Organization of Single-copy Sequences
Organization of Single-copy SequencesSingle-copy sequences are interspersed throughout the plant genome. These sequences are bounded by repeat sequences. The length of the single-copy regions varies widely among plant species. In general, two types of arrangements are recognized.
Short Period Interspersion - single copy sequences of 300-1200 bp are interspersed as islands among short lengths of repeat sequences
Long Period Interspersion - single copy sequences of 2000-6000 bp are interspersed as islands among repeat sequences
How can the interspersion type be determined? As we discussed earlier, reassociation kinetic experiments are performed with 300 nt long fragments. These experiments give a characteristic Cot curve that defines each of the components. But what happens if the fragment that is followed is of a longer length, for example 900 nt? This fragment could be considered to consist of three 300 nt fragments, and each fragment may be from any of the three components.
Indeed this is how these experiments may be performed. A tracer of a longer length (such as 900 nt) is prepared and radiolabelled, for example with H^3. This tracer is added to a normal Cot reaction in such a low concentration that the rate is not affected. The reaction is then allowed to proceed to a Cot value determined from a normal experiment with only 300 nt fragments where the repetitive sequences have reannealed, but the single-copy sequences have not. Then the amount of tracer which remains single-stranded is determined. What will be seen is a reduction in the amount of expected single-copy sequences. Why? If the 900 nt long fragment contains both single copy and repetitive sequences it will reanneal under these conditions as a repetitive sequence, and thus the amount of DNA that appears in the single-copy fraction will be reduced proportionally. Therefore, that single-copy sequence is interspersed with repetitive sequences.
If for example the single copy fraction is determined to account for 40% of the genome when 300 nt fragments are used, and we calculate that the genome has 30% single-copy sequences when a 900 nt tracer is used, 10% (40%-30%) of the 900 nt fragments contain single-copy and repeat fragments and that 25% (10%/40%) of the single-copy DNA is interspersed with repetitive sequences at an interval of 300-900 nt.
A final concern is the size of the repeat units. This can be obtained by using a long tracer (5000 nt, for example) and reannealing to an appropriate Cot such that only repeat sequences bind to the tracer. The product is then treated with an enzyme that cuts only single-stranded DNA. This enzyme is S1 nuclease. After digestion you obtain products that only contain repeat sequences. These products are then sized to give the average, mode and range of repeat sequences.
Summary of Sequence Interspersion in Plant Genomes