Genes and chromosomesI. Chromsomal elements

A. Genes: Segments of DNA that Code for(m?)RNA (re. Beadle & Tatum, 1940's, mRNA was theoriginal focus.)

Now a gene is a segment of DNA that codes for aspecific function expressed through RNA or protein.

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B. DNA molecules of cells/virus are quite long!

1. Viral (Table 24-1)a) RNA or DNAb) single/double strandedc) small (size selection pressure?) Often > than 4 kb & < 100

kb

Fig. 24-1: T2 phage, head busted open osmotically. The lineemanating from the head is DNA that is normally packaged insidethe phage head.

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2. Prokaryotic always DNA

a) Usually circularb) ~ 100 times the size of a viral genome E. coli genome is

4, 639, 675 bp. Nice artwork, pp. 982-983.c) Many bacteria contain small circular DNA’s: plasmids.

Ponder the idea of molecules “trying” to propagate. Thephrase “Selfish Gene” may be useful here. (Maybe a bit differentthan the idea in Dawkins book.

d) Plasmids may or may not help the bacterium. (β-lactamase)

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3. Eukaryotes, always DNAa) Size:

Simple eukaryote (yeast) ~2.6 times larger than E. coliDrosophila: 35 x E. coliHumans: 700 x E. coli (1.5% coding protein. See Table 24-2)

Aside: Careful with focusing too much on size/#; ferngenomes are often twice as large as the human genome, & C.elegans has close to the same number of genes as humans.

b) Nuclear chromosomes generally linearc) Organelle (mitochondrial and chloroplast) chromosomes

circular and relatively small (evolutionary history?)d) The organelle genome codes for only a small portion of

genes whose proteins function in the organelle. (< 5%for mitochondria (evolutionary history?)

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e) In eukaryotic genomes there is often not a goodcorrelation between genome size & number of genes. A.thaliana vs. us. Relates in part to what is in a genomebesides genes.

Fig. 24-5, Genetically, what’s in you! (at mitotic metaphase)

How could we determine which one this is?

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C. Eukaryotic Genes & Chromosomes are VeryComplex (1st consider genes, then chromosomes)

1. Introns: intervening sequences. Two examples ofgenes that contain introns.

Most eukaryotic genes do!2. Coding segments: Exons

3. To manage meiosis & mitosis with high fidelity:centromeres (A=T rich) are attachment sites tospindle fibers. They may not be located in the centerof the chromosome. See karyotype in Fig. 24-5 b).

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4. Telomers are a) located at the end of linear chromosomesb) allow copying at the ends of linear DNA molecules

without shorting of the molecule. c) b) relates to the primer requirement of DNA polymerase.

We discuss this in more detail later.d) TG rich and synthesized by telomerase.

See Fig. 24-8 & Table 24-3, next pages.

Fig. 24-8 Comment on BACS, YACS, & HACS. (ori?)

My favorite is the PKU gene. See: http://www.pahdb.mcgill.ca/ Then scroll down tomutation map.

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II. DNA SupercoilingThis is tricky and interesting in equal parts, and we must

understand this if we are to get a grip on packing very longmolecules into very tight spaces.

A. What is supercoiling? (The coiling of a coil.)

1. When no supercoiling is present, the DNA is“relaxed”

2. Linear DNA will generally be relaxed unless someconstraints are imposed on “twisting freedom.” (Completely unrelated to Chubby Checker.)

3. Circular DNA is clearly constrained w/ respect totwisting freedom. (relate to cyclizing an alkane?)

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If you don’t have access to an old phone, a rubberband will do. You need to see/feel it. Fig. 24-11 a)

Note that the unwinding relates to replication, some types ofrepair, and transcription.

Why do these processes require unwinding?

If the enzyme is RNA polymerase, what processis this?

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B. Most Cellular DNA is Underwound (achieved by nick-twist, nick-twist, etc.)

Comment on how living things “feel” aboutrandomness. “I think I’m just gonna let thermodynamicstake its course on that process.”?????

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0. Inherent in the double helical nature of B DNA: thestrands wrap around each other. If we unwrap themand the ends are constrained (closed-circular orotherwise), there will be strain.

Fig. 8-13

B form DNA has 10.5 bp per turn.

1. If you exerted force to “work out” the coil, thestrands would separate (see d).

2. What prevents the stands from separating?11

Look at some numbers that relat to Fig. 24-13:

Start with a closed circular DNA that is 84 bplong.

If it is relaxed: 84 bp ' 10.5 bp/turn = 8 turnsforeshadowing = Lk0

If you nick, unwind, close, the DNA will beunderwound by one turn. (Would this requirean input of energy?)

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C. DNA Underwinding is Defined by TopoligicalLinking #

1. What is topology? See Mobius Strip/Klein Bottle?

2. What is linking number? See Fig. 24-14

Number of times looping strand passes through the bluedisc from the same direction. Relates to Euler’sClosed Curve Theorem?

Note: a) and c) in Fig. 24-15 are topoisomers.

Is the winding shown above solenoidal or plectonemic?13

3. Why does a 105 bp long relaxed, circular DNAmolecule have Lk = 10?

4. If we define the relaxed Lk as Lk0, we can use Δ Lk = Lk ! Lk0 to describe the level of supercoiling of aDNA molecule.If Δ Lk is negative, the molecule is underwound; ifpositive, overwound. Confusion re. p. 956 in text?

5. More useful to have a measure of supercoiling that isindependent of molecule length. Specific linkingdifference (i.e., superhelical density) = σ

σ = Δ Lk 'Lk0 (Like normalizing?)14

On you own try Worked example 24-1, p. 989

6. Twist (Tw) & writhe (Wr) are geometric, nottopological, properties.

Lk = Tw + Wr

7. Underwinding helps stabilize formation of cruciformstructures (re. unwinding at juncture). Fig. 24-18

Where do these tend to occur?

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Genes and chromosomes part II D. Topoisomerases Catalyze Changes in the

Linking # of DNA. Types defined by enzyme mechanism:

1. Type I enzymes break one strand, and alter Lk byincrements of 1.

2. Type II enzymes break one strand, and alter Lk byincrements of 2.

Outcome, Fig. 24-19: 3. More on mechanisms?

Pharmacological significance: Remember the anthrax?

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E. DNA Compaction Requires a Special Form ofSupercoiling

1. Which decreases the length of DNA to a greaterdegree, plectonemic or solenoidal supercoiling?

2. Which of these is found in DNA derived frombiological sources?

(Don’t see much solenoidal in the absence of proteins.)

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III. The Structure of Chromosomes

A. Chromatin (contains nucleosomes, which)consists of DNA & proteins (gr, sorry!).

Fig. 24-25

B. Histones: small, basic proteins (see Table 24-4)1. Significance of small2. Significance of basic3. H3 and H4 are nearly identical in amino acid

sequence throughout eukaryotes. What does thatsuggest?

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C. Nucleosomes: the fundamental organizationalunits of chromatin, 1. Core (bead): octamer (H2A2H2B2H32H42); string part of

the DNA binds H1.Fig. 26 d) & e) nice summary

2. How does binding/coiling relate to linkage numbers?

a) left-handed wrapping (Fig. 24-27) induces negativesupercoiling in the part of the DNA bound to the histonecore. To relax, the unbound part must do + supercoiling.

b) Eukaryotic topoisomerases relax positive supercoiling.c) net effect: negative supercoiling (underwinding)

Contrast energy input with bacterial type II topoisomerases (ATP).

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3. DNA binding to the histone core appears to be atleast partially sequence specific (see Fig. 24-28).

>2 A=T bp in a row makes bending easier>2 G=C in a row does the opposite

D. Nucleosomes are packed into successivelyhigher-order structures

1. Fig. 24-29 (string of garlic?) & 31 (some plectonemic coiling?)

2. What about DNA being replicated or transcribed?

3. View animation on chromosome compaction?

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E. Condensed chromosome structures aremaintained by SMC proteinsSMC = structural maintenance of chromosomes1. Cohesins help link sister chromatids after replication.

2. Condensins maintain chromosomes in highlycompacted state going into mitosis. Figs 24-32 to 34

See pdb accession code 1w1w??? (Also 2wd5)

3. How might condensins function? Fig. 24-33

4. Cohesins/condensins in the cell cycle (Fig. 24-34)

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F. Bacterial DNA is also highly organized

1. Nucleoid: a compacted DNA structure (Fig 24-35)Brief comment on technique.

2. E. coli DNA has looped domains (Fig 24-36)

Coil type?

Boundary composition not yet fully characterized.

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