1. Mia Naglieri, Michael Kahn, Liad Elmelech, Kellianne Ticcony
Section
Mia Naglieri, Michael Kahn, Liad Elmelech, Kellianne Ticcony

2. Section 13.3 - a chromosome consists of a DNA molecule with proteins
DNA in Bacteria
The main component of the genome in most bacteria is one double stranded circular DNA molecule that is associated with a small amount of protein.
Bacterial chromosome structure is very different from a eukaryotic chromosome which consists of one linear DNA molecule associated with a large amount of protein.
In E.coli, the chromosomal DNA consists of about 4.6 million nucleotide pairs, representing about 4,400 genes which is 100 times more DNA than is found in a typical virus, but only about one thousandth as much DNA as in a human somatic cell.
Stretched out, the DNA of an E. coli cell would measure about a millimeter in length, 500 times longer than the cell
Within a bacterium, however, certain proteins cause the chromosome to coil and “Supercoil”, densely packing it so that it fills only part of the cell.

3. Section 13.3 - a chromosome consists of a DNA molecule with proteins
Eukaryotic Cells
Each eukaryotic chromosome contains a single linear DNA double helix that in humans, averages about 1.5 x 10^8 nucleotide pairs.
If completely stretched out, such a DNA molecule would be about 4 cm long, thousands of times the diameter of a cell nucleus.
In the cell eukaryotic DNA is precisely combined with a large amount of protein. Together, this complex of DNA and protein, called chromatin, fits into the nucleus through an elaborate, multilevel system of packing.

4. Section 13.3 - a chromosome consists of a DNA molecule with proteins
Chromatin
Chromatin undergoes striking changes in its degree of packing during the course of the cell cycle
Interphase cells stained for light microscopy - the chromatin usually appears as a diffuse mass within the nucleus
As cell prepares for mitosis, chromatin coils and folds up,eventually forming metaphase chromosomes
Through interphase, chromatin is generally much less condensed than the chromatin of mitotic chromosomes, it shows several of the same levels of higher-order packing
Some of the chromatin comprising a chromosome seems to be present as 10-nm fiber, but much is compacted into a 30-nm fiber, which in some regions is further folded into looped domains
Heterochromatin - the type of interphase chromatin visible as irregular clumps
Euchromatin - less compacted and more dispersed chromatin
Because of its compaction, heterochromatic DNA is largely inaccessible to the machinery in the cell responsible for transcribing the genetic information coded in the DNA

5. Exploring Chromatin packing in a Eukaryotic chromosome

6. Histones
Proteins called histones are responsible for the first level of DNA packing in chromatin
Mass of histone equals the mass of DNA
Four types of histones are common in chromatin: H2A, H2B, H3, and H4

7. Nucleosomes, or “beads on a string” (10-nm fiber)
In electron micrographs, unfolded chromatin is 10-nm in diameter.
Such chromatin resembles beads on a string.
Each “bead” is a nucleosome, the basic unit of DNA packing: the “string” between beads is called linker DNA
A nucleosome consists of DNA wound twice around a protein core composed of two molecules each of the four main histone types
The amino end of each histone extends outward from the nucleosome

8. 30-nm Fiber Next level of packing 5th histone is involved
Results from interactions between histone tails
One nucleosome, linker DNA, and nucleosomes on either side
5th histone is involved
Folds and coils when extended
Roughly 30-nm thick
Prevalent in interphase

9. Looped Domains (300-nm) The loops formed by 30-nm fibers
Attached to a chromosome scaffold
Composed of proteins
Scaffold is made up of …
One type of topoisomerase
A protein that breaks, swivels, and rejoins DNA strands
H1 molecules

10. Metaphase chromosomes
In a mitotic chromosome
Width of one chromatid is 700-nm
Specific genes end up in the in the same places
Highly specific
Precise

11. Understanding DNA Structure and Replication makes Genetic Engineering Possible!
The discovery of DNA structure marked a milestone in biology
Most notable DNA being two strands
The strands are complementary
Fundamental structure basis for nucleic acid hybridization
A technique in which single- stranded nucleic acids are allowed to interact so that complexes called hybrids
Formed by molecules with similar complementary sequences
Hybridization forms the foundation
Used in Genetic Engineering

12. : Making Multiple Copies of a Gene or Other DNA Segment
DNA Cloning
To work with specific genes, scientists use DNA cloning
A common method uses bacteria (such as E. coli)
Many bacteria have (small circular DNA molecules that replicate separately from the bacterial chromosome)
Plasmids act as a a DNA molecule that can carry foreign DNA into a host cell and replicate there
Cloning process:
Obtain a plasmid and insert DNA of different source
Formation of DNA molecule containing DNA segments from two different sources (combined in vitro)
Plasmid placed back into the bacteria →
Recombinant bacterium then reproduces and makes clones
Foreign DNA now in plasmid is replicated and passed onto clones
So what?
Many copies of a gene can a particular gene
Produce a protein product
i.e. human growth hormone can be harvested from cultures of bacteria carrying the cloned gene for the protein
Endow organisms with new traits
i.e. pest resistance
plasmi ds
cloning vector
recombinant DNA
recombinant bacterium
amplif y

13. Gene Cloning Process

14. Using Restriction Enzymes to Make Recombinant DNA
Gene cloning and genetic engineering rely on enzymes that cut DNA molecules at a limited number of specific locations
Each restriction enzyme is very specific, reorganizing a particular short DNA sequence ( ) and cutting both DNA strands at precise points
Most restriction sites are symmetric
Most commonly used restriction enzymes recognize sequences of 4-8 nucleotides
Sequences this short occur many times in a long DNA molecule
Restriction enzymes make many cuts →
The single stranded end ( ) can form hydrogen bonded base pairs with complementary sticky ends on any other DNA molecule cut with same enzyme
These associations are temporary but can be made permanent with DNA ligase producing a stable molecule of recombinant DNA
To see fragments is used
Nucleic acid fragments separated by length
DNA of bacterial cell protected from its own restriction enzymes by addition of methyl groups to adenines/cytosines
restrictive enzymes
restriction site
restriction fragments
sticky end
gel electrophoresis

15. Restriction Enzymes

16. Gel Electrophoresis

17. Amplifying DNA in Vitro: The Polymerase Chain Reaction (PCR) and Its Use in Cloning
Very Polymerase chain reaction (PCR)- Makes billions of a specific target DNA segment in a sample
Three step cycle
Reaction mixture is heated to denature the DNA
Annealing
Cooling to allow primers to form hydrogen bonds
Taq polymerase used
Advantage- does not get irreversibly denatured by heating
Extension
DNA polymerase adds nucleotides to the 3’ end of each primer
Each cycle doubles the number of DNA strands
The formula 2^n can be used to express the number of strands created
“n” is the number of cycles
DNA can be partially degraded
It just needs a complete target segment
Primers needed that hybridize only with complementary sequences at opposite ends of the target segment
¼ of molecules are identical to target segment after third cycle

18. The Polymerase Chain Reaction

19. Continued... Occasional errors limit the number of good copies
Cannot substitute gene cloning in cell
PCR used to provide specific DNA fragments for cloning
DNA primers used to add restriction site to each end of the DNA molecule to match the cloning vector
They are then cut and ligated together
Error free inserts can be selected
Often used to amplify DNA
Uses in medical field
Diagnosing genetic disorders from embryonic cells
Amplifying DNA in pre-historic animals
In crime scenes
Detecting viruses that are difficult to detect
HIV

20. Use of restriction enzymes and PCR in gene cloning

21. DNA Sequencing Use principle of complementary base pairing
Single template strand is immobilized
Polymerase and other reagents added
Allow sequencing by synthesis of complementary strand
Electronic monitors identify which nucleotide is added
New, faster techniques continue to be invented