Genetics 6 - Genes and Chromosomes

• Prokaryotic Promoter:

  • Transcription site - start of mRNA transcription
  • Pribnow box - essential for transcription initiation
  • -35 region - for high transcription rate
• Prokaryotic genes:

  • regulated by promoters and operators & up/down-stream enhancers and silencers
  • Related genes can be arranged by operons
  • Genes can be polycistronic - multiple protein encoding regions in a single mRNA
• Eukaryotic Promoters

  • Transcription start site - start of mRNA transcription
  • TATA Box - found in many eukaryotic promoters and contains TATA binding protein
  • CAAT box - site for CAAT-enhancer-binding protein
• Eukaryotic genes are regulated by :

  • promoters - core (essential) and proximal
  • up/down stream - silencers and enhancers

  Genes contain introns and exons, but introns are spliced

  • introns almost always begin with GU and end with AG
• Bacterial Chromsomes:

  • large
  • circular DNA molecules
  • contain nucleoid : where most DNA resides as well as Nucleoid-associated proteins and domains ( supercoiled loops)
• Bacterial Plasmids:

  • small
  • circular DNA molecules
  • separated from chromosomal DNA
  • replicate independently
  • contains genes for antibiotic resistance and horizontal transfer

  Plasmids are also found in archaea and eukaryotic organisms
• Genome- Eukaryotes

  75% of the genome is transcribed at some point

  Only 1 to 2 % of genome encodes proteins (exons)

  Rest of DNA consists of:

  • DNA sequences between exons (Introns)
  • DNA sequences between genes (intergenic)
  • DNA repeats
  • Transposable elements
• What are the three types of DNA repeats?

  Dispersed repetitive DNA, Satellite DNA, Tandem repeats
• Satellite DNA

  • type of DNA repeat
  • 3 types - Alpha satellite, minisatellite and microsatellite
• Alpha Satellite DNA

  • 10% of genome
  • 171 bp repeats
  • main component of functional centromeres
  • organised into chromosome-specific higher-order repeats (HORs)
  • transcribed into repetitive non-coding RNA and contributes to a large portion of the transcriptome
• What does VNTRs stand for ?

  variable number of tandem repeats
• Minisatellite DNA

  • 10-60 bp repeats
  • repeated 5-50 times
  • GC rich
  • make up many parts of the genome
  • prominent in centromeres & sub telomeric regions
  • used in genetic fingerprinting
• Microsatellite DNA - STRs ( short tandem repeats) & SSRs ( simple sequence repeats)

  • less than 10 bp repeats
  • repeated 5-50 times
  • mostly found in telomeres (human)
• What is a transposable element?

  a DNA sequence that can change its position within a genome
• Types of transposable elements

  Short interspersed elements (SINEs)

  • 90-500 bp
  • e.g. ALU repeats - 300bp, adenine rich tail

  Long interspersed elements (LINEs)

  • up to 7,000 bp
  • e.g. LINE1 (L1) - 6,500 bp and adenine rich 3' tail

  SINEs and LINEs are Non retroviral retrotransposons
• Types of transposable elements:

  LTR-retrotransposons (Long Terminal Repeat)

  • gag gene expression -> virus like particle (VLP)
  • reverse transcription of RNA → cDNA
  • mediate integration of DNA , using integrase (IN)

  Transposons

  • variable in size
  • e.g. Mariner - transposase gene
• DNA storage

  • 46 chromosomes - 23 pairs
  • 22 autosomal pairs
  • one X/Y pair chromosome
  • DNA contains 3 billion bp in haploid cells
  • In bacteria - stored as negative plectonemic supercoils
  • In eukaryotes - stored as negative toroidal supercoils
• Nucleosomes:

  • a site of DNA storage
  • 146 bp around a histone
  • 8 histone proteins
  • 2x H2A, H2B, H3, H4
  • Histone 1 Protein (or H5) links nucleosomes to form a string of chromatosomes (166 bp)
• Types of histones

  Core histones (H2A, H2B, H3, H4)

  • wrap DNA
  • form nucleosomes
  • left handed superhelical ramp
  • Amino-terminal tail extension

  Linker histones (H1 and H5)

  • high order DNA structures
  • Bind nucleosome at DNA entry/exit sites

  Requires ATP-dependent chromatin remodelling enzymes
• Histone - Modifications

  • often N-terminal 'histone tails' modified
  • Acetylation of lysines - removes lys + charge, decrease nucleosome attachment, loosen chromatin structure, allows for more action of transcription factors
  • Mono, di, and tri methylation of Lys/Arg - many mutually exclusive modifications
  • Phosphorylation of serines
  • Lysine ubiquitylation
• Reasons for modification of histones

  • Recruit other structural proteins
  • Regulation of expression - loose/tight structure
  • Cell “Memory”- Some modifications kept
• DNA Topoisomerases

  • enzymes that prevent DNA tangles
  • Topoisomerases I - at tyrosine active site, DNA nicked to allow rotation
  • Topoisomerases II - cuts DNA to allow separation for replication
• DNA Topoisomerase Inhibitors

  -chemotherapeutic

  Topoisomerase I inhibitor

  • Mainly used against colorectal and ovarian cancers
  • e.g. topotecan
  • Cons - inactivated by blood , so internal targeting requires transporter expression

  Topoisomerase II inhibitor

  • e.g Etoposide
• DNA Topoisomerase Inhibitors

  • Antibiotics that target Type II Topoisomerase in bacteria
  • e.g. DNA gyrase and Topo IV
  • Quinolones - bind to cleavage-ligation active site, leading to chromosomal breakages and excess DNA repair and can result in cell death