Outline for next 4 lectures:
Up to this point, we have been studying eukaryotic genomes from a very structural and mechanistic perspective. We have largely ignored the whole purpose for its existence, that being the storage and expression, and replication of genetic information. For the next four lectures, we will focus on the information contained in the genome, and its organization within the chromosomes.
1) Reassociation kinetics defines different
classes of DNA in genomes: single copy, middle repetitive
2) Reassociation kinetics makes it possible to determine the fraction of a genome in each kinetic class, and which transcripts are produced from each class.
3) Mapping a phenotype to a molecularly-defined locus using RFLP's
4) Probe-driven mapping of individual genes.
5) Building large-scale genomic maps.
1. Be able to work with the main equations for reassociation kinetics, and understand the units and terms for these equations.
2. Be able to interpret DNA melting curves and C0t and R0t curves.
3. Be able to explain the concept of sequence complexity X.
4. Know the characteristics of the three kinetic classes of genome sequences, and know which types of sequences are found in each class.
Cooper, GM The
Cell: A molecular approach, Chapter 4
Purine and pyrimidine bases have a peak absorbance at 260 nm. For ssDNA, the absorbance of the DNA molecule is about the same as would be predicted by adding up the absorbences of each individual nucleotide. In duplexed DNA, the total A260 is less than the sum of the absorbences of the constituent nucleotides, due to electronic interactions between the stacked bases, which decrease the amount of UV light that can be absorbed. This is referred to as the hypochromic effect.
A:T < heterogeneous < G:C
eg. polyA:polyT AAAAAAAAAA TTTTTTTTTT
- temperature at which half of the DNA is denatured.
Used to quantify base composition.
MEASURING GENOME SIZE AND COMPLEXITY USING C0t Curves
TAKE HOME EXERCISE: Reassociation Kinetics
It is possible to determine the distribution of mRNA sequences in the kinetic classes of DNA by hybridizing labeled polyA+ RNA with an excess of unlabeled DNA. The C0 t value at which a given fraction of RNA hybridizes with unlabeled DNA indicates the kinetic fraction into which that RNA falls.
Most genes coding for mRNAs fall into the single copy class (~1-10 copies/haploid genome). (The exact definition of "single-copy" DNA varies from author to author.)
= crystalline Ca10(PO4)6(OH)2
1) Bind DNA & probe to HAP at low salt.
2) Elute with increasing quantites of salt.
ssDNA, unhybridized probe elutes first. At high salt, the duplexed material elutes.
This method could also be used to isolate DNA from specific kinetic classes. It also tells you the fraction of probe that is in double-stranded or single-stranded:
amt. of radioactivity that elutes at high salt fraction dsDNA = ------------------------------------------------ total amount of radioactivity
When you add trace quantities of radioactively-labled RNA to an excess of ssDNA, most of the radioactive probe anneals to DNA (becomes ds) at high C0t values. This indicates that most of the RNA was transcribed from single copy DNA. That is, most genes are present in single copy DNA.
5S rRNA (140 - 24,000 copies)
pre-rRNA genes (140- 450 copies) Note: rRNAs for eukaryotic 18S, 5.8s and 26S rRNAs are transcribed as a single transcript and cleaved into the mature rRNAs. The rRNA genes are present as tandemly-repeated units at the nucleolar organizer regions (NOR). One or more NOR's may be located of different chromosomes.
other transcribed sequences (eg. histones, storage protein genes)
Example: The Alu1 family in primates is a 300 bp retrotransposon. These sequences have been shown to be transcribed into RNA, reverse transcribed into DNA, and inserted randomly into thousands of locations throughout the genome. AluI family sequences were discovered by annealing genomic DNA to low C0t and then treating the DNA sith the single-stranded nuclease S1. S1 degrades single-stranded DNA, leaving double stranded DNA intact. When loaded on a gel, the dsDNA exhibited a 300 bp band, against a background of thousands of other bands. When the DNA products were digested with the restriction enzyme AluI, most of the 300 bp band was shifted to two bands of 170 and 130 bp. This indicates that most of the copies of the 300 bp sequence contain an internal AluI site. Hence the name, "AluI family".
human chromosomes using an AluI family probe, and
counterstained with the DNA-specific dye TOPRO3 (red).
of All Chromosomes in Human Male Fibroblast Nuclei and
Prometaphase Rosettes Bolzer A, Kreth
G, Solovei I, Koehler D, Saracoglu K, et
al. PLoS Biology Vol. 3, No. 5, e157 (2005)
repetitive sequences are interspersed with genes
along the chromosome.
This map produced by a query to the NCBI Map Viewer
Consists largely of "satellite DNA" , simple sequences repeated >105 times per haploid genome.
extreme G+C contents, short units eg. centromeric repeats
most satellite DNA is located at centromeres and telomeres
DNAs of Drosophila virilus. Fragmented DNA was
centrifuged to equilibrium in a cesium chloride
gradient. Ultracentrifugation establishes a density
gradient within the centrifuge tube. Most of the
genomic DNA migrates as a main band at a density of
1.7. Some of the genomic DNA migrates at a lower
density. The lower density DNA is referred to as
There are many families of satellite sequences in eukaryotic genomes. An example is the As51 class of satellite sequences found in several species of charachid fishes. Note that this family of sequences is seen on several chromosomes, both at centromeric and telomeric regions.
Image displayed by hypertext link to Abel LDdS, Mantovani M, Moreira-Filho O (2006) Chromosomal distribution of the As51 satellite DNA in two species complexes of the genus Astyanax (Pisces, Characidae) Genet. Mol. Biol. 29:448-452. doi: 10.1590/S1415-47572006000300008
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