BICD 158 Problem Set 2

With the aid of a simple diagram illustrate the key steps in cut and paste transposition. [WHITEBOARD]

Steps involved:

1. Transposon produces transposase

2. Binds to inverted repeated ends of the transposon, cuts the element out from the site of the genome

3. catalyses the insertion of the transposon into the new genomic site

P-elements, which transpose according to the cut and paste mechanism, often cleanly excise from their point of origin. What is a key requirement for this to happen?

presence of intact homologous sequences (chromosome) at the site of transposon insertion

P-element transposase protein can be generated from two differentially spliced mRNA transcripts. What are the activities of these two different protein products and in what cell types are they produced?

Full-length 87 kDa transposase, encoded from. exons 1-4, produced by splicing. of intron 3 in the germline, mediates germline P-element transposition

Truncated 66 kDa transposase, encoded by exons 1-3, prouduced without splicing intron 3 in somatic cells, acts as a repressor of somatic transposition

When wild-caught male fruit flies were crossed to females from a laboratory strain, which was collected over a hundred years ago, a phenomenon referred to as hybrid dysgenesis was observed. What is the basis for this phenomenon? How do wild (P) strains differ from lab reared (M) strains? How did the wild P-strains arise? What two mechanisms are thought to contribute to P-strain males mating productively with P- strain females?

• Female fruit flies from laboratory M-strains

• No mechanism to repress P-elements

• Crossed with males of P strain

• Multiple P-elements carried by the male chromosomes mobilize through

transposase encoded by those transposons.

• These insert throughout genome leading to different phenotypes

• Reduce fertility

• DNA damage

• Cell death of germline cells

How P strains differ from lab reared M strains

P-strains carry multiple insertions of P-elements on their chromosomes whereas M-strain have no P-elements

What two mechanisms are thought to contribute to P-strain males

mating productively with P-strain females?

• Egg cytoplasm of P-strain represses transposition of P-elements due to production of interfering pi-RNAs that degrade -element transcript through Dicer-dependent mechanism

• Some P-strains, partial truncated P-elements encode shorter repressive form

of transposase

With the aid of a simple diagram illustrate insertion of a gene cassette into the genome using an integrase (e.g., fC31) [WHITEBOARD]

A plasmid carrying a transgene cassette and an attB donor site is injected along with phi31B recombinase into germline cells from a strain carrying a genomic insertion of the receiver attP site. phi31B recombinase then catalyzes recombination between the donor attB and target attP sites leading to insertion of the gene cassette into the genome.

With the aid of a simple diagram illustrate a recombinase system (e.g., FLP/FRT or CRE/LOX) for conditional deletion of a gene cassette. [WHITEBOARD]

Production of CRE recombinase in a tissue-specific

pattern (e.g., in the germline) results in excision of a

Lox-Stop-Lox cassette that otherwise prevents

expression of Cas9 from a ubiquitously acting CRM

and thus Cas9 protein production in a tissue-specific

fashion.

The tailless (t) allele in mice demonstrates a phenomenon referred to as meiotic chromosomal drive. What are the respective phenotypes of the following genotypes?

viable, and all sperm are fertile

+/t:

t/t:

How do these genotype/phenotypes explain meiotic drive of chromosomes carrying the t allele?

+/+: viable, and all sperm are fertile

+/t: viable, t-allele sperm are more fertile than + sperm

t/t: lethal

Because the t allele is preferentially transmitted to progeny by +/t heterozygotes it increases in frequency in the population when it is a rare allele. However, since t/t homozygous individuals die, t alleles are removed from the population as the allele becomes more common. An equilibrium point is reached where drive of the t allele in heterozygotes is balanced by the loss of that same allele in homozygotes.

In the lab, mice carrying the t-allele can constitute ~99% of the population when they are bred with mice of a wild-type strain. In nature, however, the t-allele often attains a much lower frequency (55 - 60%). Why might this be?

• The t-allele incurs increasing fitness over time since it is defined by a series of large chromosomal inversions that prevent recombination with the wild-type chromosome.

• Eventually these fitness costs reduce the frequency of successful transmission, survival, and/or reproduction of t/+ relative to +/+ individuals.

Explain how Homing Endonuclease Genes (HEGs) act as "selfish" genetic elements to increase their frequencies in a population. What is the key feature regarding the location of a HEG's insertion into the genome?

Homing Endonuclease Genes (HEGs) act as selfish genetic elements. HEGs get inserted into introns such that a protein is produced is a nuclease that cuts exactly at the site where the HEG was inserted into the genome. This is induces a double-stranded DNA break that is repaired by copying the unbroken allele that contains the HEG. Therefore, the HEG is copying itself into the genome at the point where it induces the break.

Are HEGs "active genetic" elements as specified by the basic definition of this term? Explain briefly (one sentence).

At the basic definition of the term active genetics, HEGs could be classified as active genetic elements because they are creating a double stranded break and repairing it with the HEG at the site that the double-stranded break was made in the genome by inserting the HEG. Following this logic, the inheritance of the HEG would become 100% when it is present in the genome, because it is changing the both homologs to contain the HEG.