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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
Prokaryotic cell
Packing ratio
104
680
40
7
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.
2.8 A 3-D structure
o
Chromatin Organization on Nuclear Matrix
• Chromatin loops (50-250 Kbp) are attached to nuclear matrix
Nuclear matrix remaining after extraction of whole cells
Nuclear matrix with DNA halo
Chromosome scaffold with DNA halo
In situ evidence for a chromatin loop organization
Loops of DNA
Protein scaffold
Chromosome painting
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)