Molecules and Cells Exam 3

in prokaryotes, gene regulation is simpler than in eukaryotes because

1. DNA is not packaged into chromosomes 2. mRNA is not processed 3. there is not a nuclear membrane separating the processes of transcription and translation

gel electrophoresis

1. DNA samples are inserted into wells at one edge of the gel 2. DNA fragments move towards the positive pole according to their size. Smaller fragments move faster and larger ones move more slowly. 3. A band represents DNA of a particular size 4 the bands in the gel become visible when viewed in fluorescent light because the bands have been dyed.

Retrotransposons

4 main families - 300-6000bp in length - flanked by long term repeats - only about 40 that are actually active - human endogenous retroviruses

chromatin is

50% wt DNA and 50% wt protein - condenses 2 m off linear DNA into a 1 um nucleus - impedes transcription

a single human cell never has

92 chromosomes, always has 46 but may have 92 copies of chromosomes in sister chromatids

plasmid origin of replication

allows plasmid to replicate independently form the bacterial chromosome

plasmid origin of replication

allows plasmid to replicate independently form the bacterial chromosome, when each bacteria divides each progeny divides and the progeny also has the plasmid

if polypoid organism contains two or more alleles, the organism is heterozygous for that

gene

somatic mutation vs germ line

germ line mutations are passed on to offspring and effects all cells in the organism - somatic cells - circumscribed area is affected

meiosis occurs in

germ/sex cells

in meiosis the amount of hereditary material is reduced by

half

gametes (products of meiosis) are

haploid

in meiosis I, cells go from diploid to

haploid

meiosis occurs in germ cells and produces

haploid gametes with half the genetic information of that organism (1n)

meiosis Ii is just like mitosis of a

haploid organism

homology means

sequence similarity

genome sequencing refers to

sequencing the entire genome of an organism

the purpose of meiosis in mammals is

sexual reproduction

there are general transcription factors in eukaryotes and

sigma in prokaryotes

replication error management

simply by the DNA polymerase in real time - DNA polymerases can proofread their work - mismatch repair enzymes can fix any error that were missed by the DNA polymerases - if proofreading doesn't catch an error, then the mismatch repair enzymes can come in and repair the damage - DNA polymerase can sense when it pairs something wrong like A-G and cut out the phosphodiester bond and bring in the new one - DNA polymerase = 1st line of defense - wrong base pair that DNA polymerase does not catch you either get a bulge or an indentation in newly synthesized strand because A-T have a specific binding, etc.

replication is the synthesis of a virtually identical

sister chromatid during S phase phase

Mendel's observations contradicted the blending-inheritance hypothesis and revealed to him

(1) the mathematical rules of heredity and (2) the phenomenon of dominant and recessive traits = Mendel's Principle of Segregation

qRT-PCR

(Quantitative real-time PCR) uses a fluorescent dye to quantify transcript abundance - using reverse transcriptas, done in the nasal swab

pedigrees document the inheritance of traits in families

- In pedigree analysis, squares are males and circles are females. Lines between people mean that they mated. - A filled in circle or square means that the person is affected with a particular phenotype, like a disease. - Historically, comparing inheritance patterns of a disease trait with other known traits/genotypes on chromosomes narrowed down the locations of disease genes. - Patterns of inheritance also reveal if the trait is conferred by a dominant, recessive, or X-linked allele.

cystic fibrosis gene deletion

- In the most common mutant proteins, three nucleotides are deleted in the CFTR gene, resulting in a missing amino acid at position 508. - the CFTR transporter pumps chloride ions out of the cell - the mutation CFTR protein is unstable and is degraded before reaching the membrane

chromosomal inversions

typically form when the region between two breaks in a chromosome is flipped before the breaks are repaired

How the coronavirus testing works

Real-time reverse transcription polymerase chain reaction (RT - PCR) tests are used to detect genetic material. These tests can be used to screen samples and to detect infection 1. samples are collected from the nose or throat of a patient using a swab. The sample is sent to the lab. 2. The sample is mixed with chemical reagents and put in a machine that duplicates the genetic material 3. If the virus exists, the copies made by this machine will confirm its presence - going to extract RNA genomes because COVID-19 has an RNA genome - turn RNA into cDNA via reverse transcriptase - conduct PCR: primers against different parts of the viral chromosome to see if they get a product - real time --> see when you start to get amplification of a product i. certain threshold for positive or negative for the virus ii. instead of a gel of these PCR iii. if you haven't started amplifying yet, then you are negative --> when in the PCR reaction, you get enough product to be read

Human Retroviruses

The existence of human endogenous retroviruses (HERVs) has been known for many years [4], but their abundance in the genome was not predicted by earlier studies. HERVs represent the remnants of ancestral retroviral infections that became fixed in the germline DNA.

in interphase (=non-dividing) eukaryotic cells,

chromatin can be packaged differentially

nondisjunction is when one of your homologous chromosome pair goes to one pole of the cell and

both of your parental homologs goes to one of the two daughter cells

LacI will not repress when

bounds to lactose

low glucose produces high

cAMP

CRP is a sensor for low glucose or high cAMP

cAMP is increased in a low glucose environment

CRP is a

cAMP receptor protein

the lac operon system in the presence of lactose is

called derepressed

microRNAs

can bind to complementary sequences on mRNA molecules either degrading the target mRNA or blocking its translation

the sister chromatids stay tethered to one another until the end of

cell division

the structure of chromatin varies along a single interphase chromosome

centromeres and telomeres do not contains genes and are constitutively heterochromatinzed - centromeres are used to divide duplicated chromosomes between cells during division

a lot of complex traits contribute to

human disorders such as heart disease

incomplete dominance looks like the blending

hypothesis of inheritance

lactoses null mutants

if we had a random mutation that would just knock out the lacI on the DNA, the mRNA, and thus not be able to produce lacI co uld be a nonsense, frameshift, or missense mutation. Thus, the lacZ and lacY proteins would be expressed in the presence or the absence of lactose. This is known as constitutive expression meaning it is always on.

lactose lac operon complementing or adding back mutation

in cells containing one mutant and one normal copy of lacI, lac Z and lacY are expressed only in the presence of lactose bwecause the functioning/normal lacI protein can function as a repressor protein for the nonmutated DNA strand and act as a repressor the mutated DNAn strand. Thus, it would prevent transcription without lactose, but in presence of lactose lac Z and lacY genes - you could use a plasmid to demonstrate this by adding back a normal operon

only germ cells do meiosis

in mammals

when embarking on a new area of scientific inquiry,

it usually works best to choose the simplest system possible to study

lacI acts as a

lactose sensor

mitosis, meiosis I, and meiosis II

look at slide 12 on meiosis power point

high glucose produces

low cAMP

Annealing (PCR STEP 2) - 50 degrees celsius

lower the temperature add DNA primers that bind wherever they are complementary to on the template DNA strand via hydrogen bonding - DNA primers bind to opposite ends of each at the 3' end of the template strand - allows us to guide DNA polymerase to specific areas

meiotic chromosome segregation errors are called

nondisjunction and can occur in either the first or second meiotic division

when there is high glucose or low cAMP

not enough CAMP binds to CRP and thus, CRP is unable to bind to the activator, and transcription does not occur

mistakes/mutations in replication would lead to alterations in the

nucleotide sequence in the DNA, and these alterations would be passed on to daughter cells when the cell divides

Monot:

there is repression of tryptophan synthase by the presence of tryptophan - if you grow ecoli in the presence of tryptophan, you can't get tryptophan synthase protein out of the cell - when tryptophan is present there is a repression of tryptophan synthase

grasshoppers are diploid meaning

they contain two homologous chromosomes

Mendel used inbred plants so

they would only produce the same traits and phenotypic appearance = all the plants were homozygous for all of their genes, two alleles for any given gene are the same

in order for the transcriptional machinery to access the DNA, chromatin must unravel to allow space for transcriptional enzymes and proteins to work

this is accomplished by chromatin remodeling in which chromatin unravels and nucleosomes change position to expose new regions of DNA

the key to allowing independent assortment

this is because what we learned in metaphase about the random alignment in the center of the cell, it does not matter what is on top and what is on bottom when they are separated - the parent gametes have ry and RY gametes which produce offspring as Rr and Yy - these F1 offspring are all RrYy and thus are made from the parental gametes of of RY and ry, thus the chromosomes within the RrYy offspring are r and y/ry and R and Y/RY which can then mix through crossing over (same chromosome) and random assortment at the metaphase plate in meiosis 1 and going to different poles contribute to the random assortment

DNA undergoes methylation on cytosine residues within CpG (CG DNA ) sequences

this is the most common chemical modification to DNA

polygenic traits

those determined by two or more genes ex. hair color, skin color, eye color, height

heritable means that the mutation is stable and therefore passed on

through cell division

sanger sequencing: because there are many more normal nucleotideds than dideoxynucleotides, each strand can terminate at a different place

thus, once the daughter strand sequence is known...the sequence of the template strand can be determined

the product of two division in meiosis is 4 daughter cells containing

one chromosome each (n). In animals, these cells become the gametes - haploid daughter cells

if polypoid gets (diploid) organism only contains

one version of the allele, that organism is homzygous

a heterozygote contains

two different alleles

pairing and crossover occur in

prophase I of meiosis (late)

all genes in DNA do not code for

proteins

not all genes make

proteins

pol

proteins required for reverse transcription and integration into host DNA such as protease, reverse, transcriptase, integrase

What do we do with that plasmid once we've engineered it?

put it in a living cell - transformation!

shot gun sequencing

putting together little fragments of nucleotide and gene sequences together --> takes a long time - sentence fragments (nucleotides and DNA) can be assembled in the correct order according to their overlaps and the original complete sentence reconstructed.

crossing over is totally

random

COVID-19 tests use

real time PCR and reverse transcriptase PCR

X-linked recessive traits are observed virtually exclusively in males and are often passed through unaffected females - autosomal dominant

recessive - you can skip generations x-linked = almost exclusively all males being affected if you have an x linked gene that is recessive, even if it is recessive it is expressed in males whereas women would have to have two recessive alleles to express it and usually they would have a dominant which would overpower

once you put something different on this plasmid than what you started with it has become

recombinant DNA

crossing over during prophase I allows genes that are on the same chromosome to

recombine and sort independently

reductive division

reducing ploidy from diploid to haploid - separation of homolgous chromosomes

this reduction in meiosis 1 is called

reductive division

tick marks on a plasmid show where the

restriction enzyme sites are and what size restriction fragment pieces you would get - if you were to run a gel of a digestion of this plasmid what would it look like

RT-PCR

reverse transcriptase PCR - a. first step that uses re verse transcriptase to make RNA into cDNA (the c is for complementary) - you can then use that cDNAn in a qPCR machine/reaction - or you can just do normal pCR in a tube and run the sample on a gel (no machine or dye necessary)

How do you make coronavirus RNA into DNA

reverse transcription - RNA is made into cDNA which is then amplified 1. reverse transcription 2. Real time PCR: Annealing, extension, denaturation 3. Repeat the process of PCR - SYBR green binds to the product and lights up after each cycle if the product is present

is it possible for two individuals to have the

same phenotype but different genotypes

bivalents undergo crossing over to exchange

segments of alleles

if you let a pea plant grow on its own, it would

self polinate - its own pollen would get stuck on the tip of the stigma and then ultimately fertilize the eggs = called selfing

the sequences of DNA fragments are analogous to

sentence fragments

during meiosis, perfect bipolar attachment and chromosome/chromatid

separation must occur during tow different metaphase-to- anaphase transitions

meiosis II

sister chromatids separate

the amount of protein encoding genes does not coincide with

size of genome

siRNAs

small interfering RNAs, turn off gene expression by directing degradation of selective mRNAs and the establishment of compact chromatin structures

small scale mutations

small nucleotide changes occuring

the Y chromosome has relatively few genes compare to autosomes

so most of the studied sex linked traits are X-linked

different genes can all respond to the same activator and repressors - operator sequences can be found on multiple locations on a chromosome so other genes, usually similar ones can be repressed in the exact same manner, by the exact same proteins

so usually similar genes can be activated and repressed in the exact same manner, by the exact same proteins

rate of mutation depends on cell type

somatic cells: cells of the body (product of mitosis) germ line cells: reproductive cells, products of meiosis - Mutations in somatic cells are passed only to the daughter cells in that area and are not transferred to future generations. (Cancer) - Mutations in germ cells are passed to new offspring. (cystic fibrosis) - In mammals, the rate of mutation per nucleotide per replication in somatic cells is much greater than in germ cells. - cancer is a somatic cell mutation - last bullet because you are dividing your somatic cells much faster than you are dividing your germ cells - you only have so many egg cells - egg cells are only dividing one time during meiosis = do not have as many germ line cells as you do somatic cells

DNA polymerase can come in and do

some fixing

heritable

stable mutation that maintains through mitosis

telophase of meiosis I

telophase - divide into two haploid daughter cells these are three decondensed chromatids from anaphase - no replication between M1 telophase and M2 prophase, just recondensing

meiosis only occurs in the germ cells located in the

testes/ovaries

autosomes are NOT sex chromosomes

the X and Y are sex chromosomes - 22 pairs of autosomes - one pair of sex chromosome

Diploid

the alignment of homologous chromosomes during metaphase I is random and crossing-over my (recombination) "mixes up" the combinations of alleles present on a single chromosome

Genome

the complete instructions for making an organism, consisting of all the genetic material in that organism's chromosomes - your entire sequence of genes and bases is called your genome - all of an organism's DNA - so much of our DNA is NOT encoding proteins

chromosome is chromatin in its condense form,

the form it is in during cell division

besides reducing ploidy (reductive division),

the most important thing that happens during meiosis is crossing over in late prophase I

during meiosis II

the orientation of chromatids relative to the spindle poles is random

What is a promoter?

the region of DNA upstream of the structural gene or genes in an operon that is bound by by the RNA polymerase to set up to begin transcription

negative regulator and silencer mean

the same thing

a cell with abnormal chromosome number is called

aneuploid

repressor

binds to the operator downstream of the promoter and stops transcription in its tracks

when tryptophan is present, the trp repressor binds to the operator, and RNA synthesis is

blocked

Repressor vs activator

both are proteins 1. bind to DNA 2. sometimes have another active site to bind to a stimulus

replication is the same thing as synthesis of sister chromatids in cells with linear chromosomes/

eukaryotic cells

a cell with one complete set of chromosomes is a haploid whereas a cell with two sets is a

diploid. Humans are diploids - homologous chromosomes are the two sets

Mendel's monohybrid cross

doing a cross when you are dealing with one gene or trait - first cross was round and wrinkled seeds - on the left is an image of the blending inheritance hypothesis (round plus wrinkled = slightly wrinkled) - then sort of wrinkled plus sort of wrinkled = sort of wrinkled F1 = first generation born from the original cross F2 = second generation born from the original cross p = parental generation the ones participating in the first cross VS. what Mendel actually observed P: round and wrinkled F1: round F2: 3 round and one wrinkled pee ratio from crossing F1 peas together from self-fertilization the peas were either one or the other, and not something in between

not all traits are determined by a single gene with simple

dominant/recessive alleles

in G1

each chromosome is a dsDNA - each G1 chromosome is just 1 long strand of double stranded DNA condensed, diploid

during sanger sequencing,

each of the possible dideoxynucleotides is labeled with a different fluorescent dye - you have no idea what nucleotides are being added except for the last dideoxynucleotide → question marks = don't know → notice dye different colors on videos → labeled nucleotide

after replication, after S

each sister chromatid contains one of the two strands from the original chromosome - replication is the process of sister chromatid synthesis - each sister chromatid contains one of the two strands from the original chromosome you give child, some of their grandfather's genes and some of their grandmother's genes from crossing over blue = DAD red = MOM

What is a transcriptional regulator?

either a positive regulator or negative regulator

encode just means

encode for

gag

encodes for components of capsid - matrix - capsid - nucleocapsid Vpr-binding protein

evn

encodes for surface glycoprotein and transmembrane glycoprotein

the effects of lactose and lacI (the repressor) are an example of

negative regulation

reciprocal translocation

reciprocal exchange of segments between two nonhomologous chromosomes

Transposons

(jumping genes) short strands of DNA capable of moving from one location to another within a cell's genetic material

Chromosomal level insertions/deletions

- A chromosome in which a region is present twice instead of once is said to contain a duplication. - A deletion is when a region of the chromosome is missing. - A deletion may result from a replication error or the joining of breaks that may have occurred on either side of the deleted region. - Because chromosomes occur in homologous pairs, a deletion in one chromosome can persist in a population. - However, in general the larger the deletion, the smaller the chance of survival.

pedigree of an autosomal dominant allele causing a disease trait

- Affected individuals are equally likely to be females or males. - Most matings that produce affected offspring have only one affected parent. - Among matings in which one parent is affected, half the offspring are affected. - in every generation approximately 50 percent of the children are affected if you get the trait, you are affected = dominant affected individuals are equally males and females - indication that it is autosomal dominant and not sex linked dominant

CpG methylation extra notes

- CpG sites are often clustered near the promoter of the gene. These clusters are referred to as CpG islands. - you have a cytosine and you add a little methyl group to it (CH3) - BEFORE TRANSLATION, a covalent modification cytosine methylation occurs in eukaryotes only in the sequence CG, where you have a CG you can add a methyl group, enzymes can add this methyl group to the cytosine when you have a group of methylated CPGs near each other → this causes a block in transcription - CPG methylation decreases/silences transcription near or in the promoter, you have a bunch of C and G grouped randomly → CPG islands - methylated CPG island will shut down the neighboring promoter

Gene Duplication and Evolutionary Divergence

- Duplication and Divergence: the formation of new genes from duplicates of old ones - The term divergence refers to the slow accumulation of differences between duplicate copies of a gene that occurs on an evolutionary time scale. - When a gene is duplicated, one of the copies can change without harm to the organism because the other copy still carries out normal function. - When a mutation in the extra copy of the gene is beneficial to the organism's survival, the extra copy can become a new gene. - you can have mutations in one of the two duplicated regions but in one of them you have normal genes - some advantageous mutations are created this way - less big of an impact since one can mutate and one can stay the same - also can badly affect your self

eukaryotic and prokaryotic transcriptional regulation summary

- Gene Expression Can Be Regulated at Many of the Steps in the Pathway from DNA to RNA to Protein - Different genes have different basal efficiencies (some genes are by default transcribed and/or translated at a higher level than others) - Transcription Is Controlled by Proteins (AKA transcription factors) Binding to Regulatory DNA Sequences - Transcription "Switches" Allow Cells to Respond to Changes in the Environment - Repressors Turn Genes Off, Activators Turn Them On A Cell Can Change the Expression of Its Genes in Response to External Signals Note: expression can change based on external signals such as extracellular signaling molecule, or nutrient

transcription reminder

- RNA polymerase is recruited to promoter by a general transcription factor and transcription gets started - prokaryotes - general transcription factor is sigma - eukaryotes - there are many general transcription factors - once transcription has gotten started, general transcription factors leave and RNA polymerase does its thing - happens until the terminator sequence is reached, and RNA polymerase and RNA fall apart from one another

reciprocal translocation

- Reciprocal translocations join segments from nonhomologous chromosomes. - In large genomes, the breaks are likely to occur in noncoding DNA, so the breaks themselves do not usually disrupt gene function. - What is this called when it occurs in homologous chromosomes? - could be seen in mitosis the centrosome just recognizes the centromere just recognizes the middle region and not the translocated regions allowing for mitosis to continue in late prophase i

pedigree of an autosomal recessive allele causing a disease trait

- The trait may skip one or more generations. - Females and males are equally likely to be affected. - Individuals may have unaffected parents. - Affected individuals often result from mating between relatives, typically first cousins. - this is a recessive autosomal - generations without the trait and then generations with it this is albinism particularly apparent in inbred populations 50/50 male or female indicates = autosomal recessive traits may skip one or more generations whereas dominant always shows up, does not skip generations

xinactivation

- X inactivation is the packaging of one X chromosome into heterochromatin in any organism with more than one X - compensate for the difference in X-linked gene dosage that would exist between males and females - if we did not have this, females would be expressing double the amount of X linked genes as males would - this would be lethal - gene expression is extremely fine tuned - has to be exactly the right amount, if you are off by a couple fold its is terrible - before the embryo is implanted into a uterus, a choice is made about which of the two X is going to be silenced and which is going to be active package the entire chromosome into heterochromatin → make inactive - at this time the embryo has a few hundred to a few thousand cells, keeps the same inactive and active X through each division - it is heritable through divisions but you are not inheriting, not inheriting the DNA but the chromosome state

when mutations go without repair

- a G is erroneously incorporated into the daughter strand opposite the T and is not corrected by the proofreading function - in the next round of replication, the G specifies a C in the opposite strand. The new C-G pair is replicated as faithfully as the original T-A pair and the mutation is now present in this cell lineage

Plasmids

- a plasmid is a small piece of replicating DNA - can be made in many copies within the bacterial cell - can be manipulated to hold different genetic components

alpha satellite DNA

- among the highly repetitive sequences of the human genome, which consists of tandem repeats of a 171-bp sequence repeated near the centromere an average of 18,000 times - this satellite DNA is essential for attachment of spindle fibers to the centromeres during cell division

Retrotransposons

- artifacts of retroviruses that have been inserted into our genomes over time - same thing as retroviruses - transposon = jumping gene - pieces of DNA that a gene can read and identify as having markings of a retrovirus gene - not really that big - computers look for the long terminal repeat - bracketing what would be an encoding region on either side - only one of these proteins is made or partially made at a very low level in one of our cells - at some point came from a virus that infected us - endogenous - bound inside of our genomes, all of us have these - retrovirus - reverse transcriptase = go backward in dogma, makes RNA into DNA, RNA gets read by reverse transcriptase to make DNA, a lot are inactive

main ideas of operons

- cells are highly efficient at making exactly the right proteins at exactly the right time - when your protein adjusts its shape --> even the lac repressor would have some type of conformation change that causes it to be unable to bind to the operator when bound to DNA

transcriptional control

- change rate of transcription - transcriptional regulation is probably the most common way of regulating genes, in part because it saves the cell the most energy - describes rate at which you transcribe a gene

XX females are mosaics for their X chromosome alleles

- clumps of cells inherited the off and on of the x chromosomes, mosaic in cats - lighter chromosome - one allele on an X chromosome - darker - comes from same gene but different allele on the other X chromosome

The gene product of the IA allele causes the surface glycoprotein to be modified by a red sugar, the gene product of the IB allele causes the surface glycoprotein to be modified by a blue sugar, and the gene product of the i allele does not cause either modification

- codominance only occurs in certain types of traits - i gene - catalyzes the addition of a terminal sugar to a carbohydrate chain that are attached to a cell surface protein - called a glycoprotein - present on your red blood cells A - you express red sugar B - you express blue sugar AB - you express both red and blue on your red blood cells lowercase i = recessive allele ii = type O - protein that catalyzes the sugar cannot do either if you are homozygous for that kind of blood type, you are type O type O is the universal blood type donor because A and B will both accept it because they both have the glucose chain Codominance of both A and B cow - codominance with their coats - if heterozygotes expresses spots of brown and white vs incomplete dominance would make a tan cow

translation control

- control how frequently an mRNA is translated into a protein - change mRNA lifespan/stability or change translation rate

How do we fix the mutations?

- during replication - post replication mismatch repair --> single mispaired base repaired by removing and replacing a DNA segment --> nucleotide excision repair: recognizes multiple mismatched or damaged bases in a region

a transcription factor is a DNA binding protein that influences the level of transcription of gene, either positively or negatively

- factors that activate transcription are called transcriptional activators - factors that inhibit transcription are called transcriptional regulators ex. CRP activator and lacI repressor

(applies to both eukaryotes and prokaryotes) genes can be expressed with different efficiences

- genes are differentially expressed based on how their sequences evolved over time - probably because cell needs more protein A then it does protein B - promoter influences it or in RNA itself - gene A could have a better promoter sequence and is better at recruiting the general transcription factors - sequences within the RNA itself can make a gene be more or less efficiently translated

the polymerase used used in PCR comes from hot spring things

- grows in really hot not great conditions, yet still works - allows for DNA polymerase to be beat up and still work - DNA polymerase prevails even in high temperatures of denaturation

next question mendel wanted to ask was how are traits inherited with respect to one another?

- if you had a parent that was green and wrinkled and a parent that was round and yellow, do the big r and big y have to stay together? Do the little r and little y have to stay together? or can they mix up/can you get some yellow wrinkled peas? - yellow is actually dominant to green this is the dependent assortment hypothesis - the F2 generation can only make a big RY gamete or a small r, small y gamete because that is how the gametes came in the first place in F1 - can only go in the same combination as what gametes the parent cell produced in F2 gamete production (dominants have to go together and recessive have to go together) = not what mendel observed!

incomplete dominance

- like the blending inheritance hypothesis - in this case, heterozygotes look like something in between - only synthesizing half as much of the pigment if you cross f1 with F1, you get a quarter homozygous dominant - ½ heterozygous - ¼ white

Histone tail modifications

- loosen chromatin - give greater access to transcription factors and other proteins.

How do we regulate how often this process is happening?

- only one RNA polymerase at the promoter at one time - the way that you make transcription increase is by loading RNA polymerase and sending it off quickly - you are really increasing the rate of initiating this process

pseudoautosomal regions of mammalian X and Y chromosomes synapse and cross over/recombine during meiosis

- pairing of the sex chromosomes happens through these tips, AKA pseudoautosomal regions - pretending to be autosomal portions of the genes that match in sequence with one another and mediate the pairing of the x and the y in meiosis reason why the x and y chromosomes believe that they are homologs during meiosis - in the pseudoautosomal regions there is pairing and there even is crossing over - in the green stuff there is no homology - they are not similar or the same genes - there is homology in the red parts cross over uses the molecular process called recombination which relies on homology or sequence similarity, only cross over in sex chromosomes is in the psuedoautosomal regions - the genes on the sex chromosomes encode for sex linked traits

basic transcriptional regulation was first described in

- prokaryotes and helped us understand eukaryotic regulation - we can understand how disease-causing bacteria regulate their virulence factors: how cholera would respond to high level of bile, know it was inside of a human gut to then burrow into mucus and then and set up colonies - this is related to quorum sensing - we can precisely drive the expression of recombinant DNA in bacteria by adding certain promoters and activators

cross between two heterozygotes

- punnett squares can be used to predict the outcomes of a cross - each possible gamete that a parent cell could make you put on the top of the square - Cross between two homozygotes: mother (rr) could make r gametes father (RR) could make R gametes - Cross between two heterozygotes: Rr and Rr each parent could make two different haploid gametes, r and R - you put your haploid gamete genotype - each possible version on the top line and on the right side ¼ of the prodigy would be homozygous recessive and ¼ of the prodigy would be homozygous dominant and ½ of the prodigy would be heterozygous dominant ***phenotpyically the ratio would be three round to one wrinkled

sanger sequence more info

- separate them based on one nucleotide differences, different dye at each end point -when you run this in a machine that separates the single daughter strand based on size, end product of each one has a label you are making a product size for each size of DNA - 1 nucleotide product that is an A, nucleotide number 1 is A - the way that you know the different sizes of these fragments is by electrophoresing them The DNA sample to be sequenced is combined in a tube with primer, DNA polymerase, and DNA nucleotides (dATP, dTTP, dGTP, and dCTP). The four dye-labeled, chain-terminating dideoxy nucleotides are added as well, but in much smaller amounts than the ordinary nucleotides. The mixture is first heated to denature the template DNA (separate the strands), then cooled so that the primer can bind to the single-stranded template. Once the primer has bound, the temperature is raised again, allowing DNA polymerase to synthesize new DNA starting from the primer. DNA polymerase will continue adding nucleotides to the chain until it happens to add a dideoxy nucleotide instead of a normal one. At that point, no further nucleotides can be added, so the strand will end with the dideoxy nucleotide. This process is repeated in a number of cycles. By the time the cycling is complete, it's virtually guaranteed that a dideoxy nucleotide will have been incorporated at every single position of the target DNA in at least one reaction. That is, the tube will contain fragments of different lengths, ending at each of the nucleotide positions in the original DNA (see figure below). The ends of the fragments will be labeled with dyes that indicate their final nucleotide. After the reaction is done, the fragments are run through a long, thin tube containing a gel matrix in a process called capillary gel electrophoresis. Short fragments move quickly through the pores of the gel, while long fragments move more slowly. As each fragment crosses the "finish line" at the end of the tube, it's illuminated by a laser, allowing the attached dye to be detected. The smallest fragment (ending just one nucleotide after the primer) crosses the finish line first, followed by the next-smallest fragment (ending two nucleotides after the primer), and so forth. Thus, from the colors of dyes registered one after another on the detector, the sequence of the original piece of DNA can be built up one nucleotide at a time. The data recorded by the detector consist of a series of peaks in fluorescence intensity, as shown in the chromatogram above. The DNA sequence is read from the peaks in the chromatogram. Sanger sequencing gives high-quality sequence for relatively long stretches of DNA (up to about 900900900 base pairs). It's typically used to sequence individual pieces of DNA, such as bacterial plasmids or DNA copied in PCR. However, Sanger sequencing is expensive and inefficient for larger-scale projects, such as the sequencing of an entire genome or metagenome (the "collective genome" of a microbial community). For tasks such as these, new, large-scale sequencing techniques are faster and less expensive.

chromatin structures

- the DNA in chromatin is wrapped around almost twice the proteins in green known as histones - histones are crucial for DNA to wrap around to form a chromatin complex - the blue is a linker histone which can bind to the side the core wrapped up DNA around histone proteins is called a nucleosome - histones are connected to one another through linker DNA: a little bit of free DNA in between the histones nucleosomes can coil on themselves to form a nucleosome looking fiber, assemble chromatin fiber called the 30nm fiber - the 30nm fibers can be looped in a protein scaffold which are packed against each other to create a mitotic chromosome

somatic mutation and cancer

- the accumulation of three successive mutation sin a lineage of colon cells results in malignant colon cancer - when looking for polyps in a colonoscopy - they are looking for build of colon cells where a mutation occurs = blue and then → benign polyp → but if you let the cell build further with the polyp → a blue cell develops another mutation is the RAS gene → it becomes a larger benign polyp → with each division, it is another chance for a mutation to occur = going back to idea of mutation rates

CRISPR/Cas9

- to make a change in genes - you are dependent on these specific RE sites. You can engineer them into DNA...but what if you could design your own RE to just cut exactly where you wanted to - in PCR and plasmids, you could cut your DNA by matching the sequence you want to cut to an enzyme that cut its or design a cut site into your primer

segregation reflects the separation of homologous chromosomes during anaphase I of meiosis

- what underlies this, is the reductiveness of meiosis - when you separate homologous chromosomes = meiosis 1 - gametes are going to be haploid - crossing over between parents two parent homologous chromosomes - grandparents DNA is what kid is getting - allows kids to get some of grandpa's dna and some of grandma's DNA per gamete

genome annotation

- wherever overhangs is what is connected together determining sequence of genes and DNA But what is in the sequence? - has to be annotated/run through a computer to determine 1. What is a gene 2. What repeats are a motif we are finding 3. What is a coding region 4. What is a promotor - computers compare the sequence to already plugged in known genes that researchers have found --> a computer identifies it --> in silico identification --> the computer actually identifies the gene

frameshift mutation

- with a one nucleotide deletion the mRNA would read differently and ribosome would produce different sequence of amino acids - throws ofd codons and amino acids for every nucleotide downstream - same is true of a single nucleotide insertion or double deletion - only way to maintain reading of the amino acids is to add or delete three nucleotides - things would stay in sync - involves insertion or deletion - an insertion or deletion that is not an exact multiple of 3 nucleotides changes the reading frame of translation. Such a mutation is called a frameshift mutation

Codominance (Blood type)

-When multiple alleles exist for a given gene and more than one of them is dominant -Each dominant allele is fully dominant when combined with a recessive allele, but when two dominant alleles are present, the phenotype is the result of the expression of both dominant alleles simultaneously -Ex. inheritance of ABO blood groups in humans, blood type is determined by three different alleles IA, IB, and i -Only two alleles are present in any single individual, but the population contains all three alleles -IA and IB are dominant to i -Individuals who are homozygous IA or heterozygous IA i have blood type A -Individuals who are homozygous IB or heterozygous IB i have blood type B -Individuals who are homozygous ii have blood type O -Individuals who are heterozygous IA IB have a distinct blood type, AB, which combines characteristics of both the A and B blood groups. -Codominance differs from incomplete dominance because in incomplete dominance the phenotype expressed is a blend of both genotypes, however, in codominance both alleles in the genotype are expressed at the same time without a blending of phenotype

A brief history of genetic engineering and biotech

1. 2000BC- breeding of dogs and plants to domesticate for agriculture 2. 1950s- Watson & Crick describe DNA structure 3. 1960s- 1st description of what mRNA is; codons figured out; 1st Restriction Enzyme discovered 4. 1972-1st recombinant DNA molecule made- cutting and ligating a piece of DNA from a virus into a bacterial plasmid 5. 1977- Sequencing of DNA. (Warner born) 6. 1983- Polymerase Chain Reaction. 7. 1990s- Transcriptomics, Genome sequencing 8. 2000s- RNAi, more genomes sequenced 9. 2010s- CRISPR/cas9 gene editing

microRNAs

1. Are transcribed just like protein-coding genes, using the same RNA polymerase and going through capping, splicing, and polyadenlyation. 2. Then the RNA folds back upon itself to form one or more hairpin structures. 3. Enzymes recognize the folded miRNAs and cleave the stems from the hairpin leaving small, double stranded fragments. 4. One strand of the fragment becomes incorporated into a protein complex called RISC (RNA-induced silencing complex) that base pairs (with some mismatches) with a region of the target mRNA. 5. Translation is inhibited.

small interfering RNA (siRNA)

1. Are transcribed, processed, and incorporated into the RISC complex just like microRNAs 2. When small interfering RNAs pair with a target mRNA, there are no mismatches. 3. The targeted mRNA is cleaved by RISC, which leads to degradation of the RNA transcript.

shotgun sequencing steps

1. Break multiple copies of genome into smaller, overlapping random fragments 2. Sequence each fragment and assemble genome based on overlapping sequences - Issue: Reassembly process can be derailed by repetitive nucleotide sequences (rare in bacteria, but a large fraction of vertebrate genomes) -- when sequence assembled incorrectly, intervening info lost --- To avoid: combined with clone-by-clone approach Figure 1 The steps involved in the whole-genome shotgun sequencing procedure, (a) Library construction. Total genomic DNA is extracted and mechanically sheared to smaller fragments. Each fragment is ligated into a cloning vector, (b) Random sequencing. About 6000 random clones per megabasepair are sequenced from both end of the insert to achieve 8X coverage, (c) The small sequences (~ 800 bp) are assembled into larger contigs using computational algorithms, such as the Celera Assembler, (d) The contigs are linked to each other during the closure phase, where the sequence is also manually edited, (e) Annotation. Using programs such as Glimmer, open reading frames (ORFs) are marked. The predicted protein sequences from these putative open reading frames are searched against nonredundant protein databases, (f) A Complete genome is obtained after manual curation of the annotation

difference between DNA and genome sequencing

1. DNA sequencing gives you a run of sequence - you use 1 primer to figure where you are sequencing - you have somewhat of an idea where to start, generally know what type of 1 primer to use PCR or Sanger sequencing 2. genome sequencing = shotgun sequencing - using random primers to amplify a piece of DNA, then sequence that piece - genome = so large and random --> use random primers --> have to break up the genome first --> put the parts of the genome on different plasmids -->sequence all different plasmids 1. library construction 2. DNA isolation 3. DNA fragmentation 4. DNA cloning with plasmids 5. Random sequencing with random primers 6. shot gun sequence assembly/closure and edidting

Recombinant DNA

1. DNA technology recombines DNA molecules from two or more different sources into a single molecule 2. the method requires a fragment of double stranded DNA that serves as the donor 3. the donor fragment may be a protein coding gene, a regulatory part of a gene, or any DNA segment of interest 4. Also required is a vector sequence into which the donor fragment is to be inserted 5. The vector is the carrier of the donor fragment, and it must have the ability to be maintained in bacterial cells 6. A plasmid is a vector

steps of polymerase chain reaction: each cycle of amplification includes three steps

1. Denaturation: a solution containing double-stranded DNA (the template duplex ) is heated to separate the DNA into two individual strands 2. Annealing - when the solution is cooled, the two primers anneal to their complementary sequence of the strands of the duplex 3. Extension: DNA polymerase synthesizes new DNA strands (complementary to the template duplex strands) by extending primers in a 5' to 3' end

recombinant DNA steps part one

1. Donor DNA and vector DNA are both cleaved with the same restriction enzyme, in this case EcoRI 2. the resulting fragments are mixed together joined by DNA ligase. The genomic and vector fragments have complementary ends.

What is the difference between a transcriptional activator/repressor and a general transcription factor?

1. Each regulatory txn factor (activator or repressor) regulates a limited set of genes that contain its specific recognition sequence in the gene's chromosomal vicinity 2. General transcription factors (GTFs) must bind to virtually EVERY gene promoter, using sequence information in the DNA (often use TATA) 3. Mediator is a large, mysterious bridging complex that somehow "interprets" the information from txn activators and repressors and conveys that info to the RNA Pol complex

Gene numbers

1. Gene number is not a good predictor of biological complexity. 2. It may seem surprising to see the relatively low number of genes in humans when compared to the other species because humans are so much more complex in cell number and type and behavior. 3. One hypothesis is that human cells can do many more things with the genes they have because of subtle gene regulation, different protein interactions, and gene splicing. --> our genome - 25,000 protein encoding genes

Mendel's experiment

1. In crossing peas, the anthers of the female parent are first exposed and then cut off to prevent self fertilization 2. mature pollen is collected from another flower and deposited on the stigma of the fame parent 3. after fertilization, a small cloth bag is tied around the fertilized flower to prevent stray pollen from entering -cut off they anthers, equivalent of castrating a flower -he would get pollen from plants that he wanted to use to fertilize and use a paintbrush to paint that pollen onto the other plants stigma = controlled meeting -cover flower with a cloth to prevent any further pollenation

After PCR, you have to make

1. Make gel using agarose, let solidify, and add samples to the wells, add electricity 2. the top of the gel is negative and the bottom of the gel in positive - All DNA molecules have the same amount of charge per mass. Because of this, gel electrophoresis of DNA fragments separates them based on size only. Using electrophoresis, we can see how many different DNA fragments are present in a sample and how large they are relative to one another. We can also determine the absolute size of a piece of DNA by examining it next to a standard "yardstick" made up of DNA fragments of known sizes. - Once the gel is in the box, each of the DNA samples we want to examine (for instance, each PCR reaction or each restriction-digested plasmid) is carefully transferred into one of the wells. One well is reserved for a DNA ladder, a standard reference that contains DNA fragments of known lengths. Commercial DNA ladders come in different size ranges, so we would want to pick one with good "coverage" of the size range of our expected fragments. - Next, the power to the gel box is turned on, and current begins to flow through the gel. The DNA molecules have a negative charge because of the phosphate groups in their sugar-phosphate backbone, so they start moving through the matrix of the gel towards the positive pole. When the power is turned on and current is passing through the gel, the gel is said to be running. - As the gel runs, shorter pieces of DNA will travel through the pores of the gel matrix faster than longer ones. After the gel has run for awhile, the shortest pieces of DNA will be close to the positive end of the gel, while the longest pieces of DNA will remain near the wells. - A well-defined "line" of DNA on a gel is called a band. Each band contains a large number of DNA fragments of the same size that have all traveled as a group to the same position. A single DNA fragment (or even a small group of DNA fragments) would not be visible by itself on a gel. - smaller fragments move faster - bigger fragments move slower - a band represents DNA of a particular size - already know what gene or DNA region you wanted to clone through pcr --> see if plasmid has the right gene or if PCR worked

DNA Repair by Mismatch Repair Enzymes

1. MutS recognizes mismatched bases in DNA and initiates the repair process 2. MutL and MutH proteins are recruited, and MuH breaks the backbone some distance away 3. An exonuclease removes successive nucleotides, including the one with the mismatched base 4. A DNA polymerase fills in the missing nucleotides and a DDDNA ligase joins the backbones

evolutionary relationships of human and simian lentiviruses

1. Once you get the whole human genome, you can look at different parts of it 2. We can sequence yeast and virus genomes 3. You can run a similarity program to determine how closely linked certain viruses are to one another 4. What percent of the genome is the same between two different organisms, how many runs of extremely similar nucleotides are there and how many different nucleotides are there --> using this you can determine and grade evolutionary relationships, where did viruses originate before crossing over to humans

Non mismatch = nucleotide excision repair

1. One or more damaged bases signals the repair process 2. An enzyme cleaves the DNA backbone at sites flanking the damage - these nucleotide issues are solved by nucleotide excision repair - red indicates damage but correct nucleotide pairing - could be interlocking with each other - could be alkaladed - other different processes can occur from a mutagen that causes them to be damaged - causes a problem when you are trying to replicate later 1. replication fork does not open up correctly 2. maybe stick to each other too much - could cause a deletion of 4 nucleotides and a frameshift - a different type of enzyme cleaves the particular piece of damaged DNA - new enzyme comes in to synthesize new DNA - were not mismatched, were damaged, and would not work in DNA replication

steps of nucleotide excision repair

1. One or more damaged bases signals the repair process 2. An enzyme cleaves the DNA backbone at the sites flanking the damage 3. region with damaged bases is removed 4. The gap is filled by new DNA synthesis, using the un-gapped strand as the template

Mendel's Key Principles

1. Principle of Segregation: Individuals inherit two copies of each gene ("particle"), one from the mother and one from the father, and when individuals form gametes, the two copies separate equally in the eggs and sperm. (Also, the allele can be dominant or recessive) 2. Principle of Independent Assortment: The two copies of each gene segregate into gametes independently of the two copies of another gene. - each gene or two alleles is a particle that gets separated from one another into the gametes and each allele can be dominant or recessive principle of segregation comes from the monohybrid cross two alleles for a single gene segregate from one another during meiosis 1 only need one gene to show that this is true the principle of independent assortment - the two copies of each gene separate into gametes independently of the second gene going into the gametes - due to crossing over= two genes on the same chromosome - random assortment at the metaphase plate, on top or bottom between different chromosomes - randomness and the genes are able to be mixed up

mutation rates per nucleotide pair per round of replication of a cell

1. RNA virus - making RNA genomes into more RNA genomes using RNA polymerase and not using DNA polymerase every replication cycle you have more of a chance for RNA viruses and retroviruses the RNA polymerase being used, has a higher mutation rate even though it had a smaller genome 2. → one mutation every replication cycle (ratio) why we have coronavirus variants leads to variants, changes in nucleotides 3. humans: largest genome and lowest mutation rate our DNA polymerase during replication has a really low mutation rate 4. problem: unlike viruses with short life and one cell or smaller, humans have many cells with a super long lifespan → we build up mutations over that time

sickle cell anemia (missense mutation) --> big fallout from single nucleotide, recessive

1. Sickle-cell anemia is caused by a single amino acid: Glu is replaced by Val in hemoglobin subunit β-globin 2. This leads to the misfolding of the hemoglobin. 3. Individuals who inherit two copies of the mutant β-globin gene have sickle-cell anemia. 4. In this condition, hemoglobin crystallizes in low levels of oxygen, causing the cell to collapse;

Insertions and Deletion (small scale)

1. Small insertions or deletions involve several nucleotides. 2. A small deletion or insertion that is an exact multiple of three nucleotides results in a polypeptide with as many fewer (in the case of a deletion) or more (in the case of an insertion) amino acids as there are codons deleted or inserted. 3. Thus, a deletion of three nucleotides eliminates one amino acid, and an insertion of six adds two amino acids.

Components of a PCR reaction

1. Template DNA 2. DNA polymerase 3. The four deoxynucleotide triphosphates 4. two primers

X inactivation, the heterochromatinization of one X chromosome, occurs in XX female mammals as a gene dosage compensation

1. The dosage of X-linked genes would be twice as great in females as in males were it not not for this dosage compensation mechanism. 2. Soon after an early embryo with two X chromosomes implants in the mother's uterine wall, one X chromosome is selected at random and inactivated. 3. as cell division continues, the inactive X stays inactive - this is called epigenetic inheritance As cell division continues, the inactive X stays inactive- this is called epigenetic inheritance. In each cell lineage, the same X chromosome that was originally inactivated remains inactive. What is being inherited is the silent chromatin state rather than DNA sequence.

recombinant DNA steps: transformation

1. The recombinant DNA molecule is introduced into a bacterial cell by the means of transformation 2. As the bacterial DNA replicates and divides, the recombinant DNA is also replicated and transferred to the daughter cells. The recombinant DNA therefore multiplies along with the bacterial DNA

PCR cycle

1. The template duplex is often longer than the amplified region 2. Primers for PCR are chosen to base pair with on or the other strand of the template duplex, with their 3' ends oriented toward each other. The primers are eded to the reaction mixture in great excess to ensure pairing with any complementary sequence Note: primers can bind to the initial template strand or a previously amplified strand 3. each round of amplification doubles the number of molecules that have the same sequence as the template duplex 4. After n cycles of amplification there are 2n copies of the template sequence Note: most of the strands after many cycles, most of the strands are doubled stranded amplified strands instead of original template plus an amplified strand - mostly get pieces defined in length by where the forward and reverse primers are bound, ton of product exactly the size of the base pairs between the primers - logarithmic over time

CRISPR and DNA editing

1. a cell with target DNA to be edited is injected with plasmid DNA with sequences for CRISP RNA and Cas9. A plasmid containing editing template for DNA is also pesent 2. CRISPR RNA combines with the Cas9 protein 3. CRISPR RNA guides Cas9 to the target DNA and the target DNA is cleaved 4. an exonuclease widens the gap in the target DNA 5. the editing template is used to repair the gap in the target DNA 6. the result is an edited DNA with altered sequence

what goes into prokaryotic gene regulation

1. a gene 2. a promoter region - upstream of the gene 3. regulators 4. a stimulus to respond to

annotated genome of a retrovirus

1. a lot of human genome is retroviral DNA 2. any retrovirus is contained by terminal repeats on each end 3. Not chalk full of proteins or open reading frames, but instead retroviruses 4. All have gag, pol, and envelop sequences

CRISPR-Cas9 steps

1. bacteria has other ways besides restriction enzymes in which it forms an immune response to invading viruses - it has sequences called CRISPR - prior you could only cut in restriction enzyme sites that matched a restriction enzyme - derived from process of how bacteria can take up foreign DNA and insert them into their own genome (if bacteria ever sees the viral DNA again, they want to cut it up and destroy it via the enzyme Cas9

What about non-mismatch DNA issues?

1. caused by oxidation/inflamation, chemotherapeutics or tobacco, UV light 2. base pairs get damaged 3. lock in with one another 4. unable to correctly base pair 5. or when DNA double helix opens up for replication it can't do so correctly because of these nucleotide issues - homologous pairs need to be the same length sister chromatids are identical copies of same chromosome = 1 chromosome, duplicated chromosome - S phase - sister chromatid synthesis homologous chromosomes are paired with one another at the metaphase plate

DNA repair by mismatch repair enzymes

1. enzyme 1/MutS recognizes bulge on strand 2. enzyme 2abc/MutL and MutH break the backbone strand some distance away 3. enzyme 3. exonuclease - removes successive the nucleotides including the mismatched base added on the newly synthesized strand 4. DNA ligase joins the backbones together after - occurs after replication has been completed - recognized because mismatch creates a change in structure of the DNA double helix - series of other enzymes come in to break the backbone, remove nucleotides including bad one, and then DNA polymerase adds correct complementary bases - ligase joins the backbone back together

chromosomes consist of regions of euchromatin and heterochromatin

1. euchromatin - transcriptionally competent: loosely packed, associated with acetylated histones 2. heterochromatin - transcriptionally silent: densely packed, associated with deacetylated histones

ploidy changes during the life cycle of a dog

1. haploids are made by diploid organisms through meiosis 2. when a gamete joins with another gamete it is fertilized and you restore ploidy to diploidy 3. in the female diploid dog, only her germ cells in her ovary undergo meiosis in order to create haploid egg gamete 4. her haploid egg will be fertilized by another haploid sperm 5. two haploids - a sperm and egg - come together through the process of fertilization to yield a diploid cell called a zygote 6. the zygote is going to divide mitotically to yield another adult dog which could be either male or female

the ara operon

1. here the repressor and activator are the same proteins 2. low arabinose = AraC proteins are repressors - looping the DNA 3. high arabinose - AraC proteins are activators - helping RNA polymerase bind

histone tails protrude from the nucleosome and undergo post translation modification, which controls what happens to the DNA

1. histone tails are poorly structured N-terminal portions of histone proteins 2. The DNA near the histone tails is regulated by them 3. Histone modifications can regulate transcription (and any DNA-dependent process!!) in different ways - sometimes they influence the way that chromatin folds; sometimes they recruit a protein that brings about a change, like increasing transcription rate.

in the ara operon, AraC can act as activators or repressors

1. in low amounts of arabanose, proteins change their structure and folds the DNA, so RNA polymerase can no longer bind 2. products of the araBAD gene break down arabinose so in high amounts the ara operon is activated

steps of recombinant DNA technology

1. isolate plasmid DNA from bacteria and purify DNA containing the gene of interest from another cell 2. piece of DNA containing the gene is inserted into the plasmid, producing recombinant DNA 3. plasmid is put back into a bacterial cell 4. genetically engineered bacterium is then grown in culture 5. cell clone carrying the gene of interest is isolated - DNA cloning is a molecular biology technique that makes many identical copies of a piece of DNA, such as a gene. - In a typical cloning experiment, a target gene is inserted into a circular piece of DNA called a plasmid. - The plasmid is introduced into bacteria via a process called transformation, and bacteria carrying the plasmid are selected using antibiotics - selectability marker. Bacteria with the correct plasmid are used to make more plasmid DNA or, in some cases, induced to express the gene and make protein.

Combinatorial control of gene expression

1. multiple transcription factors and multiple binding sites lead to complex regulatory systems 2. by having different sets of transcription factors expressed during embryonic development, you can specify the formation of different cell types 3. if you have transcription factor 2 and 4 together you will express a different set of genes than 2 and 5 4. its the groups of transcription factors that can all bind to the same gene → which can ultimately cause cells to be different cell types

lac operon mutants in the presence of lactose

1. null mutants 2. complementing or adding back mutants 3. promoter or regulatory sequence mutants

in eukaryotic cells, gene regulation occurs at many levels

1. regulatory transcription factors like activators and repressors 2. chemical modification of chromatin - methylation of cytosine bases (DNA) - histone tail modifications 3. chromatin folding state - physical structure Note: more complex than prokaryotes

Broad categories of mutations

1. small scale (nucleotide substitution or point mutation) ex. synonymous (silent) mutations, nonsynonymous (missense), nonsense mutations, frameshift mutations (small insertion deletion) 2. Large scale(chromosomal mutations) ex. insertion, deletion (of chromosome)

the three essential DNA sequence elements in a eukaryotic chromosome

1. telomere - have telomere caps that try to protect the double stranded DNA ends from being recognized as a chromosomal break 2. replication origin 3. centromere Each chromosome has AT LEAST one origin of replication, one centromere, and two telomeres. Shown schematically is the sequence of events that a typical chromosome follows during the cell cycle. The DNA replicates in interphase. In M phase, the centromere attaches the duplicated chromosomes to the mitotic spindle so that one copy is distributed to each daughter cell during mitosis. The centromere also helps to hold the duplicated chromosomes together until they are ready to be moved apart.

restriction enzymes

1. the molecules that act as scissors to cut DNA 2. originally described in the bacteria 3. recognize non-self DNA based on sequence and cut it out 4. Each species of bacteria has enzyme that DON'T digest their own chromosome 5. we have learned what enzymes recognize and how they cut 6. you can buy purified enzyme and cut your DNA by matching the sequence you want to cut to the enzyme that cuts it...OR design a cut site into your primers

(applies to both eukaryotes and prokaryotes) gene expression can be regulated at any step from transcription to

1. transcriptional control 2. translation control 3. post translation control

CRISPR steps

1. transform a cell with a plasmid containing sequences that coded for a CRISPR RNA as well as the CRISPR-associated protein Cas9 In popular usage, "CRISPR" (pronounced "crisper") is shorthand for "CRISPR-Cas9." CRISPRs are specialized stretches of DNA. The protein Cas9 (or "CRISPR-associated") is an enzyme that acts like a pair of molecular scissors, capable of cutting strands of DNA. CRISPR technology was adapted from the natural defense mechanisms of bacteria and archaea (the domain of single-celled microorganisms). These organisms use CRISPR-derived RNA and various Cas proteins, including Cas9, to foil attacks by viruses and other foreign bodies. They do so primarily by chopping up and destroying the DNA of a foreign invader. When these components are transferred into other, more complex, organisms, it allows for the manipulation of genes, or "editing." CRISPRs: "CRISPR" stands for "clusters of regularly interspaced short palindromic repeats." It is a specialized region of DNA with two distinct characteristics: the presence of nucleotide repeats and spacers. Repeated sequences of nucleotides — the building blocks of DNA — are distributed throughout a CRISPR region. Spacers are bits of DNA that are interspersed among these repeated sequences. In the case of bacteria, the spacers are taken from viruses that previously attacked the organism. They serve as a bank of memories, which enables bacteria to recognize the viruses and fight off future attacks. The Cas9 protein is an enzyme that cuts foreign DNA. The protein typically binds to two RNA molecules: crRNA and another called tracrRNA (or "trans-activating crRNA"). The two then guide Cas9 to the target site where it will make its cut. This expanse of DNA is complementary to a 20-nucleotide stretch of the crRNA. Using two separate regions, or "domains" on its structure, Cas9 cuts both strands of the DNA double helix, making what is known as a "double-stranded break," according to the 2014 Science article.

origins of CRISPR

1. viruses can invade bacterial cells 2. bacteria can take a piece of DNA from the viral DNA --> a new spacer is derived from virus and integrated into CRISPR sequence 3. CRISPR RNA is formed 4. CRISPR RNA guides molecular machinery to target and destroy the viral genome - if bacteria ever sees the viral DNAn again it knows to destroy it

lac operon with lactose

1. when the operator is not bounds by the repressor, the promoter recruits the RNA polymerase complex and transcription of the polycistronic mRNA occurs 2. in the presence of lactose, the repressor protein, lacI, is unable to binds to the operator because lacI is instnead binding the the lactose present 3. lacI acts as a lactose sensor, lackI does not become a repressor and RNA polymerase can do its job 4. this is known as a depressed system 5. lacI has two functions: 1) binding to the operator and repressing RNA polymerase and thus, transcription 2) in the presence of lactose, LacI get4s bound by lactose, acting as a sensor, and can no longer bind to the operator

Sanger sequencing

1. you are adding 1 primer, not two like in PCR 2. you need NTPS, one primer, DNA polymerase 3. The goal is to amplify a product from a template 4. You have 1 product 5. you add dideoxynucleotides to the end, and thus the creation of a daughter product is terminated there 6. you have a lot/the normal amount of deoxynucleotides and a minority of dideoxynucleotides - Dideo's have no 3' OH so no new phosphodiester bond can be createdd - fused to a fluorescent tag - different color for each dideo - ex. C is purple --> allows you to see what nucleotide was added last

meiosis II

1. you would recondense your chromosomes into sister chromatids not one long strand - they would line up in the middle - separate sister chromatids - you would get your prodigy cells each having one copy of each chromosome - meiosis of 1 cell leads to the production of 4 haploid gametes - metaphase two is random based on what poles they go to after lining up

sequence the human genome

2.91 billion base pair sequence - taking one human genome and breaking it up into bits and pieces and doding little runs of sequencing - locating genes in relation to one another on the same chromosomes

before replication you have 46 chromosomes which are also

23 pairs of 2 homologous chromosomes - after S phase, you still have 46 chromosomes total but joined sister chromatids are considered one chromosome

point mutation

A G is erroneously incorporated into the daughter strand opposite the T and is not corrected by the proofreading function - CHANGES IN A SINGLE NUCLEOTIDE

monohybrid cross

A cross between individuals that involves one pair of contrasting traits

a normal deoxynucleotided has a hydroxyl (OH) group on the 3' carbon, allowing this end to be elongated

A dideoxynucleotide lacks the 3' hydroxyl group, and it cannot be elongated because there is no hydroxyl group to attack an incoming nucleotide triphosphate.

selectable marker

A gene on a plasmid that is introduced into a cell along with a gene of interest that is being cloned. Selectable markers allow scientists to tell if the plasmid has been taken in by the cell because the marker can be seen or detected. A common selectable marker is an antibiotic resistance gene—only bacteria that have the gene will survive the antibiotic.

nonsense mutations

A nucleotide substitution that creates a stop codon is called a nonsense mutation - the type of mutation makes a truncated protein (shorter), which is then usually nonfunctional and unstable

Sanger sequencing

A procedure in which chemical termination of daughter strands help in determining the DNA sequence.

restriction site

A specific sequence on a DNA strand that is recognized as a cut site by a restriction enzyme.

Extension (PCR) - 72 degrees

A step in the polymerase chain reaction (PCR) for producing new DNA fragments in which the reaction mixture is heated to the optimal temperature for DNA polymerase, and each primer is elongated by means of deoxynucleoside triphophosphates. - bring temp up a little bit to about 72 degrees celsius and DNA polymerase bins to primer and starts synthesizing - DNA replication occurs from from 5' to 3' on each strand - going down template 3' to 5' - the purple area is what is being replicated after 1 cycle - repeat cycle by pumping temp back up to pull newly synthesized double strands apart

polymerase chain reaction (PCR)

A technique for amplifying DNA in vitro by incubating with special primers, DNA polymerase molecules, and nucleotides. - making many copies of genes - replication inside of a tube - harvest a DNA polymerase and give it everything it needs inside of a tube to do a chain reaction to amplify new pieces of DNA over and over again - guided a specific polymerase to certain parts of your genome and make little segments of your DNA

RT-PCR

A technique in which RNA is first converted to cDNA by the use of the enzyme reverse transcriptase, then the cDNA is amplified by the polymerase chain reaction. - A sample is collected from the parts of the body where the COVID-19 virus gathers, such as a person's nose or throat. The sample is treated with several chemical solutions that remove substances such as proteins and fats and that extract only the RNA present in the sample. This extracted RNA is a mix of the person's own genetic material and, if present, the virus's RNA. The RNA is reverse transcribed to DNA using a specific enzyme. Scientists then add additional short fragments of DNA that are complementary to specific parts of the transcribed viral DNA. If the virus is present in a sample, these fragments attach themselves to target sections of the viral DNA. Some of the added genetic fragments are used for building DNA strands during amplification, while the others are used for building the DNA and adding marker labels to the strands, which are then used to detect the virus. The mixture is then placed in an RT-PCR machine. The machine cycles through temperatures that heat and cool the mixture to trigger specific chemical reactions that create new, identical copies of the target sections of viral DNA. The cycle is repeated over and over to continue copying the target sections of viral DNA. Each cycle doubles the previous number: two copies become four, four copies become eight, and so on. A standard real time RT-PCR set-up usually goes through 35 cycles, which means that, by the end of the process, around 35 billion new copies of the sections of viral DNA are created from each strand of the virus present in the sample. As new copies of the viral DNA sections are built, the marker labels attach to the DNA strands and then release a fluorescent dye, which is measured by the machine's computer and presented in real time on the screen. The computer tracks the amount of fluorescence in the sample after each cycle. When a certain level of fluorescence is surpassed, this confirms that the virus is present. Scientists also monitor how many cycles it takes to reach this level in order to estimate the severity of the infection: the fewer the cycles, the more severe the viral infection is.

dependent assortment hypothesis

Alleles of two genes stay linked through and are transmitted together into gametes - alleles of two genes are sorted into gametes in the same combinations as existed in the parent

what is an operator?

Also a region of DNA upstream of the structural gene or genes in the operon but this region is bound by the transcriptional regulator to control transcription

Retrovirus

An RNA virus that reproduces by transcribing its RNA into DNA and then inserting the DNA into a cellular chromosome; an important class of cancer-causing viruses. - reverse transcriptase takes RNA, makes DNA, which gets inserted into our chromosomes this is what also causes HIV - the mRNA leaves turns back into viral RNA, makes more of itself - retroviruses are NOT HUGE, smaller than a size of a gene - very rarely, human mutations occur because a transposon jumps moves from one place to another - cells want to maintain genome in an unchanged site - in general it would benefit our cells for all of the transposons to be inactive - could there be a benefit of those ones that do not devolve?

down's syndrome = trisomy 21

Aneuploidy of chromosome 21 and sex chromosomes are relatively tolerable (non-lethal) nondisjunction events in humans - when you are born (female) your germ cells that want to be gametes are suspended in meiosis for maybe up to 50 years and then they are gonna continue, starting a car that hasn't run in 50 years, increased chance in chromosomal errors when making gametes

prediction of blending-inheritance hypothesis

At the time of Mendel's experiments, the blending-inheritance hypothesis prevailed. This hypothesis said that traits blend to form intermediate phenotypes.

Why is CRISPR better than restriction enzymes?

Both restriction enzymes and Cas9 (part of the CRISPR system) are endonucleases, meaning that they cut DNA somewhere in the middle of a strand, rather than taking bases off the end. The main functional difference is in the mechanism by which they recognize the sequence they are supposed to cut. The most important difference is that, for restriction enzymes, the cut site is "hard wired" into the protein structure. A given restriction enzyme can only cut at a particular site. In order to make a restriction enzyme that recognizes a novel site would require designing a completely new protein. Unfortunately, we do not know enough in general about how proteins fold in order to efficiently design novel enzymes. Cas9 on the other hand is guided to its cut site by an single guide RNA (sgRNA) which uniquely targets the DNA sequence to which it is complementary. This means that instead of engineering a whole new protein, if we want to target a specific site we can simply change the sgRNA sequence, the techniques for which have been common practice for the last several decades. There are some other peculiarities about each system, but that is the fundamental difference between the two, and why CRISPR has made such a huge splash. Formerly it was extremely difficult to cut DNA at a targeted sequence. Restriction enzymes were never really a feasible option for this, but techniques were developed to design specific endonucleases (TALENs and ZFNs). However, they required a lot of time and money. With CRISPR, it takes a few days. EDIT: I forgot to mention that ZFNs and TALENs (mentioned above) do often make use of the non-specific cutting domain of FokI, which is a restriction enzyme. However, the DNA recognition domain is not based on restriction enzymes.

DNA break means

Break just means DNA breakage which can happen with hUV and stuff, your cells try to repair it with a little flip large scale mutations when cell tries to fix a break - inadvertantly get a mutation due to replication --> or maybe get a complete loss of where the break --> by product of your cell trying to fix some DNA damage on either side of the red region

jacob and minot

By examining mutant strains of E. coli that exhibited defects in lactose metabolism, Jacob and Monod were able to learn how the lac operon is regulated to metabolize lactose (Jacob & Monod, 1962). The duo noted that the lac operon contains three genes that encode proteins involved in lactose metabolism. These are referred to as lac z, lac y, and lac a. The lac z gene encodes beta-galactosidase, the lac y gene encodes a permease, and the lac a gene encodes the transacetylase enzyme. Together, these gene products act to import lactose into cells and break it down for use as a food source. As in other operons, the genes of the lac operon lie along a contiguous stretch of DNA such that their expression can be easily coregulated. In addition to these so-called structural genes, the lac operon also contains other sequences that direct the bacterial gene expression machinery. - made mutant ecoli - the lac operon is regulated by lactose - genetic regulation - regulation of transcription in an organism and how it is responding to certain things - regulated as repressors and activators, regulates the expression, when a protein is going to get made when the mRNA is going to get transcribed and when it is not going to get transcribed as a result of environmental cues

the amount of DNA in your genome does not mean you are more complex organism

C value paradox - The disconnect between genome size and complexity is called the C-value paradox. - C-value is the amount of DNA in a reproductive cell, and the "paradox" is the apparent contradiction between the genome size and organismal complexity

when there is a decrease in glucose, there is an increase in CAMP which activates

CRP receptor which bind to an activator site upstream of the promoter - positive regulation in the presence of low amounts of glucose

Meiosis

Cell division that produces reproductive cells in sexually reproducing organisms

Using Restriction Enzymes to Make Recombinant DNA

Cut open the plasmid and "paste" in the gene. This process relies on restriction enzymes (which cut DNA) and DNA ligase (which joins DNA). Insert the plasmid into bacteria. Use antibiotic selection to identify the bacteria that took up the plasmid. - Grow up lots of plasmid-carrying bacteria and use them as "factories" to make the protein. Harvest the protein from the bacteria and purify it. - A restriction enzyme is a DNA-cutting enzyme that recognizes a specific target sequence and cuts DNA into two pieces at or near that site. Many restriction enzymes produce cut ends with short, single-stranded overhangs. If two molecules have matching overhangs, they can base-pair and stick together. However, they won't combine to form an unbroken DNA molecule until they are joined by DNA ligase, which seals gaps in the DNA backbone.

a plasmid is a circular piece of

DNA bound inside of bacteria which replicates on its own independent of a bacterial chromosome. Plasmids are maniputable because they are smaller than a whole chromosome so they are relatively easy to work with

heat is analgous to

DNA helicase

CpG islands

DNA regions rich in C residues adjacent to G residues. Especially abundant in promoters, these regions are where methylation of cytosine usually occurs. Near or within a promoter

transcription factor motifs

DNA sequences that bind transcription factors are often short sequences present in multiple copies near a protein coding gene 1. Some sequence motifs are detected in the double-stranded DNA. 2. Shown are two copies of a short sequence that are known binding sites for DNA-binding proteins, called transcription factors. 3. These bind to DNA to initiate transcription. - computer will look for transcription factor motifs - upstream of an open reading frame

Recombinant DNA

DNA that has been formed artificially by combining constituents from different organisms. - a piece of DNA was taken out of a virus, cut from a viral genome, and pasted or ligated into a bacterial plasmid

operon

DNA, group of genes operating together

DNA sequencing

Determining the exact order of the base pairs in a segment of DNA.

nondisjunction can lead to genetic diseases such as

Down's syndrome and Kleinfelter syndrome

Principle of Segregation

During meiosis, chromosome pairs separate into different gametes such that each of the two alleles for a given trait appears in a different gamete.

Sequencing the Genome

Each of the 46 human chromosomes was cleaved; these fragments were combined with vectors to create recombinant DNA, cloned to make many copies, and sequenced using automated sequencing machines; computers analyzed the overlapping regions to generate one continuous sequence

How do we visualize DNA?

Gel Electrophoresis

meiosis I

Homologous chromosomes separate

chromosomes are not fully condensed until

M phase

meiotic prophase 1

Meiotic prophase 1: - homologous chromosomes are going to find one another in the nuclear space - means forming tetrads or a bivalent then going to pair then do crossing over - prophase 1 is the very beginning, yet is where all the genetic mix up is happening - in late prophase - where they touch one another = where crossing over takes place

mendel pea plants

Mendel used pure breeding plants, meaning that they were inbred and homozygous for all genes/traits

Mitosis vs. Meiosis

Mitosis: one division forming 2 identical cells (clones); Meiosis: two divisions forming 4 genetically different cells

plasmid vectors

Modified versions of natural plasmids

genomic sequencing

Obtaining the entire genomic sequence from an animal or plant

sex chromosomes

One of the 23 pairs of chromosomes in the human, contains genes that will determine the sex of the individual.

independent

P1 gametes and F1 gametes do not need to match

dependent

P1 gametes and F1 gametes much match

What is ploidy?

Ploidy refers to the number of each chromosome that an organism carries. One set of chromosomes is designated n - haploid = 1n - diploid = 2n -triploid = 3n, etc.

open reading frame

RNA from a protein-coding regions contains an open reading from consisting of triplets of nucleotides that can specify amino acids - a computer will read through a find an AUG start codon how long will this protein be? Look for a stop codon - computer identifies an open reading frame or region of genome that encodes for a protein

Sister chromatid synthesis takes place during

S phase

the elongation of the daughter strands stops whenever a dideoxynucleotide terminator is incorporated at the 3' end in

Sanger sequencing because there is no OH on the 3' end so elongation cannot continue

sequencing DNA

Sequencing DNA means determining the order of the four chemical building blocks - called "bases" - that make up the DNA molecule. The sequence tells scientists the kind of genetic information that is carried in a particular DNA segment. For example, scientists can use sequence information to determine which stretches of DNA contain genes and which stretches carry regulatory instructions, turning genes on or off. In addition, and importantly, sequence data can highlight changes in a gene that may cause disease.

incomplete dominance

Situation in which one allele is not completely dominant over another allele

CRISPR editing

The CRISPR-Cas9 system works similarly in the lab. Researchers create a small piece of RNA with a short "guide" sequence that attaches (binds) to a specific target sequence of DNA in a genome. The RNA also binds to the Cas9 enzyme. As in bacteria, the modified RNA is used to recognize the DNA sequence, and the Cas9 enzyme cuts the DNA at the targeted location. Although Cas9 is the enzyme that is used most often, other enzymes (for example Cpf1) can also be used. Once the DNA is cut, researchers use the cell's own DNA repair machinery to add or delete pieces of genetic material, or to make changes to the DNA by replacing an existing segment with a customized DNA sequence.

Wrinkledness and roundness are traits conferred by different alleles (=sequence versions) of a single gene.

The Round version is dominant and denoted R; the wrinkled version is recessive and denoted r.

Mendel must have asked himself after observing the F1 generation is "What happened to the wrinkled trait???"

The answer came in the F2 generation, in which the wrinkled phenotype appeared again in ¼ of the progeny. From this confusing result (and no concept of a link between heredity and chromosome behavior during meiosis!) Mendel deduced the laws of inheritance

combinatorial control

The control of gene expression in which more than one regulatory protein is used and expression is allowed only in a specific combination of conditions.

sequence assembly

The process in which short nucleotide sequences of a long DNA molecule are arranged in the correct order to generate the complete sequence. - the sentence fragments can be assembled in the correct order according to their overlaps and the original complete sentence reconstructed

bivalent

The structure formed by the pair of homologous chromosomes during crossing over. Also called a tetrad because it consists of four chromatids.

genome sizes

There is no relationship between genome size and organismal complexity among eukaryotes. - the amount of protein encoding genes does not coincide with size of genome - here is the number of base pairs - an amoeba has a way larger genome than us - a locust and an onion have larger genome than humans

Retrotransposons

Transposable elements that move within a genome by means of an RNA intermediate, a transcript of the retrotransposon DNA.

Does each gene have its own shine delgarno sequence in the polycistronic RNA?

Yes! Shine delgarno sequences in polycistronic RNA mediate ribosome binding in prokaryotes. In mRNA, each gene has its own shine delgarno sequence in the RNA. The pattern goes stop codon, shine delgarno, start codon on the polycistronic mRNA

synonymous mutation (silent mutation)

altered codon specifies the same amino acid - a nucleotide substitution that does not change the amino acid

eukaryotic DNA is packaged as chromatin

a complex of DNA, RNA, and proteins that gives chromosomes their structure. When the chromatin is coiled, the proteins that carry out transcription cannot access the DNA. The chromatin must unravel to allow space for transcriptional enzymes and proteins to work. This is accomplished by chromatin remodeling.

a zygote is a fertilized egg

a diploid cell that gives rise to all of the cells of an organism

constitutive expression

a gene is always expressed

pea plants were Mendel's model organism because they had easily detected

alternative phenotypes (outward characteristic) that correspond to heritable traits

constitutively chromatinized

always tightly packed

the lac operon

a gene system whose operator gene and three structural genes control lactose metabolism in E. coli - the lacI is the structural gene for the REPRESSOR protein - lacZ and lacY are called structural genes because they code for the primary structure of proteins - refer to image in powerpoint 1. the large red piece is polycistronic mRNA - one piece of mRNA with two different proteins 2. Yellow and green segments are open reading frames 3. the double hatch indicates, not right upstream but somewhere far away 4. lacI is a repressor/negative regulation that has its own genes and one mRNA piece 5. the repressor protein binds to the orange operator and stop the long red RNAN from being made and the yellow and green proteins from being expressed d

lactose lac operon null mutants

a mutation in lacI (the repressor) does not produce functional repressor, so lacZ and lacY are expressed in the presence and absence of lactose

nonsynonymous mutation (missense mutation)

a nucleotide substitution that changes the amino acid is a nonsynonymous mutation (missense mutation )

reciprocal cross

a paired cross in which the respective traits of the male and female in one cross become the respective traits of the female and male in the other cross - the difference of these two reciprocal crosses is how you know it is a sex linked trait

sex-linked inheritance patterns can be observed by different outcomes o reciprocal crosses

a reciprocal cross is one in which you switch the sex off the parent affected by a phenotype

the primers of PCR define what is being

amplified - GPS of DNA polymerase ex. you can design primers to amplify your gene for eye color to pull it out and then use it somewhere else

What is an operon?

an operon is a region of DNAn that includes the coding sequence for multiple genes that get transcribed together into a single molecule of polycistronic mRNA

methylation of a CpG island can change over time or in response to environmental cues, providing a way to turn genes on or off. Repression of a gene is often

accompanied by heavy methylation of a nearby island

posttranslational control

activate of inhibit protein (by chemical modification) ex. a protein might only be made in its inactive state and have to be phosphorylated to become active

both repressors and activators have multiple

active sites

preinitiation complex

affected by chromatin folding, histone covalent modification, and DNA covalent modifications - DNA - methylation of CPG histone n- acetylation vs no acetylation chromatin - eu or hetero - these all converge to influence the efficiency by which this preinitiation complex influences the rate of transcription - all these factors are influencing the rate of which RNA polymerase is going off to influence the gene - how on or how off the gene is, very multifactorial

many of the processes (DNA replication, transcription, translation) we've discussed so far depend on reliable pairing of complementary bases, but spontaneous and random mutations occur

all of the time during the time during these processes as they are not perfect

a homozygote contains two identical

alleles

homologous chromosomes contain different sets of

alleles

independent assortment hypothesis

alleles for different genes do not stay together when gametes form

the independent assortment hypothesis

alleles of two genes are sorted into gametes in a statistically random fashion - this is what Mendel observed - no just two options - do not need to match the parent cell's gamete pairings ry and RY - instead, it is statistically random so there is RY, Ry, rY, and ry (no longer ½ to ½ ratio but not ¼ x4) - only 1/16 of the F2 prodigy yare going to get all recessive alleles - ¼ of the gametes of one parent is going to ry and one quarter of the games of the other parents is going to be ry so ¼ time ¼ equals 1/16 - the reason that there are 3/16 is because there is two possible genotypes

cells with an error in chromosome number are

aneuploids - the resulting zygote = fertilized cell from two gametes would never be able to develop into a full embryo and ever be born - majority of human miscarriages = embryo is genetically improper the cases where the baby is born alive is where there is an error in a sex chromosome - female triple x - male that is xxy males xyy - you can survive and be fine with this only other case where you can survive to adulthood is trisomy 21 - 3 copies of chromosome 21 = smallest human chromosome = has the fewest number of genes - why it is survivable - trisomy in 13 and 8 - you can survive to birth sand then usually die soon after

mutation

any heritable change in genetic material

meiosis produces 4 different gametes which

are genetically different from one another

looping occurs at many eukaryotic genes because regulatory sequences - enhancers, binding sites for transcription repressors -

are often located far from the gene promoter that they regulate - general transcription factors recruit RNA polymerase - the dark purple complex is called the mediator complex which can bind to regulatory transcription factors which are themselves bound to a recognition sequence (red) of the DNA - the recognition sequences/enhancer sequences do not have to be terribly close - break = not that close - most of the times you have to loop the DNA around to influence the mediator complex and thus RNA polymerase - mediator complex transmit information to cause RNA polymerase to activate or be repressed

Mendel's second question

are traits inherited in groups or independently of each other? independent or dependent assortment?

mutations can be fixed by they

aren't always - some mutations are small, while others are large

plasmid and vectors are two types fo self replicative DNA molecules. Plasmids are the extra-chromosomal elements, naturally occurring inside the bacterial cells. Vectors are artificially introduced DNA molecules into the cells. Plasmids do not carry essential genes for the functioning of the

bacterial cell

DMD gene is X linked

because males have only one X chromosome, duchenne muscular dystrophy usually affects males only - vary rarely affects females

one repressor/activator can respond to say heat and can

bind to multiple places on the chromosome at multiple different operons or activator sequences. Thus, there are a series of activators and repressors that can control many different things.

Small regulatory RNAs regulate gene expression by either:

binding to transcripts and blocking translation Binding to transcripts and causing degradation

activator

binds to the activator site which is upstream of the promoter and helps the RNA polymerase start transcribing or increasing transcription

positive regulator (aka activator)

binds to the binding site and increases the ability of RNA polymerase to bind to the promoter and transcribe

negative regulator (aka repressor)

binds to the operator and blocks the RNA polymerase from either binding tot he promoter or moving forward din transcription

a karyotype is the

complete set of chromosomes in a cell

large scale mutations

complete sets of genes being flipped around, inverted, completely deleted, translocated from one chromosome to another, or being inserted from one chromosome to another Note: large insertion/deletion or small insertion or deletion

But if you added a plasmid in with a completely fine operator the cell would

contain one mutant and one normal copy of lacO (operator), lacZ and lacY are expressed in the presence and absence of lactose because the mutant is always expressing lacZ and lacY and the normal is only expressing during presence of lactose, but all together they are expressing lacZ and lacY all the time

chromosomes "encode" or

contain the sequence for genes

Punnett squares are a

convenient way to analyze a cross (breeding between two genotypes) and predict the proportion of progency genotypes

crossing over and independent assortment

crossing over contributes to independent assortment of alleles on the same chromosome - Crossing over - same chromosome - contributes to independent assortment of alleles that are on the same chromosome - pink and black mixing reason for independent assortment to be observed even when the two genes are on the same chromosome

maternal and paternal alleles are mixed up by

crossing over during late prophase of M1

Bacteria have enzymes to recognize and

cut DNA that is not in their chromosome but (only cut along a very specific sequence)

instead of doing PCR, you could simply

cut your DNA

denaturation (PCR step 1) - 98 degrees celsius

denaturation = tube containing DNA, boil, split up the DNA inside our cells → 98 degrees, have our DNA split open

during chromosome remodeling, the nucleosomes are repositioned to expose

different stretches of DNA

the trp operon

different than the lacI, the tryp repressor is only able to bind to the operator when bound to tryptophan, the end product. here the repressor acts as a sensor for the end-product of these genes high tryptophan = repression low tryptophan = de-repression Note: this makes sense, if the cell has plenty of tryptophan, it would stop creating more tryptophan

The reappearance of the intact recessive "particle" in the F2 generation after its disappearance in the F1 means it must have segregated away from the dominant trait; pea plants are diploids and their gametes are haploids

everybody has two particles, alleles - synonymous with the idea that these plants are diploids - two versions of each of these genes but when the germ cell makes the gametes, these particles/alleles separate from one another - so when you make your offspring by chance, some of your offspring will get some of the wrinkled traits and some of the offspring will get the round traits - When F1 makes its gametes the round and wrinkled traits must have segregated away from one another

Plasmid: selectable marker

expresses a phenotype that distinguishes cells that contain the plasmid, and those that do not

when a mutation in the extra copy of the gene is beneficial to the organism's survival, the

extra copy can become a new gene

genomic sequencing

figuring out the order of DNA nucleotides, or bases, in a genome

an allele is just a sequence variant between two chromosomes which can code

for gene or does not have to code for a gene

the chromatin folding state, histone covalent modifications, and DNA covalent modifications influence the efficiency with which this complex

forms, ultimately determining txn level (and therefore amount of mRNA produced, and therefore amount of protein made)

the gamete cell can join with a

gamete from another by fertilization

restriction fragments

gel electrophoresis separates the resulting DNA fragments according to their size (in kb or thousands of base pairs) - the fragments can also be visualized in the gel and extracted from the gel for further analysis or manipulation

alleles are different sequence versions of the same

gene

mutations in a generation vs replication

generations over lifetime fruit fly = less cells, does not live as long

very little of what is in our genomes is protein encoding

genes

a genome is the

genetic material transmitted from parents to offspring - your genome is made up of your chromosomes - we can figure out a person's genome through sequencing

the reason we sexually reproduce is that it mixes up the

genetic pool, this mitigates the negative effect of random recessive mutations and creates a more diverse population

transcription factors have to find their binding sites in a vast

genome of sequence, can vary in length

in mammals, meiosis occurs only in

germ cells of the gonads to (ovary and testis) to generate highly specialized haploid gametes

to do a Punnett square, write each possible

haploid genotype for each parent's gametes in a line on the X or Y axis o fa grid, then fill in the coordinates

CpG methylation at a gene promoter region usually brings about

heterochromatinization and transcriptional silencing the gene

an organism can be homozygous for one gene and

heterozygous for another

green histones are part of the pink protein - float around freely and undergo many different types of post translational modifications → chemical groups are added later after transcription (methylated, acetylated, depending on what pattern of histone tail modifications there are on a particular nucleosome → can cause transcription machinery and factors to be better recruited or lesser recruited to the area

histone tail acetylation is usually associated with being more transcribable being deacetylated or not having histone groups --> being associated with being transcriptionally silent

two different versions of a chromosome containing different DNA sequence variants (alleles) are called

homologous chromosomes

the key to remembering the difference between meiosis and mitosis is that

homologous chromosomes only pair in meiosis, in mitosis homologous chromosomes ignore each other - therefore they only separate in meiosis into two haploid cells

the x and y chromosomes act as a

homologous pair during meiosis - 50 percent of sperm contain the x chromosome and 50% of the sperm contain the y chromosome - nondisjunction o sex chromosomes leads to viable aneuploids --> XXY - physically attach to one another during prophase in tetrad to allow for crossing over - the gamete of the male is going to determine the sex of the baby 100% of the time

the X and Y chromosomes act as

homologous pairs during meiosis

crossing over =

homologous recombination, where each chromosome is joined at the exact same spot to the paired homolog

in humans there are 22 autosomes and one pair of

homologous sex chromosome

transformation - putting the recombinant DNA into bacterial cell is based on the idea that

in recombinant DNA, there is some sort of selectable marker that allows you to know that the plasmid is inside of the cell

an organism can be homzygous for one gene and heterozygous for another

in this P1 generation, the parents are homozygous, RR and rr the F1 generation must be heterozygous, Rr and Rr when F1 made its gametes some were big R and some were little r 25 percent of the time, the outcome is going to be little r, little r in F1, the capital R trait is dominant over the little r traits, overpowers it in terms of phenotype

need a dihybrid cross to figure out (crossing two traits such as Y and R)

independent assortment

most polygenic traits are considered complex, meaning that they are

influenced by environmental factors such as diet

mutations of a somatic cell are NOT

inherited by the next generation

regulatory transcription factors work by interacting physically with the basal transcription machinery and influencing whether transcription

initiation occurs 1. a gene typically has several different enhancer sequences, each with its own set of regulatory transcription factors 2. general transcription factors bind to the promoter and transcriptional activator proteins to bind to enhancers 3. through looping of DNA, transcriptional activator proteins, mediator complex, RNA Pol II, and general transcription factors are brought into close proximity, allowing transcription to proceed

Our goal in cloning is to

insert a target gene (e.g., for human insulin) into a plasmid. Using a carefully chosen restriction enzyme, we digest: - The plasmid, which has a single cut site - The target gene fragment, which has a cut site near each end - Then, we combine the fragments with DNA ligase, which links them to make a recombinant plasmid containing the gene.

a vector is everything on the plasmid except the

inserted gene

lactose lac operon promoter or regulatory sequence mutations

instead of having a mutation in the lacI DNA sequence, you could synthesize a mutation in the operator region, by changing a few base pairs, and the operator would not be able to bound by the repressor protein. Even though there is a functional version of the repressor it would ddbe unable to bind. This would be constitutive operator. A mutation in lacO was (operator) prevents repressor protein from binding, so lacZ and lacY are expressed in both the presence and absence of lactose.

heterochromatin and euchromatin can be

interconverted by enzymes - extracellular signals can cause changes to your chromatin and thus, changes to your gene expression via chromatin

Most of our genome sequences are almost exclusively noncoding DNA

it is the differing amount of these noncoding sequences that in large part account for the C-value paradox - because a lot of those regions of genomes are non coding - only some genes actually code for DNA ex. introns get cut out - blue slice is only what is actually coding for proteins - more alpha satellites than protein coding genes

not all plasmids are vectors if they are not

manipulated

homologous chromosomes separate in

meiosis I

meiosis occurs in two divisions called

meiosis I and meiosis II

microRNNAs and small interfering RNAs are gene and sequence-specific ways of bringing about gene silencing post transcriptionally

microRNAs and siRNAs are examples of noncoding RNAs that will never be translated into proteins.

mutation per replication

mutation rates = chances of mutation per nucleotide during one round of replication - measure of fidelity or DNA or RNA polymerase in replication - how well DNA or RNA polymerase can repair and fix mutations generation = - combination of fidelity plus how complex the organism is and how long the cell is going to live/how many replications are going to occur

duchenne muscular is an x linked disease caused by

mutations in the dystrophin gene

independent assortment of genes in different chromosomes reflects the fact that nonhomologous chromosomes can (random orientation at metaphase plate)

orient in either of two ways that are equally likely - if you inherited from one parent dominant A and dominant B and from another parent recessive a and recessive b they could orient at the metaphase plate so that the dominant alleles are on the same side and end up in the same alleles and the recessive alleles are on the same side and end up in the same haploid gametes together BUT... it is equally likely that the dominant A would be on the same side as the recessive b and the dominant B is on the same side as the recessive a - orientation of homologous chromosomes at the metaphase plate in meiosis 1 = causing the random assortment of alleles of the dominant and recessive alleles in the F2 haploid gametes used to produce F2 diploid chromosomes

all human cells are diploids except for

our gamete cells

The lack of "blending" and discrete nature of traits was called

particulate inheritance" by Mendel. In other words, a trait maintains is integrity through generations. - particle is a gene - the particle that is being inherited is the allele - the allele is a gene - alleles are different versions of the same gene dominant allele is capital R recessive allele is lowercase r

when you see a mutation at the nucleotide level, you do not necessarily see it at the

phenotype level

codominance occurs when two alleles are

phenotypically expresses simultaneously; in other words, heterozygotes express both phenotypes

after PCR, you are not sure what the amplified DNA does so you can put it on a

plasmid and have the bacteria make ton of the DNA for you and inserted a polymerase to begin transcription and translation

remember

polycistronic mRNA - polycistronic mRNA is a mRNA that encodes several proteins and is characteristic of many bacterial and chloroplast mRNAs

you can either look inside your DNA to see if there is a restriction enzyme cleavage sequence or you can design the cut site into your

primer

in the absence of tryptophan, the pressor dissociated from the operator, and RNA synthesis

proceeds

Plasmid: promoter

used to express the gene encoded into the MCS

qPCR

using the dye to see the amplification of a product in real time - you can use the is to quantify DNA with no reverse transcription needed

mendel used true-breeding/pure breeding strains whose phenotypes had not varied generation after generation-this is why

we can assume all traits began in the homozygous state

the protein dystrophin anchors the long actin filaments to the membrane of muscle cells via glycoprotein interactions. The same glycoproteins anchor the cell membrane to the basal lamina in the extracellular regions

when there is no dystrophin; when a muscle contracts it rips open the plasma membrane from mechanical strain 8

lac operon without lactose

when there is no lactose present, the lacI repressor protein is made and binds to the operator. Thus, stopping RNA polymerase from being able to transcribe Note: when there is no lactose present, there is no need to make these proteins that are responsible for helping to break down lactose. Why waste the energy?

PCR in reality is not nearly as clean as we present it in class. There could be just a little bit of RNA that you make that might anneal partially with the COVID primers (consequently maybe I don't make that RNA), so you'd get a little bit of a product even though you have no COVID. Thus we set the machine to only look above a certain threshold (essentially trying to separate the signal from the noise). To that point, because you are taking a snapshot of the reaction at the end of each amplification cycle, you can't very well run a gel each time and see what's there- the much more efficient method is to have a dye in the tube that binds to ds DNA products- so that you can see when a product is made just by having an increase of fluorescence---however, you don't distinguish if that product is COVID or some background little amplified piece of DNA discussed above--thus you set a threshold.

why there is a threshold

mutations in X-linked genes result in predominantly male disease called

x linked diseases - mutations in x chromosomes are really bad for males because they only have one shot of it - only have one x chromosome so they are particularly vulnerable

once you have amplified a gene or got it out of a chromosome

you can ligate it to a plasmid

in PCR

you harness the power of a DNA polymerase from a bacteria

meiosis in ovary vs testes

you make 4 different haploid gametes but one of the females 4 gametes for one germ cell, 1 cell gets the majority of the cytoplasm and the other three cells get nothing, one chosen haploid egg cell gets the most cytoplasm and becomes the haploid egg oocyte

segregation

your alleles separate from one another into gametes = make haploid gametes