<-chapter_03|Chapter 03^table_of_contents|Table of Contents^chapter_05|Chapter 05->

<typo fs:x-large>Chapter 04. %%Chromosomes and sex linkage%%</typo> 

Until now our analysis of genes has focused on defining genes based on phenotypic differences brought about by different alleles or by a direct test of function – the complementation test. In Chapters 4 and 5, our analysis will be concerned with tests of gene position. 

So far in this book we’ve taken it for granted that genes reside on chromosomes, but how do we know this? The relationship between genes and chromosomes was discovered by Thomas Morgan in 1910. 

===== Chromosome theory and sex determination =====


As discussed at the end of [[chapter_03|Chapter 03]], we saw (retrospectively) the relationship between what Mendel observed and the chromosome mechanics of meiosis. This immediately and strongly suggested that genes are physically located on chromosomes. But for scientists in the early 20th century, what was needed to more definitively demonstrate that genes are on chromosomes (the idea known as "chromosome theory") was a chromosome that could be identified in the microscope and that carried an allele for a phenotype that could also be easily observed. The evidence for chromosome theory would then depend on correlating the segregation of the trait with segregation of the chromosome.

It is important to know that during this period of history, despite being able to see chromosomes under microscopes, scientists did not know what chromosomes were made of. It's also useful to know what scientists in Morgan's day knew about chromosomes and their role in sex determination (this winds up being important in our story). Drosophila, like humans, are obligate diploids (2n). While humans have 46 chromosomes, Drosophila have 8 chromosomes, or 4 pairs of chromosomes (2n=8); this includes one pair that are different in males and females. In female flies this pair looks like a regular pair of chromosomes, but males only have one of these chromosomes; instead of a second homologous chromosome as a partner, this chromosome pairs with a much smaller and different looking chromosome during meiosis in males. These chromosomes are named X and Y and also called sex chromosomes. Female flies have two X chromosomes, while male flies just have one X chromosome that pairs with the small Y chromosome during meiosis. The three other chromosome pairs in Drosophila are called II, III, and IV. Around the time of Morgan, scientists studying mealworms and butterflies already had data that strongly suggested sex determination was controlled by whether an individual carried XX vs XY pairs, although genetic evidence to support this was not discovered until after the sex-linked studies we discuss in this chapter. Humans also have an XY chromosome pair that determines sex, but the details of how sex determination works is very different between flies and mammals. While we focus on the XX/XY system in this book, not all species with sexual dimorphism have XX/XY chromosomes (birds use a system called ZZ (male)/ZW (female)), and some species don't use chromosomes to determine sex (yeast is one example of this; but fish and reptiles do not use sex chromosomes either). 

===== The $white$ mutation has unusual segregation patterns =====


[[wp>thomas_hunt_morgan|Thomas Hunt Morgan]] "proved"((It's important to note that one cannot really ever formally prove anything using the scientific method. It's more accurate to say that Morgan's work provided very strong evidence supporting his hypothesis.)) chromosome theory in 1910 using Drosophila. Wildtype (normal) flies have brick-red eyes. The first laboratory mutant for Drosophila was found by Morgan’s wife, Lillian, who worked in his lab at Columbia University in New York City. Compared to wildtype, these mutants had white eyes and were therefore named $white$ (or $w$ for short). In fact, what they initially found by sheer luck was a single male fly with white eyes. 

<figure Fig1>
{{ :fruit-flies-red-and-white-eyes.jpg?400 |}} 
<caption>
Wild type //Drosophila melanogaster// with red eyes (left) and the famous white mutant (right). Source: [[https://commons.wikimedia.org/wiki/File:Fruit-flies-red-and-white-eyes.jpg|Wikimedia]]. Licensing: [[https://creativecommons.org/licenses/by-sa/4.0/deed.en|CC BY-SA 4.0]].
</caption>
</figure>
Flies with the $white$ mutation gave strange outcomes in various crosses. Note that in [[chapter_03|Chapter 03]], we ignored the fact that to actually breed flies, you must cross male and female flies. From here on out, we also consider sex in our crosses, and use the male ♂ and female ♀ symbols to indicate sex:
  
<figure Fig2>
<div centeralign>
<WRAP center round box 60%>
$$ \begin{aligned} P: white\text{ ♂} &\times \text{red eyes ♀ (wildtype)}\\&\downarrow\\F1: \text{all} &\text{ red} \text{ (both ♂ and ♀)}\\
&\downarrow\\F2: \text{red}&:white = 3:1 \text{ (but only ♂ had white eyes)} \end{aligned}$$

</WRAP>


</div>
<caption>
The Drosophila $white$ mutant crossed to a true-breeding wildtype female (i.e., a monohybrid cross). Note that the F1 go through a sib cross to produce F2.
</caption>
</figure>

The fact that all the F1 progeny were normal (i.e., had red eyes) and the F2 progeny had a 3:1 ratio for red vs. white eyes indicated that the $white$ mutation behaves like a recessive mutation in a single gene (similar to what we saw in [[chapter_03|Chapter 03]] for $shibire$). But there was something unusual about the $white$ mutation because only the male flies in the F2 had white eyes. In fact, half the F2 males had white eyes, while the other half had red eyes. All of the F2 females had red eyes. 

Morgan then took the red-eyed F1 females from Figure {{ref>Fig2}} and crossed them to white males:

<figure Fig3>
<WRAP center round box 60%>
$$\begin{aligned} white\text{ ♂} &\times \text{red ♀ (F1 females from Fig. 2)}\\ &\downarrow\\ \text{red}&:white = 1:1 \text{ (both ♂ and ♀)} \end{aligned}$$
</WRAP>

<caption>
Crossing $white$ males to F1 females from Fig. {{ref>Fig2}}.
</caption>
</figure>

Since the monohybrid cross in Figure {{ref>Fig2}} suggests that $white$ is recessive, the cross in Figure {{ref>Fig3}} is therefore roughly equivalent to using $white$ males as testers to test cross the F1 red-eyed females. The results indicated that those red F1 females were heterozygous and further confirmed that $white$ is a recessive trait. 

The most informative cross wound up being the reciprocal (switching the sex/traits of the parents) of Morgan's original cross shown in Figure {{ref>Fig2}}:
  
<figure Fig4>
<WRAP center round box 60%>
$$ \begin{aligned} \text{red ♂} &\times white \text{ ♀}\\ &\downarrow\\ \text{all ♀ are normal and} &\text{ all ♂ have white eyes} \end{aligned}$$
</WRAP>

<caption>
"Test crossing" wildtype red-eyed males from a true-breeding line to $white$ females. This is the reciprocal (sexes switched) of the cross shown in Figure {{ref>Fig2}}.
</caption>
</figure>

This is effectively using the $white$ females as a tester to test cross normal red-eyed males. Drosophila crosses produce roughly 50% male and 50% female; therefore, ignoring the sex of the flies, it looks as if the red-eyed parent male is heterozygous in this cross - but this cannot be correct, as we used males from true-breeding wildtype stock. Furthermore, in Figure {{ref>Fig4}} the wildtype allele is only passed on to the daughters and the $white$ allele is only passed on to sons. If the $white$ mutation "behaved" the way Mendel might have expected it to, you would expect that the white eye phenotype would be split equally between males and females, which is not what Morgan observed. Clearly something unusual and "un-Mendelian" is going on here; whatever is going on with $white$, it is associated with sex. 

 
===== The $white$ mutation is sex-linked =====


Morgan explained these unusual results by hypothesizing that the eye color gene $white$ ($w$) is physically located on the sex chromosome $X$. Males only have one copy of the $X$ chromosome and females always get one copy of $X$ from their mother and one copy from their father. We can use modified symbols to show the genotypes that are directly associated, or linked, with the $X$ chromosome, and re-write Figure {{ref>Fig4}} as follows:
  
<figure Fig5>  
<WRAP center round box 60%>
$$\begin{aligned} \frac{X^+}{Y}\text{ ♂} &\times \frac{X^w}{X^w}\text{ ♀}\\ &\downarrow\\ \frac{X^w}{Y} &\text{ ♂, }  \frac{X^w}{X^+} \text{ ♀} \end{aligned}$$
</WRAP>

<caption>
Rewriting Figure {{ref>Fig4}} in more formal fractional notation.
</caption>
</figure>
	
In Figure {{ref>Fig5}}, each parent contributes one allele to the progeny; therefore, the wildtype allele for red eyes in female progeny is always inherited along with the $X^+$ chromosome from the father that carries the wild type $w^+$ allele (abbreviated as simply +), and the mutant $w$ allele is inherited from the $X^w$ chromosome from the mother. The fact that these individuals get two $X$ chromosomes (one from each parent) is what makes them females. Male progeny, on the other hand, are male because they get a $Y$ chromosome, and the only way to get a $Y$ chromosome is from their father; this means that they must get their $X$ chromosome from their mother, and their mother is homozygous $\frac{X^w}{X^w}$. Morgan named this phenomena sex linkage. 

Based on this discussion, we now have a clear definition of sex linkage in Drosophila: a gene is sex-linked if it is physically associated with a sex chromosome. The way a geneticist would say it is, "A gene is sex-linked if it maps to a sex chromosome".  The relevance of sex linkage is that this phenomena is what first allowed scientists to show that genes are associated with chromosomes. Morgan's experiments showed that at least one gene ($w$) is physically linked to the $X$ chromosome. In the next chapter, we will see that there are other Drosophila genes that are also sex linked. Subsequent experiments using Drosophila and other organisms showed that other genes are physically located on autosomes (any chromosome that is not a sex chromosome). Genes that are not sex-linked are located on autosomes and are also called autosomal genes. In the next chapter, we will also see that not only are genes physically linked to chromosomes, they also have defined physical positions on chromosomes.

Finally, let's have some further discussion on genetic notation. Note that writing $X^w$ is redundant. Since the $X$ chromosome always pairs with the $Y$ chromosome during meiosis, the presence of the $Y$ chromosome automatically implies that any genes written together with the $Y$ chromosome in fractional notation must be on chromosome $X$. Furthermore, the male and female symbols are also redundant, since the presence of a $Y$ chromosome tells you everything you need to know about the sexes of the individuals in a cross where sex-linked genes are involved. Drosophila geneticists also use the $\rightharpoondown$ symbol to represent the $Y$ chromosome. We can therefore re-write Figure {{ref>Fig5}} as:

<figure Fig6>  
<WRAP center round box 60%>
$$ \frac{+}{\rightharpoondown} \times \frac{w}{w}\\ \downarrow\\ \frac{w}{\rightharpoondown}\text{, } \frac{w}{+}$$
</WRAP>

<caption>
Rewriting Figure {{ref>Fig5}} in a simpler way. Creative laziness is a virtue. Note that "$\rightharpoondown$" is the commonly used symbol for the Drosophila $Y$ chromosome. 
</caption>
</figure>


===== Sex linkage in humans - an example =====
<figure Fig7>
{{ :pedigree_analysis.jpg?400 |}}
<caption> 
Pedigree analysis of red-green color blindness in humans. Squares represent males and circles represent females. The solid colors indicate an affected individual. Dotted lines indicate genotype unknown. Source: [[https://wellcomecollection.org/works/h6p6uqy8|The Wellcome Collection]] (original reference: Gates, R.R. (1929) Heredity in Man, Constable & Co. (London). Licensing: [[https://creativecommons.org/licenses/by/4.0/|CC BY 4.0]].
</caption>
</figure>

Sex linkage is not unique to Drosophila. Some forms of color blindness in humans are also sex-linked. If there is historical information on phenotypes in a family tree, a pedigree analysis can be used to observe this. Figure {{ref>Fig7}} shows an example of a human pedigree that contains individuals affected by red-green color blindness. In the general population, red-green color blindness is somewhat common (8% of males and 0.5% in females) but is particularly prevalent in this pedigree. In just three generations, 5 males are affected (to just one female). The underlined $X$ chromosome is a model to explain the inheritance pattern. It makes sense that individuals 1 and 2 in generation IV, who are both males, are colorblind if their mother is homozygous for a sex-linked gene that confers the color blind phenotype. We now know that this kind of heritable red-green colorblindness is caused by a mutation in the gene that codes for an opsin, a protein that is sensitive to specific colors of light in the retina of the eye. We also know that this opsin gene is located on chromosome $X$ in humans. 

<WRAP center round help 60%>
Why don't we do pedigree analysis for Drosophila? Why is it only necessary for humans?

</WRAP>

===== Closing thoughts =====


A final note on sex linkage: many students are confused by sex linkage - they think of it as some kind of special case in genetics. Technically, it is indeed a special case. But it is best to think of sex linkage more as a general case of genetics. What sex linkage historically taught us is that genes are physically associated with chromosomes. It just so happens that some genes are on sex chromosomes, and those sex chromosomes also happen to determine sex((Also note that just because a gene is sex-linked doesn't necessarily mean that this gene is involved in sex determination. There are lots of "regular" genes on the X chromosome that have nothing to do with sex determination.)). Since sex is easy to observe, it means that this fact that genes are associated with chromosomes just happened to be first discovered for genes on sex chromosomes. But the general statement that "genes are physically associated with chromosomes" is true for all genes and for all chromosomes. When thinking about sex linked genes and their inheritance, it can be easier to think about how these genes segregate with the $X$ and $Y$ chromosomes first, since chromosomes always follow the rules of meiosis. Then think about the sex of the offspring as a secondary thing. Consider chromosomes and genotypes and how they segregate first, then ask what the resulting phenotypes come from those genotypes second. Use this approach to think about all genes and chromosomes, including sex linked genes and sex chromosomes.

===== Questions and exercises =====


Exercise 1: While working in a Drosophila lab, you discover a mutant male fly with miniature wings that are much smaller than wildtype wings. You temporarily name this mutant $mini$. What experiments could you do to answer the question: is $mini$ sex linked? What would be the outcome if $mini$ was sex linked? What would be the outcome if $mini$ was autosomal? Write out your crosses in fractional notation. 

Conceptual question: You are a pediatrician working in a remote rural community where there is an unusually high frequency of children with polydactyly - they have extra fingers or toes. What could you do to try to determine if this was a genetically inherited trait? What could you do to try and determine if this was a sex linked trait?