Biology 40SMechanisms of InheritanceMiss. Paslawski

Lesson 1: DNA ReplicationTerms: Template, semiconservative, helicase, primers, DNA polymerase, leading strand, lagging strand, replication fork, Okazaki fragments

DNA Replication

DNA carries the information for making all of the cell's proteins. These pro­teins implement all of the functions of a living organism and determine the organism's characteristics. When the cell reproduces, it has to pass all of this information onto the daughter cells. Genetic material is duplicated during interphase to ensure information is passed onto the next generation of cells.

The process of DNA replication we will learn is called the semiconservative model. The result is two DNA double helices, both of which consist of one parental and one new strand. Each old strand of DNA serves as the template for the new DNA strand.

Requirements for DNA Replication

● Original DNA template - DNA is a double helix made of two complementary strands. Each strand can be used as a template to create a new DNA molecule.

● Free DNA nucleotides – needed to form the new strands.

● DNA polymerase – an enzyme that adds new nucleotides to a growing strand of DNA.

● RNA Primers – needed to start the process because DNA polymerase can only add nucleotides to an existing strand of DNA.

Replication ProcessStage 1:

● Inside the nucleus, when DNA is ready for replication, the enzyme helicase unwinds and unzips the double helix creating a replication fork. Replications begins at the origin, which varies depending on the DNA. Helicase breaks the weak hydrogen bonds between bases, which are holding the two strands together. This process occurs at several locations on a DNA molecule.

Replication ProcessStage 2:After helicase opens the DNA, a short piece of RNA called primers add nucleotides to start the replication process.The enzyme DNA polymerase moves down the DNA strands and adds new nucleotides to each strand. The nucleotides pair with the complementary nucleotides on the existing stand (A with T, G with C) and attached via hydrogen bonds. ● DNA polymerase can only add nucleotides to

deoxyribose at the (3’) ended strand in a 5’ to 3’ direction.

● This creates a problem at the replication fork because one parental strand is 5’ → 3’ while the other antiparallel parental strand is 3’ → 5’.

Leading Strand

● The 5’ → 3’ strand is called the leading strand, DNA polymerase adds nucleotides towards the replication fork.

Lagging Strand

The other parent strand called the lagging strand (3’ → 5’) is copied away from the replication fork in fragments called Okazaki fragments. RNA primers are added on the 3’, then followed by DNA polymerase adding the nucleotides. This occurs because DNA polymerase can only add nucleotides to the 3’ end.

● Okazaki fragments, each about 100-200 nucleotides are joined by the enzyme ligase.

DNA polymerase further “proofreads” the nucleotides to reduce the chance of error and correct any mistakes.

Lagging Strand

Replication ProcessStage 3:

● The two new strands twist to form a double helix. Each is identical to the original strand.

https://youtu.be/TNKWgcFPHqw

DNA Replication Storyboard

● Using the blank piece of paper, fold it into squares and create a storyboard outlining the steps of DNA replication.

● The “steps” can be whatever makes sense to you, as long as it is in the correct order.

● Write a description of what is happening on the back. ● Miss. Paslawski will be coming around to assist you and

ask questions. ● Include: DNA strands (5’, 3’), RNA primers, nucleotides,

Helicase, DNA polymerase, Ligase.

Lesson 2: Building ProteinsTerms: DNA, proteins, RNA

Building Proteins

DNA carr­ies all of the information for your physical characteristics, which are essentially determined by proteins. The order of nucleotides within a gene specifies the order and types of amino acids that must be put together to make a protein.

Proteins function in the following ways:

● Enzymes that carry out chemical reactions (such as digestive enzymes). There are thousands of enzymes that do specific functions. The suffix -ase is used in biochemistry to form names of enzymes.

● Structural proteins that are building materials (such as collagen and nail keratin)

● Transport proteins that carry substances (such as oxygen-carrying hemoglobin in blood)

● Hormones - chemical messengers between cells (including insulin, estrogen, testosterone, cortisol)

● Protective proteins - antibodies of the immune system, clotting proteins in blood

DNA and RNADNA - DeoxyriboNucleicAcid

RNA - RiboNucleicAcid

Function Long-term storage of genetic information within the nucleus

Used to transfer the genetic code from the nucleus to the ribosomes to make proteins.

Sugar The sugar portion of DNA is 2-Deoxyribose.

The sugar portion of RNA is Ribose.

Nitrogen Base The bases present in DNA are adenine, guanine, cytosine and thymine.

The bases present in RNA are adenine, guanine, cytosine and uracil.

Number of Strands double-stranded single-stranded

DNA and RNA

Lesson 3: Protein Synthesis Part 1 Terms: mRNA, codon, anticodon, amino acid, transcription, translation, tRNA

How does DNA code for Protein? - Protein Synthesis

Proteins are molecules made from chains of amino acids.Synthesis means to construct, therefore protein synthesis is the construction of proteins.

Protein Synthesis: one of the most fundamental biological processes by which individual cells build their specific proteins.

Each particular gene provides the code necessary to construct a particular protein. Gene expression, which transforms the information coded in a gene to a final gene product, ultimately dictates the structure and function of a cell by determining which proteins are made.

How does DNA code for Protein? - Protein Synthesis

There are more than 10 million proteins in the human body, that are made up of only 20 kinds of amino acids. Proteins can be as small as 20 amino acid units to thousands of amino acids long.

There are more than 10 million proteins in the human body, that are made up of only 20 kinds of amino acids. Proteins can be as small as 20 amino acid units to thousands of amino acids long.

How does DNA determine the sequence of Amino Acids?

The sequence of bases in a gene (that is, its sequence of A, T, C, G nucleotides) translates to an amino acid sequence. A triplet is a section of three DNA bases in a row that codes for a specific amino acid. This is how the language of nucleotides translates to the language of amino acids. A segment of three bases is called a codon, the equivalent triplet on an tRNA is called an anticodon.

How does DNA determine the sequence of Amino Acids?

Start and Stop Codon● DNA is the instructions to build protein, but it must

know where to start and stop the amino acid chain. ● AUG is the codon for “start”. ● UAG, UAA, and UGA are the codons meaning “stop”. ● There are 64 possible codons and only 20 amino

acids, there is some repetition in the genetic code.

How does DNA determine the sequence of Amino Acids?

● When a protein is needed within a cell the segment of DNA with the appropriate base sequence is transcribed into messenger RNA (mRNA). When mRNA leaves the nucleus and moves to the ribosome the sequence of codons is read and matched with transfer RNA (tRNA) which carries its corresponding amino acid. When complete the amino acid chain becomes a complete protein for the cell to use.

Amino Acid Chart

Determine the amino acid for:

GCA

CAG

UUG

Lesson 4: Protein Synthesis Part 2 Terms: transcription, RNA polymerase

Transcription: DNA to RNA

DNA is housed within the nucleus, and protein synthesis takes place in the cytoplasm, in order to get the message of the DNA to the cytoplasm to be translated into protein RNA is made.

RNA differs from DNA in terms of, the sugar, number of strands, and bases.

Transcription: DNA to RNA

Transcription is the process of synthesizing of a strand of mRNA that is complementary to the DNA. This process is called transcription because the mRNA is like a transcript, or copy, of the gene’s DNA code.

The strand of RNA is created by RNA polymerase from the template DNA. This RNA is called messenger RNA because of its ability to leave the nucleus as a messenger to generate protein.

Transcription: DNA to RNA

Stage 1: Initiation● A region at the beginning of the gene is called a

promoter- this sequence of nucleotides triggers transcription.

Stage 2: Elongation ● RNA polymerase unwinds the DNA segment and aligns

the correct base (A, C, G, or U) complementing the coding strand of DNA.

Stage 3: Termination● RNA polymerase is told to “stop” when it reaches the

termination signal (UAA, UAG, or UGA).

Lesson 5: Protein Synthesis Part 3 Terms: Translation, tRNA, rRNA,

Translation: From RNA to Protein

Translation is the process of synthesizing a chain of amino acids called a polypeptide. Like translating a book from one language into another, the codons on a strand of mRNA must be translated into the amino acid alphabet of proteins.

We know this language already, Valine, Serine, Cytosine etc.

Translation: From RNA to Protein

Stage 1: InitiationThe ribosome binds with mRNA at the start codon. Methionine (MET) is the first amino acid in the sequence.

http://www.biologynoteshelp.com/wp-content/uploads/2016/05/ribosome-pbworks-300x216.jpg

Translation: From RNA to Protein

Stage 2: ElongationAmino acids are brought to the ribosome by tRNA and linked together to form a peptide chain. Transfer RNA (tRNA), molecules of RNA that serve to bring amino acids to a growing polypeptide strand and properly place them into the sequence. Ribosomal RNA (rRNA), RNA that makes up the subunits of a ribosome.

Translation: From RNA to Protein

Translation: From RNA to Protein

The first, methionine-carrying tRNA starts out in the middle slot of the ribosome, called the P site (polypeptide site). **Remember that the tRNA has the corresponding anti-codon to the mRNA strand.

Translation: From RNA to Protein

The next tRNA with its amino acid attaches to the A-site (acceptor site), which acts as a landing site prior to joining the peptide chain. Peptide bonds connect one amino acid to another.

Each tRNA delivers the amino acid adding to the peptide chain as the ribosome makes its way down the mRNA strand. This shift allows the first, empty tRNA to drift out via the E site ("exit") . It also exposes a new codon in the A site, so the whole cycle can repeat.

Translation: From RNA to Protein

Stage 3: Termination This process repeats until the final stage called termination. When the ribosome encounters a stop codon the polypeptide is released into the cell.

Determine the Amino Acid sequence from DNA

DNA: TAC GGC TAT ATT

mRNA: AUG CCG AUA UAA

tRNA: UAC GGC UAU AUU

Amino Acid:

MET PRO ILE STOP

Lesson 6: Introns and Exons Terms: Introns, exons

Introns and Exons

After DNA has been transcribed into mRNA it undergoes a process called RNA processing or splicing. Prior to mRNA leaving the nucleus segments of the strand are cut out and the remaining pieces called exons are joined together. ● Introns: segments of DNA that do not code for amino

acids. Intros are removed from the DNA. Introns can range in size from 10’s of base pairs to 1000’s of base pairs.

● Exons: segments of DNA that code for amino acids that will become part of a protein.

Introns and Exons

● A key point here is that it's only the exons of a gene that encode a protein. Not only do the introns not carry information to build a protein, they have to be removed in order for the mRNA to encode a protein with the right sequence. Most genes in humans are interrupted by segments of DNA that are not part of the gene.

Introns and Exons

Remember: Introns interrupt and exons express

Example: THECATAQZURMVBNDDOGUTICXPAREBUDSMDIEJ

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

Lesson 7: Gene Regulation Terms: gene regulation, protein

Gene Regulation

Gene regulation is the process of controlling which genes in a cell's DNA are expressed (used to make a functional product such as a protein).

● Skin, eye and hair cells need the protein melanin to produce their colour, but your nails do not.

Gene Regulation

How does the cell regulate DNA expression?● Transcription- what segments of DNA are

transcribed into RNA● RNA processing - splicing of mRNA to determine

what proteins will be created. ● Translation - translation of mRNA may be

increased or decreased to produce more or less of a specific protein

● Protein activity- proteins can be modified by being chopped up or tagged with chemical groups the will affect its activity.

Gene Regulation and Disease

● Huntington’s disease, a genetic brain illness is caused by a mutation that makes proteins that attack the nervous system. This protein can be decreased to benefit patients.

Switch Off Gene in Mice to Switch on Endurance http://blogs.discovermagazine.com/80beats/2011/07/20/researchers-switch-off-gene-in-mice-to-switch-on-endurance/#.WOqDUnTytZJ

Lesson 8: Gene Mutations Terms: mutation, mutagen, silent mutations, missense, nonsense, deletion, insertion, frameshift

Gene MutationsA mutation is a change that occurs in the DNA sequence due to a mistake in DNA replication, transcription, or environmental factors. ● Environmental factors include: uv light, cigarette smoke,

radiation, and chemicals. ● Any physical or chemical agent that induces a mutation is

called a mutagen. ● A carcinogen is a substance capable of causing cancer in a

living organism. Often cells recognize potential mutations and are repaired before they cause any damage. Yet some DNA changes remain. If a cell accumulates too many changes—if its DNA is so damaged that it cannot be fixed stops dividing or it self-destructs. If any of these processes go wrong, the cell could become cancerous.

Gene Mutations

Although mutations seem to be all bad they provide a great benefit to species. Mutation creates slightly different versions of the same genes, called alleles. These small differences in DNA sequence make every individual unique. Further, mutations introduce genetic variation which is helpful as populations change over time. Variations that help an organism to survive and reproduce are often passed onto the next generation, while variations the hinder are usually eliminated from the population.

How are Mutations Inherited?

Mutations can be inherited if the mutation is present in the gametes of the mother or father. Hereditary mutations are present throughout a person’s life in virtually every cell in the body. Even though mutation are common, inherited diseases are relatively rare. This is because inherited diseases are often recessive, which means that a person must have two copies of the mutated gene to get the disease. Acquired mutations occurs during a person’s life and not present at birth.

Point Mutations

Change in one base of the DNA sequence, the substitution of one nucleotide for another.

1. Silent mutation: the substitution does not affect the amino acid it codes for. This is due to the redundancy in the genetic code.

2. Missense mutation: the altered codon now corresponds to a different amino acid. As a result an incorrect amino acid is inserted into the protein being synthesized. Altered proteins may result in changes in the protein function.

3. Nonsense mutation: the altered codon signals for transcription to stop. Thus a shorter mRNA strand is produced and the resulting protein is truncated or nonfunctional.

Frameshift Mutations

The deletion or addition of a nucleotide that causes the sequence to shift how it is read.

Mutations

Original DNA TemplateDNA: TAC ACC TTA TAT ATA GTA TTT ACT mRNA: AUG UGG AAU AUA UAU CAU AAA UGA

DNA: TAC ACC TTA TAT ATA GTA TTT ACTmRMA: AUG UGG AAU AUA UAC AUA AAU GAAA: Met Trp Asn Lie Try Ile Asn -

Mutations

Original DNA TemplateDNA: TAC ACC TTA TAT ATA GTA TTT ACT mRNA: AUG UGG AAU AUA UAU CAU AAA UGA

DNA: TAC ACC TTA TAT ATT GTA TTT ACT mRNA: AUG UGG AAU AUA UAA CAU AAA UGAAA: Met Trp Asn Lie STOP

Sickle-Cell Anemia

Sickle cell anemia is a genetic disease with severe symptoms, that affects the red blood cells ability to carry oxygen and causes sickle shaped cells. The disease is caused by a mutated version of the gene that helps make hemoglobin — a protein that carries oxygen in red blood cells. People with two copies of the sickle cell gene have the disease. People who carry only one copy of the sickle cell gene do not have the disease, but may pass the gene onto their children.

Sickle-Cell Anemia

X A a

A

a

The mutated gene is a result of a nucleotide substitution, thymine replaced by an adenine which creates the protein valine instead of glutamine. However, in African populations having the mutation for sickle-cell protects against malaria. Being heterozygous for sickle-cell was found to protect against Malaria. Malaria is a parasite carried by mosquitoes.

Cross heterozygous for sickle cell parents.

https://www.geneticliteracyproject.org/2017/03/10/sickle-cell-cure-patient-in-complete-remission-following-gene-therapy/

Lesson 9: Restriction EnzymesTerms: restriction enzyme, endonuclease, blunt, sticky, electrophoresis

Restriction Enzymes

Restriction enzymes, also known as restriction endonucleases, are specific enzymes that cut DNA. Restriction enzymes recognize specific segments of bases called restriction sites. Each restriction enzyme (and there are hundreds, made by different species of bacteria) has its own particular restriction site where it will cut DNA. The enzyme "scans" a DNA molecule, looking for a particular sequence, usually of four to eight nucleotides. The site where the restriction enzyme cuts is called the restriction site or target sequence.

Restriction EnzymesApplications● Restriction enzymes are found in bacteria. It’s

thought that restriction enzymes evolved as a defense mechanism, allowing bacteria to chop up potentially harmful foreign DNA.

● Used to insert a piece of DNA into a plasmid to make copies of the DNA.

● Used in DNA fingerprinting to identify matching DNA sequences.

Restriction Enzymes

How do Restriction Enzymes cut DNA?● The specific enzyme recognizes the sequence of

DNA where it will make the cut. ● The 5’ to 3’ base pairs are complementary to the 3’

to 5’.● The DNA molecule will be cut as many times as

that specific sequence occurs in the DNA. ● Cuts leave the DNA fragment either blunt or

sticky.

Restriction Enzymes

Example of a blunt end restriction enzyme: Hpal5'--GTT|AAC--3'3'--CAA|CCG--5'

Restriction Enzymes

Example of a sticky end restriction enzyme: EcoRI5’--G|AATTC--3’3’--CTTAA|G--5’

Gel Electrophoresis

● Electrophoresis come from the term electro meaning electricity and phoresis meaning "migration" or "movement."

● Gel electrophoresis is a technique commonly used in laboratories to separate charged molecules like DNA according to their size.

Gel Electrophoresis● Restrictive enzymes are used to create segments of DNA,

these segments vary in size by the length of the base pairs. ● DNA samples are loaded into wells (indentations) at one end

of a gel, and an electric current is applied to pull them through the gel.

● DNA fragments are negatively charged, so they move towards the positive electrode. Smaller fragments move through the gel faster and easier than larger ones.

● The result of the electrophoresis illustrates the DNA fragments seen as bands after the gel is stained with DNA-binding dye.

This method allows us to visualize DNA fragments for further analysis such as DNA fingerprinting.

Gel Electrophoresis

Example of Restriction Enzyme

● After making the cuts according to the specific restriction enzyme, count the number of DNA pieces.

● Then, count the number of bases in each piece, this information is used to determine what the gel would look like.

● Determine the type of cut - sticky or blunt.