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8. DNA Composition.pdf

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Biology (Sci)
BIOL 202
Tamara Western

DNA Composition & Using its Characteristics: DNA is not all created equally, however all chromosomes consist of some basic components:  The centromere (primary constriction) is at the center of the DNA, and binds to kinetochore proteins involved in cell division  Extending out of the centromere are 4 legs, coming from 2 sister chromatids  A series of repeated sequences at the end of the chromosome are known as telomeres, and help protect genomic integrity  As we can see from this table, the genome size can vary a lot depending on the organism, and it does not seem to follow complexity o E.g. The mouse has the same size genome as us, and the salamander beats us by approximately and order of magnitude, so the complexity does not correlate to the size of the genome  The c-value of an organism refers to its DNA content; the c-value paradox refers to the lack of correlation between the c-value and the complexity of the organism Classic eukaryotic DNA showed 3 basic types of DNA, which was shown even before sequencing was discovered:  Unique or single copy DNA sequences: o 1-10 copies / genome ; 40-70% ; average 800-1200bp; most of structural genes  Moderately repetitive DNA sequences o 10-100,000 copies / genome ; average 300 bp; genes for rRNA and ribosomal proteins; transposons of which you need a lot in the cell, so they’re transcribed back to back  Highly repetitive DNA sequences o More than 100,000 copies / genome ; satellite and non-satellite DNAs Satellite DNAs are a eukaryotic type of DNA which can be seen through density centrifugation:  In prokaryotes, centrifugation of DNA on a CsCl concentration gradient produces a single band at a given density, independent of their size o CsCl is cesium chloride  The DNA density goes up proportionally to the amount of G-C in the sequence, meaning GC rich fragments will be more dense than AT rich fragments  In eukaryotes, this same separation method produces two bands; one for the main DNA and one for the satellite DNA, which differ by they GC content o These satellite regions can then be isolated for further analysis  But where are they located? Localization of repeated sequences can be done through in situ hybridization, which means they can be carried out on a live cell sample  Mouse cells are squashed onto a slide, treated with base to denature, and then incubated with radioactive DNA probes complementary to target sequences on the mouse genome  The radioactive probes will hybridize with their specific sequence on the genome, leaving a radioactive marker at the satellite sequence, which can be viewed through microscopy  In this EM picture, we can see that mouse DNA has black dots localized at a certain region of the genome o Mouse chromosomes are telocentric, which means that one arm of the chromosome is so short you can barely see it o Therefore, the satellite DNA, present at about 10 6 copies, are all localized around the centromere In situ hybridization can also be done using fluorescent probes through a similar procedure known as fluorescent in situ hybridization, or FISH  Cells are still squashed and treated with base, but this time the cells are incubated with a probe, a telomeric repeat this time, linked to biotin, a vitamin  Once hybridization is complete, the sample is washed with a solution containing a egg protein known as avidin, which is fluorescently tagged o Avidin binds to biotin, so there will be a fluorescent tag wherever the probe hybridized to a piece of DNA, which can be seen in live cells  In this sample, a common DNA dye, called propidium iodide, stains the whole chromosome orange, while the fluorescent avidin stains yellow o Thus, all the yellow dots are telomeric regions of the chromosomes; there are 2 dots at each end for the 2 legs of the sister chromatids on either side of the centromere o In this case, the probe was for a TTAGGG telomeric repeat Many repeated human sequences are derived from transposons which have been inactivated; LINEs and SINEs make up 34% of the genome, and another 3% is taken up by DNA transposons  LINEs are long interspersed nuclear elements, the most common being L1, which have a copy number of about 20,000 – 40,000  SINEs are short interspersed nuclear elements, having a huge copy number of 1.5 million, the most common being Alu o Insertion of Alu sequences into important genes can be the cause of human diseases like hemophilia, neurofibromatosis and breast cancer (the gene BRCA2)  So perhaps the size of the genome is dependent on the number of these repeats we have in our genome o You don’t want these mobile elements to move too often  Insertion of these mobile elements rarely happen in exons, but when they do, they can disrupt the open reading frame, ORF, and thus introduce mutation which will render the final protein possible non-functional o The homogentisate 1,2-dioxygenase gene deficiency disease is known as alkoptunoria (black urine) but is NOT caused by TE movement; it is caused by a single point mutation
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