Bio 402 502 advanced cell developmental biology i
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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.

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BIO 402/502 Advanced Cell & Developmental Biology I

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Bio 402 502 advanced cell developmental biology i

BIO 402/502 Advanced Cell & Developmental Biology I

Section IV: Dr. Berezney


Lecture 1

Lecture 1

The Cell Nucleus and its Genome


Organization of eukaryotic 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.

Renaturation (Hybridization) of DNA

  • 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

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. 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


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 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


Bio 402 502 advanced cell developmental biology i

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


F luorescence i n s itu h ybridization fish procedure

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


F luorescence i n s itu h ybridization fish

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].

Telomeres


Chromosomal aberrations

  • Inversion:resealing of a double break in the reverse direction. This leads todeletions/duplicationsfollowing 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 calledtransposonsand associated transposon factors

Chromosomal Aberrations

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

Genome Organization in the Interphase Cell Nucleus

Packing ratio

104

680

40

7

1

Prokaryotic cell


3 d structure of the nucleosome

  • 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

o

2.8 A 3-D structure


Chromatin organization on nuclear matrix

Chromatin Organization on Nuclear Matrix

  • Chromatin loops (50-250 Kbp)are attached to nuclear matrix

Loops of DNA

Protein scaffold

Chromosome painting

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


Bowl of spaghetti model for organization of chromatin in the interphase cell 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 Territory Model for Organization of Chromatin in the Interphase Cell Nucleus

Chromosome 1 (red),

Chromosome 9 (green)


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