Microbiology 314
MICROBIOLOGY 314
DNA PACKAGING AND REPLICATION
1. Genomes of prok. = single “chromo”
2. Genome = circular, double-stranded DNA molec
3. Sizes of bacterial genomes vary
4. E.coli ~1.3mm long (extended form), chromo more compact
5. Phages & plasmids much smaller
GENOME PACKAGING
Prokaryote Eukaryote
Single “chromo” Multiple chromo
In vivo compact gnm DNA density ~
~ 100mg/ml
10mg/ml
Nucleoid = 80% DNA, 20% prot Nucleus = 50% DNA, 50% prot
Packaging ratio vs extended DNA 1000-2000x - 7000x in mitotic div
Prot assoc w DNA genome identified eg. HU & H (basic prot, like histones in euk.)
↳ DNA coils around HU prot, forms nucleosome-like struct
Integrative Host Factor (IHF) charactersitics similar to HU, but specifically involved in lambda
integration. HLP1 binds to DNA - conformation affected by its absence
DNA binding proteins in E.coli:
Eukaryotic
Protein Composition number/cell Locus
equivalent
αand βunits
HU 50 000 dimers histone H2B hupA, B
9 kDaeach
2 identical
H 30 000 dimers histone H2A Not known
28 kDaunits
αunit (10,5 kDa)
IHF 15 000 dimers HMG himA, D
β unit (9,5 kDa)
H1
15 kDaunit 20 000 copies Not known Not known
H-NS
NUCELOID
= round/oval mass w coiled domains, extend from center. -ve
supercoil of 1 -ve superhelical winding for every 20 windings
(200bp/winding) & packed in loop-shaped domains (100-200 in
a gnm). Bottom ends held tog by prot. Break in doub stranded
DNA, -ve supercoil of 1 domain will unwind, adjacent domains
will be retained. - vely coiled domains each ~40kb DNA
= Repetitive DNA seq = REPs (Repeated Extragenic
Palindromes_ ~ 70bp long; 100-200x in gnm; absent w/in genes
DNA binding prot assoc w REPs bring loop-like domains tog &
DNA gyrase binds near REPs - provides necessary -ve supercoiling
Shannon Goodchild
072 827 0597
, Microbiology 314
GENE ORGANISATION
Certain genes in grps (eg 7rrn loci - contains r/tRNA genes, found near OriC)
Studies where hisD-gene placed on diff parts E.coli gnm = transcr. 3x more actively near
OriC (probs why 7rrn loci are there)
Inversions (Hin syst) - HIn recombon - rotation of 980bp section, allows cntrl expression
alternating flagella genes
EUKARYOTIC DNA ORGANISATION
More specialised - 5 diff levels
Sim to prok = DNA packed by means of prot & -ve supercoiling maintained
Higher level packing in euk = req higher % prot found in chromo
ONE GENE: ONE POLYPEPTIDE HYPOTHESIS
Beadle & Ephrussi examined mutants of fruit fly w abnormal eye clr to solve biosynth p/w for
red eye clr pigment
Should be possible to gen mutants which known biochem rxn not occur
Beadle & Tatum gen mutants of bread fungus Neurospora crassa - expose fungus to UV
irradiation
↳ mutants tested for inabil grow on min media that original, wild-type able to grow on
↳ mut req certain nutritional components ∴ rep blocks in metabolic p/w ∴ rep absence of
certain enz
Lead to 1 gene:1 enz hypothesis - enz = prot
1957, Ingram proved mut in gene for haemoglobin responsible for sickle-cell anemia which
confirmed this hypothesis
Later, known enz & other struct prot consist +1 polypep ∴ adjust to 1 gene : 1 polypep:
“One gene codes for the AA of one polypep chain - the basic struct unit of prot.”
Shannon Goodchild
072 827 0597
MICROBIOLOGY 314
DNA PACKAGING AND REPLICATION
1. Genomes of prok. = single “chromo”
2. Genome = circular, double-stranded DNA molec
3. Sizes of bacterial genomes vary
4. E.coli ~1.3mm long (extended form), chromo more compact
5. Phages & plasmids much smaller
GENOME PACKAGING
Prokaryote Eukaryote
Single “chromo” Multiple chromo
In vivo compact gnm DNA density ~
~ 100mg/ml
10mg/ml
Nucleoid = 80% DNA, 20% prot Nucleus = 50% DNA, 50% prot
Packaging ratio vs extended DNA 1000-2000x - 7000x in mitotic div
Prot assoc w DNA genome identified eg. HU & H (basic prot, like histones in euk.)
↳ DNA coils around HU prot, forms nucleosome-like struct
Integrative Host Factor (IHF) charactersitics similar to HU, but specifically involved in lambda
integration. HLP1 binds to DNA - conformation affected by its absence
DNA binding proteins in E.coli:
Eukaryotic
Protein Composition number/cell Locus
equivalent
αand βunits
HU 50 000 dimers histone H2B hupA, B
9 kDaeach
2 identical
H 30 000 dimers histone H2A Not known
28 kDaunits
αunit (10,5 kDa)
IHF 15 000 dimers HMG himA, D
β unit (9,5 kDa)
H1
15 kDaunit 20 000 copies Not known Not known
H-NS
NUCELOID
= round/oval mass w coiled domains, extend from center. -ve
supercoil of 1 -ve superhelical winding for every 20 windings
(200bp/winding) & packed in loop-shaped domains (100-200 in
a gnm). Bottom ends held tog by prot. Break in doub stranded
DNA, -ve supercoil of 1 domain will unwind, adjacent domains
will be retained. - vely coiled domains each ~40kb DNA
= Repetitive DNA seq = REPs (Repeated Extragenic
Palindromes_ ~ 70bp long; 100-200x in gnm; absent w/in genes
DNA binding prot assoc w REPs bring loop-like domains tog &
DNA gyrase binds near REPs - provides necessary -ve supercoiling
Shannon Goodchild
072 827 0597
, Microbiology 314
GENE ORGANISATION
Certain genes in grps (eg 7rrn loci - contains r/tRNA genes, found near OriC)
Studies where hisD-gene placed on diff parts E.coli gnm = transcr. 3x more actively near
OriC (probs why 7rrn loci are there)
Inversions (Hin syst) - HIn recombon - rotation of 980bp section, allows cntrl expression
alternating flagella genes
EUKARYOTIC DNA ORGANISATION
More specialised - 5 diff levels
Sim to prok = DNA packed by means of prot & -ve supercoiling maintained
Higher level packing in euk = req higher % prot found in chromo
ONE GENE: ONE POLYPEPTIDE HYPOTHESIS
Beadle & Ephrussi examined mutants of fruit fly w abnormal eye clr to solve biosynth p/w for
red eye clr pigment
Should be possible to gen mutants which known biochem rxn not occur
Beadle & Tatum gen mutants of bread fungus Neurospora crassa - expose fungus to UV
irradiation
↳ mutants tested for inabil grow on min media that original, wild-type able to grow on
↳ mut req certain nutritional components ∴ rep blocks in metabolic p/w ∴ rep absence of
certain enz
Lead to 1 gene:1 enz hypothesis - enz = prot
1957, Ingram proved mut in gene for haemoglobin responsible for sickle-cell anemia which
confirmed this hypothesis
Later, known enz & other struct prot consist +1 polypep ∴ adjust to 1 gene : 1 polypep:
“One gene codes for the AA of one polypep chain - the basic struct unit of prot.”
Shannon Goodchild
072 827 0597
