Molecular Genetics

Levene (1910)

  • Levene isolated two types of nucleic acid: ribonucleic acid (RNA) and deoxyribonucleic acid (DNA)

  • these molecules were made up of long chains of individual units called nucleotides

  • nucleotides are made up of a sugar, a phosphate group and one of five nitrogen-containing bases

    • adenine (A), thymine (T), cytosine (C), guanine (G) and uracil (U)

RNA

  • single stranded

  • ribose sugar

  • A, U, C, G bases

  • small molecules used for synthesis of proteins (rRNA, tRNA, mRNA)

DNA

  • double stranded

  • deoxyribose sugar (missing an oxygen)

  • A, T, G, C bases

  • large molecules used to store genetic information

Frederick Griffith (1928)

  • studied pathogenic bacteria that caused a pneumonia epidemic in London

  • discovered the transforming principle in his experiments when injecting mice with different forms of pneumonia causing bacteria

  • found that hereditary information passed from dead bacterial cells to live bacterial cells were transformed from a harmless form to a disease causing form

Oswald Avery, Colin MacLeod, Maclyn McCarthy (1944)

  • repeated the mouse studies but first separated the components (lipids, RNA, carbohydrates, proteins, DNA) of the hear-killed pathogenic bacteria before adding it to the mice containing non-pathogenic bacteria —> only mice injected with DNA died

  • therefore, DNA contains the information necessary to turn harmless bacteria into killer bacteria

Erwin Chargaff (1950)

  • noticed a pattern in the amounts of the four bases in DNA: adenine, guanine, cytosine, and thymine

  • he took samples of DNA from different cells and found that the amount of adenine was almost equal to the amount of thymine and that the amount of guanine was almost the same amount of cytosine —> A=T and G=C

  • this later became Chargaff’s rule

Chargaff’s rule states that adenine only bonds with thymine and guanine only bonds with cytosine.

2 Types of Nitrogenous Bases

  1. purines - double ring structures

    1. adenine and guanine

  2. pyrimidines - single ring structures

    1. cytosine, thymine, and uracil

Alfred Hershey and Martha Chase (1952)

  • later experiments by Hershey and Chase using a strain of virus known as a bacteriophage - consists of a protein coast surrounding a length of DNA

  • found that viral DNA, not viral protein, enters the cell causing the host to produce virus particles instead of carrying out its own functions —> DNA is the genetic information

Rosalind Franklin and Maurice Wilkins (1952)

  • Franklin was able to crystalize the DNA molecule and capture a photo of it which revealed the helical shape of the molecule - Photo 51

  • Wilkins shows this photo to James Watson without Franklin’s knowledge

James Watson and Francis Crick (1953)

  • Watson and Crick published a paper on the structure of DNA - the double helix, by using information gained from x-ray diffraction

  • Watson, Crick and Wilkins were awarded the Nobel Prize in 1952

  • Franklin died in 1958 from ovarian cancer and has never been recognized for her crucial contribution in solving the structure of DNA

DNA Structure

  • DNA is a double helix molecule

  • made up of two antiparallel strands of nucleotides

  • this means that the 5’ end of one strand is paired wit the 3’ end of its complimentary strand, complimentary base pairs

  • each nucleotide is composed of a five carbon sugar (deoxyribose) and a phosphate group (together these two are the backbone) and one of four nitrogen-containing bases (the rungs of a the ladder)

  • nucleotides are linked to each other by their phosphates groups which bind the 5’ end of one sugar to the 3’ end of another sugar

  • two strands linked by hydrogen bonding between complimentary nitrogen base pairs

    • A and T via 2 H-bonds

    • C and G via 3 H-bonds

  • it is estimated that the human genome contains about 3 billion base pairs of DNA in 46 chromosomes

  • uncoiled, each chromosome averages about 5 cm in length wrapped around histones

DNA Replication

  • occurs during S-phase

  • nucleotides required to build new DNA strands come from ingesting food

  • DNA replication is called semi-conservative - when DNA is replicated each new molecule of DNA contains one strand of the original DNA molecule and one of the new daughter strand

  • replication starts at a specific nucleotide sequence called the origin of replication and continues in both directions at once

  • eukaryotes have multiple origins of replication, forming multiple replication bubbles

Enzymes for Replication

  1. DNA Helicase

    1. separate the two strands of DNA at the replication forks, unwinds that DNA double helix

    2. binding proteins attach to the separated strands to prevent them from twisting back up

  2. Primase

    1. used to make a shot segment of RNA (around 12 nucleotides) which is used to get replication started

  3. DNA Polymerases

    1. responsible for elongation of new DNA at the replication fork

    2. elongates strands in 5’ to 3’ direction

    3. can only add nucleotides to an existing 3’ end of an existing strand, needs a primer

    4. the leading strand is built 5’ to 3’, following the replication fork and will elongate continuously

    5. the lagging strand is built in the direction moving away from the replication fork so it must grow in length by the addition of short segments discontinuously called Okazaki fragments

    6. a different DNA polymerase also removes the RNA primer and replaces it with DNA

    7. also proofreads/double checks to make sure mistakes aren’t made during DNA replication (a mistake happens around once every 100 000 nucleotides)

      1. if an incorrect nucleotide is added, the mismatched base is displaced and removed and DNA polymerase then adds the correct nucleotide

      2. mistakes that are not corrected are mutations

  4. DNA Ligase

    1. joins the fragments together when synthesis is complete

How Genes Work

  • DNA replication is vital for cell to pass on their genetic information to future generations

  • the order of the nucleotides in the DNA sequence encodes genes that are used to direct the roles of the cell —> to produce proteins

  • proteins are the working molecules of the cell

  • proteins are made up of subunits - amino acids

  • the order of nucleotides in DNA is known as the genetic code

  • genetic code contains only four bases found in DNA (T, A, G, C) and has to encode 20 amino acids

  • the sequence of bases on the DNA determine the sequence of amino acids which determine the function of the protein

    • 3 bases code for 1 amino acid

  • short segments of a protein - polypeptides

  • in 1960 it was confirmed that 3 bases are used to determine and amino acid

  • DNA never leaves the nucleus and protein synthesis takes place in the cytoplasm

    • the intermediate used is mRNA

Transcription

  • DNA does not leave the nucleus, therefore a copy of the DNA is required to make proteins

  • the copy is called messenger RNA (mRNA)

  • the template strand is the side of the DNA molecule that stored the information that is transcribed into mRNA

  • the other strand is the non-template strand or coding strand and it has the same nucleotide sequence as the mRNA

  • similar to DNA synthesis except”

    • RNA is a much shorter strand

    • RNA is single stranded

    • RNA contains no thymine, it has uracil that bonds with adenine instead

    • sugar backbone is made from ribose sugar instead of deoxyribose sugar

    • RNA polymerase is the only enzyme required to make mRNA

  • promoter sequences (the TATA box) and terminator sequences tell the RNA polymerase where to start and stop - these sequences are before and after the gene that needs to be made

RNA Processing

  • eukaryotic genes are initially copied into nonfunctional RNAs called primary transcripts

  • these primary transcripts need modification called RNA processing to generate mature and functional RNA

  • removal of introns and the joining together of exons is known as splicing and occurs in the nucleus

  • in eukaryotes alternative gene splicing means that one gene can produce different combinations of exons leading to different polypeptides

  • 20 000 genes —> 100 000 polypeptides

  • following transcription the mRNA moves to the cytoplasm where it is used for protein synthesis on the ribosomes

Nucleotide - basic subunit of nucleic acid (DNA or RNA) made of sugar, phosphate and nitrogen base

Gene - a sequence of nucleotides on DNA which encode for a polypeptide

Triplet - a group of three nucleotides on DNA

Codon - a group of three nucleotides on mRNA

Transcription Unit - the genes required to make a functional protein

Translation

  • translation is the process by which proteins are made using the genetic code copied onto mRNA

  • translation takes place on the ribosome or rER which is made of protein and rRNA (ribosomal)

  • there are 64 codons but only 20 amino acids, therefore there is redundancy in the genetic code

  1. Initiation

    1. the mRNA attaches to the ribosome at the start codon (AUG) which encodes for the amino acid methionine

    2. codons are three nucleotides on mRNA that encode for an amino acid

    3. a tRNA (transfer) delivers this amino acid to the ribosome

    4. tRNAs have two specific ends - one that compliments the mRNA codon (called the anticodon) and the other that attaches to the specific amino acid translating the message

  2. Elongation

    1. tRNAs continue delivering amino acids to the ribosome according to the mRNA code

    2. these amino acids are joined together via a peptide bond

  3. Termination

    1. construction of the polypeptide ends once a stop codon (UAA, UAG, UGA) is read on the mRNA

    2. the stop codons encode for a protein release factor that releases the finished polypeptide and causes the ribosome to break apart

Point mutations - a chemical change that involves one or a few base pairs in a gene

Types of Point Mutations

  1. Substitution

    1. replacement of one nucleotide and its partner from the complimentary DNA strand with another pair of nucleotides

    2. some substitution mutations are silent - they have no effect on the coded protein

    3. usually are missense mutations which still codes for an amino acid that makes sense, but not necessarily the right sense

    4. can be nonsense mutations - if the substitution changes the amino acid codon into a stop signal then this leads to nonfunctional proteins

  2. Insertion and Deletions

    1. addition or loss of one or more nucleotide pairs of a gene

    2. usually have a more disastrous effect

    3. may alter the reading frame (triplet grouping) of the genetic message

    4. called a frameshift mutation

Chromosomal Mutations

  • chromosomal mutations affect large portions of the genetic code or entire genes or chromosomes

Mutagens

  • physical, chemical or biological agents that interact with DNA to cause mutations

DNA Technology - Genetic Engineering

  • the science of manipulating genes that carry hereditary information

  • recombinant DNA, combines the DNA from two different sources

    • a gene is cut out of one organism’s genome and inserted into the genome of another organism

    • once inserted, that DNA will be replicated, transcribed and translated into functional proteins

DNA Cloning

  • uses recombinant DNA to transfer a DNA fragment of interest from one organism to a self-replicating genetic element, such as a bacterial plasmid

  • used to make multiple copies of a gene

Cohen-Boyer Experiment

  • conducted the first genetic engineering experiment in 1973 by transferring frog DNA into a bacterial plasmid

Uses of DNA Technology

  • to produce a protein product such as human growth factor or insulin

  • to a endow a particular organism with a metabolic capability it did not previously possess

    • pest resistance in crops, bacteria that digest oil, fish that are fluorescent

  • to create more copies of the gene itself so that it can be structured further - DNA cloning

Tools Used

  1. Restriction Enzymes

    1. enzymes originally found in bacteria that were used as a defense mechanism

    2. role is to cut up intruding DNA from other organisms

    3. the key to much of the advances in molecular genetics

    4. restriction enzymes cut DNA into pieces when they recognize specific sequences of nitrogen bases

    5. when they cut up DNA the ends are not cut evenly so the ends are sticky, sticky ends can be combined with other sticky ends

  2. Ligase

    1. used to rejoin sticky ends of DNA cut with restriction enzymes

  3. Vectors

    1. DNA engineered in a test tube and must be returned to a cell in order to function

    2. vectors are either bacteria plasmids or viruses, since they are able to transform their DNA into host cells

Applications for Recombinant DNA

  • gene therapy - providing “fixed” genes to people with faulty genes, must use a vector to deliver these genes

  • high tech selective breeding - instead of crossing individuals with desirable traits hoping for a suitable offspring, they can take an offspring and specifically insert genes to promote resistance to disease or increase productivity

  • biological warfare - insert genes for harmful toxins into harmless bacteria, transfer it into food and water to infect with bacteria immune to antibiotics

Gel Electrophoresis

  • other DNA is cut with restriction enzymes, the DNA fragments are separated according to size and charge using gel electrophoresis

Polymerase Chain Reaction (PCR)

  • used to amplify DNA by repeated cycles of heat, primer, DNA polymerase and nucleotides

DNA Sequencing

  • the Human Genome Project has three basic goals

    • identify all of the 20 000 - 25 000 genes in human DNA

    • determine the order of nucleotides for 3 billion chemical base pairs that make up human DNA

    • store this information in databases

  • mostly completed in 2006 when the sequence of the last chromosome was published in Nature - the complete sequence of all chromosomes was published in Science in March 2022

  • the Human Genome Project uses probes, restriction enzymes and DNA sequencers to do their work

DNA Fingerprinting

  • on average about 99.9% of the DNA between two humans is the same

  • the remaining 0.1% is what makes us unique (unless you have an identical twin)

  • these 3 million base pairs contain short sequences (10-69 base pairs) of repetitive DNA called mini-satellites that show variation between people

  • DNA fingerprinting detects lots of mini-satellites in the genome to produce a pattern that is unique to an individual - DNA fingerprint

DNA Not Part of the Human Genome

  • some organelles such as the mitochondria and chloroplasts contain their own DNA - their jobs are such that they need to be able to function somewhat on their own

  • all mitochondria comes from the mother, therefore mitochondrial disorders are inherited from the mom

  • mitochondrial DNA is much more limited and has a structure more like that found in a bacteria