Unit 3 - Molecular Genetics

Question 1

Griffith’s experiment

Answer 1

In 1928, pneumonia was a deadly virus at the time, and Frederick Griffith was studying creating a vaccine. He injected mice with two strains of it; a smooth (S-) strain, and a rough (R-) strain. The S-strain was virulent and killed the mouse, but the R-strain was non-virulent, and when injected, the mouse still lived. Griffith also recognized that he could heat-kill the R-strain; rendering it non-virulent. When a mouth was injected with the S-strain and the heat-killed R-strain, however, the mouse still died. Griffith theorized that the heat-killed virulent bacteria of the R-strain passed on its traits to the living non-virulent bacteria of the S-strain; rendering it deadly. He labelled this, the “transforming principle” and it suggested that bacteria were capable of transferring genetic information.

Question 2

Hershey-Chase experiment

Answer 2

Alfred Hershey and Martha Chase were a microbiologist team in 1952 who were determined to figure out whether DNA or proteins contained genetic code. They used bacteriophages; viruses that infect bacteria - specifically the T2 bacteriophage strain of a virus, which contains a protein coat that surrounds DNA. To study the role protein and DNA alike play in T2 infection, Hershey and Chase used radioisotopes to trace each molecule. They used a radioisotope of sulphur to trace for proteins (as proteins contain sulphur, but DNA doesn’t), and they likewise used a radioisotope of phosphorus to trace for DNA (as DNA contains phosphorus, but proteins don’t). The bacteriophages (both phosphorus-labelled and sulphur-labelled) injected E. coli bacteria, which were then agitated with a blender and centrifuged, and when the infected bacterial cells and the liquid phage “ghosts” were separated, Hershey and Chase found that most of the radioactivity in the phosphorus-labelled phage solution was found in the bacterial cells, but most of the radioactivity in the sulphur-lablelled phage solution was found in the phage shell medium. With this data, the two scientists concluded that the viral DNA was transferred into the bacterial cells, and was therefore was contained the genetic information.

Question 3

DNA

Answer 3

• hereditary molecule that passes traits on to the next generation
• contains information (nucleotide sequences) that codes for proteins (genes)
• polymer composed of nucleotides
• hydrogen bonds hold complementary bases together in double-stranded DNA

Question 4

genome structure

Answer 4

• diameter = 2 nm
• 1 base-pair = 0.34 nm
• 1 revolution = 10 base-pairs
• 1 revolution = 3.4 nm

Question 5

nucleotide

Answer 5

• composed of a phosphate group, pentose sugar, and nitrogenous base (is a phosphorylated nucleotide)
• sugar-phosphate backbone linked by phosphodiester bonds
• pentoses and nitrogenous bases linked by glycosyl bonds

Question 6

nucleoside

Answer 6

composed of a pentose sugar and nitrogenous base

Question 7

purines

Answer 7

• adenine and guanine
• composed of 2 rings

Question 8

pyrimidines

Answer 8

• thymine, cytosine and uracil
• composed of 1 ring

Question 9

DNA vs. RNA

Answer 9

DNA

• composed of deoxyribose sugars (hydrogen in carbon 2)
• more stable
• used for CSI and archaeology
• found in the nucleus

RNA

• composed of ribose sugars (hydroxyl in carbon 2)
• less stable; degrades much faster over time than DNA
• is an umbrella term; there are many different types, each with their own structures, functions, and locations (mRNA, rRNA, mtRNA, tRNA, iRNA, gRNA, etc.)

Question 10

The Central Dogma

Answer 10

a dynamic (non-unidirectional) process that states how DNA contains instructions for making a protein:
DNA ⇄ mRNA → proteins

1. DNA replication (DNA → DNA)
2. DNA transcription (DNA → mRNA) and reverse transcription (DNA ← mRNA)
3. DNA translation (mRNA → proteins)

Question 11

codon

Answer 11

• a triplet of bases
• RNA-based
• results in the transcription of one amino acid
• there are 64 different codons in total (4 bases and 3 positions; 4³ = 64)
• start codons (AUG) signal the beginning of protein synthesis
• stop codons (UAA, UAG, UGA) are non-translatable, and simply signal the end of protein synthesis

Question 12

DNA replication

Answer 12

• a.k.a. natural cloning (in vivo)
• occurs in the nucleus
• takes place at multiple origins of replication - within “replication bubbles” - in eukaryotes, but at only one origin of replication for prokaryotes
• semi-conservative nature of DNA synthesis

1. Gyrase/topoisomorase unwinds supercoiled DNA.
2. Helicase disrupts hydrogen bonds to make single-stranded DNA via denaturation; forming replication forks.
3. Single-stranded binding proteins prevent hybridization.
4. RNA primers bind to DNA, via primase, and act as a starting point for DNA polymerase III.
5. DNA polymerase III synthesizes DNA, from 5’ to 3’ direction (on the leading strand, this is done continuously; on the lagging strand, this is done in chunks and forms Okazaki fragments between primers).
6. DNA polymerase I checks for errors in DNA, and replaces RNA primers with the appropriate nucleotides (uracil to thymine).
7. DNA ligase joins remaning gaps together with phosphodiester bonds.

Question 13

DNA transcription

Answer 13

• occurs in the nucleus for eukaryotes; occurs in cytosol (the nucleoid) for prokaryotes
• coupled with DNA translation in prokaryotes

1. Initiation: RNA polymerase binds to a promoter region. (The promoter region is an A=T-rich area, as A=T has two hydrogen bonds, which are easier to break down than the three hydrogen bonds between C≡T. This is therefore energetically favourable for RNA polymerase.)
2. RNA polymerase unwinds the double stranded DNA; exposing the template strand (a.k.a. sense strand), and the complimentary coding strand (a.k.a. anti-sense strand).
3. Elongation: mRNA is synthesized within the “transcription bubble”, in 5’ to 3’ direction. It has the same sequence as the coding strand, but with uracil instead of thymine.
4. As RNA polymerase moves along DNA, mRNA is continuously synthesized, and DNA that has already been synthesized is rewound.
5. RNA reaches the end of the gene (the “termination sequation”).
6. Termination: RNA synthesis ends; mRNA is released, and DNA and RNA polymerase are recycled to form more mRNA.

Question 14

post-transcriptional modifications

Answer 14

• occur in the nucleus
• added to the primary transcript (mRNA without modifications) to form the full mRNA transcript (mRNA with modifications)

5’ capping

• 5’ cap of modified guanine nucleoside triphosphate added on the 5’ end of mRNA
• protects the primary transcript from being broken down by nucleases and RNAses

poly-A addition

• poly-A polymerase adds a tail of 200-400 A’s to the 3’ end mRNA
• acts as a guidance mechanism to rough endoplasmic reticulum or ribosomes

splicing

• spliceosomes excise introns (non-translatable mRNA regions that stay in the nucleus), and join together exons (translatable mRNA regions that exit the nucleus)

Question 15

DNA translation

Answer 15

• made possible with ribosomes, mRNA and tRNA
• one mRNA can result in thousands of polyptides
• amplication factor of greater than 10⁶ cells
• coupled with DNA transcription in prokaryotes

1. mRNA is pulled through the ribosome; the start codon establishes the reading frame.
2. tRNA is brough into the P-site (peptidyl site); the anticodon of the tRNA is complimentary to the codon of the mRNA and carries the amino acid coded by the start codon.
3. A second tRNA enters the A-site (aminoacyl site). A peptide bond forms between the two amino acids.
4. The ribosome translocates one codon over, and the next tRNA brings in the appropriate amino acid into the A site; the first tRNA exits the ribosome, and is recycled for later use.
5. This process is repeated until the ribosome reaches a stop codon, for which no tRNA exits.
6. The ribosome-mRNA complex is dismantled, and the polypeptide chain is released.

Question 16

tRNA

Answer 16

• full name is “transfer RNA”
• has a phosphate at the 5’ end and a hydroxyl at the 3’ end
• hairpin loops stabilized with hydrogen bonds
• 2D shape of a clover leaf; 3D shape of a boomerang with wings
• the anticodon arm holds the anticodon; a complimentary triplet of the mRNA codon it is coding for

Question 17

ribosome

Answer 17

• the smallest and most numerous organelle
• not membrane-bound
• made up of proteins and ribosomal RNA (rRNA)
• composed of a large subunit (60S), and a small subunit (40S)

Question 18

post-translational modifications

Answer 18

• occur in Golgi bodies
• make proteins functional

methylation: adding methyl (-CH₃ to polypeptides)

phosphorylation: adding phosphates to polypeptides

glycosylation: adding sugar molecules to polypeptides

Question 19

mutations

Answer 19

• changes in the DNA sequence
• can be lethal or non-lethal
• can work in favour for species’ survival (contribute to genetic variability)
• include point mutations and chromosomal mutations

Question 20

point mutations

Answer 20

silent mutation

• occurs when a single base-pair is substituted, but there is no amino acid change
• the insertion or deletion of three nucleotides causes no frameshift mutation, but may result in a loss of function

missense mutation

• occurs when a single base-pair is substituted, and there is a subsequent single amino acid change
• may result in loss of function

extensive missense mutation

• occurs when a single base-pair is inserted or deleted, and the following amino acids are all changed
• frameshift mutation; loss of function occurs

nonsense mutation

• occurs when a single base-pair is substituted, and a stop codon is formed
• results in premature termination and a truncated polypeptide

immediate nonsense mutation

• occurs when a single base-pair is inserted or deleted, and a stop codon is formed
• frameshift mutation; results in premature termination and a truncated polypeptide

Question 21

chromosomal mutations

Answer 21

• affect many genes
• higher phenotype abnormalities
• multifactorial diseases (e.g. Trisomy 21; down syndrome)
• include translocation, inversion and deletion

Question 22

regulation of gene expression

Answer 22

done via operons in prokaryotes; collections of genes that are regulated simultaneously

Question 23

lac operon

Answer 23

• full name is “lactose operon”
• is composed of the promoter, the operator, β-galactosidase (breaks up lactose into glucose and galactose), permease (allows the sugars to enter the cell), and transacetylase (converts galactose into glucose)
• lactose is the inducer (trigger): when not present, the Lacl protein binds to the lac operator, covering part of the promoter, and blocking transcription; when present, it binds to the Lacl protein, changing its shape, and disabling it from binding to the lac operator
• when food is available, transcription occurs

Question 24

trp operon

Answer 24

• full name is “tryptophan operon”
• is composed of the promoter, the operator, and five genes that encode the enzymes that produce tryptophan
• tryptophan is the inducer (trigger): tryptophan acts as a corepressor and binds to the tryptophan repressor; this complex can bind to the operator and block transcription; a lack of tryptophan inactivates the repressor and allows transcription
• when tryptophan isn’t available, transcription occurs

Question 25

biotechnology

Answer 25

* refers to bio-scientific technologies that are used to make products and services for human wellness * not necessarily a molecular biology science; it's interdisciplinary (has a little bit of everything)

Question 26

gel electrophoresis

Answer 26

* molecular biology technique that uses electrical current to resolve (separate) molecules * can be characterized by molecular mass (further distance travelled = smaller molecular mass), concentration (thicker molecule = greater concentration), and charge (towards the cathode (top) = positive; towards the anode (bottom) = negative) **agarose gel electrophoresis** * separates nucleic acids and pigments * uses agarose gel as a medium; an agar derivative that acts like a sieve * lower concentration of agarose means more permeable and quicker migration * sizes in kilobases (kb) **polyacrylamide gel electrophoresis (P.A.G.E.)** * separates proteins, and DNA for sequence analysis * sizes are in kilodaltons (kDa)

Question 27

plasmid

Answer 27

short, circular piece of extra chromosomal (outside the chromosome) DNA

Question 28

bacterial transformation

Answer 28

1. Restriction enzyme cuts within the plasmid. 2. Foreign DNA is added. 3. DNA is cloned as bacteria cells duplicate via binary fission.

Question 29

restriction enzymes

Answer 29

* recognize specific sequences and act as "molecular scissors"; cutting DNA at precise points * include endonucleases (cut phosphodiester bonds internally) and exonucleases (cut phosphodiester bonds from the ends)

Question 30

examples of genetic engineering

Answer 30

**bacterial transformation** * focuses on mostly DNA, rather than RNA * e.g. pGLO plasmid produces the green fluorescent protein (GFP) **CRISPR technology** * uses blunt end cuts and the insertion of nucleic acids to correct mistakes * done via guide RNA (gRNA) * focuses on RNA, rather than DNA

Question 31

sequencing

Answer 31

* determining the base sequence of an unknown segment of DNA * vital for The Human Genome Project (HGP)

Question 32

Sanger dideoxy sequencing

Answer 32

* used to read sequences of a region of a genome * the higher the ddNTP concentration, the more likely *Taq* polymerase will grab it, and the greater the concentration of shorter bands 1. DNA primer is added onto the end of the template DNA. 2. dNTPs, template DNA, *Taq* polymerase, and 1% of the respective ddNTP is added into tube A (ddATP), T (ddTTP), C, (ddCTP), or G (ddGTP). 2. ddNTPs terminate DNA synthesis, and therefore generate varying lengths of DNA. 3. Gel electrophoresis is used to visualize the lengths of DNA; the rest of the unknown DNA sequence can then be found by reading the varying lengths.

Question 33

polymerase chain reaction

Answer 33

* a.k.a. DNA cloning in vitro * dNTPS, DNA primers (oligonucleotides 10-20 bases long) and *Taq* polymerase (from the bacteria *thermus aquaticus*) is necessary 1. The test tube is brought to about 90°C, and the DNA is denatured. 2. The test tube is brought to about 55°C, and annealling occurs; the DNA primers bind to the DNA strands. 3. The test tube is brought to about 70°C, and*Taq* polymerase binds to the DNA polymerase, and synthesizes DNA from 5' to 3' direction, using the dNTPs. 4. The amount of copies is equal to the amount of cycles (n^th cycle = 2ⁿ copies)

Question 34

DNA fingerprinting for identification

Answer 34

* combines PCR and electrophoresis * has uses in CSI, paternity/maternity tests, and DNA tests such as AncestryDNA and 23andMe

Question 35

restriction fragment length polymorphism

Answer 35

use non-coded regions of DNA called short tandem repeats (STRs) in DNA analysis

Question 36

biotechnological applications

Answer 36

**medicine** * diagnosis of various genetic diseases and conditions/behaviours * single nucleotide polymorphism (SNP) analysis or DNA microarray (a.k.a. "DNA chip") (e.g. BRCA-1 = familial breast cancer gene) * personalized medicine: everyone has a unique SNP profile - the presecription for similar conditions will depend on your SNP profile - and personalized medicine will result in decreased side effects **agriculture** * bioremediation * creation of genetically modified (GM) foods (e.g. bit corn, golden rice (enriched rice with vitamin B), salmon (carries the anti-freeze gene) **forensic science** * usage of biotechnological techniques by police services to solve crimes (PCR and electrophoresis use in CSI, STR analysis to verify identities, and RFLP testing for paternal/maternal testing)