Unit 4 - Genetics Flashcards
Describe 2 unplanned or chance event that led to a major discovery in the history of early DNA research.
- Griffith discovering that the combination of two non-lethal strains of bacteria could produce a fatal case of pneumonia in the mice
- James Watson being allowed to see Rosalind Franklin’s unpublished X-ray crystallography results, enabling him to realize which of his own models was probably right
Oswaled Avery, Colin MacLeod, and Maclyn McCarty showed
that Dna, not proteins, transformed the properties of cells
Erwin Chargaff discovered arrangement on )
nitogen bases in DNA vary but the bases always occurred in a one-to-one ratio (AT-GC)
Roaslind Franklin and Maurice Wilkins used xray diffraction to show
helical nature of DNA
Hershey and Chase proved
DNA is genetic material
Watson and Crick discover and publish
Dna double helix structure
Matthew Meselson and Franklin Stahl proved
the semi-conservative model of DNA replication using nitrogen isotopes N15 and N14 in E.Coli bacteria
Frederick Griffitch proved
transfer of Genetic Material from bacteria (initially thinking it was through protein transfer)
Phoebus Levene described structure of
nucleotides
Friedrich Miescer discovered and isolated
nuclein
who used bacteriophages which are composed of DNA and proteins, to show that DNA enters host bacteria after infected by bacteriophage
hershey and chase
who Isolated nuclein and first to identify DNA as a distinct molecule
miescher
who Discovered the fundamental laws of inheritance
mendel
who Proved that nucleotides were composed of a phosphate-sugar-base-complex and proposed the ‘polynucleotide’ model for how DNA molecules were put together
levene
who Identified there is a one-to-one ratio between Adenine-Thymine and Cytosine-Guanine through experiemtnation
chargaff
who Described the structure of DNA as a twisted double helix
watson and crick
3 Components of Nucleotides:
Nitrogen bases = Adenine, Guanin, Cytosine, and Thymine
Deoxyribose = sugar base
Phophate group
Pyrimidines have
a single ring structure and can only bond to purines (Cytosine and Thymine)
Purines have
a double ring structure and can only bond to pyrimidines (Adenine and Guanine and Uracil)
why do purines bond better with pyrmidines
- hydrogen bond positions
- single to single ring pairs are too far part while double to double are too close
why can adenine only bond to thymine, and guanine to cytosine
- position of hydrogen donors and acceptors
how many hydrogen bonds are between an AT and GC pair?
2 with an AT and 3 with a GT
Deoxyribose
A cyclic five carbon sugar found in nucleotides
where does deoxyribose attach to the nitrogen base and what kind of bond
glycosidic bond on 1st prime carbon
where does deoxyribose attach to the phosphate group and what kind of bond
phosphodiester bond on 3rd prime carbon
why are dna strands labeled 5 prime and 3 prime?
because the first nucleotide has a DNA strand attached to the 5’ end, and the opposing end has a hydoxyl group on the 3’ end.
Isolating DNA from the tissue is called
DNA extraction
4 steps to DNA extraction
- Physically breaking tissue to make it easier to get at DNA
- Adding a lysis solution or buffer to break remaining cells apart, releasing DNA from nucleus and organelles. (Heating up the solutions will breaks down any enzymes that degrade DNA)
- DNA is physically separated from solution through a filtering process (i.e. filter paper or centrifuge)
- DNA can be purified by washing with water and filtering to result in a clear fluid containing water and dissolved DNA.
why are strawberries ideal for isolating DNA?
Since they are octoploid (contain 8 copies of their DNA) while mose organisms are diploid containing only two sets of DNA, one from each parent.
- Ripe strawberries in particular already have the enzymes pectinases and cellulases that break down cell walls making it easier to extract the DNA.
describe the conservative model of DNA replication
the parent or original DNA molecule produces an exact copy of itself but remains intact - with one new strand of the same, and the original strand of the same
describe the semi-conservative model of DNA replication
= parent DNA splits apart with each strand creating it’s daughter strand. These two new strands would have one half of its strand from the original strand and it’s other half new strands
describe the dispersive model of DNA replication
= parental DNA is broken into fragemsn and that each daughter DNA is made up of a random mix of parental and new dna, with the same nucleotide sequence of the parent
Prokaryotes dna has:
- circular strands of DNA
- contain about a thousand times fewer nucleotides than eukaryotes
- Replication is 10 times faster because DNA is not packaged in Chromatin
3 main phases of replication
Initiation - DNA is unwound and separated to each strand, it is done at multiple points for speed. Eukaryotic DNA can only add 80 nucleotides per second. So hundreds or thousands of replication sites are needed.
Elongation - Enzymes attach complementary nucleotides onto the 3’ end of each strand in a linear sequence and check for errors
Termination - Nucleotide addition stops, enzymes are removed, and newly formed strands of DNA coil back into the double helix shape.
why is DNA in eukaryotes shorter every time and prokaryotes is not
At termination of replication the RNA primer is removed with DNA polymerase ; because the gap left by the RNA primer at the end of the DNA can’t be filled, the unpaired bases break off causing a short DNA molecule each time. Prokaryotes DNA is circular so not an issue.
Origins of Replication -
Origins of Replication - multiple sites on DNA where replication will begin ; located with lots of A-T base pairs because these nucleotides are held to gether with only two hydrogen bond – easier to pull apart than C-G base pairs that have 3
Helicase
Helicase - enzyme that goes to each origin of replication to unwind and separate dna using ATP; breaks the hrydogen bonds
Replicaiton Fork
Replicaiton Fork - Where the two strands split into a fork after helicase splits the hydrogen bonds
Replication Bubbles
- the space between the replication forks where DNA is split that forms a bubble; eventuallly they grow wide enough that they merge together
Topoisomerase
- an enzyme that moves ahead of the helicase to cut the DNA and relax the tension on the DNA coils and prevent supercoiling further ahead ; in prokaryotes and SOME eukaryotes the particular tupe of topoisomerase is called gyrase.
SSbs
Single-Stranded DNA binding proteins (SSbs) - proteins that prevent the two parent strands from rebonding until they can bind with the new nucleotides; danger is the two parent strans will re-anneal/join back together ; after helicase separates the strands the SSbs bind to the exposed bases.
DNA polymerase I
- Enzyme that is active near the end of DNA replication, removes the RNA primer
DNA polymerase III
DNA polymerase III - enzyme responsible for joining the nucleotides together and checking for errors; continuously adds nucleotides to lead strand
dNTP
Deoxyribboneculoside triphosphates (dNTP) - nucleotides (A, T, G, C) with two extra end phosphate groups that form dATP, DTTP, dGTP, and dCTP respective that provide energy required for DNA formation ; during the joining reaction which requires energy, the two end phosphates beak off from the dNTP to release energy that creates the phsphodiester bond that connects the nucleotide to the chain.
Primase
Primase - Enzyme that forms RNA primer; released once RNA primer is formed and attached to the DNA template
RNA primer
RNA primer - a starting sequence of nucleotides for DNA polymerase II to attach to formed RNA with the help of primase ; about 10-100 base pairs long
Leading Strand
Leading Strand - The strand that DNA polymerase 3 can continuously add nucleotides from the initial RNA prime in a 5’ to 3’ direction
Laggin Strand
- Created discontinuously in short fragments in the opposite direction to the leading strand (away from fork) with use of many RNA primers and formation of Okazaki fragments and DNA ligase
Okazaki Fragments
Okazaki Fragments - 100 nucleotide long fragments formed form laggief strand between RNA primers
DNA Ligase
- enzyme that join the Okazaki Fragments in lagging strand
which enzyme proofreads the new DNA
DNA polymerase III
5 Steps for Lagging Strand
- Make RNA primer; an initial chain of nucleotides using RNA primase to attach to complimentary exposed bases on the template strand.
- Add nucleotides to RNA primer; DNA polymerase 3 adds dNTPS on those the free 3’ hydroxyl (-OH) end of the rna primer, with the extension running in the opposite direction to the replication fork. The fragment of DNA are called Okazaki fragments after Tsuneko Okazaki who discovered them in 1960s.
- Remove RNA primer- once the Okazaki fragment has grown long enough to bump into the next fragment along the template, it stops growing and DNA polymerase III is released. DNA polymerase I attaches at this location and removes the RNA primer and rplaces the RNA with DNA nucleotides.
- Join Okazaki fragments ; DNA polymerase I is released and the adjacent OKazaki fragmeents are joined, using ligase to complete the strand
- Proofreading - DNA polymerase III prevents point mutations by detecting the number of hydrogen bonds a nucleotide forms. If hydrogen bonding does not occur the bases are mismatch and the DNA replaces the incorrect base with the correct one. ; process is so effective only about one error per billion base pairs occurs
Point mutations=
= a mutation affecting only one of very few nucleotides in a gene sequences
Telomeres -
found at end of DNA where primers are removed after replication, human gene sequence is TTAGGG ; evelvoled to help deal with genetic loss after replication ; several thousand nucleotides made up of a simple repeated sequence that coest not code for proteins; humans sequence is TTAGGG ; a buffer region of expendable DNA that can be gradually lost over repeated replications without damaging the active genetic code. ; of finite length so division is a certain number of times ;
Telomerase
- Enzyme helps form telomeres at end of DNA molecules after RNA primers have been removed; particularly reproductive cells need to replicate their DNA more times so they produce telomerase that builds and extends telomeres, adding extra pieces of DNA each time a cell divides. .
CHROMOSOME
- In the nucleus
- Made up of two identical strands of DNA, each called the Chromatid
- Centromere joins the two chromatids together which fibres attach during cell division
- Telomeres are end of of chromosomes
Cancer -
when telomerase is overactive repairing telomeres and allowing cancer cells to replicate indefinitely
3 Phases of Transcription Stage (protein synthesis)
3 Phases = Initiation (of mRNA transcription), Elongation of mRNA, Termination of mRNA
Template strand
- one of the two strands of DNA used for protein synthesis, aka the sense strand.
Untranscribed strand
- aka the anti-sense strand that doesn’t code for proteins
what does RNA polymerase do in the transcription stage
separates the two strands, and binds onto the template strand at the starting point for the gene to be read
promoter sequence
point on template strand where RNA polymerase first attaches located several nucleotides up from where the transcription begins, which are are mostly adeninde and thymine/ TATA box due to weak bonds
what direction does RNA polymerase move during elongation
3-5 direction
what nucleotide does RNA polymerase add when it sees adenine
uracil
why would another RNA polymerase attach to the same gene after an RNA polymerase already transcribed it
to make multiple of that protein
transcription bubble
small portion of unravelled DNA that RNA polymerase creates
describe elongation phase of transcription
RNA polymerase unwinds the DNA and begins creating the mRNA strand until it ends
GC hairpin
-first step in termination of mRNA transcription
-its the termination sequence that stops RNA polymerase from continuing to build mRNA
a loop of guanine-cytosine pairs that acts as a bump to dislodge RNA polymerase
what must happen to mRNA before it leaves the nucleus and why
- post transciptional modifications must happen or else enzymes in cytoplasm will attack introns
- guanine triphosphate/GTP cap is added at 5’ end
- long series of repeating adenine nucleotides called a poly-A tail is added to 3’ end
- it also helps ribosomes know what they are attaching to
introns
stretches of nucleotides that do not get translated to proteins
-must be removed before translation can occur
exons
nucleotides that do get coded to proteins (inbetween introns)
spliceosomes
cut out introns and join the exons in mRNA
mRNA transcript
the series of exons left after the introns have been removed
3 phases of translation stage (protein synthesis)
Iniation - when tRNA moves the mRNA in cytoplasm, ribosomes attach to it to to begin attaching amino acids together via tRNA. The large and small subunits work together to prepare the elongation process using the sites E, P, and A
Elongation - amino acids are brought to acceptor sites (A) and is charged by aa-tRNA complex, amino acids is bonded to between peptidyl (P) site and A site to create polypetide chain, after it is bonded the amino acid in the P site loses it charges and moves to the exit/E site, and the A site amino aid moves to the P site and a new amino acids is moved to the A site and is charged with another aa-tRNA and continues until the protein is formed
Termination - when the stop codon is reached at mRNA transcript, elongation ends, and a special protein release factor reads it and terminates it
The Central Dogma of genetics
DNA-transcription-RNA-translation-Protein
proposed by francis crick
a one way process
Gregor Mendel published his work on inheritance of
physical trains in a pea plan, showing discrete genes were responsible for discrete phenotypic characteristics. First demonstration of a relationship between genotype and phenotype
genotype
(genetic makeup of an organism)
phenotype
(observable characteristic on an individual)
Archibald Garrod hypothesized that protein was
- determined by genes
- by noticing certain illnesses were more common in some families that others. Indicating that the illnesses were inherited.
- Garrod studied patients suffering from a rare genetic disease that cause their urine to turn black when exposed to air.
one gene can code for more than one
polypetide (protein)
Mutations in genes can cause diseases first shown by Vernon Ingram in 1956, by
copying amino acid sequences of protein hemoglobin in people with health red blood cells vs those with a genetic disease called sickle cell anemia.
- Using electrophoresis to determine amino acid sequence of polypeptides that make up the protein.
Marshall Nirenberg and Har Gobind Khorana were able to identify
the triple letter sequences/codons that coded for each of the 20 amino acids
3 codons that do no code for any amino acid:
UAA, UAG, UGA (stop codons)
start codon
AUG/methionine
iniation factors
proteins that combine with two ribosome subunits to form an iniation complex to stablize mRNA to make sure its in the correct position
iniaitor tRNA-amino acid
beginning amino acid to enter ribosome at start of translation
- met-tRNAmet complex
- tRNA bonded with methionine the start codon
aminoacyl-tRNA synthetase
enzyme that helps form the aa-tRNA bond, bonds between tRNA and the corresponding amino acid
when a tRNA is normally carries an amino acid (aa-tRNA) it is said to be
charged
which site does the iniator tRNA-amino acid/met-tRNAmet complex attach to at beginning of elongation in translation stage
the P/peptidyl site
why is reghulation of protein synthesis important
- it requires energy so control is required
- not all proteins are needed all the time, but at key points in a persons life
four different levels of protein synthesis control
- Transcriptional - determines which genes get transcribed or controls the rate at which transcription occurs.
- Post-transcriptional - when mRNA does not have it’s poly-A tail or cap, it can be blocked
- Translational - Regulatory proteins in the cytoplasm can bind near the 5’ cap of an mRNA transcript. The small ribosomal subunit is then unable to attach to the mRNA transcript, because the proteins have covered the first few nucleotides where it would normally attach. These proteins can be removed to allow translation to restart.
- Post-translational levels. - Once the polypeptide is released from the ribosome, it can be modified by chaperone proteins. These proteins affect the way in which the polypeptide folds into its final protein shape or can add additional chemical groups to it. These modifications can inhibit or activate its function.
operon
is a group of genes under the control of a single regulatory protein. The result is that the genes contained in the operon are either expressed together or not at all. In the same way that you can use a power bar to turn many plugs on and off with one switch, one operon can turn many genes on and off using one regulatory protein.
Operons can operate using
Negative regulation: involves the binding of a regulatory protein (“repressor”) to the operator to prevent transcription.
Positive regulation: involves the binding of a regulatory protein (“activator”) to an area close to the promoter region to stimulate transcription. The lac operon shows both types of regulation.
Mutagens
environmental factors (for example, UV light) that can cause DNA mutation
what coudl chemical agents impact that would cause DNA mutations
nucleotide sequences
somatic cell mutations
mutations that occur in the body cells
germ cell mutations
mutations that occur in the sex cells and can be passed to next generation
two classes of mutations
Gene-level mutations: mistakes in the base-pair sequence of genes
Chromosome-level mutations: are mistakes in the arrangement or number of genes on a chromosome.
two major types of gene-level mutations:
Point Mutations (Substitution Mutations) Frameshift Mutations
Explain Point mutations and it’s 3 types
substitution of one nucleotide, but it only affects the codon in which it occurs. It has no effect on neighbouring codons in the gene.
3 point mutations: Silent (has no change in amino acid), Missense (one amino acid is replaced), Nonsense (when amino acid changes to a stop codon and elongation ends causing polypeptide to be nonfunctional)
Explain frameshift mutations and it’s 2 types
Either an insertion or deletion of a nucleotide that can cause entire reading frame of gene to be shifted and affects all neighboring codons
Explain chromosome level mutations, it’s difference between gene level mutations and it’s two types
- mutations that occur during meiosis (reproduction of gamete cells)
either translocation (a relocation of groups of base pairs (AT/GC) on one part of the chromosome to the other or inversion (when a segment of chromosome is cut out and then reinserted backwards)
The Barcode of Life Project
- Canadian developed tool of genetic sequencing for biodiversity conservation
- Most species have short segments of genetic code (approximately 648 base pairs) that are nearly identical across all individuals of that species, but which are different from other species, so the project aims to identify the distinction of species via their DNA
- helps identify false food products, endangered species
Why is biotechnology often shown as being controversial in media reports?
Because biotechnology has implications for many different considerations in society, such as legal, ethical, economic, political, and environmental factors, these considerations can sometimes be contradictory. For example, a new biotechnology may have a medical benefit, but its production could harm the environment, so it’s difficult for society to decide whether it is a good or bad invention, overall.
sodium dodecyl sulphate (SDS) uses for biotechnology
to break lipid rich membranes apart, usually in household detergents
why does cold protect DNA from degradation
it slows down the destructive action of the enzymes
why can alcohol be used to filter DNA
because DNA is not soluble in alcohol but the other cell components are
restriction endonuclease (RE) enzyme
- cut individual genes from the chromosomes by recognizing and cutting specific sequence at a restriction site
- each RE can target different sequences
recombinant DNA
Recombinant DNA is the result of removing genetic material from one genome and inserting it into another genome. This is often done across species to create transgenic organisms.
How did the discovery of restriction enzymes allow biologists to work with DNA samples?
Restriction enzymes (RE) allow biologists to cut DNA at known locations into short sequences. The DNA fragments can then be analyzed, removed, or inserted into the DNA of other individuals (even individuals of other species). RE makes genetic engineering possible.
Gel Electrophoresis
- technique for determining whether your sample contains the right fragments of DNA
- Negatively charged DNA molecules are forced to pass through a gelatin-like material called agarose, which slows them down. The negatively charged molecules are pulled towards the positively charged end of the agarose gel.
- large fragments move slow, small move fast
- The result is that the different sizes of DNA molecules are spread out across the gel in stripes or bands
- The image it shows is are in lanes and ladders
What roles do the electrical current and the agarose gel play in sorting DNA samples according to their different sizes? =
Because DNA is negatively charged, it will move towards the positive end of the gel when an electric current is applied to the gel. The electric current provides the “pull” that drags the DNA fragments through the gel. Agarose gel is like an obstacle course that slows the larger DNA molecules down, as they pass through it. The smaller fragments will move faster than the larger ones, so they will reach the positive end of the gel the fastest.
What is the purpose of having a DNA ladder run alongside the other samples?
= The DNA ladder provides a known reference of fragment lengths spread along the gel. You can compare a fragment from another sample to the ladder, to determine its number of base pairs.
polymerase chain reaction (PCR)
a method of replicating specific sequences within a DNA sample
- amplifies a particular sequence of DNA using a customized DNA primer that only binds to specific DNA, then replicates that sequence similar to DNA replication
- high temperatures speed up the replication, but cools also allow for slow down for positionality, requiring a rapid cycling for the replication
- Taq polymerase found in Thermus aquaticus bacteria helps in replication
Describe the unique feature of Taq polymerase that makes PCR possible.
The unique feature of the Taq polymerase enzyme that makes PCR possible is that its optimal activity occurs at a high temperature (around 72°C). Most enzymes denature (split apart) above 40°C. Therefore, the DNA sample being amplified can be separated cleanly by raising the temperature, while the activity of the enzyme is improved. This speeds up the replication process greatly.
Explain why PCR needs to be carried out at three different temperatures.
PCR requires three steps and each one occurs at a different optimal temperature. The first step is to denature or split apart the DNA molecule. At 94°C, this will happen naturally, without the use of enzymes. The next step is the attachment of the primer molecule to the exposed DNA templates. This is called annealing and happens most effectively at around 60°C. The final step is the addition of new nucleotides to the growing DNA strand. This elongation step occurs with the help of Taq polymerase, which is optimally active at 72°C.
chain termination sequencing
- a way of DNA sequencing to determine order of bases
- creation of many short segments of the original DNA fragment called DNA oligonucleotides
- these fragments are replicated with dideoxynucleotide triphosphates (ddNTPs) that can be added to the oligonucleotides and stops elongation
- results in many fragments at different legnths, when ordered from shortest to longest, can be read in order
- this is useful for forensic evidence as then
electrophosphoresis can be used to compare fragments
plasmid
structures in prokaryotic cells that contain genetic material and which can be easily extracted and then transferred to other bacteria to create recombinant DNA
Describe one useful product produced by recombinant DNA.
Insulin is a hormone needed by millions of diabetics around the world. Producing insulin in transgenic bacteria creates a cheap, plentiful supply that does not cause the same allergy problems as the old source of insulin obtained from animals.
What is the role of restriction enzymes in the process of making recombinant DNA?
Restriction enzymes are designed to cut the DNA at particular locations. They are like “molecular scissors” that can be designed to cut out specific genes or fragments of genes.
