CHAPTER 35

• DNA

  • DNA mostly in the nucleus
  • 23 pairs of chromosomes
  • 1% DNA found in the mitochondria
• Deoxyribonucleic Acid

  • A double helix
  • HUMAN GENOME

  • 3.5 billion base pairs of DNA divided into 23 chromosomes

  • 99%: Non-Protein- coding

  • <1%: Protein coding
• HUMAN CHROMOSOMES

  • Possess a 2-fold symmetry
  • Centromere
  • Connects 2 sister chromatids
  • A-T rich region with repeated DNA sequence
  • Kinetochore – provides the anchor for the mitotic spindle
  • Nucleosome + histone H3 variant protein CENP-A + other centromere binding proteins
• Ends of each chromosome is called telomeres
• Each sister chromatid contains one (1) double stranded DNA
• INTERSPERSED

  • Dispersed throughout the genome, between and within genes
  • Alu sequence - repeated 500,000x in the genome
• TANDEM (Satellite)

  • 5-500 bp repeats

  • Usually located in centromeres and telomeres

  • Transcriptionally inactive
• MACROSATELLITE

  • Exists as both dispersed and grouped tandem

  • 2-6bp repeated up to 50 times

  • Most common: dinucleotide or trinucleotide

  repeat sequences
• TANDEM REPETITIVE SEQUENCES (SATELLITE DNA)

  • DNA sequences that comprise short tandem repeats incorporating specific motifs.
  • 1/3 of DNA repeats
  • Ex. macrosatellites, minisatellites, mucrosatellites
  • Repeated many times adjacent to each other
• INTERSPERSED REPEATS

  • LINES (long interspersed elements) = 15% of human genome

  • inserted randomly as retrotransposons

  • Function as polymorphisms

  • SINES (short interspersed elements) from LINES = 10%

  of the human genome;

  • mostly ALU REPEATS

  • Repeated many times and scattered throughout the genome
• Macro-satellite

  • Repeated small units of DNA in tandem

  • VNTR(variable number of tandem repeat)

  • Large polymorphism in centromere and telomere
• MINISATELLITE

  • Fewer
  • Repeat units larger than macrosatellite
• Microsatellite

  • Small tandem (2-6 bp in size)
  • Simple sequence repeat
  • short tandem repeat
  • used in gene discovery by linkage analysis for identification purposes ( paternity testing and forensic DNA testing)
• • “split genes” – composed of exons separated by introns

  • Exons – encode information; protein coding genes

  • Introns – non-coding regions
• Gene structure: mRNA

  • Introns - removed from the primary transcript

  Exons - ligated together in sequence to form the mature mRNA – RNA splicing
• HISTONE: total = 400 bp; exon = 400 bp

  B-GLOBIN: total = 1,660 bp; exons = 990 bp

  HGPRT: total = 42,830 bp; exons = 1263 bp

  FACTOR VIII: total = ~186,000 bp; exons = ~9000 bp
• CHROMATIN STRUCTURE

  • Very long double-stranded DNA (dsDNA) molecules

  • Nearly equal mass of histones (small basic proteins)

  • Smaller amount of non-histone proteins (acidic and larger than histones) and a small quantity of RNA.

  “Beads on a string” appearance
• NUCLEOSOMES

  • Composed of a core - histones

  • Dense spherical particles, approximately 10 nm in diameter and connected by DNA filaments;

  • Composed of DNA wound around an octameric complex of histone molecules

  • Left-handed helix (negatively coiled)
• Nucleosome

  • 1 3⁄4 turns of DNA around the histone octamer

  • 146 base pairs of DNA

  • The core particles are separated by 30 bp of DNA (linker DNA)

  • Give rise to the bead-on- a-string appearance
• HISTONES

  • Small proteins

  • Positively charged at physiologic pH due to high content of Lysine and Arginine

  • Assist in condensing the DNA and can participate in gene regulation

  • Molecules that comprise chromatin that condenses the DNA

  • Integrally participate in gene regulation, and contribute to all DNA-directed molecular transactions.
• HISTONES

  • Two molecules each of H2A, H2B, H3, and H4 form the structural core of the individual nucleosomes

  • Also called the Histone Octamer

  • Histone H1 is not found in the nucleosomes core, but binds to the DNA chain between the nucleosomes beads
• HISTONES

  • 4 major types of histones in nucleosomes: H2A, H2B, H3, and H4 (core histones) – highly conserved between species

  • Other variants exist and have specialized function.

  • Carboxyl terminal two-thirds: Hydrophobic Amino Acids

  • Amino terminal thirds: Basic Amino Acids.
• Acetylation (H3 & H4)-– associated with the activation or inactivation of gene transcription.
• Acetylation of core histones is associated with chromosomal assembly during DNA replication.
• Phosphorylation (H1) – associated with the condensation of chromosomes during the replication cycle.
• ADP-ribosylation of histones is associated with DNA repair
• Methylation – correlated with activation and repression of gene transcription.
• Monoubiquitylation – associated with gene activation, repression, and heterochromatic gene silencing.
• Sumoylation of histones (SUMO; small ubiquitin-related modifier) is associated with transcription repression.
• METHYLATION

  • Methylation of DNA and histones causes nucleosomes to pack tightly together.

  • Transcription factors cannot bind the DNA

  • Genes are not expressed.
• ACETYLATION

  • Histone acetylation results in loose packing of nucleosomes.

  • Transcription factors can bind the DNA.

  • Genes are expressed.
• PROPERTIES OF THE HUMAN GENOME

  NUCLEAR

  • The haploid human genome has 3.5 billion DNA base pairs

  • < 1% protein coding 99% non protein coding

  • Genes are composed of 1 to >75 exons

  • Genes vary in length from <100 to >2,300,000 bp

  MITOCHONDRIAL

  • Makes up 1% of total cellular DNA

  • Circular, containing 17,000 bp of ds DNA

  • Encodes 13 protein subunits of the respiratory chain
• CENTRAL DOGMA

  •DNA is the genetic material within the nucleus

  •The process of replication creates new copies of DNA

  •The process of transcription creates an RNA using

  DNA information

  •The process of translation creates a protein using RNA information
• DEOXYRIBONUCLEIC ACID

  Transcription

  • Source of information for the synthesis of all protein molecules of the cell and organism

  Replication

  • Provides the information inherited by daughter cells or offspring
• • Within a particular gene in the dsDNA molecule, the genetic information resides in the sequence of nucleotides on one strand, the template strand.
  • This is the strand of the DNA that is copied during RNA synthesis, the non-coding strand.
  • The opposite strand is considered the coding strand because it matches the sequence of the RNA transcript but contains uracil instead of thymine; this encodes the protein.
• REPLICATION AS A PROCESS

  1. Double-stranded DNA unwinds
  2. The junction of the unwound molecules is a replication fork.
  3. A new strand is formed by pairing complementary bases with the old strand.
  4. Two molecules are made. Each has one new and one old DNA strand.
• PROKARYOTIC REPLICATION Basic requirements for DNA synthesis

  1. Substrates 2. Template 3. Primer 4. Enzymes
• Basic requirements for DNA synthesis

  1. Substrates

  • Deoxyadenosine triphosphate (dATP)

  • Deoxyguanosine triphosphate (dGTP)

  • Deoxycytidine triphosphate (dCTP)

  • Deoxythymidine triphosphate (dTTP)

  2. Template

  • Each strand of parental DNA

  3. Primer

  • Prepares the template strand for the addition of nucleotides

  4. Enzymes

  • DNA-dependent DNA polymerases
• Helicase unwinds parental double helix
• Ss Binding proteins stabilize separated Strands by preventing premature reannealing of dsDNA

  Primase (Dna G)

  Synthesis of RNA primers for priming DNA synthesis