1. Retroviruses and Trans(retro)posons

2. Transposons Mobile DNA (elements) in the genome Jumping genes
DNA fragments (elements) with the capacity to move in the genome
Mobile DNA (elements) in the genome
Jumping genes
Function: structural
~ 50% genomic in mammals.
Clasification:
DNA transposons
Retro (RNA) transposons

3. DNA transposons IS elements (insertion sequence)_bacterias
> 20 types
Frequency of movements (1/ a 1/ per generation)
Gene inactivation
F plasmids
Mechanism:
Cut and paste
Structure:
Inverted repeats
Target repeat
Sequence coding region-Enzyme “Transposase”

4. ADN transposones
Elementos IS (insertion sequence) bacterianos (eucarioticos)
Más de 20 tipos distintos
Movimiento poco frecuente (1/ a 1/ por generación)
Pueden inactivar genes
Pueden transferirse a otras bacterias mediante su transposición a plásmidos o a viruses
Estructura de los elementos IS･ Repeticiones invertidas en los extremos
Region codificante central: Transposasa

6. Holliday junction

7. The Holliday Model: single strand break model

8. Recombination
In eukaryotes, crossing over is associated with the formation of the synaptonemal complex during prophase of meiosis I (Chiasmata).
Crossing over involves the breakage of parental chromosomes and rejoining of the parts in new combinations.
The Holliday model and the double-strand break model are two explanations of the molecular basis of recombination.
© John Wiley & Sons, Inc.

9. The Recombination Intermediate is a Chi Structure
© John Wiley & Sons, Inc.

10. The Recombination Intermediate is a Chi Structure
© John Wiley & Sons, Inc. 10

11. The Holliday Model: single strand break model
--DNA-dependent ATPase.
--can hold a single strand and
double strand together.
--DNA synapsis reaction
between a DNA double helix and a
homologous region of single
stranded DNA.
--catalyzes branch migration begins. 11

13. Gene conversion is the process by which one DNA sequence replaces a homologous sequence such that the sequences become identical after the conversion event.
(Non) Homologous recombination
perfectly complementary
Seq.
Not perfectly
complementary seq.
Synthesis Dependent
Strand Annealing
Non-crossovers
intrinsically mutagenic
Double Holliday junction
Crossovers

14. Retro (RNA) transposons
Mostly in Eukaryotes
Yeast
Drosophila
Mammals (humans y ~8%)
Mechanism:
~ retroviruses (copy and paste)
Types:
---Retroviruses like (LTR)
---No related to retroviruses
Structure:
Inverted repeats LTR (Long Terminal Repeats) ( pb)
Sequence coding region-Enzymes Reverse Transcriptase (RT), Integrase (IN) and other proteins.

15. Types:
---Retroviruses like (LTR)

16. Figure 22.12 Types: ---No related to retroviruses Absence of LTRs
Mostly in Eukaryotes
Two types:
LINES (Long Interspersed Elements) ~6 Kb
SINES (Short Interspersed Elements) ~300 bp
Structure:
Target-site direct repeats
A/T rich regions
Sequence coding region- RNA binding proteins (ORF 1) and Enzymes ~Reverse Transcriptase (RT) and endonuclease (ORF 2)

18. Transposons Function: Alterations in the patterns of gene expressions
DNA fragments (elements) with the capacity to move in the genome
Mobile DNA (elements) in the genome
Jumping genes
Function: structural
Function: Alterations in the patterns of gene expressions

19. DNA transposon-based gene vehicles – scenes from an evolutionary drive

20. Introduction Retrovirus life cycle
--Retrovirus (two copies of RNA packaged in a capsid) attaches to cell membrane (Env) and fuses (Env), allowing entry of RNA into cell.
--RNA is reverse transcribed into dsDNA and integrated into the host genome as a provirus.
--The host transcribes the provirus, making all the proteins necessary for assembly of the virus which buds out of the cell as the complete retrovirus.
Figure 22.1

21. The Retrovirus Life Cycle Involves Transposition-Like Events
1-A retrovirus has two copies of its genome of single-stranded RNA.
2-An integrated provirus is a double-stranded DNA sequence.
3-Polyproteins are initially produced and then processed (proteases) to give all the viral protein products necessary for retrovirus reproduction.

22. A retrovirus generates a provirus by reverse transcription of the retroviral genome.
---Retroviral RNA (plus strand) is reverse transcribed (Pol – reverse transcriptase) into ssDNA (minus strand).
---The complementary strand of DNA (plus strand) which adds LTRs (long terminal repeats) to the 5’ and 3’ end, the linear dsDNA is inserted into the host genome (Pol-integrase).
---Transcription of the provirus (integrated retrovirus sequence) into RNA (plus strand) by the host RNA polymerase.
Figure 22.2 v vm

23. Retroviral Genes Codes for Polyproteins
A typical retrovirus has three genes: gag, pol, and env.
Gag and Pol requires either stop codon
suppression or a frame-shift.
This reduces the amount of Pol produced.
Env is translated from a separate viral mRNA
that is generated by splicing.
Each of the three protein products are
processed by proteases to give multiple
Proteins.
Full length viral mRNA is packaged as
the viral genome.
The terminal region of the viral mRNA
contains specific sequence (U and R
segments)
mRNA is capped 5’ and polyadenylated 3’
Figure 22.3 U3 U5
vmRNA

24. Retrovirus encoded polyproteins that must be processed
vmRNA U5 U3
Retrovirus encoded polyproteins that
must be processed
Gag - structural proteins in matrix, capsid, and nucleocapsid.
Pol – protease, reverse transcriptase, and integrase.
Env – surface protein, transmembrane protein for recognition and fusion

25. What is the source of the viral membrane?
Figure 17.4
What is the source of the viral membrane?

26. Influenza Virus Must “Uncoat” in Cytoplasm
H5N1 hemagglutinin (HA) type 5 and neuraminidase type 1

27. Viral DNA Is Generated by Reverse Transcription A short sequence (R) is repeated at each end of the viral RNA.
1-Reverse Transcription
2-Integration
3-Transcription
Figure 22.5
Virus RNA structure
U5 – unique sequence at 5’ end ( bp)
U3 – unique sequence at 3’ end ( bp).
A short sequence (R): is repeated at
each end of the viral RNA, so the 5’
and 3’ ends are R-U5 and U3-R.

28. Viral DNA is generated by reverse transcription
-RT converts RNA to DNA
-RT shows DNA polymerase activity
-RT has RNAase H activity
Reverse transcriptase starts synthesis when a tRNA primer binds to a site 100 to 200 bases from the 5′ end.
(Synthesis= 5’ to 3’ direction and requires a double stranded region to initiate)
(tRNA is a “viral transcriptional RNA “?”)
When the enzyme reaches the end, the 5′-terminal bases of RNA are degraded.
This exposes the 3′ end of the DNA product.
The exposed 3′ end base pairs with the 3′ terminus of another RNA genome
Strand switching event
Synthesis continues, generating a product in which the 5′ and 3′ regions are repeated.
This gives each end the structure U3-R-U5.
Quality and Quantity---Control
U3 R U5
Figure 22.6

29. Quality and Quantity---Control
Similar strand switching events occur when reverse transcriptase uses the DNA product to generate a complementary strand.
-Synthesis continues, generating a product in
which the 5’ and 3’ regions are repeated,
giving each end the structure U3-R-U5 (LTR)
(Long Term Repeat)
-Similar strand switching events occur when
reverse transcriptase uses the DNA product
to generate a complementary strand.
-Strong stop plus strand DNA jumps to 3’
end of cDNA. This adds U3 to 5’ end of
dsDNA and completes the formation of
the LTR.
U3 R U5
U3 R U5
U3 R U5
Quality and Quantity---Control
Figure 22.7

30. Strand switching is an example of the copy choice mechanism of recombination.
-Positive-sense vRNA
(vmRNA) (HIV)
-Negative-sense vRNA
(Ebola)
Figure 22.8

31. Viral DNA Integrates into the Chromosome
The organization of proviral DNA in a chromosome is the same as a transposon.
The provirus is flanked by short direct repeats of a sequence at the target site.
Linear DNA is inserted directly into the host chromosome by the retroviral integrase enzyme.
Two base pairs of DNA are lost from each end of the retroviral sequence during the integration reaction.

32. RNA has direct (inverted) repeats, linear DNA form of virus
has Long Terminal Repeats (LTR) and the integrated
DNA form (provirus) has LTRs that are shortened by 2 bp
And a 4-6 bp repeat of host DNA.
Integrase, a viral product, acts on both viral DNA
and target-host DNA.
-It targets random sites by making staggered cuts at the
selected-host DNA sites.
-it targets end of the viral DNA converting the blunt ends
into the recessed ends by removing bases beyond the CA
dinucleotide sequence.
-it needs sticky ends
-1 to 10 copies can be introduced into host cells.
(amplification and rearrangement of DNA)
U3 region of each LTR carries a promoter and enhancer
( initiates transcription)
Figure 22.9

33.
Non-germline vs germline?

37. How are the Replication-defective viruses generated?
Figure 22.10
Transducing retro-viruses: a virus
variant containing cellular RNA.
Replication-defective: a virus variant in
which a viral sequence has been replaced.
Helper virus: a retro-virus that provides
the missing viral function to a replication-
defective virus. U5 U3 U3 U5
How are the Replication-defective viruses generated?

38. Retroviruses May Transduce Cellular Sequences
Transforming retroviruses are generated by a recombination event:
A cellular RNA sequence replaces part of the retroviral RNA.
Model for the formation of the transforming viruses.
1-virus integrates near a gene
(c-gene)
2-transcription of the viral RNA
copy #1 (deletion)
3-transcription of the viral RNA copy #2 (no-deletion)
4-packaging of both copies of the viral RNA
Figure 22.11

39. Why is the addition of v-onc into the viral genome a problem?
Figure 22.10 U3 U5 U3 U5
Why is the addition of v-onc into the viral genome a problem?

40. Figure 30.14
Human retroviruses?

41. Figure 30.13
Replication-defective virus

42. Yeast Ty elements resemble retroviruses
Figure 22.12
Yeast Ty elements resemble retroviruses
What features are similar between retroviruses and Ty elements?
Translation of TyA and TyB involves stop codon suppression and frame shift.
TyA and TyB are similar to gag and pol.
Transposition of Ty is through an RNA intermediate.
Direct repeat delta () elements at both ends of Ty like LTR.
No Env genes ….so non-viral particles are generated.

43. 22.7 Yeast Ty Elements Resemble Retroviruses
Ty transposons have a similar organization to endogenous retroviruses.
Ty transposons are retroposons, with a reverse transcriptase activity.
They transpose via an RNA intermediate (splicing reaction)
Figure 22.13

44. 22.8 Many Transposable Elements Reside in Drosophila melanogaster
Copia is a retroposon that is abundant in D. melanogaster.
What features are similar between retroviruses and Copia elements?
Copia is a single gene, similar to gag and pol.
Transposition of Copia is through an RNA (messenger)
Direct repeat elements (LTR-like) at both ends.
No Env genes so non-viral particles are generated.
Figure 22.15

45. Viral vs Non-viral particles
Some retroposons directly resemble retroviruses in their use of LTRs. Others do not have LTRs.
Other elements can be found that were generated by an RNA-mediated transposition event; But they do not themselves code for enzymes that can catalyze transposition.
No Env. genes?
Viral vs Non-viral particles
Mammalian
genomes
Yeast
D. Melanogastes
Human
Figure 22.17

46. 22.9 Retroposons Fall into 3 (or 2) Classes
Viral
Figure 22.16

47. Transposons and retroposons constitute almost half of the human genome.
LINES: long interspersed elements
SINES: short interspersed elements

48. 22.11 Processed Pseudogenes Originated as Substrates for Transposition
A processed pseudogene (not active) is derived from an mRNA sequence by reverse transcription. (i.e., LINES)
Figure 22.19: Reverse transcription + insertion = pseudogene.

49. b-terminal repeat in A/T-rich region
LINES
a-6,500 bp long
b-terminal repeat in A/T-rich region
c-Two ORF (1/2)
-RNA binding protein
-RT
-endonuclease
Functional
transposon
Figure 22.21: LINES proteins are cis-acting.

50. 22.10 The Alu Family Has Many Widely Dispersed Members (SINES)
A major part of repetitive DNA in mammalian genomes consists of repeats of a single family:
organized like retro-transposons
No Rev. Trans.
Alu I sensitivity
<300 bp repeats
derived from RNA polymerase III transcripts
Sequence related to 7SL RNA, a comp. of SRP/SRP-Receptor.
5S rRNA, tRNA and U6 snRNA
(A)n= 5' AG/CT 3';