Molecular genetics test
Nucleotides
a single unit of nucleic acid
Nucleotide sequences
Make up genetic code. They link together via a condensation reaction between the phosphate group (attatched to the 5' Carbon of the sugar) and the hydroxl group in the pentose sugar (3' carbon of the sugar). This is called a phosphodiester bond, which also produces a water molecule.
Leading strand
the new DNA polymer at the opening of the replication fork (DNA is building from the 3' end
Lagging strand
the new DNA polymer that is opposite the opening of the replication fork (building from 3' end)
Purines
Guanine and Adenine. Purines are double ring structures that always form complementary pairs with pyrimidines through hydrogen bonds. Adenine binds to thymine via two hydrogen bonds. Ctyosine bonds with guanine via three hydrogen bonds.
pyramidines
Ctyosine, Thymine, and Uracil. SIngle ringed structures that always form a complementary pair with purines.
Hydrogen Bonding in DNA
Groupchat (Guanine, ctyosine) = 3 bonds, in a groupchat there are multiple people
Chargaff's rule
Using paper chromatography, Chargoff found that the frequencies of the four nitrogenous bases were not equal in amounts. In DNA, the percent of adenine is the same as thymine (60%, 30% of each), and the percent of cytosine and guanine were the same (40%, 20% of each)
Why does Eukaryotic DNA need to be supercoiled?
To pack genetic material into the nucleus, to organise DNA to allow for cell devision, allow cells to specialize, promote or inhibit transcription, and package DNA for meiosis and mitosis
Nucleosomes
A condensed structure formed when double stranded DNA wraps around a group of 8 histone proteins. They protect DNA and allow it to be packaged. The DNA wraps twice around the 8 histones and is attracted to them because they are slightly positive (DNA is slightly negative)
H1 Histone
histone H1 is a separate, linker histone that binds to the nucleosome's DNA entry/exit points and the linker DNA between nucleosomes. the combination of DNA and histones that make a nucleosome is secured by the H1 linker protein and forms the 30 nm fibre. This leads to further packaging of DNA, allowing it to be supercoiled. Nucleosomes are then joined together by DNA that run between them called linker DNA.
Viruses (hershey chase)
Viruses are made up of proteins and nucleic acid (allowed them to figure out which was the genetic material), they inject their genetic material into a host cell to reproduce. Amino acids containing radioactive isotopes to label the virus. Sulfur for the protein coat, and phosphorus for the DNA. The bacteriophage was then combined with E-coli bacteria. When separate with a centrefuge the virus remained a liquid and the bacteria formed a pellet.
Virus replication (Lyctic cycle)
The phage attatches to a host cell and injects its DNA, the phage DNA circularizes, new phage SNA and proteins are synthesized and assembled into phages. The cell lyses, releasing phages
Virus replication (lysogenic cycle)
phage DNA integrates into the bacterial chromosome, becoming a prophage. The bacterium reproduces normally, copying the prophage and transmitting it to a daughter cell. Many cell divisions produce a large population of bacteria infected, ocasionally a prophage exits the bacterial chromosome, initiating a lyctic cycle.
tracking protein (hershey chase)
The viral proteins in the bacteriophage were tagged with sulfur. A blender was then used to separate the infected bacteria and extra virus parts. Then a centrefuge was used, being more dense, the bacteria cells were isolated because they formed a pellet at the bottom of the tube. Radioactive protein molecules were not present in the bacteria cells.
Tracking DNA (hershey chase)
The DNA in the virus was tagged with phosphorus. Blender and centrefuge used. The radioactive DNA molecules were present in the bacteria cells. This led to the conclusion that the molecule that was passed on to the next generation of virus was DNA and not protein.
What is DNA replication?
DNA replication occurs before cell division and is required for reproduction, growth, and tissue replacement. The Original DNA strands serves as a template for the creation of a new strand. DNA polymerase 3 builds the new strand by reading the template and adding the complementary DNA nucleotide.
Caesium chloride (Meselson and Stahl)
Showed that DNA replication is semi-conservative. A solution of caesium chloride was placed in a centrefuge for 20 hours, causing the more dense caesium ions to move to the bottom (this created a concentration gradient). Any substance centrefuged with the caesium chloride beecomes concentrated at a level corresponding with its density
Meselson and Stahl
E.coli bacteria were grown with heavy (15N) and light (14N) nitrogen respectively which entered their DNA. This DNA was extracted after allowing it to replicate for several hours. Using Caesium chloride, they measured the density of the DNA. After one replication, the resulting DNA contained both light and heavy nitrogen. After a second replication, the DNA was either all light or hybrid.
Semi-conservative replication
During DNA replication, the two strands of DNA separate and each one serves as a template for the building of a new strand. The new strands are made by adding complementary nucleotides one at a time and linking them together. The resulting two DNA molecules each have an original strand and a new strand
Initiation (DNA replication)
begins at a specific nucleotide sequence called the origin of replication (creates replication bubbles all over the DNA) . Origin recognition complexed bind to the DNA to begin unwinding it. DNA helicase unwinds the helix by breaking hydrogen bonds. Single stranded binding proteins keep the separated strands apart so that nucleotides can bind. Topoisomerase 2 helps relieve the strain on the helix. Initiation causes a replication fork to form.
Helicase
an enzyme formed from multiple polypeptides and doughnut in shape. The lagging strand passes through the centre of the enzyme while the leading strand passes on the outside, seperating the dna into two strands. ATP is needed to help the helicase move along the DNA molecule towards the replication fork (5' to 3') and break hydrogen bonds. It gets this from the cleaving of the 2 extra phosphate heads from a nucleotide.
Origin recognition complexes
bind to origins of initiation, allowing DNA Helicase to attach. Prokaryotes only have one replication fork, while eukaryotes have many to make replication faster and more efficient
DNA polymerase 3 (initiation)
an enzyme that cleaves off the extra 2 phosphate heads from nucleotides to create energy. DNA polymerase cannot initiate the process of DNA replication, there has to be a 3' end to which a free nucleotide can be added.
Elongation
Synthesis of new DNA strands. RNA primase adds around 10-60 RNA nucleotides to the template strand (RNA primer), this will be removed later. DNA poly 3 adds free nucleotides (deoxynucleoside triphosphates) by cleaving off the heads. DNA poly 3 can only synthesize new DNA in a 5' to 3' direction towards the replication fork.
Lagging strand (DNA replication)
synthesized discontinuously in short fragments away from the replication fork. Needs primers continuously at replication fork along the DNA parent strand and DNA poly 3 builds in short fragments called okazaki fragments. Eventually DNA poly 1 removes the RNA primer and fills it in with DNA bases. DNA ligase joins the fragments together. DNA poly 3 adds nucleotides moving away from the replication fork
Leading strand (DNA Replication)
3' to 5' template is called the leading strand. Continuously built towards the replication fork
Termination
as soon as the new strands are formed, they rewind automatically into their double helix shape. The two DNA molecules separate from each other and the replication machine (origin replication complex) is dismantled
Errors in DNA replication
DNA polymerase 3 proofreads the newly formed strands of DNA. When they find mistakes (e.g mispairing or additions/deletions of nucleotides) they fix it. Errors that still remain after are then considered mutations in the genome once cell division occurs
Telomeres
In order for DNA polymerase 1 to remove the RNA primer and nucleotides, there needs to be a sequence on the other side of the primer. Since there is none on the end, the lagging strand is slightly shorter. Because of this, repetitive sequences called telomeres are added to the end of the chromosomes by enzymes called telomerases. As we get older, telomerase activity slows down and the lagging strrand continues to get shoter after every replication, information from the coding portion of chromosomes may be lost, leading to aging.
Polymerase chain reaction
PCR is the direct method of making multiple copies of a desired gene or section of DNA (making large amounts for analysis is called DNA amplification. PCR is carried out in thermocycler machines that change temperature quickly at set times. First, the hydrogen bonds break and strands separate at high temperatures, and then cooled to allow primers to stick to the template DNA. Then it is heated again so that Taq polymerase (bacterium that withstands high heat) can build more complementary strands using free nucleotides that were added to the solution.
Short tandem repeats
In the non-coding regions of the genome there is satelite DNA made up of repeating elements called short tandem repeats. Individuals are likely to have a different number of repeats, generating unique DNA profiles
Recombinant DNA
A molecule of DNa composed of genetic material from different sources. Made using restriction enzymes which have the ability to cleave viral DNA in prokaryotes
Restriction endonucleases
specific restriction enzyme that cleaves double stranded dna within the interior of DNA strands rather than the ends. Done by disrupting the phosphodiester bonds between nucleotides. Most of the sites/target sequences it binds to are spelt the same forward and backward (palindromic)
Gel electrophoresis
Amplified DNA samples are cut by restriction enzymes and fluorescent markers bind to them so they can be seen. The gel is submerged in a buffer solution to maintain its pH. The samples are then added and an electric current passed through. The DNa will travel towards the positively charged electrode. Smaller fragments go further than heavier ones, creating a banding pattern for each DNA sample.
VNTRS
variable number tandem repeats have longer base sequences repeaded few times. These are inherited from both parents, therefore no one will have the same VNTR (except for siblings)
What is transcription?
The synthesis of RNA, using DNA as a template. it takes place in the nucleus, copying the gene sequence into the messenger RNA. It only occurs along one of the two strands of DNA, and genes can be transcribed repeatedly (happens all the time, vs DNA replication that only happens during cell division)
Messenger RNA
Made in the nucleus, travels into the cytoplasm. Determines the amino acid sequence of a protein. A transcript compy of a gene used to encode a polypeptide.
Transfer RNA (tRNA)
contains an anticodon that base-pairs with a codon on the mRNA and has the corresponding amino acid attached to it according to the genetic code.
Ribosomal RNA (rRNA)
Associates with a set of proteins to form ribosomes.
Small nuclear RNA (snRNA)
involved in splicing pre-mRNA to form mature mRNA
Micro RNA (miRNA)
involved in RNA silencing, prevents translation, no protein (bonds to mRNA, preventing translation)
Small interfering RNA (siRNA)
involved in RNA silencing (bonds to mRNA and degrades it, preventing translation)
RNase
an enzyme that prepares tRNA for translation
Promoter region
a DNA sequence near a gene that serves as the binding site of RNA polymerase (non-coding). The promoter however, is not transcribed. At the end of the promoter region there are several A-T bonds (TATA Box) because they are easy to break and it signals the RNA polymerase that it is reaching the end of the promoter. The binding of RNA poly to the promoter is mediated by transciption factors close to (proximal control) or far from (distal control) the promoter
Coding sequence
After the RNA polymerase has bound to the promoter, it causes the DNA strands to unwind and separate. This region that is transcribed by the RNA poly is called the coding sequence
Terminator
RNA polymerase will continue to tanscribe the DNA until it reaches a terminator sequence
initiation (transcription)
The strand transcribed is called the antisense/template strand. The strand not transcribed is called the sense/coding strand, which has the same sequence as the mRNA with thymine instead of uracil. RNA Polymerase 2 binds to the DNA at a promoter sequence with the help of transcription factors (upstream of the gene). It unzips the DNA as it constructs an RNA transcript of the DNA. The binding site of RNA polymerase only recognizes the promoter region and can only bind upstream of it.
Elongation (transcription)
RNA Polymerase builds the single stranded mRNA after it binds to the promoter in the 5' to 3' direction, by covalently bonding ribonucleotide triphosphates to create energy. When RNA polymerase leaves the promoter region another RNA polymerase can bind. Elongation starts as soon as RNA Poly binds to the promoter. If an error occurs, only one protein will be affected vs all of them in DNA. MRNA does not stay bonded to the DNA, it becomes displaced and the helix reforms
Termination (transcription)
RNA poly reaches the terminator sequence and dissociates with the template strand. The double helix of DNA reforms and the primary RNA transcript is produced (pre-mRNA) it is not ready to leave the nucleus just yet.
Transcriptome
the full range of RNA types made in a cell. Different cells and tissue types have different transcriptomes, and they can change as cell activity changes
Introns
non-coding nucleotide sequences found in eukaryotes which interupt exons (the coding sequence in a gene). They are found in a wide range of genes. When proteins are formed from genes that contain introns, introns are removed during RNA splicing and the remaining exons are joined together
Methyl guanosine capping (5' capping)
mRNA capping is highligh regulated and allows for matures mRNA to undergo translation. A methylated guanosine nucleotide is added onto the 5' end of the mRNA, and regulates nuclear transport, prevents degradation by nucleases, and promotes translation
Polyadenation (poly-A tail on 3' end)
the poly-A tail is a long chain of adenine nucleotides added to the mRNA after transcription on the 3' end by the enzyme poly A polymerase. This makes RNA more stable, prevents degradation, and encourages further modification of the mRNA.
Splicing
proteins called spliceosomes (combinations of mall nuclear RNAs/snRNAs and snRNPs) work together to cut out the introns from mRNA, leaving only the exons and creating the mature mRNA that leaves the nucleus.
Alternative splicing
particular exons might be included or exluded from mature mRNA during splicing. E.g cardiac troponin (gene active in heart muscle) keeps a particular exon in the fetus but is not included in the gene of an adult, making the fetal cardiac muscle more sensitive ot calcium.
What is translation?
the process of protein synthesis in which the genetic information encoded in mRNA is translated into a sequence of amino acids in a polypeptide chain
structure of the ribosome
Translation takes place in free membrane bound ribosomes using the mRNA strand from transcription. Rbosomes are composed of two subunits (larhe and small) which are made of long strands of rRNA. The two subunits lock together when synthesizing a new protein with an mRNA trapped in the space between. The ribosome walks down the mRNA three nucleotides at a time to build the protein
A Site
holds the tRNA carrying the ext amino acid to be added to the polypeptide chain
P site
holds tRNA carrying the growing polypeptide chain
E site
site from which tRNA that has lost its amino acid is discharged
structure of tRNA
tRNA moves through three binding sites (A to P to E), then delivering amino acids to the growing polypeptide chain in translation. It picks up new amino acids when activated by a specific tRNA activating sequence, there are 20 of these that correspond to the 20 amino acids. This costs ATP. tRNA has a complementary anticodon for the 20 seuqences, and the energy in the bond linking the tRNA to the amino acid is used in translation to form peptide bonds between amino acids.
Codons
there are 64 different codon combinations, codons are a triplet of bases that codes for amino acids. The coding rehion always starts with a START codon (AUG), and the coding region of mRNA terminates with a stop codon (UAA, UAG or UGA), causing the release of the polypeptide
Initiation (translation)
the 5' end of the mRNA binds to the small subunit of the ribosome. The small subunit of the ribosome moves along the mRNA molecule in a 5' to 3' direction until it reaches the start codon. A molecule of tRNA complementary to the start codon (UAC) binds to the p site, and the large subunit of the ribosome binds to it as well as the small subunit
Elongation (translation)
a second tRNA (with amino acid) complementary to the second codon on the mRNA binds to the A site of the ribosome. The amino acid carried by the first tRNA is transferred to the second, causing the polypeptide to increase in length and the dipeptide being attached to the second tRNA. The ribosome then moves one codon along the mRNA and the first tRNA is moved to the E site, the second moving to the P site. Another tRNA binds that is complementary to the next codon and the process keeps repeating.
Termination (translation)
when a stop codon is reached at the A site translation is stopped. A protein known as the release factor then fills the A site. It helps to break the bond linking the tRNA in the P site with the polypeptide chain. This causes the ribosome subunits to dissassemble, releasing the mRNA and the protein. The new protein is then folded and modified to the targeted areas of the cell where it is required
Modification of polypeptides into their functional state
after translation, polypeptides are modified so they are fully functional. This can happen through changes to the side chain of amino acids, folding of the polypeptides, removing part of the polypeptide chain, combining polypeptides (quarternary structure), or forming conjugated proteins by adding a non-polypeptide component
Formation of insulin
the precursor to insulin is known as pre-proinsulin which is produced in the beta cells of the pancreas. A signal peptide (short attached peptide chain destined for secretion) is removed as pre-proinsulin enters the endoplasmic reticulum to produce proinsulin. This is then exposed to enzymes that break peptide bonds, removing a section of peptides (C peptide), producing mature insulin. Then it is packaged by the golgi apparatus and secreted from pancreatic cells via exocytosis
molecular chaperones
play a role in protein folding, but dont actually fold proteins. They provide protection from interfering conditions.
Glycosylation
the addition of carbohydrate side chain to a polypeptide to prevent protein chains from sticking or clumping together.
Silent mutation
no effect on the amino acid produced
missense mutation
one different amino acid is produced (e.g sickle cell)
nonsense mutation
caused a stop codon to be produced, so the polypeptide produced is shortened
sickle cell disease
a single base substitution that leads to the production of valine instead of glutamine. Leads to the production of abnormal red blood cells. One copy of the gene produce some abnormal Hb but are fine, but two faulty copies means all abnormal Hb and is dangerous
Sickle cell anemia
a disorder that makes all red blood cells a sickle shape, reducing their ability to carry oxygen since it affects the hemoglobin.
Malaria and sickle cells
Mosquitoes have a plasmodium cell that invades red blood cells and causes them to burst. However sickle cells cannot be affected and those with it are resistant to malaria, meaning in areas where malaria is more common, so is sickle cell
proteome
the entire set of proteins that can be expressed by a cell, tissue or organism.
Proteases
can break down proteins in the lysosomes if they are damaged, miss-folded or no longer functional and need to be recycled. These proteins are tagged by smaller proteins called ubiquitin that act as signals. They are then recognized by regulatory subunits at either end of the proteasome and unfolded and fed into the central chamber by a subunit that uses ATP. Proteins are then broken down into short chains of amino acids which are then broken down into amino acids by the cytoplasm.
What is gene expression?
the process by which information from a gene is used in the synthesis of a functional gene products that enables it to produce end products, proteins or non-coding RNA, and affect a phenotype
Epigenesis
the patterns of differentiation in the cells of a multicellular organism
Epigenetics
the study of heritable changes in the gene activity not caused by changes in the DNA base sequences
Epigenetic tags
molecules that either bind ot DNA or histones that regulate gene expression, they are chemical modifications of chromatin
Geneome
the whole of genetic information of the cell, including the coding and non-coding regions
epigenome
the sum of all epigenetic tags
Methylation
the addition of methyl groups to the DNA (specifically cytosine), it helps to inhibit transcription, and bind the DNA more tightly to the histone to make it less accessible to transcription factors (in the promoter region)
Acetylation
the addition of acetyl groups to histones to promote transcription. It does this by binding the DNA more loosely to make it more accessible to transcription factors
Methylation of histones
Helps promote and inhibit transcription. Whether or not a gene is expressed depends on where it is methylated. Methylation of lysine 4 (near the tip) causes transcription, and methylation of lysine 9 or 27 (along the tail) causes the gene to be silenced
Epigenetics inheritance through changes to gene expression
E[igenetics allows a genome to be applied different ways in different situations. Methylation patterns can be influenced by experiences (can increase likelihood of certain diseases and behaviours), these can be reversible, but some tags are passed down to the next generation
Factors contributing to methylation patterns
Human hair and skin colour can be affected by temperature and sunlight exposure, pollution can affect the expression of proteins that regulate the immune system, air pollution is most damaging during pregnancy. As well as the environment of a cell
Morphogens
They help regulate the expression of genes that determine body patterns, they diffuse across the surface of cells from a concentrated source, meaning different embryonic cells get different concentrations of morphogenes, causing transcription activation and inhibition to be different in different cells and genes
Imprinted genes
Genes that bypass the empigenetic reprogramming process, methylated differently in male copies of the gene vs female copies (why you can't fuse two egg or sperm). They are silenced in only one of the two copies of the gene (maternal or paternal), and can have higher mutation rates as well as higher rates of evolution
Imprinting in lions and tigers
Male lions have sperm containing imprinted genes that promote growth as they want to have the largest offspring in a litter. However female lions have imprinted genes in their eggs that restrict growth to prioritize the survival of all cubs. This cancels out and creates a normal sized lion. However, tigers only produce cubs from a single male, meaning they don't develop imprinted genes
Ligers
the lion sperm is combined with the tiger egg, resulting in large offspring
tigons
the tiger sperm was combined with the lion egg, resulting in normal sized offspring
Methylation in monozygotic twins
DNA methylation patterns will differ between twins and continue to diverge over time as a consequence of environmental exposure. DNA methylation patterns in twins can be used to identify genes involved in the development of specific diseases that are present in a single twin. Variation increases in the levels of hypermethylation (high levels) and hypomethylation (low levels) with age.
regulatory proteins
They bind to DNA sequences outside of the promoter and interact with transcription factors. Repressor proteins bind to the silencer sequences and decrease the rate of transcription. Activator proteins bind to enhancer sites and increase the rate of transcription
Control elements
the DNA sequences that regulatory proteins bind to. Most genes have multiple control elements. Transcription factors typically bind to proximal control elements, while regulatory proteins typically bind to distal control elements.
Steroid hormones (transcription inducing ligand)
They are lipophilic and can freely cross the plasma membrane to induce changes in gene expression. They bind to receptors in either the cytoplasm or nucleus, forming an active receptor-hormone complex that moves into the nucleus, binding to the DNA, acting as a transcription factor
Protein hormones (trannscription inducing ligands)
Since they are lipophobic they cannot directly induce changes in gene expression by crossing the plasma membrane. Instead, they bind to transmembrane receptors on the plasma membrane, which activate intracellular molecules called second messengers. These then initiate changes to the patterns of gene expression
Post transcriptional regulation
Regulating the amount of mRNA that persists after transcription influences the amount of protein produced. To prevent a protein being made, mRNA can be degraded before it is translated. Enzymes called exonucleases remove nucleotides on at a time to degrade the mRNA with the help of the decapping complex that removes the protective 5' cap, and the deadenylase complex that removes the poly A tail. Hormones like estrgen can also affect the stability and rate of transcription in the mRNA, influencing its degradation rates