Transcript BIO 402/502 Advanced Cell & Developmental Biology

BIO 402/502 Advanced Cell
& Developmental Biology I
Section IV: Dr. Berezney
Lecture 1
The Cell Nucleus and
its Genome
Organization of Eukaryotic Genome
• Contrasting features of prokaryotic and eukaryotic
genomes with respect to size, percent of coding region
and number of genes
Renaturation (Hybridization) of DNA
• DNA renaturation plots for prokaryotic versus
eukaryotic DNA demonstrate that: prokaryotic DNA
is a unique sequence of DNA whereas eukaryotic DNA
is composed of highly repetitive, moderately
repetitive and unique sequences.
• Simple sequence DNA such as satellite DNAs are
separated by CsCl density gradients due to major
changes in the AT versus CG content (A-T rich DNA has
a lower density than GC rich).
Alpha Satellite DNA
• The human alpha satellite sequences at the centromere is
an example of tandemly repeated sequences where two
chromosomes are held together and connected by
spindle fibers for separation of chromosome during
mitosis.
Gene Structure
• Introns and Exons : Most of transcribed DNA is intron (~ 90%
of the gene sequence), e.g. the chicken ovalbumin gene
contains 8 exons & 7 introns in over 7.7 kb of DNA. The exons
(mRNA) total only 1.9 kb or about 25% of the total transcript,
while the factor VIII blood clotting factor gene is 186 kb with
26 exons that compose only about 9 kb or about 5% of the
total sequence.
Gene Families & Pseudogenes
Globin gene family; gene amplification: e.g, human type 1 interferon gene
cluster is 480 kb in size and is composed of dozens of repeating genes and
pseudogenes. Gene duplication or amplification is a result of “unequal
crossover” during meiosis & is a general mechanism of evolution of tandemly
repeated DNA sequences. This is due to misalignment on the two homologous
chromosomes. This also leads to gene deletions.
Fluorescence In Situ Hybridization (FISH)
For detection of specific DNA sequences (e.g., genes) in the
nucleus of cells and chromosomes on metaphase spreads
Four step procedure
• Prepare labeled DNA probes for DNA sequences
of interest (e.g., genes, centromeric DNA, etc)
• Hybridize labeled probes to sample on cover slip
• Label with fluorescent probes
• Detect and collect images
Fluorescence In Situ Hybridization (FISH)
Procedure
• Prepare DNA probes
Gene 1  biotin-dNTPs  biotin labeled gene 1
Gene 2  digoxyigenin-dNTPs  dig labeled gene 2
• Add to cover slip following DNA denaturation
• Renature DNA
• Detect with alexa 488 (green) strepavidin and anti
dig-alexa 594 (red); collect images on microscope
Fluorescence In Situ Hybridization (FISH)
For detection of specific DNA sequences (e.g., genes) in the
nucleus of cells and chromosomes on metaphase spreads.
Telomeres
• Telomeric sequences occur at ends of chromosomes and
are essential for the replication of end DNA by
telomerase.
• Loss of telomeric sequences (telomerase knockout)
leads to huge chromosome aberrations [chromosome
fusion].
Chromosomal Aberrations
•
Inversion: resealing of a double break in the reverse direction. This leads to
deletions/duplications following meiosis (unequal cross-over) and loss of viability.
•
Translocations: A piece of one chromosome becomes attached to another non
homologous chromosome (characteristic of human cancers especially leukemias).
•
In chronic mylogenous leukemia (CML) chromosome #22 is shortened (“Philadelphia
Chromosome”) not due to a deletion but a translocation in which the missing piece of
#22 is translocated to chromosome #9. This occurs within an essential gene of #9 that
codes for a protein kinase (c-abl) involved in cell proliferation.
•
DNA sequence organization is also very dynamic as revealed by DNA transposition
mediated by mobile DNA elements called transposons and associated transposon factors
9
Chromosome 7 (red) / 12 (blue)
Translocation
Philadelphia Chromosome
Genome Organization in the Interphase Cell Nucleus
• Eukaryotic cells: DNA is
folded in the cell nucleus as
a hierarchy of organization
from nucleosome to the
complete chromosome.
• Prokaryotic cells: DNA is
highly folded in nucleoid
structures
Packing ratio
104
680
40
7
Prokaryotic cell
1
3-D Structure of the Nucleosome
• DNA (146bp) is wrapped (about 1.7 turns) around an octamer of core
histones H2A, H2B, H3, H4 with H1 histone in between the nucleosomes
and linker DNA of 15-55 bp between individual nucleosomes.
o
2.8 A 3-D structure
Chromatin Organization on Nuclear Matrix
• Chromatin loops (50-250 Kbp) are attached to nuclear matrix
Loops
of
DNA
Protein
scaffold
Nuclear matrix remaining
after extraction of whole cells
Nuclear matrix with DNA halo
Chromosome painting
Chromosome scaffold with DNA halo
In situ evidence for a
chromatin loop organization
Chr #18 & 19 in human
lymphocyte interphase
nucleus
Bowl of Spaghetti Model for Organization of
Chromatin in the Interphase Cell Nucleus
Chromosome Territory Model for
Organization of Chromatin in the
Interphase Cell Nucleus
Chromosome 1 (red),
Chromosome 9 (green)