1.2 Nature of the Gene and the Genome Flashcards
Genome complexity
- Can be partially determined by DNA denaturation
- GC content determines melting temperature (Tm)
- more GC = more hydrogen bonds to break = higher melting temp
DNA Denaturation
Cot = Initial [DNA] mol/l X time (sec)
Co = initial DNA [ ] t= time of incubation
Prokaryotes:
the larger the genome, the lower the [ ] of complementary fragments in solution and the greater the time required for renaturation
Eukaryotes:
Steps to DNA Reannealing
- Extract DNA
- Dissolve DNA in buffer
- Shear DNA to uniform lengths
- Denature DNA
- Incuabate DNA and allow to reanneal
- the larger the genome, the lower the [ ] of complementary fragments in solution and the greater the time required for renaturation
- Complexity (number of genes) related to length of DNA
Viral and Bacterial Genomes
Vs.
Eukaryotes
Cot = concentration of DNA x time of reassociation
Viral and Bacterial Genomes:
-the smaller the genome, the greater the number of genomes present in a given weight of DNA and the greater the change
Eukaryotes DNA Renaturation
- DNA reaneels are different rates
- various nucleotide sequences in a preparation are present at markedly different [ ]s
- 3 distance steps which correspond to 3 broad classes of DNA sequences reannel at different rates because they differ with the number of times their nucleotide sequence is repeated within the population
- highly repeated, moderately repeated and non repeated
Three Classes of Eukaryotic DNA: types and examples
•Highly Repeated Satellite, Minisatellite, Microsatellite DNA’s •Moderately Repeated tRNA and rRNA ones •Nonrepeated (Unique) Single genes
Satellite DNA
- Monomer length from several to several hundred base pairs
- Large clusters of up to millions of base pairs
- Normally localized to centromeric and telomeric regions of chromosomes
Minisatellite DNA
- Monomer repeats from 12 to 100 base pairs
- Clustered repeats
- Up to 3000 monomers per cluster
- Repeat length varies between individual
- occupy consideralblt shorter stretches of genome than satellite
- unstable (copies increase or decrease in generations due to crossing over)
- minisatalie forms the basis for DNA fingerprinting!
Microsatellite DNA
- Simple sequence repeats (1-5 base pairs)
- Small clusters of 50-100 base pairs
- Scattered through genome
- Highly variable between individuals
- Monomer repeat number varies
- Used for population and forensic studies
- shortest
- used for analysis of dif human populations
Satellite DNA 3 functions
- CenP-B sequence in human satellite DNA binds CEN-B protein
Helps form kinetochore - Uniformity of satellite DNA length (170 to 200 bp)
May be related to nucleosome phasing - Satellite DNA Transcription
May form ribozymes
Initiate histone H3 methylation
Heterochromatization
In Situ Hybridization
Used to localize specific sequences to chromosomal location
- make into s.strand by treatment with hot salt soln to denature DNA
- incubate with biotin-ladled s.s sat DNA which binds selectively to complementary strands of mobilized sat DNA located in chromo
- incubate your now d.s fDNA hybrid with florescently labled avidin to reveal location of bound labeled DNA probe. Counter stain chromo so they appear red
Where is satellite DNA in the chromo?
sat is in centromere region of chromo
Triplet Expansion Diseases two categories
- Type I- CAG repeat (neurodegenerative)
Coding regions of genes
due to expansion of CAG in Coding regions of genes - Type II- variety of repeats
Non-coding regions of genes
has a variety of repeats and is found in area that doesn’t code for a.a also effects dif parts of body not just brain
Huntington’s Disease
•Tp I CAG repeates
•Neuordegenerative disease
•Hungtingtin gene normally has 6-35 copies of CAG repeat (polyglutamine)
•Affected individuals have more than 35 copies
•Huntingtin protein forms aggregates
•Dominant genetic disorder (Hh or HH)
•Exhibits genetic anticipation
Severity increases from generation to generation/it strikes at an earlier age
Fragile X Syndrome
- Typ II
- Normal gene has 5-55 copies of CGG in FMR1 gene (function unknown)
- Carriers have 60-200 repeats
- Affected individuals have >200 repeats
- X-chromosome highly unstable (fragile)
- Loss of function mutation
