BIOL 3010 - Exam 3 Review

What is the expected frequency of missense mutations based on the codon table? What is the expected frequency of synonymous mutations based on the codon table? In most natural population, missense mutations are less frequent than synonymous mutations. What does this discrepancy tell you about the difference between the de-novo mutation rate (rate at which new mutations arise in each generation) and the standing mutation rate (frequency of mutations that are already present in a population)?

Frequency of missense mutations = probability of nucleotide mutation 3 number of codons that encode a different amino acid

Frequency of synonymous mutations = probability of nucleotide mutation * number of codons that encode the same amino acid - 1 (since one codon will encode the original amino acid)

Missense mutations are more likely to be deleterious and removed by natural selection than synonymous mutations, which may be more likely to be neutral or even slightly beneficial. Therefore, missense mutations may be less likely to become established in a population and may be removed more quickly, leading to a lower standing frequency of missense mutations compared to synonymous mutations.

In a mutant screen, why do you need to wait until the 2nd or 3rd generation after mutagenesis to screen for mutants?

To allow for the completion of meiosis - occur in germ cells

To allow for homozygosity or heterozygosity of mutations - most are typically heterozygous in the first generation, so it would be difficult to identify and isolate mutants

If two independently derived EMS eye color mutations are caused by the same nucleotide change, what phenotype do you expect in their F1s? What phenotype do you expect in the F1s if the aberrant eye color are caused by different mutations? What phenotype do expect if they are caused by the different mutations in the same gene? Or in different genes?

WT eye color phenotype -- same mutation will be present on both copies of the gene

Heterozygous -> WT eye color -- each mutation is recessive

Heterozygous for both mutations (aberrant eye color) -> mutations are likely to be loss-of-function mutations

WT eye color -- each mutation is likely to affect a different gene or pathway

What are the pros and cons of being able to modify your mutation rate? What about if you are a bacteria? Or, if you are an elephant?

Pros: adaptation to changing environments, evolutionary potential, repair of DNA damage

Cons: increase in deleterious mutations, error-prone replication, risk of cancer

If you are a bacteria, it would be more beneficial than an elephant due to quick generation times and high reproductive rates

Are you a transgenic organism?

Yes - we have genes from parents and relatives and in our gut from food consumption

What are the benefits of a binary UAS-Gal4 system?

It kills (or ablates) specific classes of cells

A gene trap vector is designed to create a truncated endogenous protein and also express GFP where and when that endogenous protein is expressed. What molecular features do you need to have in that vector?

Promoter, splice acceptor, reporter gene, truncation cassette, selection marker

Are you concerned about GMO alleles "leaking" out into non-GMO crops or wild plants? Do you think that GMOs are safe?

Gene flow/transgene escape may lead to environmental and economic impacts but they're generally seen as safe as their non-modified counterparts

What is the roles of poly-linkers?

Synthetic pieces of DNA that contain different cut sites and restriction enzymes

In our generic vector, we have a selectable marker and the inserted gene. If the inserted gene is GFP, do we need to have miniwhite? What features of the promoter sequence would cause you to say YES or NO?

No -- if promoter sequence is strong enough or if reporter genes (mCherry or RFP) are better

Spontaneous mutations

Mutation accumulation in the lab: looking for "mutant" phenotype

Sequencing DNA following meiosis - sperm

Sequencing genome from families

Measuring levels of standing genetic variation in population or divergence between species

Spontaneous mutation rates vary

Mammals -- age

Microorganisms -- environment

Across species

Across genes: the sequence of the gene itself can be mutagenic

What causes mutations?

Copy errors: errors in replication, strand slippage, etc..

Chemical decay: deamination, depurination

MMutagens: x-rays (delations), EMS

What is a transgenic organism? Why is transgenesis important?

Transgenic organism -- contains a gene from another individual of the same species or a different species

Transgenesis happens in the wild and has been critical for the diversification of eukaryotes

What are transgenes?

Transgenes are constructed and synthesized using bacteria, plasmids, and enzymes -- inserted into eukaryote organism through germ line transformation, somatic transformation, and somatic expression

What are plasmids?

Plasmids are necessary for bacteria to share genes -- involved in horizontal gene transfer of antibiotic genes -- can be transferred into cells through chemical transformation (heat shock - 42 degrees for 30 seconds) and electroporation (15 kV/cm, 5usec pulses)

Molecular cloning

Get a single plasmid in one E. coli cell

Grow liquid culture derived from one cell with plasmid

Harvest E. coli, extract plasmid

What are the possibilities of basic genetic engineering?

Complementation

Enhancer trap

Binary expression systems

Recombination mediated cassette exchange

Conditional knockouts

What are the practical applications of genetic engineering?

Roundup blocks the action of a nuclear encoded enzyme that is transported to the chloroplast and is involved in amino acid production

Roundup ready is a modified version of this enzyme that is not susceptible to Roundup

What is Bacillus Thuringiensis (Bt)?

Common organic pesticide -- endotoxin that destroys the insect gut

How does the Somatic Gene Therapy process occur?

A new gene is inserted directly into a cell. A carrier (Vector) is genetically engineered to deliver the gene. An adenovirus introduces the DNA into the nucleus of the cell, but the DNA is not integrated into a chromosome

Does not necessarily involve incorporation into the genome

Vector borne DNA is transcribed into mRNA and then translated

How do you cut DNA in specific spots in the genome?

Restriction enzymes:

Pros: many different types that cut at different motifs

Cons: motifs are found throughout the genome

Programmable Zinc-Finger Nucleases

Pros: attach the scissor part of the RE to synthetic zinc finger proteins that bind specific sequences to DNA

Cons: ZF binding not entirely predictable, thus very difficult to work with since they vary from species

CRISPR-Cas9

Pros: ease of RNA design for new targets; use of a single, constant Cas9 protein; mutate many targets simultaeously with multiple guide RNAs

Cons: some off-target mutations

CRISPR-Cas9

Microbial adaptive immune system that has a memory with storage of genetic sequences of old viruses/infections to better fight viruses -- transmit to future generations

How does CRISPR/Cas 9 work?

Adaptation -- bacterial incorporation of DNA fragments into the CRISPR array

Processing -- CRISPR array transcripts get processed to generate small crRNAs that derive from the spacers

Interference -- targeting and cleavage of an invading genetic element, or its transcripts by Cas protein-crRNA complexes

What happens when Cas9 cuts 3-4 basepairs 5' of the PAM site?

NHEJ - non-homologous end joining

Homologous recombination - often knock-in DNA gets inserted on a separate plasmid from CRISPR plasmid

Somatic therapy

Destroy the splice acceptor using NHEJ induced insertion/deletion in Leber Congenital Amarurosis

Point mutation engineered o the homologous repair template co-injected with the CRISPR/sg-RNA

NHEJ repairs double strand breaks that occur naturally in cells. 95% of the time, NHEJ repairs the double strand break (DSB) perfectly. NHEJ results in small indel mutations 5% of the time during the repair of normal DSBs. Researchers observe that CRISPR-Cas9 will produce indels 100% of the time when NHEJ is used to repair DSBs made by CRISPR-Cas9. Why?

If NHEJ repairs the break perfectly, CRISPR-Cas9 will recognize the DNA site ad cut again and will keep doing so until an intel is formed, causing it to no longer recognize the DNA site

The PAM sequence is required 3' of the protospacer for CRISPR to cut. Will you ever find the PAM sequence at the 3' end of the spacer sequence at the CRISPR locus?

No -- it's at 5' NGG

What are the key features of the transgenic locus that makes CRISPR based mutagenic chain reaction different work?

Homology arms, Cas9, gRNA, payload gene

How does Sanger sequencing differ from sequencing by synthesis (Illumina)?

Sanger -- permanent terminators

Illumina -- reversible terminators

Why is particle restriction digest necessary to make overlapping BAC clones?

Generates compatible ends between BAC clones -- provides flexibility

How is sequencing vector different from transgenic vector?

Sequencing -- plasmid vector -- insertion of DNA fragments that need to be sequenced into vector

Transgenic -- introduced new genes into cells or organisms

How can you make a cDNA library without knowing the gene sequence?

Process: random priming

Isolate the mRNA from the cells or tissue of interest.

Use a reverse transcriptase enzyme to synthesize cDNA from the mRNA.

Add random primers to the reaction mixture, which anneal to the cDNA and allow for initiation of synthesis at multiple sites.

Amplify the resulting cDNA using PCR or other methods to create a library of cDNA fragments.

Why is gene content less variable than genome size in eukaryotes?

The gene content in eukaryotes is less variable than the genome size because genes are essential elements of the genome that carry out specific functions necessary for the survival and reproduction of the organism. While the size of the genome can vary greatly due to differences in the amount of non-coding DNA, such as repetitive sequences and transposable elements, the core set of genes required for basic cellular functions is generally conserved among related organisms.

This conservation of gene content is largely due to the fact that changes to essential genes can have detrimental effects on the organism's fitness, making them less likely to be passed on to future generations. Additionally, many genes are subject to functional constraints, meaning that changes to the gene sequence could disrupt the protein's structure or function, also reducing fitness.

Why can gene duplication lead to gene decay between paralogs? Why might gene duplication lead to gene divergence between paralogs?

Psuedogene -- after a gene is duplicated, one copy may become redundant and start to accumulate deleterious mutations due to the relaxation of selective pressure.

Gene divergence -- duplicate copy is free to accumulate mutations without the risk of compromising the function of the original gene. Over time, these accumulated mutations can lead to changes in the sequence and function of the duplicate gene, resulting in a new gene with novel functions

Illumina sequencing (NGS) can identify large insertions or deletions by assessing read depth. Why does read depth relate to copy number?

Read depth -- number of times a given base in the genome is read by sequencing technology; proportional to number of copies of that region. If there is a heterozygous deletion, the read depth in the affected region will be approximately half of that in unaffected regions.

Why do inversions prevent recombination?

Orientation of genetic material is changed -- pairing of homologous chromosomes is disrupted

How can you detect duplications with PCR?

Compare the sizes of PCR products -- duplication results in more PCR product from the reference regions

Why do inversions generate supergenes?

They bring together co-adapted sets of genes that function together in a specific way, allowing them to act as a single unit and enhance the fitness of the organism