Lab 2

Learning Objectives

1. Explain relationship between genotype and phenotype

2. Define terms gene and allele, use them appropriately

3. Know that different alleles of a gene can exhibit different phenotypes

4. Predict phenotypes of progeny that arise from a genetic cross of parents containing mutations in linked and unlinked genes

5. Predict genotypes of progeny that arise from a genetic cross of parents containing mutations in linked and unlinked genes

Learning Objectives 1-3

1. Explain relationship between genotype and phenotype

2. Define terms gene and allele, use them appropriately

3. Know that different alleles of a gene can exhibit different phenotypes

Learning Objectives 4-5

4. Predict phenotypes of progeny that arise from genetic cross of parents containing mutations in linked and unlinked genes

5. Predict genotypes of progeny that arise from genetic cross of parents containing mutations in linked and unlinked genes

Classical genetics is used to

perform genetic screens in order to identify genes functioning in a biological process.

Location of gene on genetic map instrumental in identifying gene that contained mutation

What gene are we tagging today

unc-32,

was isolated in a genetic screen for mutations that cause worms to move abnormally (uncoordinated).

We also use genetic principles to

generate strains containing multiple mutations and visible markers.

The strains we are using for our experiment contain

one mutation and 4 transgenes inserted into a chromosome.

Nomenclature

nomenclature style and rules different for all commonly studied organisms

unification between fields unlikely to exist, we are too entrenched in our ways

C. elegans relatively straightforward bc more recent history as model organism

Gene names, in C. elegans, how is it written/formatted, how is it named

3-5 letters, a hyphen, a number

letters and number italicized

The letters chosen are often an abbreviation of a longer descriptor for the phenotype caused by a loss of function in that gene (such as dpy for dumpy or unc for uncoordinated).

number follows letter to distinguish, for example, unc mutations

how many unc loci

132

allele, includes

A gene can have many different alleles, or versions.

The name of a mutant allele contains

letter(s) that indicates the lab of origin, number

included in parentheses after the gene name.

For example, e53 and e152 are both alleles of unc-5 isolated at the MRC in england. We would write them out as unc-5(e53) and unc-5(e152).

Confusion with alleles

because if you see only “unc-5” with no allele, you don’t necessarily know whether it refers to the wild-type version of unc-5 or a mutant version. In a well-written paper, context should make that clear.

homozygous

homozygous for a mutation, that means the allele on each chromosome is the same. We usually indicate a homozygous genotype as simply e.g. ‘unc-5(e53)’. Sometimes when writing out crosses, it is helpful to indicate both alleles, and we would write ‘unc-5(e53) /unc-5(e53) ’.

heterozygous

that means the alleles on each chromosome are different and unless stated otherwise, we assume one allele is wild-type. A heterozygous genotype can be indicated as “unc-5(e53) / +” or “unc-5(e53) / unc-5(+)”

Protein names in C. elegans

are indicated in all caps. For example, the gene unc-32 encodes the protein UNC-32.

not italicized

Phenotype

refers to the outward manifestation of a genotype.

The shorthand for phenotype in C. elegans is indicated by a 3-5 letter non-italicized code, with the first letter capitalized. For example, the phenotype of unc-5(e53) is ‘Unc’.

In reality, phenotype is rarely so simple. We can describe phenotype at many levels. The Unc phenotype indicates the worm moves in an abnormal way. We could also describe the phenotype at the cellular level – for example, that axons are not connected correctly. We could describe the phenotype with respect to the localization of other proteins in mutant worms.

Karyotype

refers to the chromosomal make-up of an individual.

C. elegans has six chromosomes: five autosomes (I-V) and one sex chromosome (X). Hermaphrodites are diploid for all six, whereas males are diploid for the autosomes but are haploid for X (designated XØ).

Hermaphrodites contain 2 alleles of genes on the X chromosome (the sex chromosome),because they are XX;

males contain one allele of genes on the X chromosome because they are XØ

how we indicate the genotype of a worm, linked

if the two genes are linked on the same chromosome, we write both gene’s names together with no separator, e.g. unc-5(e53) dpy-13(e184).

unlinked

different chromosome we indicate that by separating the 2 genes with a semicolon, e.g. unc-5(e53); dpy-5(e61).

null

if a gene results in a complete loss-of-function of a gene, we call that allele a “null” allele or null mutation.

For example, if a gene encodes a protein and a mutation creates a stop codon near the start of the protein, that allele is likely a null allele.

hypomorphic alleles

Alleles that cause only a partial loss-of-function of a gene are called “hypomorphic” alleles.

Usually, hypomorphic alleles exhibit less severe phenotypes than null alleles.

Some mutations can cause a gain-of-function

For example, a mutation that causes too much of a protein to be made or a mutation that prevents the inhibition of a protein (so that it is always “on”) could both cause the protein to function more than it normally does. Sometimes gain-of-function alleles can exhibit the opposite phenotype of a loss-of-function allele.

penetrance

proportion of individuals carrying a particular variant (or allele) of a gene (genotype) that also expresses an associated trait (phenotype)

Ex. If 70% of worms homozygous for mutation show trait, 70% penetrance

Expressivity

refers to how strong the phenotype is

all N2s

descend from same worm

phenotype of worms

N2s can have slight variations in phenotype despite their identical DNA sequence.

The phenotype of mutant worms, again with identical DNA sequence, can vary quite a bit.

sister chromatid

identical copies (chromatids) formed by the DNA replication of a chromosome, with both copies joined together

homologous chromosomes

a set of one maternal and one paternal chromosome that pair up with each other inside a cell during meiosis.

products of meiosis

germ cells

In prophase I

the four homologous chromatids (produced by the duplication of each parental autosome) join together to form a bivalent.

The exception is the X chromosome in males, for which there are only two chromatids

Crossing over in C. elegans

In general, a single crossover event will occur between just one of the two pairs of parental chromosomes.

The pair of maternal and paternal chromosomes that undergo the exchange will consequently contain both maternal and paternal sequences.

The other maternal-paternal chromatid pair will remain in their original form.

crossing over part 2

At low frequencies, two spatially separated crossover events can occur between a single pair of chromatids. When such double exchanges occur, they generally take place at opposite ends of the chromosomes because the crossover points (called chiasmata) discourage other nearby crossover events.

crossing over point

chiasmata

consequences of crossing over mechanism in C. elegans

The practical consequence of this phenomenology is that we are almost always safe in assuming that the recombinants that we isolate are the result of a single crossover event, particularly if we are targeting crossovers within relatively small regions of the genome.

Recombination frequency can be used to order loci along a chromosome, or “map” them. Being aware of recombination is also important so that you can predict the genotypes of all possible progeny.

genetic crosses in C. elegans

C. elegans hermaphrodites can self-fertilize, but generally, we do not refer to their self-fertilization as a cross. A genetic cross requires mating, and all matings males. That presents a [minor] challenge when working with C. elegans, because the vast majority of worms on plates will be hermaphrodites. Unless spontaneous non-disjunction of the sex chromosome (X) occurs during meiosis, progeny of hermaphrodites will all be hermaphrodites.

Non disjunction in C. elegans, frequency, how do we do crosses

Non-disjunction of the X occurs at a low frequency (~0.2%) in wild- type hermaphrodite populations.

Therefore, a C. elegans researcher doing genetics maintains a stocks of males by placing about 3-5 males on a plate with 2-4 hermaphrodites [See the PostLab Questions].