Test 3 Transposable DNA elements

Question 1

Two major classes of mobile elements?

Answer 1

• DNA Transposons
  
  • Cut & Paste
• Retrotransposons
  
  • Copy & Paste
  • RNA intermediate
  • Subtypes
    
    • Long Terminal Repeats
    • Non-Long Terminal Repeats
      
      • LINE
      • SINE

Question 2

Bacterial Insertion Sequence (IS)

Answer 2

• Bacterial form of DNA Transposons
  
  • Structure:
    
    • Inverted Repeats on both ends
    • Protein coding region
      
      • This region can be transcribed into mRNA and then translated into a working enzyme:
        
        • Enzyme is Tansposase : Machinery of the transposition.
    • Target - Site Direct repeat on both ends
      
      • Not on the transposon but is the target site of the transposon.

Question 3

Insertion Sequence (IS) Steps

Answer 3

1. Excision of IS element by transposase.
   
   • Target site is staggard cut aswell.
2. Target site is random
3. Ligation IS element into target site
4. DNA polymerase “cleans up” producing direct repeats

Question 4

Are there any active LTR in humans?

Answer 4

NO

Question 5

Retrotransposons make up 40% of human genome?

Answer 5

YES

Question 6

DNA Transposons make up 5% of the human genome?

Answer 6

NO

Make up 3%

Question 7

Where are retrovirus genome encoded? and what does it mean?

Answer 7

SS RNA transcriptase

Means that it is capable of synthesizing DNA using RNA as template.

Question 8

Do Retrotransposons move to the new cell?

Answer 8

No

Question 9

Role of RNase H

Answer 9

Digests away RNA of an RNA - DNA duplex in retrotransposition.

Question 10

Steps of Retroviral genomic transcription and integration, and important enzymes.

Answer 10

• Synthesize a DNA strand
• Digest away a RNA hybrid
• One jump to other end
• Synthesize the other strand.
• Further digestion of RNA hybrid
  
  • Leaving just the short construct
• Extended by DNA polymerase
• Second jump and then both ends are extended via DNA polymerase.
• Important enzymes:
  
  • Reverse Transcriptase
  • RNase H

Question 11

What did the RNA intermediate experiment test and prove?

Answer 11

• Used Ty mobile elements in yeast.
• Showed that this gene must be turned on in order to undergo transposition.
  
  • Most likely caused by an mRNA construct.
• When turned on the transposed element had its intron removed.
  
  • Must be the result of an RNA construct causing RNA splicing.
• Sum: RNA intermediate must have been used since evidence of mRNA construct and RNA splicing.

Question 12

Describe Non-LTR retrotransposons

Answer 12

• Termed: Non-viral retrotransposons
• Two major types:
  
  • LINES
  • SINES
    
    • Parasite of LINE elements, in that they require active LINE elements to move about the genome.

Question 13

What are the main proteins of LINE reverse transcription?

Answer 13

• ORF1 and ORF2
  
  • ORF2 has reverse transcriptase activity.

Question 14

What type of site does LINE look for?

Answer 14

AT rich site and it nicks at both sides

Question 15

What acts as a primer in LINE?

Answer 15

Poly-A tail act as primer for synthesis of New LINE DNA.

Question 16

How often does a full LINE element make it?

Answer 16

Seldom

Question 17

What is the classic example of SINE, explain.

Answer 17

• ALU1 recognition site: 5’ - 3’ = AGCT
  
  • Samecode for a restriction enzyme known as ALU1
    
    • Hotspot for recombination
    • Genetic signposts, scattered all over genome.
  • Structure:
    
    • TSD (Target site duplication) found on both sides of the element.
    • A+B = RNA pol 3 promoters.

Question 18

Explain Exon Shuffling via recombination?

Answer 18

Double crossover between two ALU elements in two different genes.

Question 19

Why do we care about SINEs and especiialy Exon shuffling?

Answer 19

This is the process of genome evolution.

Question 20

Explain Exon shuffling by DNA transposons?

Answer 20

If two DNA transposons are close to one another the transposase may not cleave them out individually but cleave from the 5’ end of the first and the 3’ end of the second. This would bring anything in between with it.

Question 21

Explain Exon Shuffling by LINEs

Answer 21

Since LINE elements have a weak Poly(A) signal; if there is a strong poly A signal nearby then it may be mistaken as the end of the LINE element causing both the LINE element and the other gene being transposed to the insertion site.

Question 22

Explain Duplication of DNA sequence at Target Site?

Answer 22

The target DNA segment may be repeated on both ends of the newly inserted DNA sequence due to the Staggard cuts made during splicing.

Question 23

Most exon shuffling or duplication is beneficial?

Answer 23

False, most is deletarious.

Question 24

What GENE converts Codeine to morphine and what do you know about it?

Answer 24

• CYP2D6​
  
  • Some people do not have this gene due to gene deletion
    
    • This makes them poor metabolizers and the drug a placebo.
  • Some have 12-16 copies of the gene
    
    • These are ULTRA metabolizers
      
      • ​When given the correct dose of codeine they can make too much morphine too fast causing overdose.