| M W F; 10:10 - 11:00 pm |
Molecular Biology |
Douglas W. Smith |
| York 2722 |
BIMM 100 |
5254 Muir Biology Building |
| Fall, 2000 |
x42620; dsmith@ucsd.edu |
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Readings: Brown, 6:137-141; 13:359-364
Outline:
Bacterial Genetic Mobile Elements: Insertion Seqs (IS) - Transposons (Tn)
Eukaryotic Genetic Mobile Elements:
Transposons (Tn) - Retrotransposons
Bacterial and eukaryotic Transposons are similar in mechanisms
of transposition
Retrotransposons or retroposons transpose via an RNA intermediate,
and thus mimic retroviruses in their genomic duplication: need
Reverse Transcriptase activity
... [Brown, Section 6.3.2]
Genetic elements that are Mobile - can self-transport or translocate or transpose from one location to another on the organismal DNA molecules, or ... "hopping" genes
Often "hop" into other genes: source of Mutations
Genome evolution: acquire new sequences (plasmids, phage) or rearrange existing ones
Transposons can cause deletions, insertions, inversions, rearrangements
A. Insertion Sequences:
IS
... [Brown, Fig. 6.21]
Discovered via E. coli mutants that occurred at high frequency (1 in 105), were polar, and were stable ... electron microscopy of heteroduplex DNA molecules showed insertions in the mutants.
Short (~1 kb) DNA elements
with Inverted Terminal Repeats (ITRs) of 10-50 bp
...[Brown, Fig 13.31]
Most encode a single gene whose product is the Transposase: catalyzes transposition
Transposition causes duplication of 5-10 bp of host DNA: Direct Repeats
Structure of DNA containing an IS element (Direct Repeats, adjacent to Inverted Repeats) is diagnostic for presence, either now or in the past, of an Insertion Sequence orTransposon
Transposase recognizes the ITRs, uses these for Transposition ...
Transposition frequency: 10-3-10-4 per element per generation
Precise excision: 103 less frequent ... Requires excison of the host DNA duplication !!!
B. Transposons ... [Brown, Fig 6.21]
Transposons are either Composite
Mobile Elements or Non-composite Mobile Elements
Examples: Composite - Tn9, Tn10; Non-composite -TnA family
Composite Mobile Elements contain:
Drug resistance gene, flanked by IS elements, as inverted or direct
repeats, which THEMSELVES have Terminal Inverted Repeats ... IS
elements identical, or near so
The IS elements provide Transposase; one is often inactive
Thus: Transposon is two IS elements, with DNA inbetween ...
Usually the ITRs that are "internal" (the right ITR of the left IS, the left ITR of the right IS) in the Transposon are heavily mutated and inactive for transposition.
Example of Hopping between DNA molecules to create a New Transposon:
When internal ITRs ARE functional, and the Tn hops to a small genome, e.g. a plasmid, a second hop to the chromosome can use the initially internal ITRs, creating a new "Inside-Out" Transposon with the IS elements reversed and with the plasmid DNA substituted for the DNA found between the IS elements of the initial Tn, as shown in the following Figure:
C. DNA Rearrangements - Tn mediated Mutagenesis Examples
Reciprocal Recombination between
Direct Tn Repeats => Deletions between Tn Repeats
Reciprocal Recom between Inverted
Tn Repeats => Inversion between Tn Repeats:
D. Transposition Mechanisms
General Mechanism: Staggered nicks, insert and join Tn, fill in gaps, ligate
Basic route: Replicon Fusion with (replicative) or without (nonreplicative) CoIntegrate formed
1. Replicative Transposition
... CoIntegrate Intermediate:
... [Brown, Fig 13.33]
Tn is duplicated during transposition, copy transposed .. Copy number of Tn increases
Need 2 enzymes:
Transposase - acts on ends of parent Tn
Resolvase - acts on ends of copied, daughter Tn
Four nicks, from Transposase, 2 in donor at ends of Tn, 2 in target, staggered at each 5' end of Target site ...
The latter defines the size of the resulting Host/Recipient DNA Tandem Duplication
Tn 5' ends in Donor ligated to Host 3' ends of the Tn: Crossover Structure
DNA Repair Synthesis by DNA Polymerase THROUGH the Tn AND one Direct Repeat, to yield CoIntegrate with Reciprocal Direct Repeats
Resolvase executes site-specific homologous recombination at the Res Site in the CoIntegrate, yielding a Tn with Direct Repeats at either end in BOTH the Donor DNA and the Target DNA
Resolution of the CoIntegrate,
via Resolvase, releases daughter replicons, each with one copy
of the Transposon.
2. Conservative Transposition: Phage Mu; Tn5; Tn10 ... [Brown, Fig 13.33]
True Hopping, Tn moves to new site ... original site is repaired ... Tn no longer in Donor DNA
Only Transposase is needed
Same as above, but 2 more nicks in Donor DNA in Crossover Structure, followed by DNA repair to repair the Tandem Repeats ... sometimes called "Cut and Paste" Transposition
E. Transposon Examples
1. TnA family: Non-Composite Transposon Examples - Tn3 and Gamma-Delta
Figure of Tn3:
NOT composite Transposons, no IS elements ... 38 bp Inverted Terminal Repeats target Direct Repeat of 5 bp ... Ap-resistance
tnpA gene - Transposase
tnpR gene - Repressor and Resolvase
TnpR Resolvase: binds to promoter region of both tnpA and tnpR at 3 sites
This region is called res, for resolution of the CoIntegrate
If only one site is bound, Resolvase is thought to act as the Repressor
If all 3 sites are bound and the DNA structure is correct, a large nucleoprotein complex is formed, Resolvase introduces nicks spaced 2 bp apart in a TTATAA sequence in the res site, rearranges DNA ends, and ligates appropriate ones together to yield the resolved DNA products
2. Tn10: Composite Transposon Example: 9300 bp; IS10R, IS10L (nonFunc) inverted; Tc-resis
Figure of Tn10:
IS10: 1300 bp, 22 bp ITRs; Host Target: 9 bp, NGCTNAGCN "hot spots"
Frequency of Tn10 Transposition tightly regulated:
IS10R: Pin - Pout overlapping Promoters: Pout stronger than Pin, mRNA more stable
1. MultiCopy Inhibition: RNA from Pout of Tn10 on plasmid is an Antisense RNA, limits expression from Pin, which expresses the Transposase ... Limits hopping
2. Transposase "cis-preference": functions best on Tn from which it is expressed
3. GATC hemimethylated state needed at 2 sites: GATC in Pin; GATC in ITR ... enhances Transposase transcription and binding ... coupling to Replication
Daughter Tn10 with Template Strand GATC methylated: 1000x more active ... Daughter Tn10 with other Strand GATC methylated: 3x more active
F. Eukaryotic Transposons:
1. Drosophila P Element ... [Example of use: Brown, Research Briefing 11.2, page 288]
Transposes as Transposon ... transposition accounts for most of Drosophila spontaneous mutations ...
2. Corn Ac and Ds elements:
Ac - Activator element - like a bacterial Insertion Sequence - active Transposase
Ds - Dissociative element - like Ac element but has no active Transposase due to internal deletions - requires presence of Ac element (and its Transposase) for transposition.
G. Retrotransposons
... [Brown, Section
6.3.2, Fig 6.19, Fig 13.34]
Two classes of Retrotransposons:
1. Viral Retrotransposons - contain an LTR (Long Terminal Repeat) at each end of the Retrotransposon - LTRs are characteristic of Retroviruses (see below) - [Brown, Fig 6.19B]
Examples: Yeast Ty elements, Drosophila Copia element
2. Nonviral Retrotransposons - no LTR present, but transpose via an RNA intermediate ...
Examples: LINES and SINES ... [Brown, Fig 6.19C,D] ... classified as Medium Repetitive DNA, due to abundance in eukaryotic genomes ... recall Cot Curves
LINES - Long INterspersed ElementS - 6-7 kb long
SINES - Short INterspersed ElementS - ~ 300 bp
Highly abundant in the human genome: once every 5 kb => 500,000 copies per human genome (5% of total DNA!)
Most SINE elements contain an AluI restriction site ... hence called Alu repeats
| BIMM100 | Syllabus
| Sections / Off Hrs | Grading
Policy | DNASYSTEM
|
| Lectures | Journal
Articles | Study Qs | Lab
Techniques | Exams |
If you have problems or comments, send email to Doug Smith