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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.
Section IV: Dr. Berezney
The Cell Nucleus and its Genome
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.Renaturation (Hybridization) of DNA
Introns and Exons:Most of transcribed DNA is intron (~ 90% of the gene sequence), e.g. thechicken ovalbumin genecontains 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 thefactor VIII blood clotting factor geneis 186 kb with 26 exons that compose only about 9 kb or about 5% of the total sequence.Gene Structure
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 amplificationis 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 togene deletions.Gene Families & Pseudogenes
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
Gene 1 biotin-dNTPs biotin labeled gene 1
Gene 2 digoxyigenin-dNTPs dig labeled gene 2
dig-alexa 594 (red); collect images on microscope
For detection of specific DNA sequences (e.g., genes) in the nucleus of cells and chromosomes on metaphase spreads.
Telomeric sequences occur at ends of chromosomes and are essential for the replication of end DNA by telomerase.
Inversion:resealing of a double break in the reverse direction. This leads todeletions/duplicationsfollowing meiosis (unequal cross-over) and loss of viability.
Chromosome 7 (red) / 12 (blue) Translocation
Eukaryotic cells:DNA is folded in the cell nucleus as a hierarchy of organization from nucleosome to the complete chromosome.
DNA(146bp)is wrapped(about 1.7 turns)around anoctamer of core histonesH2A, H2B, H3, H4 with H1 histone in between the nucleosomes and linker DNA of 15-55 bp between individual nucleosomes.3-D Structure of the Nucleosome
2.8 A 3-D structure
Loops of DNA
Chromosome scaffold with DNA halo
Nuclear matrix remaining after extraction of whole cells
Chr #18 & 19 in human lymphocyte interphase nucleus
Nuclear matrix with DNA halo
In situ evidence for a chromatin loop organization
Chromosome 1 (red),
Chromosome 9 (green)