Current Lecture Notes Up to Fri. April 9

Exam 3 New Material
Questions about DNA replication on this test will be detailed, not just a “put the steps in order” question
Steps of DNA replication
1) Origin of replication – spot on DNA where replication starts. Bacteria (single, small circular chromosome) only have 1 origin of replication. Humans have bunches of origins of replication per chromosome (depends on chromosome size). Expect to find lots of A and Ts at origin of replication (weaker hydrogen bonds than C & G). (abbreviation for origin of replication: ori)
2) DNA helicase – unwinds/uncoils supercoiled DNA just locally (at origin of replication – helicase attaches to origin of replication) – not enough space in cell for all DNA to be unwound at the same time. One little part of DNA has to be pulled out/unwound from rest of coil. Moves ahead of DNA polymerase with respect to leading strand. DNA helicase uncoils and unzips double stranded DNA.
3) Where DNA unwound, double strands separate (“unzip”) (hydrogen bonds between complementary base-pairs are broken). Strands want to join together again, but we can’t have that. So, single-stranded DNA covered by single-strand binding protein (SSB) – prevents strands from reattaching (re-annealing)
4) RNA primase – enzyme that makes (synthesizes) a small chunk of RNA that’s complementary to small chunk of unzipped DNA (ssDNA – single stranded DNA). This small chunk of RNA is called RNA primer. DNA polymerase is enzyme that synthesizes DNA (make new DNA strand). DNA polymerase can’t attach to single stranded DNA. It can only attach to ssDNA that has a RNA primer attached to it. DNA polymerase is the “hand” in the book diagram.
DNA polymerase synthesizes new strand of DNA complementary to original strand. Accomplishes this by bringing in nucleotides (really nucleotide triphosphates – ATP, CTP, GTP, TTP) which are complementary to existing strand. Can attach 3 phosphates to A, C, G, and T = all act as energy currency. nTPs – nucleotide triphosphates – have phosphates broken off, providing energy to make new phosphate backbone (broken off phosphates provide energy). A, C, G, and T which were attached to phosphates become the bases – are complementary to existing strand attached to backbone. Don’t need energy to form hydrogen bonds between bases. Use energy from phosphates to create sugar phosphate backbone – these atoms are covalently bonded. DNA polymerase can only go in 5 prime to 3 prime direction along a DNA strand (creates leading and lagging strand). Leading strand can continue to be unwound, unzipped, and then replicated while other strand has to stop and go because DNA helicase is NOT moving ahead of it. (The lagging strand has to wait for DNA to be unwound.) Second stand (lagging strand) has Okazaki fragments due to stop and go. Leading strand – polymerase can attach to RNA primer and “just keep trucking” until the whole strand has been synthesized.
5) Okazaki fragments must then be patched together with DNA ligase. Leading strand has no fragments. DNA ligase removes RNA primers on lagging strand and patches Okazaki fragments together.

Telomere – region of repetitive sequences on each end of a linear chromosome. Don’t code for proteins – can afford to lose them. Help chromosomes from breaking apart. Telomeres are needed because wherever RNA primer was on end of linear chromosome, DNA can’t be replicated (EX RNA Primer was at the end of the chromosome. A new DNA strand was created after DNA polymerase attached to the primer. Can’t form another primer “upstream” from the primer at the end, meaning the DNA here can’t be replicated. So, this region is called a telomere). Chromosome gets shorter with each round of replication because DNA can’t be replicated where RNA primers are located – new primer has to be formed with each round of replication. Enzymes can’t copy end of chromosomes because of RNA primers. After too many rounds of replication, the DNA strand gets too short, and the cell commits apoptosis (kills itself off) – if DNA gets too short, it can’t code for proteins properly anymore. DNA polymerase can only move in 5 prime to 3 prime direction
Why haven’t things with linear chromosomes gone extinct?
Sex cells have the ability to regenerate telomeres. No somatic cells have this ability. Telomerase (enzyme) in gametes regenerates telomeres.
Why can bacteria divide indefinitely?
Bacteria have circular chromosomes. DNA polymerase can go all around chromosome in 5 prime to 3 prime direction without stopping. There is no loss of DNA during bacteria cell replication.
Repair to errors in DNA replication
3 ways to correct errors of DNA replication: DNA proofreading, mismatch repair, excision repair
Because DNA polymerase is copying DNA strand really fast, sometimes wrong base paired between new and original strand
Mismatch repair system/Excision repair:
Enzymes scan length of DNA, identifying mismatched swatches (look for missing hydrogen bonds between bases – kink in double stranded DNA)
On new DNA strand, enzymes snip out (break sugar-phosphate backbone) swatch of DNA which contains mismatched base (bases surrounding mismatched base are snipped out). New swatch is synthesized. Then, DNA ligase comes in to make the strand whole (like with Ozaki fragments)
How does the enzyme know which strand is the original strand and which is the old strand? methylation
Methylated DNA is older
Methylation: methyl (carbon with 3 hydrogen atoms) groups are added to some bases of the DNA. Happens after every other step of DNA replication. Because of methylation, enzyme can tell which strand is the original. Methylated strand is original strand.

Proofreading:
DNA polymerases usually have “proofreading” abilities. While copying strand, might synthesize wrong base. DNA polymerase can “tell” if they’ve placed an incorrect base on the new strand – hydrogen bonds don’t form (forming a “kink”). If an incorrect base has been placed, DNA polymerase stops and backs up 1 base, “snips out” wrong base, and then continues on by replacing wrong base with correct base.
Because of proofreading ability, spontaneous mutation rate is only about 1 mutant base pair per 10 billion base pairs copied (really low)
DNA polymerases without proofreading ability have a spontaneous mutation rate of about 1 X (per?) 10^5 bases copied
DNA polymerase – enzyme (ase). Makes new DNA strand. Polymer = many parts / molecules connected together
Size of genomes (genome - all of the DNA within one cell)
Prokaryotes – range in size of 500 thousand base pairs (bp) to 8 million base pairs
Ex E. coli has 5 million base pairs (bp) in genome
Eukaryotes
Ex Humans 80 million bp
How many spontaneous mutations would occur after 1 human cell divides one time? 80 million/10 billion = .008 (Where did the 10 come from?)
How many rounds of replication would have to occur for 1 mutation to occur? 125 ( Total copied/Mutation rate) (If DNA polymerase didn’t have proofreading ability, mutations would occur more often and in greater numbers)
PCR (polymerase chain reaction)
Technique we’re going to use in lab
Relies on the fact that DNA polymerase will copy strand of DNA given other correct circumstances (DNA polymerase needs a RNA primer or enzyme that makes primer, strand to be replicated (template DNA), and a pool of nucleotide triphosphates)
PCR: way of copying DNA without a cell
What PCR needs to work: DNA polymerase, RNA primer, template DNA, & pool of nucleotide triphosphates
DNA strands will unzip themselves without breaking sugar phosphate backbone if they’re heated to 90 degrees C. Sugar phosphate backbone connected via covalent bonds while bases connected via hydrogen bonds. Covalent bond stronger than hydrogen bonds.
EX: heat up double strand of DNA to 90 degrees C, strands unzip. Let strands cool to 50 degrees C. Perform these two tasks in the presence of DNA polymerase, ntps, primers, and a template DNA. DNA polymerase is a protein. (WAIT- HOW IS DNA POLYMERASE A PROTEIN? ISN'T IT AN ENZYME B/C IT ENDS IN -ASE?) Proteins don’t like being heated so high – they die. DNA isn’t killed by heat. Heating samples doesn’t break covalent bond, only hydrogen bonds. When strand splits into two, one strand will likely “wander off,” and the strands won’t get together. When DNA cools, RNA primer attaches followed by DNA polymerase which attaches to primer.
PCR works if DNA polymerase of organisms which live in hot springs is used – discovered by Kary Lolis (guy who is usually stoned)
OUR PCR:
Template DNA from S. aureus samples (started in lab on 3/11)
Primers specific for mec A and pvl genes (primers have bases that match genes)
Also, pool of ntps will be needed
DNA polymerase from bacteria that live in hot springs of Yellow Stone - bacteria: Thermus aquaticus (live at 90 degrees C)
If bacteria have genes we’re looking for, DNA will be copied since we’re only providing primers for those genes.
PCR is the “go-to” technique. On CSI, they perform PCR to create duplicates of single celled evidence.
3/12/10
Gene – sequence of nucleotides (DNA bases) which codes for a particular protein (polypeptide – some genes don’t code for polypeptides). Gene for tRNA, but tRNA is not coded for.
We’re mostly made out of protein, but DNA codes for protein through a process
Gene expression – how particular genes in a cell follow instructions to turn on/turn off the making of certain proteins at certain times. 1st step is transcription.
All cells in an organism have the same DNA in them (except with a mutation here and there)
BUT, cells don’t all make the same protein all the time
Proteins made by skin cells different than proteins made by liver cells
During summer, skin makes melanin – trigger is large amount of sunlight

DNA to RNA to protein
DNA codes for RNA, then RNA codes for protein
Transcription: when DNA codes for RNA
Purpose: make single stranded RNA copy of DNA gene
Only 1 strand of double stranded DNA gene is copied into RNA
Have to unwind & unzip DNA strand locally – where DNA is going to be copied into RNA
Complimentary RNA strand is made like complementary DNA strand during DNA replication
Translation: when mRNA codes for protein
DNA vs RNA – KNOW FOR TEST!!
DNA: deoxyribose-phosphate backbone, double stranded, uses thymine (shares double hydrogen bond with adenine), very stable (not affected by heat or extremely cold temperatures)
RNA: ribose-phosphate backbone, either single stranded (mRNA) or forming complex structure (clover leaf structure), doesn’t use thymine – uses uracil instead (uracil shares a double hydrogen bond with adenine), single stranded; RNA very fragile, RNA in general acts likes an enzyme even though it’s not a protein
Different kinds of RNA
(mRNA (messenger RNA – single stranded RNA that is transcribed into protein; its sequence of bases determines sequence of amino acids in protein it codes for. Proteins are coded for in ribosomes. Ribosomes are NOT made out of protein; they’re mostly made out of ribosomal RNA (rRNA). rRNA acts as an enzyme to link amino acids together even though it’s not a protein. tRNA (transfer RNA) – works with rRNA to make amino acid chain based on mRNA template. tRNA embeds rRNA into amino acid chain)
mRNA, rRNA, and tRNA are all originally transcribed from DNA genes
Central Dogma of biology: DNA to RNA to protein
Dogma – statement of belief that can’t be “shaken/broken/ refuted”
Refutations of central dogma
RNA
RNA viruses (influenza, HIV) – RNA to DNA
Prion – protein that without using DNA or RNA can change the form of another protein (mad cow disease) (Do we need to know this?)
Things required for transcription
You’re going to make cold potato soup in the summer instead of chili. There are environmental signals (hot weather) to undergo transcription. In the cook book, there are bookmarks. Each bookmark is a transcription factor. Bookmark is telling you which recipe you want to make.
Every time cell divides, it gets a cookbook of all the recipes for every protein in the organism. As cells differentiate into tissues, only a few of the recipes are accessible (unlocked).
RNA polymerase – enzyme that makes polymers of RNA that are complimentary to single strand of DNA gene (transcribes - makes RNA copy of the DNA template). RNA polymerase does NOT require primer to start copying.
Unzips double stranded DNA as it moves down the template strand (only 1 of the 2 DNA strands)
Brings in complementary RNA bases for DNA template strand and attaches bases together with ribose (sugar) phosphate backbone. ATP is needed as an energy source (RNA uses uracil, not thymine)
Accessory proteins (transcription factors) allow RNA polymerase to attach to DNA. These activate gene expression by binding to particular sequences of DNA bases upstream of region that will be transcribed. RNA polymerase attaches to transcription factors which are bound to DNA – transcription begins. Transcription factor attaches to DNA. Definition: protein that binds to DNA at promoter sequence
Sigma factors (1 category of transcription factors) They are used by bacteria. Subunit of RNA polymerase. Particular protein. Bacteria make different sigma factors under different environmental conditions, allowing different genes to be expressed under various conditions.
Lab info:
Bacteria we’re working with have the ability to make the pvl protein, but the gene is only expressed when bacteria are in human body at temp of 37 degrees C (99 degrees F). Only at 37 degrees C will S. aureus make sigma factor specific to pvl promoter.
TBP (tata-box binding protein), others (eukaryotes). Many more transcription factors in eukaryotes than in prokaryotes. Humans are complicated – cells in liver need to make different proteins than proteins made by cells in skin. All genes in eukaryotes will use TBP (tata-box binding protein). Some other transcription factors then bind to TPB, allowing RNA polymerase to attach.
Promoter sequences – Specific sequence of bases on DNA that transcription factors bind to; where transcription starts. Definition: sequence of bases upstream of where transcription starts (has to be upstream
One example of transcription factor: TBP
TATA box attaches to TATAAT (promoter sequence)
Lots of A & Ts here (all promoter sequences? yes)
Technical name is TATA Box – where replication begins
TATA box located 10 bases from where actually transcription occurs
Another promoter region about 35 bases away from start of transcription which requires different, specific transcription factors

Steps of transcription: initiation, elongation, termination
Initiation
Transcription factors (proteins) bind to promoter region of gene to be expressed. At minimum, have TBP bind to TATAAT (promoter region).
RNA polymerase binds to TBP (transcription factors.) Once RNA polymerase binds to TBP, RNA polymerase can unwind and unzip DNA. RNA polymerase does this in order to create a DNA template in which to create a complementary RNA strand.
Elongation
RNA polymerase moves in 3 prime to 5 direction down DNA strand – that’s how it’s decided which strand is synthesized. New strand synthesized is 5 prime, 3 prime RNA.
RNA polymerase pairs RNA bases with DNA template strand. Bases are connected by a ribose- phosphate backbone. ATP is used as the energy to create the backbone.
Termination
In prokaryotes
“hairpin loops” – DNA sequence that will base-pair with itself (EX: AAACGTTTT – As can pair with Ts & C can pair with G)
RNA polymerase can’t go past double stranded “hair pin.” So, it detaches. RNA polymerase takes mRNA with it
Used by both eukaryotes and prokaryotes (really only way prokaryotes can terminate transcription)
Eukaryotes have many ways to end transcription besides “hairpin loops”
Termination proteins: like transcription factors in that they bind to specific DNA sequences. BUT, termination proteins are different than transcription factors in that they STOP RNA polymerase from continuing with transcription
Indicate end of gene
These proteins detach RNA polymerase from DNA template strand
mRNA (messenger RNA) is the end result of transcription
Use mRNA in translation (2nd step of gene expression) to make protein (amino acid sequence)

mRNA processing
In prokaryotes: mRNA is transcribed into cytoplasm (no nucleus in prokaryotes); do NOT have to do any processing to mRNA before it’s translated into protein in the ribosomes (located in the cytoplasm)
Proteins are made in the ribosomes (ribosomes are made of rRNA, not protein)
In eukaryotes: ribosomes are in cytoplasm – proteins are translated from mRNA in ribosomes. mRNA is located in the nucleus (where DNA is located). Lots more processing needed in eukaryotes to get mMRA out into cytoplasm to ribosomes . mRNA shipped out of nucleus via nuclear pores
GTP added to 5 prime end of mRNA (guanine cap added to5 prime end)
GTP only applies to mRNA, not DNA
Long strand of adenines added to 3 prime end of mRNA
GTP and adenines tell/signal proteins in nucleus to ship mRNA out of nucleus to cytoplasm
This needs to happen with ALL genes in eukaryotes
Single stranded RNA divided into introns and exons (intron between 2 exons)
Intron – only occur in eukaryotes – part of mRNA that was transcribed, but isn’t going to be translated into protein (amino acid sequence). Has to be cut out of single stranded RNA before protein can be coded for. Chopped out when mRNA is still in nucleus. DO NOT NEED TO KNOW PROCESS OF CHOPPING OUT INTRON.
Exon: part of single stranded RNA which codes for proteins
Transcription regulation: Why aren’t all genes expressed the same by all cells with same DNA?
Different cells make different transcription factors (takes time)
As cells differentiate, some genes, “recipes,” get locked away.
This is done by adding methylation or changing interaction of DNA of gene with histone it’s wrapped around (particular genes are stopped from being unwound from histones)
All of our cells have the same DNA in them. Each cell is given a full cookbook when it’s first created. As a cell specializes, certain “recipes” get locked away. Ex: lung cells only have access to recipes for lung cells; they don’t have recipes for brain, liver, etc cells
EX Brain cells don’t grow back. This recipe is locked away. Biologists are trying to remove the lock.
Many kinds of RNAs are involved in translation
mRNA (messenger RNA): single strand of RNA that’s “created” from transcription; acts as template for amino acid sequence in translated protein
tRNA (transfer RNA): involved in gathering the appropriate bases; has the shape of a clover leaf/ lower case t. complementary base pairing on each arm/branch. At the very top, there is a region of bases which are not complementary base paired - called an anti-codon (interacts with codons on mRNA.) Amino acid attached to arm opposite anti-codon. Aminoacylated/ “charged” tRNA – tRNA attached to amino acids
rRNA (ribosome RNA): makes ribosomes (ribosomes links amino acids together to create proteins)
Initiation (translation)
Prokaryotes
Ribosome attaches to mRNA
This happens at sequence of bases on mRNA called Shine-Dalgarno sequence
Shine-Dalgarno sequence – sequence of bases on all prokaryotic mRNA which is complementary to part of RNA; this allows ribosome to attach to mRNA
After ribosome attaches to mRNA at Shine-Dalgarno sequence, ribosome moves down mRNA one base at a time until it recognizes a start codon
Open reading frame – Bacteria transcribe lots of genes at the same time into a single transcript. This mRNA is translated into separate proteins.
This long strand of different genes is called polycistronic mRNA
Eukaryotes transcribe and then translate ONE gene at a time
Eukaryotes
Ship mRNA out of nucleus via nuclear pores
5 prime end guanine cap
mRNA scanning
ribosome binds to methylated-guanine on 5 prime end of mRNA
ribosome moves down mRNA one base at a time until it reaches a start codon (start codon is the same for prokaryotes and eukaryotes)
Elongation (translation)
Definition: Ribosome moves down mRNA, bringing in matching tRNAs. Ribosomes link amino acids on tRNAs into a protein
Elongation factor (factor = protein that does the word in front of factor) – protein that enables building of amino acid chain (interact with ribosome to provide energy to build chain – ATP is energy source)
Ribosome large subunit sites
Aminoacyl (A) site – first place ribosome goes to. Where tRNA anticodon initially paired with mRNA codon
Peptido (P) site – where peptide bond (covalent bond) (essentially a link) is formed between two amino acids which are attached to tRNAs
Exit (E) site – where tRNA detached from amino acid
Termination (translation)
Stop codons: UAA, UAG, UGA
No matching tRNA anticodon (stop codon can’t pair with tRNA anticodon)
Release factors – protein which releases ribosome from mRNA and amino acid chain, and tRNA. Attaches to stop codons.
In prokaryotes: RFI and RF2
In eukaryotes, there’s just one release factor

March 29, 2010
Change in DNA leads to chain in protein DNA codes for
Mutation – any change to DNA sequence of an organism
Most mutations are neutral (have no effect on phenotype)
Eukaryotes have a LOT of noncoding DNA
Mutations can happen to noncoding and / or coding DNA
Many of mutations to coding DNA are neutral
More than one way to code for any given amino acid (more than 1 codon/amino acid)
64 possible codons, 20 amino acids
Kinds of mutations: cell type affected
Somatic cell mutations
Somatic – body cell /not a gamete
Mutation to somatic cell can’t be passed down to the next generation of organisms, but the mutation can be passed down onto next generation of cells within the original organism
Germ line mutations
Germ line - Sperm or egg
Only kind of mutations that can be passed down onto the next generation of organisms
Sperm are more likely to mutate than eggs
Female mammals: Eggs are made before birth (divide slowly) – females born with 300 eggs. No more eggs are created after birth.
Exposed to something that causes mutations? Don’t have to worry – eggs already made
Male mammals: about 1 billion sperm created every 24-48 hrs
Exposed to something that causes mutations? Do have to worry – sperm are continuously being made
Kinds of mutations: causes of mutations
Spontaneous mutations: occur when DNA polymerase does NOT make perfect copies
Induced mutations: caused by mutagen agent (gen – to start) – something that causes mutations by causing DNA polymerase to screw up
Ex: tanning In a DNA strand, two thymines bind together via a covalent bond as a result of short UV rays of tanning bed. When DNA polymerase copies DNA strand, it doesn’t know what to do at covalently bonded TTs. So, it just throws in a random base at TTs.
Cancers start as mutation in cell – not all of these mutations lead to cancer
Carcinogen – cancer causing
If something is a carcinogen, it’s a mutagen
Kinds of mutations: Change to a single nucleotide
Point mutation - change to just one nucleotide (base) of an organism’s DNA
EX sickle cell anemia
Missense mutation –point mutation; causes change in amino acid sequence of protein coded for
Frame-shift mutation – addition/deletion of 1-2 bases (NOT THREE (3)) in reading frame of a gene. Results in different codons.
Nonsense mutation – point mutation; results in early stop codon, so protein is a lot shorter (has fewer amino acids) than expected/normal
Silent mutation - point mutation; has no impact on (NO change to) amino acid sequence of protein. Changes DNA sequence, but not amino acid sequence. More than one codon codes for a specific amino acid.
April 6, 2010
Kinds of mutations: chromosomal rearrangements (chunks of DNA within chromosomes)
Common among eukaryotes
Tend to occur during crossing over during meiosis
Deletions and duplications occur at the same time
Deletions
Part of an entire chromosome disappears – not just one or two bases, but a chunk of chromosome. One gamete has chromosome with missing parts
Duplications
Other gamete which results from crossing over in meiosis has extra chromosome part (gets the part that was chopped off from first gamete)
Inversions
Part of a chromosome reverses direction relative to the rest of chromosome. All the genes are present, but they’re just in a different order (originally ABC, now CBA). Doesn’t affect how the chromosome works
Translocations
Part of one chromosome is moved to be part of another new chromosome (EX part of chromosome 21 becomes part of chromosome 23 – new chromosome 23 doesn’t pair up correctly in fertilization)
Genes don’t know what number they are, so duplications and translocations don’t affect the phenotype – no part of the gene is missing
Deletions have the worse immediate negative effect on the phenotype of an organism
Duplications are driving force in evolutionary change
Duplications have the greatest effect on the phenotype of an organism. Duplications make cells with extra copies of genetic info, can allow for evolution of new genetic traits.

Protein folding
Protein – word ends with the letters “-in”
EX Cdk = cyclin-dependent kinase.
Allows cell cycle to start by disabling RB, by adding phosphate to RB
Cdk can do this because it has right shape to add phosphate to RB
All proteins have functions they do because of their shape (WILL BE ON TEST)
If the amino acid sequence changes, the shape of the protein changes.
If the shape of the protein is changed, the function of the protein is changed.
The shape of a protein is determined foremost by the sequence of amino acids that make it up
Shape also affected by pH, temperature, and osmolarity (water concentration of solution) of environment
Osmalarity – chemistry of amino acids affected by amount of water
EX: Heat hamburger to kill E. coli. The heat changes the shape of the meat’s protein. Changing the proteins’ shape kills them.
EX: Soaking raw fish in acidic solution changes proteins’ shape which kills the bacteria on the outside of the fish.
Chaperone proteins influence protein folding
Defin: proteins that assist other proteins in folding in the right shape/shape they’re meant to be
Act as enzymes
How they work: hold new amino acid sequence in certain shape until “natural” events occur such as the formation of a bond between certain amino acids
Lose ability to make chaperone proteins as we get older – why we age. Need red wine and dark chocolate to make chaperone proteins
EX: Prions
Cause mad cow disease; cause brain proteins to misfold – lose brain function; can’t cook them out of meat cause proteins are already “misfolded”

Protein structures
Primary structure of any protein is its amino acid sequence
If on test asked for primary structure of protein, figure out the amino acid sequence
Secondary
Alpha-helices – spiral structures (look like telephone cords)
Beta-sheets – flat structures
Amino acid sequences spontaneously fold into one of these structures during certain environment conditions
Tertiary
Form as a result of the interactions between alpha-helices and beta-sheets
A single protein has both alpha-helices and beta-sheets
Quarternary
Interactions between 2 or more proteins which form 1 large protein
EX TATA Box interaction with transcription factors (proteins)
Interactions that influence folding
Hydrogen bonds & Covalent bonds - amino acids could form either bond – explain why beta sheets and alpha-helices form
Hydrophobic interactions – (hydrophobic = water hating) - 1 of the most important interactions between amino acids; hydrophobic amino acids fold in towards the middle/inside
EX oil

WOW!!!!!!! That's pretty much amazing. lol. Thank you for taking your time to post this; it's very helpful!

I read through these while giving plasma (don't tell Robson), and thanks for posting them!

jeaze………… thanks a lot this is going to be handy

um….wow

Nice!! thanks a lot! this will help a ton for the test!

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