a.j. pierce mi615 university of kentucky genomic architecture and inheritance of human ribosomal rna...
TRANSCRIPT
A.J. Pierce MI615 University of Kentucky
Genomic Architecture and Inheritance of Human Ribosomal
RNA Gene Arrays
Meiotic and Mitotic Instability
MI615
Andrew J. Pierce
Microbiology, Immunology and Molecular GeneticsGraduate Center for Toxicology
Markey Cancer CenterUniversity of Kentucky
A.J. Pierce MI615 University of Kentucky
Adapted from: Morgan T.H., Sturtevant A.H., Muller H.J., and Bridges C.B., "The Mechanism of Mendelian Heredity", 1915By http://www.accessexcellence.org/RC/VL/GG/comeiosis.html
http://biology-pages.infoPhotomicrograph by Prof. Bernard John
Meiosis Requires Genomic Restructuring
Crossing-over and RecombinationIn Meiosis
Gametes
A.J. Pierce MI615 University of Kentucky
Complex structure of selected low-copy repeats (LCRs). Horizontal lines represent specific genomic regions with the centromere toward the left and telomere to the right. At the right are listed abbreviations for the disease manifested through common deletions of the regions. The colored regions refer to LCRs with the orientation given by the arrowhead. Note complex structure of LCRs consisting of both direct and inverted repeats. (a) LCRs in chromosome 2q13 responsible for rearrangements associated with familial juvenile nephronophthisis 1 (NPHP1). (b) LCRs7 flanking the Williams–Beuren syndrome (WBS) chromosome region 7q11.23. (c) LCRs15 within the Prader–Willi syndrome/Angelman syndrome (PWS/AS) chromosome region 15q11.2. (d) Smith–Magenis syndrome (SMS) repeats within 17p11.2. (e) LCRs22 within the DiGeorge syndrome (DGS) chromosome 22q11.2.
Some Genomic Disorders Mediated by Repetitive Sequences
Stankiewicz P, Lupski JR. Genome architecture, rearrangements and genomic disorders. Trends Genet. 2002 Feb;18(2):74-82.
A.J. Pierce MI615 University of Kentucky
60S
40S
Ribosomal Composition
5S, 5.8S, 28S
18S
4 RNAmolecules
… and 80+ proteins too
Nobel Prize 2009!
A.J. Pierce MI615 University of Kentucky
unit repeat
Repeat Array
bulk genomic DNA bulk genomic DNA
Experimental Strategy
1. Isolate very high molecular weight genomic DNA from human peripheral blood
2. Liberate rDNA arrays by digesting with restriction enzymes that cut external to the array
3. Separate arrays by size on pulsed-field gels
4. Detect rDNA specific bands by Southern blotting
i. Length of the arrayii. Fraction of cells in the population
containing any given sized array
Band intensity is proportional to:
A.J. Pierce MI615 University of Kentucky
400
300
200
50
500
KB
100
5S rDNA Arrays from Anonymous Human Donors
• Everyone has two different sized arrays
• No two people have the same array lengths
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Stults et al. Genomic architecture and inheritance of human ribosomal RNA gene clusters. Genome Research 2008 18(1):13-18.
A.J. Pierce MI615 University of Kentucky
5S rDNA Arrays from Human Families – Inheritance
400
300
200
100
50
25
KB
500
ARDO PYFI VAGR THAE
MO
M
DA
DGIR
L
BO
Y
MO
M
DA
D
MO
M
DA
DBO
Y
BO
Y
BO
Y
Pa
t. GR
AN
DM
A
Pa
t. GR
AN
DP
A
Ma
t. GR
AN
DM
A
MO
M
DA
DGIR
L
BO
Y
Stults et al. Genomic architecture and inheritance of human ribosomal RNA gene clusters. Genome Research 2008 18(1):13-18.
A.J. Pierce MI615 University of Kentucky
5S rDNA Arrays – Inheritance and Somatic Mosaicism
l llHindIII NcoI PvuII
l5
l4
l
MO
M
DA
DBO
Y
BO
Y
MO
M
DA
DBO
Y
BO
Y
MO
M
DA
DBO
Y
BO
Y
Meiotic rearrangement: Band length not represented in either parent( dotted circle )
Somatic mosaicism: Band of reduced intensity relative to length( square bracket )
Stults et al. Genomic architecture and inheritance of human ribosomal RNA gene clusters. Genome Research 2008 18(1):13-18.
A.J. Pierce MI615 University of Kentucky
5S rDNA Arrays – Human Statistics( N = 27 )
Each HaploidArray
DiploidTotals
Average number of repeats 98 ± 35 195 ± 51
Fewest repeats observed 35 51
Most repeats observed 175 299
Stults et al. Genomic architecture and inheritance of human ribosomal RNA gene clusters. Genome Research 2008 18(1):13-18.
A.J. Pierce MI615 University of Kentucky
http://www.pathology.washington.edu/galleries/Cytogallery/main.php
45S Ribosomal DNA in Human Karyotypes
A.J. Pierce MI615 University of Kentucky
Ribosomal DNA Repeats
• generally structured in tandem head-to-tail arrays• unit repeats completely sequenced
Intergenic Spacer18S 28S
5.8S43 kilobase pairs
Tandem Array of Repeats( example: 4 x 43kb )
45S rDNA repeat
A.J. Pierce MI615 University of Kentucky
13 14 15 21 22
45S rDNA Arrays and the Human Genome Project
A.J. Pierce MI615 University of Kentucky
45S rDNA Arrays from Anonymous Human Donors
6000
4000
3000
2000
500
5000
KB
wells
1000
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Stults et al. Genomic architecture and inheritance of human ribosomal RNA gene clusters. Genome Research 2008 18(1):13-18.
A.J. Pierce MI615 University of Kentucky
45S rDNA Arrays from Human Families – InheritanceVAGRARDO PYFI THAE
6000
4000
3000
2000
1000
500
5000
KB
wells
MO
M
DA
DGIR
L
BO
Y
MO
M
DA
D
MO
M
DA
DBO
Y
BO
Y
BO
Y
Pa
t. GR
AN
DM
A
Pa
t. GR
AN
DP
A
Ma
t. GR
AN
DM
A
MO
M
DA
DGIR
L
BO
Y
Stults et al. Genomic architecture and inheritance of human ribosomal RNA gene clusters. Genome Research 2008 18(1):13-18.
A.J. Pierce MI615 University of Kentucky
High Resolution 45S Arrays – Inheritance and Mosaicism
1000
800
600
400
200
100
50
KB
wells
VAGRARDO PYFI THAE
MO
M
DA
DGIR
L
BO
Y
MO
M
DA
D
MO
M
DA
DBO
Y
BO
Y
BO
Y
Pa
t. GR
AN
DM
A
Pa
t. GR
AN
DP
A
Ma
t. GR
AN
DM
A
MO
M
DA
DGIR
L
BO
Y
Stults et al. Genomic architecture and inheritance of human ribosomal RNA gene clusters. Genome Research 2008 18(1):13-18.
A.J. Pierce MI615 University of Kentucky
Four Sets of Identical Twins
45S rDNA 1Mbp - 6Mbp 45S rDNA 50kb - 1Mbp
5S rDNA
Stults, Killen, Pierce and Pierce (unpublished results)
A.J. Pierce MI615 University of Kentucky
Conclusions
• Array lengths vary from person to person
• Array lengths are not coordinated between chromosome homologs
• Individual arrays are usually inherited in Mendelian manner but undergo meiotic recombination with a frequency of ~10% per array
• Mitotic recombination is capable of altering rDNA architecture in different cells in the same person
A.J. Pierce MI615 University of Kentucky
Killen et al. Loss of Bloom syndrome protein destabilizes human gene cluster architecture. 2009 Human Molecular Genetics 18(18):3417-3428.
Mitotic Recombination: Chromosomal Instability Syndromes
A.J. Pierce MI615 University of Kentucky
Killen et al. Loss of Bloom syndrome protein destabilizes human gene cluster architecture. 2009 Human Molecular Genetics 18(18):3417-3428.
Human Cells are (Mostly) Stable
A.J. Pierce MI615 University of Kentucky
Killen et al. Loss of Bloom syndrome protein destabilizes human gene cluster architecture. 2009 Human Molecular Genetics 18(18):3417-3428.
BLM Cells Restructure rDNA Every Cell Division
A.J. Pierce MI615 University of Kentucky
Amor-Gueret M. Bloom's syndrome. Orphanet Encyclopedia. February 2004
Normal cell metaphase Bloom's syndrome cell metaphase
Bloom's Syndrome
• proportionate pre- and postnatal growth deficiency• sun-sensitive• telangiectatic• hypo- and hyperpigmented skin• chromosomal instability, predisposition to malignancy
BLM protein in RecQ-helicase family with Werner Syndrome (WRN), Rothmund-Thomson Syndrome (RecQ4), and two other non-disease-associated members RecQL, RecQ5b
A.J. Pierce MI615 University of Kentucky
Th
ibo
de
au
SN
, B
ren
G,
Sch
aid
DM
icro
sate
llite
inst
ab
ility
in c
an
cer
of
the
pro
xim
al c
olo
n.
Sci
ence
. 19
93
Ma
y 7
;26
0(5
10
9):
816
-9.
Killen et al. Loss of Bloom syndrome protein destabilizes human gene cluster architecture. 2009 Human Molecular Genetics 18(18):3417-3428.
BLM Primary Cells Show Instability
MSI
A.J. Pierce MI615 University of Kentucky
rDNA Clusters Restructured in Half of Solid Tumors
Lung & Colorectal Cancer:
(39 tumors analyzed)
45% stable 30% preclonal alteration25% active restructuring
Stults et al. Human ribosomal RNA gene clusters are recombinational hotspots in cancer. 2009 Cancer Research 69: 9096-104.