14 terms

BIOL 202 Lecture 10

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C-value
The DNA content of an organism
C-value paradox
The lack of correlation between genome size and the biological complexity of an organism.
Classes of eukaryotic DNA sequences
1. Unique or single copy DNA sequences
2. Moderately repetitive DNA sequences
3. Highly repetitive DNA sequences
Highly repetitive DNA sequences
Class of eukaryotic DNA sequences
(more than 100,000 copies / genome ;
satellite and non-satellite DNAs)
Moderately repetitive DNA sequences
Class of eukaryotic DNA sequences
(10-100,000 copies / genome ; average 300 bp; genes for rRNA and ribosomal proteins; transposons)
Unique or single copy DNA sequences
Class of eukaryotic DNA sequences
(1-10 copies / genome ; 40-70% ; average 800-1200bp; most of structural genes)
o 1-10 copies, mainly structural genes, make up 40-70% of genome size
o on average, about 1000 bp
Satellite DNA
In prokaryotes, centrifugation of fragmented DNA on a CsCl gradient produces a single band at a given density, irrespective of the size of the fragments. (DNA density will increase with increasing percentage of G:C)
In prokaryotes, always got a single band.
In eukaryotes, the same experiment lead to 2 bands: the main density band and satellite band
Eukaryotes have satellite DNA
Satellite DNA consists of very large arrays of tandemly repeating, non-coding DNA. Satellite DNA is the main component of functional centromeres
Localization of repeated sequences: In situ hybridization (radioactive probe)
In situ hybridization (ISH) is a type of hybridization that uses a labeled complementary DNA or RNA strand (i.e., probe) to localize a specific DNA or RNA sequence in a portion or section of tissue
- What we do is isolate the satellite band and use enzymes to make copies of the DNA
- These enzymes will also add radioactive compound such as P32
- We take the copies grown in the culture and squash cells on slide
- NaOH will then denature the DNA
- You incubate it with radioactive DNA
- You wash the excess radioactive DNA
- You put the slide on an emulsion and develop an autoradiograph
An autoradiograph is an image on an x-ray film or nuclear emulsion produced by the pattern of decay emissions (e.g., beta particles or gamma rays) from a distribution of a radioactive substance
Tells us where the satellite DNA is found
Mouse satellite DNA found near centromeres but most mouse DNA found near telomeres
FISH
FISH (fluorescence in situ hybridization) is a cytogenetic technique developed by biomedical researchers in the early 1980s[1] that is used to detect and localize the presence or absence of specific DNA sequences on chromosomes.
Fluorescent probe
Used for in situ hybridization to locate where DNA sequences are found.
- At the end of the telomeres, there is repeated sequence of TTAGGG
- We take this sequence and link it to biotin
- Biotin and Avidin link together
- These two molecules link with fluorescent dye, forming fluorescent probe
Transposons
3 types: DNA Transposons, LINES, SINES
LINES are autonomous and include L1
SINES are nonautonomous and include Alu elements
Insertion of Alu sequences into genes (transposons) is the cause of human diseases such as hemophilia
Human genes contain many transposable elements but rarely insert into exons (negative selection)
Ex. Homogentisate 1,2-dioxygenase gene deficiency in alkaptonuria (black urine), but mainly due to point mutations
The C Value paradox resolved in plants
- Why does rice have such a large genome?
- 70 million years ago there was ancestral grass that gave rice to barley, rice, sorghum and maize
- Has lots of repeats in it which made it larger
- As the number of repeats increase, the size of the genome increases
- This is why the salamander has more DNA than humans
- It has more repeated elements
Putative functions of highly reptitive DNAs
1. Structural organization role in chromosomes.
2. Involvement in chromosome pairing during meiosis.
3. Involvement in crossing over or recombination.
4. Protection of important structural genes (rRNA, etc).
5. Repository of unessential DNA for future evolution.
6. Junk DNA.
Gel electrophoresis
Separates DNA fragments according to size
Such digested DNA fragments can be separated, according to their sizes, on a agarose gel, stained with ethidium bromide, and seen under UV light