CRIPSR, cancer, polyploidy, gene dosage, aneuploidy

forward genetics answers.. what is the process?

what genes are involved in biological process + function?

mutagenenisis screen

1. chemical induces mutation (parent)

2. self fertilized progeny (may be induced

pros and cons of forward genetics

p: easy to create new mutations

c: no specitivity, hard to determine which gene is mutated, need to look thru 100s to find phenotype

reverse genetics- what does it find out, what is the process?

what biological processes does a gene play a role in + function of gene

targeted mutagenenisis

1. introduce plasmid with mutant sequence

2. recombination, gene is in chromosome

pros and cons of reverse genetics

p: specific, discovers new functions

c: challenging to synthesize DNA seq

What was observed in Duchenne muscular dystrophy

• small deletion (one nucleotide), reading frame changed, early stop codon= severe phenotype, protein truncated, LOF

• large deletion (multiple of 3) but reading frame intact: onset, much less severe phenotype

• when exon 23 cut out, restored reading frame= mild symptoms

oncogene

mutated gene, presence stimulates cancer GOF, derived from proto-oncogene which regulates cell growth

• 1 mutated copy (+ / -) = dominant negative and haploinsufficient

tumor supressor gene

absence promotes cancer

2 mutated copies (- / -) = lsot of inhibitory factors, uncontrolled cellular division

loss of heterozygosity

inactivation of functioning allele in heterozygotes, so only mutant is present

translocations on chromosomes can cause cancer promoting mutations, how? reciprical translocation

translocated next to enhancer for B cells, hella antibodies, + extra abilities

philadelphia chromosome (BCR/ABL fusion protein)

makes a hybrid oncogene/ fusion gene, half of one and another, new function makes cancer (BCR/ABL fusion protein)= overactive kinase that promotes uncotnrolled cell division, expressed in WBC

euploid, diploid, polylpoid

n, 2n, 3n4n5n…

why is polyploidy more common in plants than animals

can survive but result in sterility, interferes with meiosis

why are polyploidy tissues and plants larger

increased number of chromosomes, increases the amount of DNA in the nucleus, which often causes the nucleus and overall cell to enlarge + higher gene dosage can lead to higher production of proteins and enzymes,

aneuploidy (trisomy) vs polyploidyl (triploidy) in humans

Trisomy involves an extra copy of one specific chromosome (47 total),

triploidy involves an entire extra set of chromosomes (69 total). 

• infertile because chromsomes can’t divide evenly during meiosis

autopolyploidy

has 2+ sets of homologous chromosomes, all derived from same species

error in mitosis. meiosis, cause chromosome sets to double, whole genome duplication and unreduced gametes can happen

partially fertile

allopolyploid

polyploid with multiple sets of chromosomes that were derived from different species (hybridization)

• two diploid species make hybrid, sterile because chromosomes cant pair properly,

• if hybrid produces unreduced gametes (2n=10) = restores fertility

euploid

correct number of chromosomes

haploid n =23

diploid 2n=46

polyploid banana 3n=33, whole number multiple of base set (n=11)

polyploidy and reproductive isolation

• polyploids can’t produce viable with others offspring due to mismatched chromosome numbers

• polyploids can only breed with others of same ploidy level = reproductive isolation and speciation

comapred to XY individuals, XX individuals would have twice the dosage of X linked genes, hwo is this corrected?

• XCI lionization: during embryogeneisis, one X chromosome becomes barr body (inactivated chromsome, heterochromatin), ensures proper dosage so X and Y have equal expression of X linked genes

  • mosaic cell: cell that is geneticlaly different from other cells in same individual

    • occurs in XX individuals due to random X chrosomosome inactivation, XX people are mosaics for X-linked gene expression- mix of normal adn affected cells

mutation types from DSB in different parts of a gene

promoter/ regulatory:

start codon:

early coding sequence (exon)

middle of coding region

end of gene, stop codon (last exon)

promoter/ regulatory: reduce/ stop transcription (LOF)

start codon: no translation (LOF)

early coding sequence (exon): frameshift, nonsense (LOF)

middle of coding region: missense, delete protein segments (partial LOF)

end of gene, stop codon (last exon) protien mostly fucntional (mild LOF/ no major effect)

mutation types depending on repair pathway

NHEJ:

HDR:

NHEJ: DNA ends joined directly, with errors- indels, frameshift, gene knockout LOF

HDR: use of provided template to repair break, percise edit, gene correction, GOF

if there's a deletion in the UTR does that cause frameshift

no! frameshift mutations occur in coding regions, so will not directly affect AA sequence, but can impact gene expression

5’ UTR: can affect ribosome binding for translation initiation, regulatory elements- LOF

3’ UTR: determines cleavage and polyadenylation, mRNA stability- LOF

give an exmaple of mosiacism with anhydrotic dysplasia

When X+X+ crossed with XaY, girls are XaX+, in embryogeneisis (early mitosis) random inactivation (barr body)= msoaic expression in cell populations of females, some express normal, other expresses mutant,

• once X is inactivated in cell, daughter cells maintain inactivation pattern

• phenotypically: if cirtical number of genes have mutant, sweatglands obersvable

barr body formation

before eymbryogensis

• transcription from one X chromosome per cell

• long non coding RNA (lnRNA) premotes heterochromatin and silencing

• Whichever X chrom expresses Xist first becomes Barr Body

why does gene dosage matter

leads to imbalanced pathways- too much promoter/ inhibtion

how are chromosome deletions and duplications formed

• 

1. highly repetitive sequence

2. crossover event

3. if sisters are imporperly aligned with sequences, can lead to deletion and duplication

4. more seen in x chromosomes

   

paracentric

inversion (flipped sequence DEF, FED) does not include centromere

paricentric

inversion includes centromeret

reciprocol translocation:

transfer of DNA between non homologous chromosomes

not losing info, just moved t o new chromosome, oftenWT phenotype because not disrupted gene dosage

inversion loops at meiosis- pericentric inversion

homologous chromosome: help inverted chromosomes to seperate by paring wiht homolgous regions

paracentric inversions can lead to deletion products. how?

homo chromosomes, if crossover event within inversion loop doesnt properly segretate, dicentric bridge breaks randoly, acentric fragments get deleted

how does reciprovcal translocation happen?

breaks near centromere and double stranded breaks on chromsome

how does robertsonian translocation happen, what does it look like on the karyotope, what is the disease associated with this

acrocentric chromosome- centromere is located near one end, resulting in a very short arm (p) and a long arm (q)

break near centromeres, form metacentric chromsomes, small fragment is lost

genes are now in different location, BUT GENE DOSAGE IS THE SAME

,

what are the gametes from robertsoain translation

what is down sydrome caused by

tirsomy 21

why are almost all trisomies and monosomies lethal- except x chromosome

trisomy: bar body, only expression of one x chromosome, neutralizes

monosomy: only one X is expressed anyway

why does risk for down syndrome and other trisomies increase by maternal age

• a phase that begins in the fetal ovary and is only completed decades later, during ovulation

• cohesions can weaken

• spindle fibers that properly segerate chromosomes in meiosis

do inversions result in change in gene dosage

no

role of NAHR and repetitive sequences

non-allelic homologous recombination sequences that are not from same location on chromosome, share high degree of similarity=

• repeitative DNA sequences misalign leading to unequal crossing over

deletions

• phenotypic effects

• meitotic effects

• evolutionary implications

strong effect ebcause loss of dosage can lead to haploinsufficiency, foten deleterious and selected againstdupl

duplications

increase doasage= may alter gene regulation, major soruce of new genetic material

difference between sporadic and familial down syndrome

sparadic: trisomy 21 due to non disjunction in meisosis, not inerhited

familial: due to translation of chromosome 21 onto chromosome 14, can be passed down

hallmark characteristics of cancer

• constant proliferation, no growth supressors, resisting cell death, activating telomerase for immortalility

insertions happen when

DNA poly slips on newly synthesized strand, the second round of replication is used as template and bp are inserted

slippage happens when

mononucletie, dinucletodie, tadem repeats

how must mutations happen?

second round of replication- incorporated into DNA

non reciprocal

non-homologous chromosomes: one chromosome gaining genetic material while the other loses it, loss in gene dosage

two ways translocation can make mutation

change of expression

• can relocate to new area near enhancer

fusion gene

• can make hybrid