Genetics Lecture 22, 10/22 - Eukaryotic Chromosome Structure, Problems with Packing DNA

Eukaryotic Chromosome Structure

• Dependent on the cell cycle
• Interphase --> chromatin --> less condensed form of DNA
• mitosis --> chromosome 10,000 fold condensation of chromatin to form distinct structures.
  
• overall organization of a eukaryotic chromosome is greater than the organization of the prokaryotic chromosome.
• E. Coli has a single chromosome with a length of 1,200um. Humans have multiple chromosomes (46) and the length is 19,000 to 73,000um.
• Two protiens that associate with DNA during condensation
• histones - major protein responsible for packing
• overall charge on a histone is positive. DNA has a negative charge.
• non histone proteins

(6:10) Evidence for how the packing of DNA in eukaryotic chromsomes occurs

• 1.) digestion of chromatin with an endonuclease. Yields fragments that are consistently 200bp in length. Suggests that the DNA in chromatin is in a repeating unit
  
• 2.) microscopic visualization shows that chromatin fibers are linear arrays of spherical particles. Look like "beads on a string"
• 3.) observed that histone proteins could interact with each other. In this interaction that formed a tetromere structure and proposed that one tetromere associated with 200 BP of DNA.
• 4.) refined endonuclease digestion data. 146 BP of DNA that associates with two tetromer of histones.

PACKING

• (12:30) First level of chromatin packing. Nucleosom core particle or the 11nm fber. 146 BP of DNA wrap around two tetromere of histones. End result is DNA with a diameter of 11nm.
• second level. 30nm fiber. Solenoid structure - you have a number of nucleosome core particles. These align around a central histone called the H1 histone. Creates a DNA molecule with a diameter of 30nm, chromatin exists as teh 30nm fiber.
• Mitotic chromosome with a diameter of 1400nm

(19:45) PROBLEMS WITH PACKING DNA

• packing leaves the DNA inaccessible to certain non-histone proteins. Ways to get around this problem is called Chromatin remodiling.
• (22:45) Chromatin remodeling: allows the packing of DNA to be temporarily relaxed so it can be replicated or transcribed.
• this is done by modifying amino acids on the histone proteins to weaken their association with DNA:
  
• Acetylation: add an acetyl group to lysine. this removes the positive charge on lysine.
• methylation: add a methyl group to lysine and arginine thus altering charges to allow brief access
• phosphorilation: add a phosphate to either serine or histidine. END RESULT: weaken the association between the histone and the DNA.
  
• (28:40) Within a chromosome there are different regions - two different levels of DNA packing
• Euchromatin - DNA that is undergoing normal packing. The genes in those regions can be expresed. APX. 90% of the DNA in the cell.
  
• Heterochromatin - DNA that has undergone extreme levels of packing. Highly condensed in comparison to euchromatin. Because of this the genes in these regions are not expressed.
• Unique features of heterochromatin:
• 1.) regions of DNA that are heterochromatin are gentically inactive.
• 2.) regions of heterochromatin are replicated later in the cell cycle (S phase). It is proposed that heterochromatin is important to the structural integrity of the DNA.
  
• 3.) Heterochromatin is unique to Eukaryotes.
• (34:55) What's composed of heterochromatin
• centromere, telomeres - sequences on the end of linear chromosomes, the majority of the Y chromosome
• Often see that regions adjacent to heterochromatin exhibit position effect. Genes in these regions are not expressed.

(38:35) REPETITIVE SEQUENCES ON CHROMOSOMES

• centromere - location of chromsome attachment during mitosis and meiosis, spindle fiber attachement. Within this there is a CEN sequence which is composed of three parts.
  
• 1st and 3rd - regions that are found on all chromosomes with high similarity.
• 2nd region is unique from chromosome to chromosome. BUT they're similar in homologous chromosomes.
• (42:20) Did mutational analysis on these sequences --> found that the 3rd is most critical to the function of the centromere
• (43:30) Telomere - repetitive sequence found on the end of linear chromosomes. Contains a number of repeats of the sequence GGGATT. Telomeres vary in length. All the individuals have different lenghts of telomeres. Role of telomere: protect the DNA at the end of teh chromosome from degredation. Only 5-10% of the DNA in an organism is usde in genes.

47:20 - whats on the test for 10/24

Genetics Lecture 21, 10/20 - DNA organization into chromosomes,

DNA organization into chromosomes

• Viral and bacterial chromosomes relativley simple in comparison to eukaryotic chromosome
• typically we have a single piece of nucleic acid without associated proteins. By comparison eukaryotes typically have multiple chromsomes complexed with a number of proteins.
  

• Viral chromosomes: come in variety --> DNA or RNA and these can be single stranded or double stranded, furthermore they can be linear or circular.
• Size range of viral genome: 2-52 micrometers in length. The viral genetic material undergoes packing to fit into the phage head.
• (6:00) Bacterial Chromosomes - always double stranded DNA. The DNA lives in the nucleoid region (an area in the bacterial cell where the DNA congregates). No membrane surrounding the nucleus of the DNA.
  
• (8:35) E. Coli
• Single circular chromosomes - 1.2 mm in length. It is associated with a few DNA binding proteins. Circular chromosomes have the ability to supercoil. How was this figured out?: 1965 they took viral DNA from a mouse and did a density gradient centrifugation - Take something we want to seperate based on size and put it in a tube and the lighter the molecule the faster it travels. Upon doing this they found 3 distinct species of DNA in the centrifuge. One moved slowly and thus it was believed to be linear DNA. The other two moved more quickly. One of the factions was underwound circular DNA and the other was relaxed circular DNA.
• (17:00) Relaxed circular DNA - has the standard number of twists for that pieces of DNA. Circular DNA can be underwound.
• (18:10) Underwound DNA - DNA is unwound (fewer twists) and creates stress on DNA molecule. WHen this happens it allows for the DNA to supercoil. This supercoil relieves the stress the DNA feels of being unwound. Overwound DNA has the same effect in that it causes stress and results in supercoiling.
• (21:10) How we determine when and if supercoiling will occur?
• Linking # --> predicted # of turns for a DNA molecule. Dividing total # of BP = 10.4 # of BP/turn
  
• EXAMPLE: SV40 virus - 5200 BP in its genome
• 5200/10.4 = 500 (predicted)
• Actual linking number = 475
• To determine the number of supercoils you take the actual linking # and subtract the predicted linking #. This gives us 25 negative supercoils in our DNA
• Topoisomers - two DNA molecules of the same DNA with two different structures.
• (27:25) Topoisomerase - Enzyme responsible for converting DNA from one topoisomerase to the next.
  
• (28:50) Specialized Chromosome Structures
• Polytene chromosome: very large chromosomes readily viewed under a microscope. Found in the cells of a number of tissues. They are seen as a series of alternating bands (DNA) and interband regions.
• (31:40) How do we get polytene chromosmes?
• These chromosomes are always found as homologous chromosomes pairs. Typically chromosomes only pair during mitosis and meiosis.
  
• During DNA replication, these chromsomes replicate. But the DNA is not dispersed into new chromosome pairs/new cells. A chromosome with 5000 strands of DNA stacked together puffing occrus when teh DNA strands seperate to allow activity to occur
• (35:50) Lampbrush chromosome - received its name because it looks like a lampbrush (used to clean kerosene lamps in the 19th century)
• found in the oocytes of sharks and a number of vertebrates. Also in spermadocytes in insects.
  
• It is found during prophase 1
  
• Lampbrush chromosome functions to direct metabolic activities during meiosis I