Genetics 16: 'Introduction to Drosophila: genotypes, recombination and balancer chromosomes'

These are my notes from lecture 16 of Harvard’s Genetics 201 course, delivered by Mitzi Kuroda on October 27, 2014.

Intro to Drosophila

Why study flies?

• Easy to work with
• Short life cycle (~2 weeks)
• Great phenotypes (if you like looking at fly eyes)
• Great chromosome analysis
• Easy to cross

From egg fertilization and embryogenesis, Drosophila spend a total of ~4 days in three larval stages, then ~5 days in prepupa and pupa stages. Then the adult emerges from the pupal case, an event called eclosure, and they become reproductively active within 8 hours after. They can live 45-60 days under good conditions.

A few classic phenotypes are Curly wings (Cy; dominant), white eyes (w), yellow body (y), Stubble (Sb), and irregular facets in the eye (If). Flies are often used for studying development, chromosome structure, neurobiology, and behavior. Morgan and Muller did the earliest famous work on Drosophila. Drosophila have polytene chromosomes in some tissues, which made the chromosomes large enough to see even with the microscopes of yesteryear. Lewis, Nüsslein-Volhard & Wieschaus won the 1995 Nobel Prize in Physiology or Medicine for studying embryonic development in Drosophila and discovering Hox genes. Anntennapedia is a gain-of-function mutation where legs, instead of antennae, grow out of the head.

Comparison to other organisms

In contrast to model organisms we’ve recently studied in this class, Drosophila come in male (♂) and female (♀) only (no hermaphrodites ⚥) and are always diploid. Here are some other comparisons:

Drosophila have chromosomes X, Y, 2, 3 and 4. Females are X/X and males are X/Y. Chromosome 4 is small and contains hardly any genes. Like most organisms, Drosophila have the annoying property that most genes are named after their loss-of-function phenotype, i.e. the opposite of what they do. For instance, the white gene makes eyes red. However, you can’t count on this always being true. For instance, cinnabar (cn) and brown (bw) are two genes which encode cinnabar-colored and brown-colored pigments, such that a cn bw / cn bw double mutant is white.

Nomenclature

A singed (sn; crooked bristles, recessive, on chrom 2), cinnabar (cn) and brown (bw) (both recessive, both on chrom 3, this combination gives white eyes), Serrate[d] wing (Ser; dominant, hence Initcaps, located on chrom 4) fly would be written:

sn/sn ; cn bw / cn bw ; Ser/+

By default, sn means the mutant gene, while wild-type genotypes are simply omitted. If you need to emphasize for clarity, you can write the wild-type allele as sn+ and the mutant as sn-. If you have multiple singed mutants you could write them sn¹, sn², etc. Genes known to be on the same chromosome are written in one fraction. Reference is Flybase.org

Chromosomal position and phase are represented in this fraction nomenclature, such that cn bw / + + has the cinnabar and brown mutations in cis, while cn + / + bw has them in trans.

Protein products are written in Initcaps, no italics. Flybase has full nomenclature rules.

Balancer chromosomes

A challenge of Drosophila is they cannot be readily frozen or archived - mutants have to be continuously propagated. In addition, many of the most interesting genes in Drosophila are essential for life, and have to be propagated as heterozygotes which have no phenotype. This gives you two problems: first, it leaves you blind as to whether you’re still propagating the mutation. Second, as you cross your heterozygotes to one another, you lose all the mut1/mut1 offspring, and this selection reduces the allele frequency of your mutation of interest in every generation. This is a problem in all constitutively diploid organisms, including mice. In Drosophila, the solution for studying mutations that are recessive lethal or recessive sterile is as follows:

1. “Balance” the mutation with another lethal/sterile marker, so for instance all your flies are of a mut1 + / + mut2 genotype, such that only hets for both mutations can survive.
2. Add a visible dominant marker in cis with one of the two balancing mutations: mut1 + + / + mut2 Curly
3. To prevent recombination from short-circuiting your system, you additionally introduce multiple inversions into the trans chromosome. This gives you a multiply inverted balancer chromosome which contains mut2 and Curly. A downside is that this doesn’t completely prevent recombination - in fact, when recombination does still occur, it results in crazy copy number variations because parts of the multiply inverted chromosome will be gained or lost.

Example

DTS is a dominant temperature sensitive mutation that causes flies not to develop at 29°C (their preferred temperature is around 22°C but wild-type flies can go from 19°C to 29°C). DTS is recessive lethal at any temperature. CyO (“Curly O”) is a balancer chromosome which is wild-type at the DTS locus and also has a recessive lethal mutation. Implicit in the nomenclature DTS/CyO is that each chromosome is wild-type for the other chromosome’s mutation. In a cross:

The DTS/DTS and CyO/CyO are both recessive lethal, so you have successfully propagated only the parental genotype, DTS/CyO.

Because Drosophila have only three chromosomes of appreciable length, there are only three balancer chromosomes in common use.

How to get mutants

Usually you mutagenize males with EMS or X-rays. Males are used because they don’t need to be virgins. Also they are more resistant to DNA damage, which means they can tolerate enough damage to get useful mutations. Note that you only care about mutations in the germline, not soma. Then you cross to females. Each F1 will have a unique heterozygous mutation inherited from the father’s germline. At the F1 stage, you can only identify viable dominant mutations. (An exception is if you mutagenize females instead of males, you can find X-linked recessive mutations in the male F1s). If your viable dominant mutation is sterile, you’re out of luck, because you can’t propagate it beyond that one male. This motivates a need for more complicated screen designs, which will be covered later this week.