Biochemistry 441 Lecture 14 March 10, 2000 Ted Young

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• Know then thyself, presume not God to scan.
• The proper study of mankind is man.
• Placed on this isthmus of a middle state,
• A being darkly wise and rudely great.
• -Alexander Pope from Essay on Man

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Announcements
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Biochemistry 441Lecture 5January 13, 2006Ted
Young

• Chromosome structure DNA condensation in
  chromosomes
• A typical human cell has 2 meters of DNA in a
  nucleus with a diameter of 10-5 meters. How does
  the DNA get packed into it? (Is there enough
  space? Do the calculation!)

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Views of the nucleus stained for DNA

• Chromosomal DNA is present in two different
  states in the interphase nucleus condensed,
  transcriptionally inactive heterochromatin (the
  dark regions), and decondensed, transcriptionally
  active euchromatin.

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Disrupted nucleus shows extended DNA fibers
NB ends are never seen in these Ems gt
chromosomes are made up of a single DNA molecule.
Fig 24-31
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Metaphase chromatin is densely packed

• Paired human chromosomes at metaphase. Note the
  diameter of fibrils-much thicker than a single
  DNA molecule.

Constriction at centromere always at the same
position of each chromosome
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Histones
Histone Residues Arg Lys Evolutionary
stability (UEP) H1 215 1 29 8 H2A 129
9 11 60 H2B 125 6 16 60 H3 135 13 10 330 H4
102 14 11 600 UEP unit evolutionary period
the time in megayears for a proteins amino acid
sequence to change by 1 after two species
diverge.

• Conclusions Histone amino acid sequences are
  remarkable stable. Implication the sequences
  have been conserved for functional reasons only
  this amino acid sequence will work.

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Calf thymus histone H4

• Red Arg/Lys underlined post-translationally
  modified by acetylation or methylation of Lys or
  Arg, phosphorylation of Ser, and ubiquitylation
  of lysine. Most modified amino acids are in the
  amino terminal 20 residues. What effect on
  histone charge would these modifications have?
• Pea Lys77gtArg Val60gtIle only two changes,
  implying strong selection for maintaining the
  exact amino acid sequence.

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Structural conservation of histones

• Each histone has a very similar structure (but a
  very different sequence) a long central a-helix
  flanked on each end by a turn and two a- shorter
  helices. Many other transcription-related
  proteins have a very similar structure and may
  have evolved from the same ancestral gene.

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A histone code?

• Modifications of histones (usually the amino
  terminal tails) convey epigenetic information
• Types of modifications
• Acetylation and methylation of lysine.
• Methylation of arginine
• Phosphorylation of serine
• Ubiquitination of lysine.
• The histone code hypothesis posits that serial
  modifications provide a blueprint for reading
  chromatin -for transcription, replication,
  repair, and recombination.

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Evidence for chromatin organization in the nucleus

• 1. Stoichiometric amounts of H2A, H2B, H3, and H4
  (H1 1/2). 2. X-ray
  analysis 10nm fibers.
  3. EM beads on a string.
  4. Micrococcal nuclease digestion
  periodic cleavage along the DNA. 5.
  Chemical cross-linking of (H3/H4)2.

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Micrococcal nuclease digestion of chromatin
Units Of nuclease
MW marker

• A ladder of DNA fragments differing by 200 bp

30 20 10
bp of DNA
800
600
400
200
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High resolution structure of a nucleosome core
particle
Note that the DNA tracks around the outer surface
of the nucleosome like the tread of a tire.
Arg/Lys
H3/H4
H2A/H2B

• Modeling of the DNA along the histone octamer
  surface of a nucleosome.

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Structure of a nucleosome core particle
containing DNA
Successive minor grooves have different widths.

• DNA contacts histones on only one side.

DNA
histones
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Higher order structure of nucleosomes?
Figure 24-30.
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Identifying and cloning functional units of
chromosome behaviour

• 1. Replication origins DNA sequences that allow
  initiation of replication
• 2. Centromeres DNA sequences that insure equal
  chromosome partition at mitosis and meiosis
• 3. Telomeres DNA sequences at the ends of
  chromosomes that insure complete replication
• Identification of each of these elements required
  the construction of artificial chromosomes in
  bakers yeast-a model eukaryotic organism whose
  chromosomes have all of the hallmarks of those of
  larger eukaryotes but are 10-100X smaller.

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Artificial yeast chromosomes
Centromere (CEN 100 bp)
Circular
Gene for selection in yeast(HIS, TRP,
URA) (encoding an enzyme for synthesis of an
amino acid or pyrimidine)
Ori (yeast 60 bp)
Gene for selection in E. coli
(Amp)
poly-linker (unique restriction enzyme sites)
Ori (Ec)
Linear
Ori (yeast)
URA
CEN
Telomere (TEL)
Telomere (TEL 600 bp)
2000 - 20,000 bp
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Identifying and cloning functional parts of a
yeast chromosome
1. Cloning a yeast gene that complemented a
bacterial mutation a. yeast library in a
bacterial plasmid-----gtE. coli his-
mutant --------gtselect rare E. coli His
transformants-------gt b. characterize yeast DNA
insert it is homologous to E. coli HisG gene.
Therefore had cloned a yeast gene that functioned
in E. coli by complementation of the genetic
defect. 2. Transform a yeast His- mutant with
this plasmid. At a very low frequency His
transformants were found. The yeast cells had
taken up the plasmid and been genetically
transformed. ((Why the E. coli intermediate???-bec
ause the cloning step is inefficient and E. coli
is transformed more efficiently than yeast))
Where is the plasmid DNA in the cell? Two likely
possibilities1. Replicating as a plasmid or 2.
Integrated into a chromosome. 3. Isolate DNA from
His transformants and do a Southern blot
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Southern blot of yeast DNA
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Sal1
HIS3 gene
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1.5
5
Sal1
2
4.5
radioactive bands on the nitrocellulose membrane
Probe for Southern blot
Conclusion the plasmid had integrated into the
yeast chromosomal DNA, implying that it was
unable to replicate on its own in yeast.
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Replication origins and centromeres
Other yeast genes were cloned in an analogous
manner, but some behaved differently. Increased
transformation efficiency was due to an origin of
replication (known in yeast as an
ARS-Autonomous Replication Sequence) stability
during mitotic growth was conferred by a
centromere.
Yeast gene Transformation Location of
Stability during efficiency in yeast plasmid
DNA mitotic growth HIS3 0.001 chromosomal
very stable LEU2 0.001 chromosomal
very stable TRP1-ARS1 1.0 non-
chromosomal unstable TRP1-ARS1-CEN 1.0 no
n-chromosomal very stable
Why is the efficiency of transformation
different? (Because Recombination into the
chromosome occurs rarely)
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Centromeres add mitotic stability to artificial
chromosomes
Centromeres enhance chromosome stability by
insuring that each daughter cell inherits one
and only one chromosome at each cell division
and also insure proper segregation at meiosis.
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Southern blot of yeast DNA
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Sal1
HIS3 gene
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1.5
5
Sal1
2
4.5
( ) TRP1-ARS1 gene
Probe for Southern blot
Conclusion the plasmid containingthe TRP1-ARS1
region replicates autonomously (therefore it is
has an origin of replication associated with it).
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Replication origins, centromeres and telomeres
have unique DNA sequences

• Yeast origins of replication
• Yeast centromeres
• Telomeres Yeast (TG(1-3))n n200-300
• Human (T2AG)n n2000

(ATCAT)n
(ACCTA)n
(ATATAT)n
TG(1-3)n
TG(1-3)n
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ORIGINS OF REPLICATION, CENTROMERES AND TELOMERES
RECRUIT SPECIALIZED PROTEIN COMPLEXES
0.1-10X106 bp
105 bp
ORCORIGIN RECOGNITION COMPLEX (many)
TELOMERASE COMPLEX (2)
CENTROMERE BINDING COMPLEX (1)
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Summary

• The basic structural unit of chromosomes is the
  nucleosome
• A nucleosome contains an octamer of histones-a
  tetramer of (H3/H4), and two H2A/H2B dimers, and
  146 bp of DNA wound around the outside of the
  histones
• Chromatin remodeling occurs in two ways 1.
  covalent modification of the histones-mainly the
  N-termini by histone acetyl-transferases and
  deacetyltransferases, and 2. ATP-dependent
  remodeling that alters nucleosome
  positions/conformation on the DNA
• The functional units of the chromsomes-origins of
  DNA replication, centromeres, and
  telomeres-consist of specific and unique DNA
  sequences that bind multi-subunit protein
  complexes