1. DNA structure and function

Who was Friedrich Miescher and what did he discover?

• Isolated phosphorus-rich substance from nuclei of WBCs in pus (1869)

• Later separated into protein and acid components —> termed nucleic acid, now known as DNA

Who was Walther Flemming and what did he discover?

• Discovered certain class of dyes revealed thread-like material in nucleus - coined ‘chromatin’ (1879)

• Prepared slides showing series of changes inside nucleus during cell division, observed distinctive chromosomes - coined ‘mitosis’

Who were Boveri and Sutton and what did they discover?

• Boveri - studied nematode worms and sea urchins

• Sutton - studied grasshoppers

• Both independently proposed chromosomal theory of inheritance (~1902)

  • That chromosomes carry hereditary info and that behaviour during cell division explains Mendel’s laws

Who was Frederick Griffith and what did he discover?

• Discovered genetic information could be transferred between bacteria - coined ‘transformation’ (1928)

• Later led to proof that DNA carries genetic information

What did Frederick Griffith use in his experiments?

Streptococcus pneumoniae

• R strain, IIR: rough, non-virulent

• S strain, IIIS: smooth, virulent

Describe Frederick Griffith’s experiments

1. Injected mice with heat-killed IIIS bacteria —> did not cause disease

2. Combined harmless IIR with harmless heat-killed IIIS —> caused disease, blood sample from mice contained living IIIS

3. Concluded that IIR bacteria must have taken up a chemical ‘transforming principle’ allowing them to ‘transform’ into IIIS bacteria and become virulent

Who were Avery, McCarty and MacLeod and what did they discover?

• Identified small amounts of Griffith’s ‘transforming principle’ - evidence suggested this was DNA (1944)

• “Nucleic acid of the deoxyribose type is the fundamental unit of the transforming principle”

• Cautious in interpreting results - potential for contamination —> debate continued

Describe Avery, McCarty and MacLeod’s experiments

1. Began with large cultures of heat-killed IIIS bacteria

2. Lysed IIIS and extracted cellular components

3. Treated different extracts with different enzymes to determine which macromolecule responsible for transformation

4. Protein- and RNA-degrading enzymes had little effect on transforming ability

5. DNA-degrading enzymes eliminated transforming ability

6. Elemental composition closely resembled DNA with ratios of N and P

Who were Hershey and Chase and what did they discover?

• Discovered that DNA is the material that carries genetic information through studying bacteriophages (1952)

What did Hershey and Chase use in their experiments?

Bacteriophages

• Batch A - prepared in presence of 35S (radioactive isotope of sulfur)

• Batch B - prepared in presence of 32P (radioactive isotope of phosphorus)

Describe Hershey and Chase’s experiments

1. Prepared 2 batches of bacteriophages, batch A (35S) and batch B (32P)

2. Each batch used to infect different culture of bacteria, then blended to remove any bacteriophage/parts from the surface of the cells

3. Cultures centrifuged to separate bacteria from phage debris

4. Bacterial pellet found to contain large amount of 32P, whereas almost all 35S found with phage material

5. Since S found in many proteins and not DNA, and P is found in DNA and not proteins, proved that DNA and not protein was injected into the bacterial host cells by bacteriophages and that it was DNA that made up their genetic material

Who was Maurice Wilkins and what did he discover?

• Took the first clear images of DNA using X-ray diffraction (~1950)

Who were Crick and Watson and what did they discover?

• Discovered double helix model of DNA (1953)

• Predominantly used other scientists’ experimental data

What evidence did Crick and Watson base their discovery on?

1. DNA molecules contained repeating deoxyribose sugar and phosphate groups

2. A&T, C&G always appeared in same ratio

3. High-resolution X-ray images including photo 51, viewed without consent

Who were Franklin and Gosling and what did they discover?

• Gosling took photo 51, very clear X-ray diffraction image of B-form of DNA under supervision of Franklin (1952)

• Published paper on structure of DNA immediately following that of Crick and Watson, with consistent findings (1953)

What were Franklin and Gosling’s contributions to determining the structure of DNA?

• Photo 51 enabled calculation of basic dimensions of DNA molecule

• “Structure probably helical”

• Phosphate groups lie on outside of molecule

• “Diameter of helix consistent with 10bp per turn of the helix”

• “Probably consists of two coaxial molecules”

What are nucleic acids?

• Long chains of nucleotides

• DNA, RNA

What are nucleotides made up of?

• Pentose sugar: deoxyribose in DNA, ribose in RNA

• Phosphate group: attached to 5’ C of sugar

• Nitrogenous base: attached to 1’ C of sugar

Which carbon of the pentose sugar is the phosphate group attached to?

5’ C

Which carbon of the pentose sugar is the nitrogenous base attached to?

1’ C

What makes up the backbone of nucleic acids, and what holds them together?

• Pentose sugars

• Phosphate groups

Held together by strong phosphodiester bonds

Which carbons on the pentose sugars do the phosphate groups bond to in nucleic acid backbones?

5’ C of one, 3’ C of other

What is found at the 5’ and 3’ ends of a nucleic acid strand?

• 5’ = phosphate group

• 3’ = hydroxyl group (attached to sugar)

What are purines, and which bases are considered purines?

• Double ring structure

• Adenine and guanine

What are pyrimidines, and which bases are considered pyrimidines?

• Single ring structures

• Cytosine and thymine (uracil in RNA)

Which complementary bases form 2 hydrogen bonds?

A - T

Which complementary bases form 3 hydrogen bonds?

C - G

Describe the structure of DNA

• Double-stranded

• Strands run antiparallel

• Double helix structure

• Phosphodiester bonds in backbone

• H-bonds between complementary base pairs

What are major grooves in DNA?

• Wide, deep depressions in double helix

• Make base edges easier to reach

• Easier for transcription factors to bind

What are minor grooves in DNA?

• Smaller, shallower depressions than major grooves

• Make base edges more difficult to reach

• Harder for transcription factors to bind

What are the different structural forms of DNA, and what do they describe?

• A-form: wider, shorter right-hand coils; forms in dehydrating conditions

• B-form: normal form; right-hand coils; hydrated

• Z-form: rare form; narrow, left-hand coils with zigzag backbone

How is genetic information typically found in prokaryotes?

• Circular chromosome (single, continuous, double-stranded DNA molecule)

• Plasmids (small, extrachromosomal DNA rings carrying non-essential genes)

Which organisms have no introns?

Prokaryotes only

How are prokaryotic genomes typically packaged?

• Supercoiling

• Over- or under-winding of double helix to twist the molecule into a compact, strained shape

• Allows it to fit inside the cell

How is genetic information typically found in eukaryotes?

• Linear chromosomes (stored in nucleus)

• Organelle genomes (mitochondrial DNA, chloroplast DNA)

What is chromatin and what is its function?

• DNA-protein complex

• DNA wrapped around histone proteins

• Helps compact long DNA and fit it inside the nucleus

Describe the charge on histone proteins and why this is important

• Histone proteins positively charged

• Allows them to closely adhere to negatively charged DNA molecules

What is a nucleosome, and what links them together?

• Repeating subunit of chromatin

• DNA wound 1.65x around a core of 8 histones

• Nucleosomes linked using histone H1 —> forms chromatin fibre

What are scaffold proteins and their function?

• Non-histone architectural proteins

• Form framework/axis to anchor chromatin —> further condense the chromatin

• Organise genetic material into compact structures

Describe the 2 main forms of chromatin

1. Euchromatin:

   1. Loosely packed

   2. Rich in active genes

   3. Allows for active gene expression

2. Heterochromatin

   1. Dense, compact

   2. Peripheral

   3. Less accessible to transcription factors

   4. Contains transcriptionally inactive genes

What is the centromere of a chromosome?

• Constricted region

• Region linking sister chromatids

• Site of spindle fibre attachment

What are the 2 chromosomal arms?

• P-arm: short arm

• Q-arm: long arm

What are telomeres?

Protective caps made of specialised proteins found at the end of chromosomes

What are G bands?

• Giemsa banding

• Banding visible in each chromosome

• Used to name chromosomal regions