled “golb13 mutation” shows where PCR was successful (+) and where it failed
(), resulting in either millions of copies of that particular DNA sequence or in no copies at all. Based
on the pattern of PCR amplification in golb13, what type of mutation is present in golb13 animals?
Explain your answer. (Recall that PCR fails when a stretch of chromosome is completely missing.)
2. What is the effect of the golb13 mutation on the slc24a5 gene product (i.e. the resulting RNA or
protein)?
a. Are any other genes affected by this mutation? If so, which ones?
Below is a small portion of the slc24a5 gene from the wild-type zebrafish and the golb1 mutant.
Nucleotides are grouped into codon triplets, which each code for an amino acid.
3. Using a codon table (https://en.wikipedia.org/wiki/DNA_codon_table) translate this portion of the
slc24a5 allele for both types of fish.
Wildtype:
golb1 mutant:
4. How does the golb1 mutant differ from the wild-type? Are the two sequences the same? Look at
both the nucleotides and the translated amino acids and explain the differences.
5. What type of mutation do golb1 zebrafish have in this portion of the slc24a5 gene?
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6. Do you think the golb1 variant of the slc24a5 gene would still make a functional protein? Why or
why not?
7. Contrast the type of mutations in golb1 and golb13 mutants and explain the difference between the
two.
Human SLC24A5 alleles and evolution
The slc24a5 gene in zebrafish has a counterpart in humans called SLC24A5, which has a very similar
function. This suggests that the gene is well conserved and probably existed in a common ancestor that
– like humans and zebrafish – had pigment-producing cells. This most recent common ancestor of
humans and zebrafish is thought to have lived roughly 420 million years ago.
There are two different alleles of SLC24A5 that are most common among modern humans. Once the
alleles are translated into a sequence of amino acids to form a protein, they have different amino acids
at position 111 of the protein (see Fig. 2 below). In one, the amino acid is alanine (ala111 allele); in the
other the amino acid is threonine (thr111 allele).
Figure 2. Amino acid sequences encoded by a portion of the zebrafish slc24a5 gene (top row) and the two
most common versions of the human SLC24A5 gene (bottom rows). The most commonly shared amino acids
between zebrafish and humans in these sequences are highlighted in black. Position 111 is marked with an
asterisk. (Modified after Fig. S5, Lamason et al. 2014, Supplementary Materials)
8. Based only on the alignment of the human and zebrafish slc24a5 amino acid sequences, can you
conclude without any doubt which amino acid was present at position 111 in the common ancestor
of humans and zebrafish? Why or why not?
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Below are additional amino acid sequences of slc24a5 genes from a few more vertebrates (Fig. 3).
Figure 3. Partial amino acid sequences encoded by various vertebrate versions (or orthologs) of the zebrafish
slc24a5 gene. As in Fig. 2, the most common, shared amino acids are highlighted in black, and position 111 is
marked with an asterisk. (Modified after Fig. S5, Lamason et al. 2014, Supplementary Materials)
9. Can you now determine with more confidence which amino acid was present at position 111 in the
common ancestor of humans and zebrafish? Explain your answer.
10. What other parts of the shown amino acid sequences were probably also encoded by the ancestral
slc24a5 gene? Recall that amino acids highlighted in black are shared by all vertebrates with a black
highlight at that position.
11. Can you predict from the amino acid sequence whether a change from alanine to threonine at
position 111 in humans would affect the function of the SLC24A5 protein? Why or why not? You can
go back to the article to see what the researchers did to find the answer.
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The authors found that the derived thr111 allele is
mostly found in Europeans while Africans and
Asians mostly have the the ancestral ala111 allele.
On the left are images of a wildtype zebrafish
and three golb1 zebrafish mutants. Two of the
three mutants were injected with either wildtype slc24a5 mRNA from zebrafish or human
mRNA from the European thr111 allele when they
were embryos (Fig. 4). A control zebrafish that
did not receive any mRNA is shown for
comparison.
Figure 4. Results of so-called rescue experiments in
which golden zebrafish mutants were injected with
mRNA from wildtype zebrafish slc24a5 alleles (J
and K) and human SLC24A5 alleles that contained
the thr111 mutation (L and M) to test if they would
restore pigmentation. (Modified after Fig. 2,
Lamason et al. 2014)
12. Can the human SLC24A5 mRNA bring back (or “rescue”) pigmentation in golden zebrafish?
a. What does this tell us about the remaining function of the SLC24A5 gene with the thr111
mutation?
b. Would you expect the result in M if there was no remaining function of the thr111 form of
SLC24A5? What if it had some remaining function? (Hint: In the most severe forms of human
albinism, for example, there is no dark pigment in either the eyes or the hair.)
13. The thr111 European allele is associated with pigmentation loss. Based on the rescue experiment
with human thr111 mRNA, what can you conclude about the function of the thr111 protein in zebrafish
versus humans?
AUTHORS
Brett Robison, PhD, University of California, Berkeley
Revised and edited by Sandra Blumenrath, PhD, HHMI and AAAS
Reviewed by Keith Cheng, MD, PhD, Pennsylvania State University College of Medicine
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