Genome organization

1. Genome organization

2. Nucleic acids • DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) store and transfer genetic information in living organisms. • • DNA: • – major constituent of the nucleus • – stable representation of an organism’s complete genetic makeup • • RNA: • – found in the nucleus and the cytoplasm • – key to information flow within a cell

3. Determining the Chemical Composition and Structure of DNA . In the early 1900s, Phoebus Levene isolated two types of nucleic acid: RNA and DNA. In 1919, he proposed that both were made up of individual units called nucleotides. Each nucleotide was composed of one of four nitrogen-containing bases, a sugar, and a phosphate group.

4. The Chemical Composition of the Nucleotides, DNA, and RNA In later years, other scientists confirmed and extended Levene’s work. DNA and RNA are both made up of a combination of four different nucleotides. Nucleotides are often identified by referring to their bases: • DNA has the nucleotides adenine (A), guanine (G), cytosine (C), and thymine (T). • RNA has the nucleotides adenine (A), guanine (G), cytosine (C), and uracil (U).

5. UNIT 3 Chapter 5: The Structure and Function of DNA Section 5.1 The Chemical Composition of the Nucleotides, DNA, and RNA The general structure of a DNA nucleotide includes a phosphate group, a deoxyribose sugar group, and a nitrogen-containing base. Nucleotides in RNA have the same basic structure, except a ribose sugar group is used. The sugar groups differ by a hydroxyl group at the 2′ carbon. Both DNA and RNA contain the same purine bases and the cytosine pyrimidine base. However, thymine is only present in DNA, and uracil is only present in RNA.

6. UNIT 3 Chapter 5: The Structure and Function of DNA Section 5.1 The Modern DNA Model: The DNA Double Helix • The two strands are complementary due to complementary base pairing of A with T and C with G. Hydrogen bonds link each complementary base pair. • Each strand has a 5′ end and a 3′ end. The 5′ and 3′ come from the numbering of the carbons in the deoxyribose sugar. • The two strands are antiparallel, where the 5′ end from one strand is across from the 3′ end of the complementary strand. • The sequence of a DNA strand is written in the 5′ to 3′ direction.

7. UNIT 3 Chapter 5: The Structure and Function of DNA Section 5.1 The DNA Double Helix

8. The Structure and Organization of Genetic Material To relate DNA’s primary structure (how nucleotides are linked) and secondary structure (how two strands of nucleotides form a double helix) to DNA function, it is necessary to consider: • how DNA is organized in the cell, and • how its genome (the total genetic material of an organism) is arranged into distinct functional regions on DNA The functional unit of DNA is a gene, which is a specific sequence of DNA that codes for proteins and RNA molecules. The majority of DNA in an organism’s genome does not contain genes and, instead, has non-coding regions. These regions may contain regulatory sequences, which are sections of DNA that regulate the activity of genes. 

9. DNA of Prokaryotic Cells In prokaryotes such as bacteria, the bacterial chromosome consists of a circular, double-stranded DNA molecule. More than one copy of the bacterial chromosome may exist in a bacterial cell. Since prokaryotes do not have a nuclear membrane, each bacterial chromosome is packed in the region of the cell called the nucleoid. The DNA of prokaryotic cells such as E. coli is packed near the centre of the cell in the nucleoid, which is not segregated by membranes from the rest of the interior of the cell. Continued…

11. UNIT 3 Chapter 5: The Structure and Function of DNA Section 5.1 DNA of Prokaryotic Cells Prokaryotic DNA must be tightly packed so that it can fit in the nucleoid. DNA packing is achieved through coiling, compacting, and supercoiling. (A) The circular chromosomal DNA molecule can be compacted through (B) the formation of looped structures. (C) The looped DNA can be further compacted by DNA supercoiling. Note: the coloured balls represent proteins involved in supercoiling. Continued…

12. DNA of Prokaryotic Cells DNA supercoilingis the formation of additional coils in the structure of DNA due to twisting forces. It is controlled by the enzymes topoisomerase I and topoisomerase II. Antibacterial drugs have been developed to specifically block these enzymes and inhibit bacterial survival. Some prokaryotes also have plasmids, which are small, circular or linear DNA molecules that often carry non-essential genes. Plasmids are not part of the nucleoid. They can be copied and transmitted between cells or incorporated into the chromosomal DNA and reproduced during cell division. Continued…

13. Recombinant proteins Includes DNA isolation Cuts transformation

14. Four Steps: • Cell disruption by a lysis solution • Removing membrane lipids by a detergent • Removing proteins by a protease • Precipitating the DNA with an alcohol

15. Denaturing agents • Ionic detergents, such as SDS, disrupt hydrophobic interactions and hydrogen bonds. • Salts associate with charged groups and at low or moderate concentrations increase protein solubility. • Heat disrupts hydrogen bonds and nonpolar interactions. • Some DNA purification methods incorporate proteases such as proteinase K to digest proteins.

16. DNA isolation DNA must be separated from proteins and cellular debris. Separation Methods • Organic extraction • Salting out • Selective DNA binding to a solid support

17. Organic extraction • DNA is polar and therefore insoluble in organic solvents. • Traditionally, phenol:chloroform is used to extract DNA. • When phenol is mixed with the cell lysate, two phases form. DNA partitions to the (upper) aqueous phase, denatured proteins partition to the (lower) organic phase. • DNA is a polar molecule because of the negatively charged phosphate backbone. • This polarity makes it more soluble in the polar aqueous phase.

18. Genomic DNA isolation: phenol extraction 1:1 phenol : chloroform or 25:24:1 phenol : chloroform : isoamyl alcohol • Phenol: denatures proteins, precipitates form at interface between aqueous and organic layer • Chloroform: increases density of organic layer • Isoamyl alcohol: prevents foaming

19. Genomic DNA isolation: phenol extraction

20. Ethanol to precipitate • Measure at 260nm/280nm • A ratio of ~1.8 is generally accepted as “pure” for DNA; a ratio of ~2.0 is generally accepted as “pure” for RNA

21. RECOMBINANT DNA TECHNOLOGY : cuts Treatment of a plasmid with an unique EcoR1 site. This restriction enzyme will open the plasmid and make it amenable for manipulation.