Genetics 7

These flashcards include key vocabulary terms and their definitions related to the concepts of forward and reverse genetics, DNA delivery methods, and gene regulation mechanisms discussed in the lecture.

Forward Genetics

A research approach that starts with a PHENOTYPE to find the gene responsible for it.

Reverse Genetics

A research approach that starts with a GENE to determine what phenotype it affects.

Mutagenesis

The process of creating a pool of random mutants, often used in forward genetics.

Complementation Analysis

A method used to determine whether mutations are in the same or different genes by crossing mutants.

Auxotrophic Yeast

Yeast mutants that cannot synthesize a specific nutrient, leading to a specific phenotype like leu–.

Selection

A method where only organisms with the desired phenotype survive.

Screening

A method to assess all variants manually to identify those with the target phenotype.

Zygotic vs. Maternal Effect

In diploid organisms, zygotic gene expression comes from the embryo's genome, while maternal effect is determined by the mother's genome.

RNAi

RNA interference; a biological process where RNA molecules inhibit gene expression, typically by causing the destruction of specific mRNA molecules.

CRISPR

A system utilized for genome editing that offers adaptive immunity against foreign DNA.

NHEJ (Non-Homologous End Joining)

A DNA repair process that is error-prone and can result in insertions or deletions (indels).

HDR (Homology-Directed Repair)

A DNA repair process that uses a donor template for precise edits, allowing for gene corrections or insertions.

sgRNA (Single Guide RNA)

An engineered RNA that combines crRNA and tracrRNA for genome editing in CRISPR systems.

Transfection

The introduction of nucleic acids into eukaryotic cells, often via chemical, physical, or viral methods. (viral or non-viral)

Transformation

The uptake of external DNA by bacteria, archaea, or yeast, which can be natural or artificially induced.

Transduction

The process of transferring DNA into prokaryotes via a bacteriophage or virus. Bacteriophage/virus injects DNA into the host

Conjugation

A method of genetic transfer in prokaryotes involving direct cell-to-cell contact.

complementation

mutations are in DIFFERENT genes → different complementation groups

non-complementation

mutations are in the SAME gene → same complementation group

zygotic screen

(F0 to F4) F0 breed F1, F1 intercross to F2 then F2xF2 to make F3 and then look for abnormal/dead F3 embryos

maternal screen

(F0 to F4) look at phenotype of F4

mutational tagging

find mutant, identify which gene is mutated by tagging gene and sequencing it to compare to reference genome

natural competence

Transformation - some bacteria naturally take up DNA from their environment

Chemical (artificial) competence

Transformation -   treat cells with Ca²⁺ (CaCl₂) then heat shock (42°C)

Electroporation (artificial)

Transformation - wash cells to remove salts/ions, then apply electrical pulse

co-supression

Both the transgene AND the endogenous gene were silenced

c. elegans

Microscopic roundworm with fully mapped cell lineage — every cell division is known

LIN-14

Transcription factor; high in L1, off by L2

lin-4

Encodes the FIRST miRNA ever identified (Lee, Feinbaum & Ambros, Cell 1993)

siRNA Mechanism

PERFECT MATCH, DESTROY mRNA - dsRNA appears and a virus is introduced, the dicer cuts short siRNA pieces and loads it onto the Ago plate so they form RISC. We keep one strand as a guide strand and then there is perfect base pairing with the target mRNA and the RISC cuts mRNA and degrades it soooo mRNA is gone

miRNA Mechanism

IMPERFECT MATCH, BLOCK TRANSLATION Cell makes a small hairpin-shaped RNA (pri-miRNA) and it gets processed into pre-miRNA and dicer cuts it into small short miRNA, its loaded onto AGO and turned into RISC. It sticks to mRNA that it doesn’t match with perfectly which bocks translation. The mRNA is still there but can’t be translated well.

three steps of CRISPR immunity

spacer acquisition, crRNA processing, interference

• involves a short RNA fragment that guides the Cas9 protein to target DNA for cleavage.

Type 1 CRISPR

uses a multi-protein Cascade complex to recognize foreign DNA and then recruits the Cas3 enzyme to unwind and degrade the target DNA

Type 2 CRISPR

uses a single protein, Cas9, guided by RNA to directly cut specific target DNA sequences

dead Cas9

cas9 binds but does not cut

Episome

autonomously replicating circular DNA that replicates independently of the host chromosome

Plasmid curing

loss of a plasmid by the host cell when selection pressure is removed

Random insertion

sequence-independent integration of DNA anywhere in the chromosome; donor DNA is non-replicative (no ori) or linear

Transposon-mediated insertion

hijacking transposon cut-and-paste machinery to insert DNA randomly into the chromosome; requires transposase + inverted repeats on separate plasmids

Virus-mediated integration

engineered virus delivers and integrates foreign DNA into host genome

Homologous recombination

targeted, sequence-dependent chromosomal integration guided by 5' and 3' homology arms flanking the donor DNA

Site-specific recombination

targeted insertion using a recombinase enzyme (e.g., Cre/lox) that swaps a plasmid recombination site with one already in the genome

Functional complementation

introducing a functional gene to rescue a mutant phenotype back to wild-type; used to confirm gene identity and map essential regions via mutational analysis

Gain-of-function

enhancing an existing activity or introducing a completely new function via DNA delivery

Gene knockout

replacing or disrupting a target gene to eliminate its function; achieved via homologous recombination or CRISPR

Disruption vs. deletion

disruption = marker inserted into the gene; deletion = marker fully replaces the gene

Functional genomics

systematic knockouts of every gene in an organism to determine which genes are required for any given phenotype