Molecular genetics

Molecular Genetics - chapter 5 

  1. DNA contains all the genetic information for any living organism 

  2. Proteins control a wide variety of cellular process 

  3. Genetic research and biotechnology have social, legal, and ethical implications

    1. A Lot of memorization 

    2. Scientific method of historical experiments

    3. Make sure to know the name of ALL the people and experiments 

Determining Material of Heredity

  • How was the material of heredity discovered?

    • We know that DNA is an material of heredity 

    • The structure of DNA was discovered in 1953, so how was it discovered?

      • There were experiments on what cellular substance resulted in inherited traits 

      • Was it DNA protein or was it something else?

Nucleic Acids 

  • Only known about DNA for about 75 years. 

    • DNA was accepted as hereditary material during 1952

    • Ensocymbothic theory - Chloroplast and mitochondria have their own DNA independent of the nucleus because they are ancestors of independently living prokaryotes 

      • Plasmid DNA, not chromosomal 

      • Mitochondrial DNA usually larger in animals than plants 

  • DNA cannot exist in long strands

    • Chromosomal DNA is packed into chromosomes around proteins 

    • In order to wrap the DNA you need to prevent sharp corners. 

    • Histones are used to protect the code from being damaged 

  • DNA undergoes many levels of packing. 

    • Each DNA molecule has been packages into a mitotic chromosome that is 50000x times shorter than its extended length

    • Its like wrapping a package over and over again until the sharp corners are rounded 

    • Has to unpack and repack it every time a segment of DNA is needed to be copied 

  • Structure of DNA 

    • Consist of two antiparallel strands 

    • 5’ to 3’ (5 prime to 3 prime)

    • 3’ to 5’ (3 prime to 5 prime 

    • The C5 attached to the PO4 is with the ester bond 

    • C1 attached to N base is with a glycosyl bond 

    • Cannot have two of the same type (double ring, single ring) back to back, this causes the structure to shift and change 

      • Like having stairs of a staircase but all the staircase are all equal length, which makes the stairs unusable 

    • Bases are bonded with hydrogen bonds, in a single strand of DNA, there are alot of hydrogen bonds holding it together, meaning its a pretty strong structure 

      • The location of hydrogen bonds are specific.

        • 3 hydrogen bonds between C and G

        • 2 hydrogen bonds between A and T 

          • C and G are always and forever together 

          • A and T are always and forever together

            • Makes it easier to remember because if you only have one side of code, you will know the other side of the code because these letters can only be with one partner 

  • DNA vs RNA

    • RNA is single sided, and Uracil replaces thymine. 

      • Uracil is more stable compared to thymine without a partner 

      • RNA has the extra hydroxyl group for ribose sugar 

      • There are three types of RNa: mRNA, tRNA, rRNA 

      • RNA will leave the nucleus, find a ribosome, attach to it, find an amino acid and produce a protein

        • More detail will be talked about later on 

DNA Replication 

  • Overall process:

    • Gyrase - relieves any tension during unwinding 

    • Helicase - “unzips” the two sides of DNA by breaking the H-bonds making a “replication fork” 

      • Where the two strands are connecting into one

      • Basically unraveling the strands into two  

    • Single-stranded binding proteins - attach to the template strands to prevent the H-bonding from reforming 

    • Free nucleotides are structurally different from those in a strand of DNA 

      • They have 3 phosphates similar to ATP 

    • Nucleotides comes from the food we eat, or anything else that has been broken down 

  • Enzymes are extremely picky, the enzymes that add the new bases only work in one direction 

    • One template side, one strand will be made continuously while the other strand needs to be made in fragments and sealed

      • Leading strand (with light blue line) = strand made continuously 

      • Lagging strand (will a lot of fragments of enzymes) = strand that needs to be sealed 

        • The short fragments are called Okazaki fragments 

  • Process for lagging strands 

    • Primase (enzyme) lays down RNA primers to act as starting points 

      • RNA can serve as primers because they are single stranded 

    • DNA polymerase III adds the nucleotides to the 3’ end of the new strand, the energy from the phosphates is what drives this process

    • DNA polymerase I takes out of the RNA primers and fills the gaps with the appropriate nucleotides 

      • Primers should have the ratio of 2 bonds for every base rather than 3 because 2 bonds takes less energy to break compared to 3 (hydrogen bonds)

        • This is why it's only A and T founded in RNA because of the smaller bonds its has (2 bonds) 

    • DNA Ligase joins the gaps between Okazaki fragments by creating phosphodiester bonds 

    • DNA polymerase I and DNA polymerase III proofread by taking out any incorrectly paired nucleotides and adding the correct ones 

    • Finally, the two strands twist automatically

Prokaryotic vs Eukaryotic 

  • Similarities 

    • They both have continuous leading and fragmented lagging strands 

    • Elongation for both occurs in the 5’ to 3’ direction

    • Both require the use of primers

    • Both use DNA polymerases however they are different in number and structure 

      • 5 are used in prokaryotes and 13 are used in eukaryotes 

Prokaryote 

Eukaryote 

  • Prokaryotes have shorter pieces of DNA, only one replication fork is necessary and replication is much faster 

  • Eukaryotes have longer sequences copying from one end to the other is not efficient 

    • Thousands of replication bubbles open up on eukaryotic DNA so the copying can occur simultaneously 

  • Have more complex enzymes and more sophisticated proofreading mechanisms 

  • DNA also contains telomeres to prevent loss of sequences at the end of chromosomes 

    • Overtime, the telomeres gets shorter and shorter as you age 

Dispersive - each strand has new and old segments (double stranded) 

1. Why is a nitrogen label a good tool for studying DNA?

Found in bases 

2. What other molecules in a cell have nitrogen in them?

Proteins 

3. What’s the difference between 14N and 15N at the atomic level?

N15 is radioactive 

4. What’s the term for two atoms of the same element with different molecular masses?

Isotopes 

5. Give an example of another element that has atoms of more than one molecular mass.

Carbon, hydrogen, phosphorus, daws

Protein Synthesis

  • Step 1: Transcription 

    • mRNA made from DNA in nucleus 

  • Step 2: Translation

    • Polypeptide made from mRNA in cytoplasm 

    • DNA is too big to leave the nucleus

      • Advantageous of the cell to protect the code 

    • When the RNA leaves the nucleus, it will find a ribosome 

  • Only segments of DNA that code for proteins are transcribed and translated 

    • Short sections of DNA can be transcribed to mRNA multiple times to produce identical polypeptides simultaneously at different ribosomes 

3 types of RNA 

  • mRNA 

    • Transcribed code from DNA, small enough to fit through nuclear pores

    • Strand is as long as required, linear 

  • tRNA

    • Carry amino acids to mRNA, it is in a t-like shape 

    • 70-90 base pairs 

  • rRNA 

    • Structural with proteins

    • 2 subunits clamp mRNA to tRNA 

Step 1 - Transcription 

  1. Initiation, it has to start 

    1. RNA polymerase binds to DNA at a promoter near the beginning of a gene 

    2. Promoters have more A and Ts. RNA polymerase uses less energy because A and T only have 2 H bonds 

    3. Template strand would be 3’ 5’ 

  2. Elongation

    1. Starts building the mRNA sequence 

    2. Primers are not specifically required as in DNA replication

  3. Termination

    1. After the RNA polymerase passes the end of the gene, it recognizes a single to stop transcribing and stops 

    2. Signal is a sequence and is different between prokaryotes and eukaryotes 

Step 2 - Translation

  • How does the DNA code for each amino acid 

  • You look at a table to determine the amino acid that is associated with the mRNA 

  • The reading frame is set on the mRNA read in the 5'3 direction. Each codon is 3 bases and codes for an amino acid

Steps:

  • Step 1: initiation

    • Ribosome recognizes a sequence on mRNA and binds to that site 

    • Start codon is “AUG”. thus all polypeptides begin with methionine 

      • Methionine hanging off of tRNA 

  • Step 2; elongation

    • tRNA delivers amino acids that correspond to the codons on mRNA 

    • tRNA with corresponding anticodon attaches to A site on ribosome 

    • A peptide bonds form between amino acids on adjacent tRNAs and the first one detaches its RNA 

    • Ribosomes moves forward, tRNA without an amino acid leaves 

  • Step 3;

    • The amino acid chain keeps on going until a stop codon is reached 

    • There is no tRNA for stop codons 

    • Release factor binds to the A site instead. It breaks the bonds between the last tRNA and the polypeptide

Mutations

  • There are 64 codons to ensure that if there is a mistake, it won't be too drastic to affect the outcome. 

What are mutations?

  • Changes in the sequence of DNA. 

  • Can occur in somatic cells, if mutations are in somatic cells, they cannot get passed on. 

  • Can be spontaneous or due to environmental conditions, radiation and chemicals can cause mutations in the genome 

  • The things that cause the changes are called mutagens, they can be physical, chemical or biological. 

Types of mutations 

  1. Point mutations 

    1. Involve only one base pair in substitution in a single gene. 

    2. 3 main types, silent, nonsense, and missense 

      1. Silent mutations does not affect the product (does not alter the amino acid)

      2. Nonsense mutation is when it is changed to a stop codon, causing the whole protein to be unusable 

      3. Missense mutation is when it is changed to obtain an brand new different amino acid, split into two types, conservative and nonconservative 

        1. Missense mutations are thought to be useful in normal functioning of the immune system resulting in increase variety of antibodies 

    3. Point mutations - being replaced by something else 

  2. Frameshift mutations - added on or deleted 

    1. A mutation that causes a change in the reading frame of codons. All codons and amino acids after the mutations are usually different. 

    2. Insertion and deletion are types of frameshift mutations 

    3. Ex. if one letter is deleted, the copied one will still copy the other letters, it would just be read wrong and the pairs of three will be different due to the missing one 

      1. If three nucleotides are added then reading is not affected but the shape can be 

  3. Chromosome mutations 

    1. Affect serverage genes 

      1. Deletion - deletes a whole chunk of the chromosome 

      2. Dupliations - sections of the chromosome are duplicated 

      3. Inversions - a segment becomes inverted (turned around)

        1. Depending on the location and length of the segment, it can cause inactive genes 

      4. Translocation - similar to inversion, but it is switching to a different chromosome entirely. Two chromosomes are involved. 

        1. Called “jumping genes”, they can disrupt proteins depending on the position. 

The work of Beadle and Tatum

  • Responsible for the one-gene/one-polypeptide hypothesis

  • Metabolic pathways often involve multiple steps and enzymes

  • DNA codes for the enzymes and thus each enzyme would be governed by its own game. 

  • Any mutation in ant of the enzymes would result in no growth in minimal medium 

    • however, if the growth medium contained the missing compound that the enzyme helped produce, the mutation gene could be identified 

      • Refer to the lesson slides for pictures. 

    • Growth medium is what the mold would grown on, menial medium is when no extra ingredients are added 

  • To figure out which enzyme was effective, they take out the specific enzyme and test if the mold will grow. 

  • The observed that mutant strains could not grow on minimal medium because they could not synthesize required biomedical compound 

  • Some isolated mutant strains that grew when arginine or its specific intermediates were added to the medium 

  • Identified 4 genes responsible for 4 enzymes in the arginine synthesis pathway. 

    • They deduced the order in the pathway

    • Each enzymes has a associated gene 

    • In between step is the on that is affected 

      • Refer to the lesson slides on graphic with table 

Biotechnology

  • Use living systems or living things to create an product 

  • Techniques in Biotechnology

    • Recombinant DNA 

      • Sequences or fragments from at least 2 different sources 

      • Purpose is the change the genetic of an organism so they produce useful products 

        • Usually the organism will be a bacteria 

    • Restriction Endonucleases 

      • Can be called restriction enzyme 

      • Act as “chemical scissors”, they cut the DNA and the dna fixes it self together connecting the two different things 

      • Naturally found in many prokaryotes to defend themselves against viral DNA sequences. These enzymes cut up foreign DNA 

      • Methylases are used by biotechnologists to protect genes that they do not want to cut 

      • Each restriction enzymes has its own recognition site and are usually palindromic and 4-8 nucleotides long 

        • Palindromic means it can read forwards and backwards 

        • Palindromic sequences are used because they are not common. Therefore, genes can be isolated without cutting them. 

        • We use the exact same enzyme for both organism so when they connect, they fit together perfectly 

      • Two ways these enzymes can cut 

        • Sticky ends, blunt ends 

          • Sticky ends can easily join fragments together as long as the same restriction enzyme is used with DNA ligase.

          • Enzymes that produce blunt ends are not as useful

          • So we want sticky ends, not blunt ends 

  • Plasmids 

    • Genes from other courses placed into a bacterial genome so that the cellular resources can be used to produce protein in large quantities 

      • Like insulin

      • Plasmids are 1000-2000 b.p. long, lacks a protein coat, and can be passed to other bacteria. 

        • Natural function is to carry genes for resistance to heavy metals antibiotics 

        • Can be replicated independently, so you can use them to create large amounts of whatever you need, like insulin 

    • Artificial plasmids 

      • Designed to contain only one recognition site for many restriction enzymes so that it is predictable and easy to insert a gene 

      • The plasmid shown has the different restriction enzymes recognition sites mapped 

      • Thuse genes can be easily inserted and the plasmid can be used as a vector 

  • DNA fingerprinting

    • DNA is treated with restriction enzymes to produce fragments of different lengths 

      • Restriction enzymes are used in every type of biotechnology

    • DNA is negatively charged because each nucleotide has a phosphate group with a negative charge 

    • The differences in molar mass amongst nucleotides are negligible 

      • The only difference between the fragments are only the size of the fragments 

    • Individuals have sections that repeat. the number of repeats varies between individuals, this is why everyone's DNA are different 

      • Called VNTRs or variable number tandem repeats. 

      • This creates the differences in the mass of fragments 

    • If someone's entire genome were to be run on a gel, it would not create discrete bands, rather a large smear would appear.

      • “Short tandem repeats” STRs are focused on, because there is more valuable information to gain, higher concentration of what is needed (DNA) 

    • You can also use this in relations within family, not only for crime scenes. 

      • You look at the amount of bands that are in the same position of the other 

    • Steps in DNA fingerprinting

      • DNA is treated with glycerol and a dye so it can be seen

      • DNA is loaded into the well of an agarose gel chamber with a buffer solution. 

      • Current is passed through the gel, causing fragments to move to the positive end 

      • Shortest garments will migrate faster, therefore further. 

      • The sizes of the fragments can be determined by comparing them to markers which are fragments of known sizes that run on the same gel

  • PCR (Polymerase chain reaction)

    • This is usually done before DNA fingerprinting, these two are done about the same time because PCR makes more DNA taken from a small sample 

    • Involves repeated cycles of the same steps producing copies of DNA in each type 

      • Was very laborious due to the enzymes denatured with every heating and cooling cycle. 

  • DNA Sequencing 

    • This method is a little outdated, but important to know the concept 

    • We need “altered” g t a and c’s 

      • The DNA will stop copying with theses altered nucleotides 

    • Can be done manually or with computers 

    • Dideoxy chain termination method uses DNA replication producing fragments of different lengths ending with dideoxy nucleotides 

      • These nucleotides lack hydroxyl groups, meaning the next nucleotide cannot be added 

      • Should be enough fragments so that every length is represented and the sequence can be read off a gel 

      • We read these from shortest to longest, so bottom to top

      • One base difference between each fragment