PLNT3140 Introductory Cytogenetics 

Lecture 14

October 26, 2017

GLOBAL MODELS OF GENOME ORGANIZATION 

Outline for next 4 lectures:


Learning Checklist:
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.


Suggested reading:


I.REASSOCIATION KINETICS

A. Melting of DNA

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

 heterogeneous GGTACTACCAT CCATGATGGTA

 polyG:polyC GGGGGGGGGGG CCCCCCCCCCC

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Tm - temperature at which half of the DNA is denatured. Used to quantify base composition.

Melting points (Tm) of DNA and their relationship to content of G-C pairs.

[Redrawn from P. Doty in D.J. Bell and J.K. Grant (eds.), "The structure and biosynthesis of macromolecules.  Biochem. Soc. Symp., 21:8, Cambridge University Press, London, 1962.]

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B. Reassociation of DNA

MEASURING GENOME SIZE AND COMPLEXITY USING C0t Curves

TAKE HOME EXERCISE: Reassociation Kinetics

II. IDENTIFICATION OF THE KINETIC CLASSES OF GENOMIC DNA

A. Most mRNA sequences are found in single-copy DNA: R0t curves

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.)

1. Measuring C0t values of sequences using labeled probes

                 amt. of radioactivity that elutes at high salt
fraction dsDNA = ------------------------------------------------
                         total amount of radioactivity

2. Hybridization of labeled RNA to genomic DNA

B. Middle repetitive fraction - This is a very heterogeneous class of sequences, present in copy numbers from 101 - 105 copies per haploid genome.

examples:

1) SINES - short interspersed repeats ~500bp between copies, as many as hundreds of thousands of copies

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".

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FISH of human chromosomes using an AluI family probe, and counterstained with the DNA-specific dye TOPRO3 (red).

These data indicate that members of the AluI family can be found on all human chromosomes,  dispersed among thousands of chromosomal locations.

From:

Three-Dimensional Maps 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)
doi:10.1371/journal.pbio.0030157


 


Middle repetitive sequences are interspersed with  genes along the chromosome.

Example of AluI sequence distribution on human chromosome 15.  Locations of  members of one subclass of the AluI SINE family, AluSq, are shown at right. The center column show the locations of all annotated repeats in this region. The leftmost column shows the location of annotated genes in the same region. Note that some of the repeats are even found within introns eg. LIPC.

Only part of the map is shown.

This map produced by a query to the NCBI Map Viewer
http://www.ncbi.nlm.nih.gov/projects/mapview/maps.cgi?TAXID=9606&CHR=15%7CHuRef&MAPS=genes,rpts[38872726.21%3A39658033.79]&QSTR=SINE&QUERY=uid(13886539)&ZOOM=10.0000

B. Highly repetitive fraction

Consists largely of "satellite DNA" , simple sequences repeated >105 times per haploid genome.


Satellite 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 satellite DNA.


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 [Redrawn from J.G. Gall and D.D. Atherton, J. Mol. Biol. 85 (1974):633-634.]  



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 
http://www.scielo.br/scielo.php?pid=S1415-47572006000300008&script=sci_arttext



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