LEC31: Recombination, Transposition, Genomic Repeat Sequences

Question 1

what does homology dependent DNA repair fix? what does it require?

Answer 1

fixes break in double stranded DNA

requires another chromatid and 2 double stranded DNAs

Question 2

what might might cause DNA to come out of its linear sequence?

Answer 2

homology-based generalized recombination of chromosomes

Question 3

what is generalized recombination?

what catalyzes it?

Answer 3

recombination between 2 homologous (similar sequences in the same orientation) chromosomes

Rad proteins (i.e. Rad 51) catalyze it

Question 4

what does homolgous mean?

Answer 4

share a high percentage of similartiy

in context of chromosomes, have similar sequences in the same orientations

basis for recombination

Question 5

why aren’t children of the same parents identical?

when do processes causing this occur?

Answer 5

because there is random assortment of chromosomes AND because of recombination between pairs of chromomes

recombination occurs before formation of gametes

Question 6

what is recombitant DNA?

Answer 6

when some paternal DNA is linked to maternal DNA and vice versa

product of homologous recombination between 2 double stranded DNA molecules

Question 7

what is holiday junction?

Answer 7

1 model of recombination btwn 4 strands of DNA

Question 8

what are all possible outcomes of recombination?

Answer 8

1) equal crossing over
2) unequal crossing over
3) inverted repeats on the same chromosome
4) exogenous DNA recombining w/ host chromsome

Question 9

what is equal crossing over?

what is the outcome?

Answer 9

homologous recombination btwn similar sequenecs on 2 chromosomes

occurs b/c maternal and paternal have copies of same gene to contribute

result: diversity in sequences of the 2 chromosomes, no change in function overall although may have some ntd changes

results in 2 chromsomes

Question 10

why does unequal crossing over occur? what is it?

what is the result?

Answer 10

2 or more copies of the same gene can be be present on a chromsome

happens if homolgous copies of gene align out of register and undergo recombination

result: unequal distribution of gene copies (i.e. 3 gene copies on 1 chromosome, 1 on the other); may cause disease

Question 11

what is alpha-thalassemia? what causes it?

Answer 11

alpha-thalassemia = chromic anemic condition, manageable, but not good!

unequal crossing over of chromsomes w/ alpha globin genes

result is 1 chromosome w/ 3 alpha genes, 1 with 1 alpha gene

Question 12

what does homologous recombination between 2 chromosomes yield?

Answer 12

2 products!

Question 13

what is the breakdown of chromosomal DNA between repeated sequences and unique sequenecs?

Answer 13

50% unique

40% repetitive sequences

10% simple repeat sequences

Question 14

where does homologous crossing over within a chromsome occur?

Answer 14

between regions of repeated sequences

Question 15

what causes hemophilia A?

Answer 15

crossing over between inverted repeats on the same chromosome

Question 16

what does Factor VIII gene do?

how does homologous recombination occur w/ the Factor VIII gene?

Answer 16

X-linked gene involved in blood clotting that, if deficient, results in hemophilia

end of X chromsome has 123 sequence, 4000 bp away is Factor VIII gene, and in middle of INTRON 22, repeated 123 DNA sequence

DNA of chromosome can loop around so that the 2 homolgous 123 sequences are near each other, and in same orientation

result is 1 nonfunctional Factor VIII gene, b/c exons 23-26, needed for function, are disconnected from the other exons

Question 17

how does exogenous DNA recombining w/ a host chromsome work? when does it work, when does it fail?

Answer 17

if have mutate (beta globin, for ex.) gene, can introduce a wild type copy into a plasmid DNA, get homology between wild type B-globin and the nonfunctional version on chromosome

via homologous recomination, should get double crossing over from host chromosome into exogenous DNA, and vice versa

now should have recombinant molecule w/ wild type beta-globin which replaces mutant beta-globin

works in bacterial cell, not human cell, b/c get illegitimate recombination where our inserted DNA sequence ends up in another chromsome

Question 18

why doesnt exogenous DNA recombination into a host chromosome work in some cases?

beta globin example

Answer 18

does not work when do it in human cell

b/c introduced gene does not express when out of its normal context

i.e. if introduce functional, wild type B-globin into human DNA, no expression continues after several weeks

Question 19

what are 2 methods of gene therapy whereby can have wildtype gene insertion in chromosome?

Answer 19

1) direct delivery
2) cell based delivery

Question 20

how does direct delivery method of gene therapy work?

Answer 20

take your cDNA of interest mRNA

put into a viral vector, usually lenti virus

directly introduce this into tissue target - i.e. liver

Question 21

how does cell-based delivery of gene therapy work?

Answer 21

take bone marrow cells out of body

do genetic modifications to cells out of body

then put genetically modfiied cells back into body

Question 22

what are the problems w/ doing gene therapy by direct delivery and cell-based delivery?

Answer 22

1) locus size of gene to insert may be too large for packaging into a viral vector
2) **position effect: **cannot guarantee that homologous recombination occurs - illegitamate recombination may - gene can go somewhere heterochromatic, therefore won’t be expressed
3) insertional mutagenesis: b/c we don’t control where exogenous DNA inserts into chromosome, can get effects that turn off a gene we need or inappropriately turn on a gene (i.e. turn on an oncogenic gene and cause cancer as side effect)

Question 23

what is gene addition gene therapy?

Answer 23

gene addition:

stem cells w/ a genetic mutation are pre-stimulated to prepare them for manipulation;

insert a functional copy of the gene into a retroviral vector;

do semi-random integration into cells;

express that vector in a person;

get normal gene product in person

Question 24

what is gene repair gene therapy?

Answer 24

gene repair:

treat cells w/ a nuclease engineered to cut around mutated site

DNA template for functional sequences is added to pre-stimulated cells via a viral vector

mutated sequence is replaced w/ wildtype via homologous recombination

so try to replace the mutant gene w/ a wildtype copy of the gene, and this in turn expresses normal gene product

works if you treat cells w/ factors that prevent early differentiation (dmPGE2 and SR1) & treat with signalling molecules, cytokines, that decrease the cells’ sensitivity to the toxic effects of the nuclease

result: turn on normal gene expression

Question 25

what is transposition?

where and when does it occur?

what catalyzes it?

Answer 25

movement of a unit of DNA, a “transposon,” from 1 site to a new target site

occurs in absence of homology, at random sites

transposaze enzyme catalyze transposition

Question 26

what encodes for transposaze?

Answer 26

a gene located on the transposon itself

Question 27

what does uncontrolled transposition cause?

Answer 27

havoc on genome structure!

thus it is highly controlled process

Question 28

describe process of cut-and-paste, non-replicative bacterial transposition

Answer 28

ends of transposon unit in donor DNA are recognized by transposase

transposase makes cuts at both ends of donor DNA & staggered cuts in a target DNA site

transposon DNA is inserted in target site

target may be same DNA molecule as donor or different DNA molecule

Question 29

what is complex transposition of a bacterial transposase?

Answer 29

carrying more genes than just the transposase gene into the target

i.e. antibiotic resistance gene can be carried, jump into plasmid DNA, and then recipient cell can receive antibiotic resistance gene which is not good

Question 30

what is a simple “insertion sequence” versus “complex transposon”

Answer 30

bacteiral transposon can be

1) IS: insertion sequence; just the transposase coding sequence is carried, i.e. IS3
2) complex transposon: carries transposase coding sequence AND gene for antibiotic resistance, for ex. (i.e. Tn3 ampicillin resistance gene, Tn10 tetracycline resistance gene)- accounts for multiple drug-resistant strains in hospitals!

Question 31

what is mechanism of bacterial versus eukaryotic transposon movement?

Answer 31

bacterial: DNA only
eukaryotic: RNA intermediate!

Question 32

how does eukaryotic retrotransposon integration work?

Answer 32

transposon DNA sequence is transcribed by RNA polymerase

this creates an RNA copy of transposon sequence

then reverse transcriptase converts RNA intermediate to double-stranded DNA

double-stranded DNA integrates into target DNA via activity of an integrase protein

retroposon sequence includes coding sequence for reverse transcriptase & gene for txn of DNA into target

Question 33

what kinds of sequences are found in the genome? examples of each?

Answer 33

1) unique sequences (may occur 2x since we are diploid)
2) repetitive sequences (i.e. rRNA genes, histone protein genes)
3) HIGHLY repetitive sequences (LINES, SINES, VNTR)

Question 34

what kind of sequences encode for rRNA genes and histone protein genes? why, re: function?

Answer 34

**repetitive sequences **

need more than 1 18S rRNA gene so we can make enough RNA for all our ribosomes

need more than 1 H2A gene, for ex., or would never have enough protein for entire nucleosome

thus have dozens of copies of each rRNA gene

Question 35

what are LINES?

Answer 35

long interspersed elements; a type of highly repetitive sequence

6000 bp long element

L1 is a LINE

Question 36

what is L1 an example of?

what is L1?

Answer 36

a long interspersed element (LINE) that codes for retrotransposon, which can undergo retrotransposition itself:

makes an RNA copy of itself, makes that into DNA via reverse transcriptase, and jumps elsewhere in the chromosome

have 500,000 L1 elements in our genome, but only 50 are active

2) provides region of homology where recombination can take place

they are **highly repressed via hyper methylation **b/c can cause disease

Question 37

what are SINES? how are they transposed? what is a major example?

Answer 37

short interspersed sequences

transposition depends on assistance of an L1 element

major type: AluI; 1 million copies (!) in genome; contains no coding sequence

Question 38

what is a VNTR?

Answer 38

variable number of tandem repeats

small DNA sequence (9-80 bps) present in multiple contiguous copies in same orientation

diff individuals may have diff number of a certain tandem repeat at a particular locus

PCR takes advantage of VNTR for forensics

Question 39

how do forensic studies use VNTRs?

Answer 39

can differentiate between 2 people based on the VNTR:

diff ppl have different number of repeats of the same sequence

thus can use primer to garget unique sequence outside the repeat region; size of PCR product will depend on number of repeats; if do this for several repeat regions simultaneously, unique personal pattern is generated; basis of forensic ID

Question 40

is an AluI sequence going to be an intron or exon?

Answer 40

intron, b/c doesn’t code for anything

Question 41

Explain why crossing-over events in meiosis generally have no consequence or generate diversity without affecting human health.

Answer 41

Because equal crossing over involves homologous recombination between similar sequences on 2 chromosomes, and the result is 2 chromosomes that have genes of the same function. You do not get loss of function through this kind of homologous recombination, you just get diversity because maternal and paternal genes are mixed into resultant recombinant chromosomes.

Question 42

There are multiple copies of ribosomal RNA genes in the chromosome. Why would unequal crossing over between these genes probably not result in serious consequences?

Answer 42

Multiple copies means redundancy!

We have so many copies of rRNA genes precisely because losing one would be devastating to that cell otherwise.

The sections of chromosomes with these genes generally have many copies in a row; unequal crossing over leaves many functional copies within that stretch of repeats (e.g. 40 repeats -> 30 or 50 repeats on the resulting chromosomes).

Question 43

Reverse transcriptase activity is required for transposition in eukaryotes but not in prokaryotes. Explain.

Answer 43

Transposition in eukaryotes occurs via an RNA intermediate; transposition in prokaryotes occurs directly from the DNA w/ the transposase gene to the target DNA

Question 44

Overuse of antibiotics has led to widespread antibiotic resistance. How do transposons play a role in this phenomenon?

Answer 44

Complex transposons present the transposon gene AND another gene into the target

They often carry antibiotic resistance genes