Molecular genetics Flashcards

Terms Definitions
Enhancers
DNA sequencesorientation independentact at long distancescontain several binding sites for multiple transcrription factorsassociated with DNase I HS sites Have specific binding sites transcription factors that are only produced in that cell.
ribosomes: eukaryotes
40s+60s=80s ribosomal subunits(18s)+(5s+5.8s+28s) rRNAs
introns
a non-coding, intervening sequence within a eukaryotic gene
subviral pathogens
mostly found in plants
Types of alternative splicing?
see script
ribose
a white, crystalline, water-soluble, slightly sweet solid, C5H10O5, a pentose sugar obtained by the hydrolysis of RNA.
 
 
 
How does mRNA differ from DNA?
 
PEV
position effect variegation- the same gene in genetically identical cells can have different expression patterns. Due to heterogeneous patterns of chromatin structure and modification.
HDAC
Histone deaetylases act to decrease gene expression
frameshifting
-ribosome moves back (or sometimes forwards) one nucleotide while reading the mRNA-induced by things such as hairpin loops, RBS site upstream, or a codon at the frameshift site that calls for a tRNA with a wobble site that creates a weak codon-HIV uses this to create long and short proteins from one RNA
What are they?
Initiation, elongation, and termination
What is GWAS?
Genome wide association study.
Responsible for many of the new discoveries in complex diseases

Families not needed, since not looking for “linkage” (co-segregation of alleles and phenotype)

Summary data consists of p-values for selected phenotypes, allele frequencies, etc.

All results are added into dbGaP database.

Recently, privacy issues have caused this data to be pulled offline.
Hershey, Chase
1952- Used radioactive material to label DNA and protein; infected bacteria passed on DNA; helped prove that DNA is genetic material not proteins
Epigenetic effects
changes in gene expression patterns that are heritable but do not involve changes in the DNA sequence of the genes involved.
gene-related DNA sequences
pseudogenes, gene fragments, introns, and UTRs
virusoids
small circular ssRNA like viroids but are dependent on a helper virus for replication-hepatitis delta virus-rolling replication, producing a concatomer RNA
X-ray crystallography
-producing highly pure protein in large quantities suitable for crystalization-exact location of every atom
slo proteins
-determine the auditory properties of the hair cells in the cochlea-40,000 different splice patters
RNA polymerase, what are the subunits?
 
Binds where?
-->holoenzyme:
 
see script
 
--> binds the promoter with help of sigma factor.
What is RT AKA?
RNA-Dependent DNA polymerase (telomerase)
What type of process is binary fission?
Asexual
Mitosis
- Consists of production of identical daughter cells.
- Chromatids are dragged into daughter cells by their centromeres.
- Daughter cells are diploid with normal chromosome number of 46.
What is FISH?
Fluorescence In Situ Hybridization. Can be used to identify a wide variety of syndromes.

Each probe is specific to one region of a chromosome (pair), and is labeled with fluorescent molecules throughout it's length.

Step 1 - break apart (denature) the double strands of DNA in both the probe DNA and the chromosome DNA so they can bind to each other.

This is done by heating the DNA in a solution of formamide at a high temperature. 

Step 2 - the probe is placed on the slide and a glass coverslip is placed on top.

The coverslip is sealed with rubber cement.
The slide is placed in a 37 C incubator overnight to allow the probe to hybridize with the target chromosome.
        
protein
any of numerous, highly varied organic molecules constituting a large portion of the mass of every life form and necessary in the diet of all animals and other nonphotosynthesizing organisms, composed of 20 or more amino acids linked in a genetically cont
promoter
region of DNA that indicates to an enzyme where to bind to make RNA
Genomic imprinting
a form of epigenetic regulation that regulates transcription of a homologous pair of alleles based on which parent the gene was inherited from.
Affinity purification
Determines what protein is binding to the specific sequence by using a column.
filamentous virions
-have coat proteins coating the viral nucleic acid helix-other coats protect a dsDNA core, attached to a proteinaceous tail (T4) or without tail fibers (lamda)
functional domains
delineated by treating a region of purified chromatin with DNAse I which, being a DNA-binding protein, cannot gain access to the more compacted regions of DNA
Nuclear pore
-allowing mRNAs among other things to come in and out of the nucleus
What did Ham Smith discover?
Specific restriction endonuclease (HindII)
Steps in DNAse footprinting:
1.Attain strand of DNA2.Bind Protein3.Add DNAse (low conc) then remove protein and denature DNA4.Use electrophoresis to see the gel5. locate sequence where protein boundDNA-BAEL
For example?
There might be cleavages and or additions at the terminal ends of the chain
Types of Variation
- Insertions and deletions
- Short tandem repeat polymorphisms (short runs of simple DNA sequences 1-4 bp in length. Similar to CNV, often used as markers.
- SNPs. Young SNPs are those that have arisen recently in human history, and will have a low frequency of the least common allele.
- Copy Number Variation involves large (>1kb) segments that vary in number from one to several copies. These show up again in anticipation diseases.
What is xeroderma pigmentosum?
Relatively rare autosomal recessive disease caused by a defect in DNA nucleotide excision repair. Caused by mutations in any of 7 XP genes.

Sunlight sensitivity as well as cigarette smoke carcinogen sensitivity.

High incidence of skin cancer.

Neurological abnormalities.
rrna
a type of RNA, distinguished by its length and abundance, functioning in protein synthesis as a component of ribosomes.
Gel Electrophoresis
DNA fragmens are applied to a gel, electricity is used, smaller fragments move quickly, this creates bands
lytic cycle
-host cells are starved and the viral sequence inserts itself into the host genome (a prophage) and is released
Initiation in eukaryotes
-eIF-2, GTP, and initiator tRNA bind to the mRNA-then binding of the 5' cap-eIF-4G serves as a bridge between eIF4-A, eIF4-E and eIF 3-poly (a) tail also helps with initiation via association to 5' end
Besides RE and ligase a simple gene cloning experiment needs
A Plasmid
When pHpI?
pH +Charge of protein A
pH>pI --> -Charge of protein A
How big is the melted region?
about 12 nucleotides
What forms as replication proceeds in a given direction?
A replication fork
What is the process of viral replication and extrusion in animal viruses called?
A productive cycle
Why Study Mendelian Disorders?
- Numbers are large
- Relatively straightforward
- Have high predictive value for identifying systems involved in particular disease. Identifying the protein coded for by a particular mutant gene and the system(s) in which it participates suggests other candidates for disorders with similar phenotypes, or other genes involved in the same complex trait.
What is the most efficient point to control protein expression?
Transcriptional control.
Vancomycin & Vancomycin Resistance
- Vancomycin, like pen., targets cross-linking of the peptidoglycan side chains. Unlike pen. it binds the terminal D-ala-D-ala directly and sterically prevents its cleavage by the transpeptidase.
- Gram- bacteria are innately resistant to vancomycin b/c it's too large to pass through the outer membrane.
- Resistance occurs from a multigene operon, the net effect of which is to replace the terminal D-ala with D-lac. Crosslinking can still occur, but vancomycin no longer binds.
Outline the steps in translation
Initiation: ribosome binds to nRNA strand. translation begins when the ribosom reaches the start codon
Elongation: transfer RNA is responsible for carrying the amino acids. methionine is carried to the p site. the second tRNA enters the A site and a peptide bond formss between methionine and the second amino acid. this process continues until it reachesn the stop codon
Termination: ribosome reaches stop codon. a release factor recognizes the stall form the ribosome and causes the subunit to detach the nRNA and the new polypeptide is released
Genome percentages
30 percent of the genome is known, by comparitave genomics another 30 % was tentatively identified
controlling gene expression at initiation checkpoint
-determining which genes are switched on-not as many control points because secondary regulation involves all steps after transcription initiation
What does X-Gal do?
A colorless compound that is cleaved by B-galactosidase into a blue dye Xgal-->Galactose + Blue
What are some advantages to using bacteriophages?
1.Infect cells more efficiently2.clones are not colonies, but plaques on a lawn of bacteria 3.Replace some phage genes with foreign DNA4. can accept larger pieces of DNA 5. Often used to make genomic libraries
Does Sigma actually get recycled?
Maybe not... there is another model
What are episomes?
They are plasmids that are capable of integration into the bacterial genome
What does each amino have?
It has its own aminoacyl-tRNA synthetase
What Makes an Allele Recessive?
- Usually passed from asymptomatic carrier parents to one or more children.
- May result from loss of function, often of an enzyme which can serve its purpose even if only 50% is present.
- Each of us are estimated to be carriers of 5-10 extremely deleterious (lethal) recessive alleles.
- Can be multiple recessive alleles at a locus, allowing for homoallelic or heteroallelic abnormal individuals.
Southern Blotting
- A DNA sample is treated with a specific restriction enzyme.
- Gel electrophoresis is used to separate DNA fragments of different sizes (large fragments migrate slower b/c they bump more).
- Transfer the separated pieces to a membrane by changing direction of electric field.
- Expose the membrane to a labeled hybridization probe.
- See whether your fragment of interest (mutation, say) is present in your original DNA sample.
Prader Willi syndrome (uncontrolled eating) is...
A deltion in paternal chromosome 15.

If the deletion is on maternal chromosome 15 the result is angelman ("happy puppet") syndrome.
What type of bond exists between the base and sugar of nucleic acids?
N glycosidic bond
Cis-Acting Elements Regulating the Average Transcription Unit
- Promoter
- Enhancers
- LCR
- Splice donor and acceptor sites
- Cleavage/Poly-adenylation site
Name two things the process mitosis is used for.
cell division and cloning
helix-turn-helix DNA binding motif
-alpha helix on the surface of the protein in an orientation that enables it to fit inside the major groove-turn allows for positioning of the alpha helices
What is another method to get more BP cloned?
use a bacteriophate.
What is the basis of Ion-Exchange chromatography ?
Separate substances (usually proteins) according to charge via a resin
What can initiate transcritpion immediately?
Tight complexes b/w the Holoenzyme and T7 DNA
What happens to retroviral DNA?
It becomes integrated into the host DNA
What is messenger RNA?
mRNA carries the complement of a DNA sequence and transports it from the nucleus to the ribosomes, where protein synthesis
What does it do?
It is necessary for the initiation of DNA synthesis
What is a chromatosome?
The bead.

a nucleosome + H1 +166 bp to complete a complete turn since the DNA in the nucleosome only covers 1 3/4 of a turn.
How do transcription factors actually bind DNA?
Involves hydrogen bonds, ionic bonds, hydrophobic interactions, etc.

Certain DNA-binding motifs from TFs have been identified:

Helix turn helix motif:
Composed of two a-helices connected by a turn. The so called recognition helix is the part that actually binds the major groove and is on the carboxylic acid end of the molecule.
Some helix-turn-helix TFs have two motifs that bind to adjacent major grooves.

Zinc finger motif:
Characterized by two b-sheets, a turn, and one a-helix. Also contains a single zinc atom and is named for the aa that hold the zinc. CYS-CYS-HIS-HIS is a common one.
The alpha helix is the part of the zinc finger that actually binds the major groove.

Other motifs include b sheets, leucine zipper, helix-loop-helix, etc.
How to Isolate Mutants
- The easiest way is direct selection: pour on antibiotics, and only those bacteria that mutate a resistant gene will survive.
- You can use successive cycles of enrichment to select for auxotrophs, as penicillin will not kill any bacteria that are not dividing (b/c they can't synthesize whatever AA is absent from the medium).
- Replica plating: Grow colonies with a supplementary compound, use velvet to replica plate them onto plates w/o that compound, and see which colonies fail to grow.
5' cap on a eukaryote
-just a GTP added backwards onto the 5' end of an mRNA-5'-5' phosphodiester bond-guanyl transferase, followed by guanyl methyltransferase-the methyl is the cap-all RNA are capped in eukaryotes
Gene cloning Step 1: ___A YGI from among thousands of genes 2. ___B YGI to bacterial (or phage--virus taht infects bacteria) plasmid DNA3. ___c recombinant plasmid into bacterial (or phage) host by ______d 4.___E individual cells/clones5. ____F sufficient
A. IsolateB. LinkC. IntroduceD. TransformationE. SeparateF. Make
In Gel filtration Chromatography, what molecules move slower?
Smaller molecules b/c they have access to space larger ones dont
Why is Leading edge fret useful?
Decrease in FRET efficiency if sigma is released. If sigma is not released, FRET will be increased in energy(probes get closer. If sigma is released Fret will be decreased (distance increases) FRET goes down.
What happens to the leading strand?
It is continuously synthesized by DNA polymerase in the 5’ ‡ 3’ direction
What is copy number variation?
Certain regions of the genome vary in the number of copies of specific DNA sequences.

Can range in size from 10kb to 5Mb.

May be involved in disease susceptibility.
What are the molecule sinvolved in the export of mRNA to the cytoplasm?
Heterogeneous ribonuclear proteins are bound to the RNA in the nucleus when it is mature.

One of these is the nuclear export receptor which bind snear the poly A tail and another is the CBC protein which binds to the 5' cap.

The complex is transported through the nuclear pore by an ACTIVE process.

In the cytosol, CBC is displaced by initiation factors for translation.

The nuclear export receptor is also displaced and recycled to the nucleus from the cytosol.
Gram+ vs. Gram- Bacterial Envelope
- Look at picture from notes.
- Gram- bacteria have two membranes, the second of which contains LPS. LPS contains Lipid-A (the cell's endotoxin and a permeability barrier to large or hydrophobic molecules), a core, and a species-specific repeating polysaccharide sticking out into the medium (O-antigen).
- Gram+ bacteria have super-thick peptidoglycan wall, which prevents gram stain (purple) from escaping. They have lipoteichoic acids that are involved in antigenic identification.
- Gram+ bacteria have teichoic acids extending out from both the membrane and the peptidoglycan, which is immunogenic.
transcription is much slower in action than on paper
-due to pauses and working around nucleosomes
What passes through faster in Gel filtration chromatography? Small or Large?
Large molecules move faster b/c they are excluded and emerge at void volume
Describe the DMS melting experiment thoroughly. 7 steps
1. Start with T7 labeled promoter. 2. Bind RNA polymerase3. Melt a particular region forming an open complex by adding a polymerase.4. Add DMS (this will methylate all the free A's ) 5. Remove polymerase6. Use S1 to cut single stranded DNA 7. Run a gel to see where the S1 nuclease cuts the single stranded DNA
What is mRNA synthesized from?
A DNA template in a process similar to DNA replication
Linkage, the Exception to Mendel's Law of Independent Segregation, and LOD
- Linkage is when a parental combination of traits occurs more often than expected among F2 generation.
- Genetic distance between genes A and B = number of recombinant gametes / total gametes * 100%, with 1 map unit (cM) as a recombinant frequency of 1%.
- Linkage analysis is an attempt to link a phenotype (disease) to a DNA marker whose position is known.
- Log Of the Odds is used to compare the heredity of a phenotype and a marker in a particular family and predict whether they're linked.
- LOD = log10 (likelihood of data if two loci are linked by a certain distance) / (likelihood of data if they're not linked). LOD of 3 or higher is considered definite evidence of linkage, and the distance producing the highest LOD is taken as the map distance.
What do we mean when we say that siblings are concordant?
They both have the trait.
What does HindII Create (blunt or sticky ends)?
Blunt end b/c it cuts in the middle
What is the first step in Ion-Exchange chromatography ?
Determine the charge of the protein using 1 positive and 1 negative column and figure out where the protein sticks.This is called ANION/CATION Exchange
Why is Trailing edge fret useless for our purpose?
Whether sigma is released or not released doesnt matter because the distance b/w the donor and acceptor will increase regardless (6.13a) Increase distance=decreased FRET. Either way, FRET is decreased and this is useless
What happens once viral progeny are assembled?
They may be released either by lysis of the host cell, or by extrusion, a process similar to budding
Synthesizing DNA and Site-Directed Mutagenesis
- We can synthesize DNA of up to ~100 bp in length.
- A growing nucleotide chain is anchored to resin. We wash a bunch of nucleotides over, each which has a lbocking group so only one NT is added.
- Wash off the excess nucleotides, remove the blocking groups, and repeat with the next nucleotide in the chain. Repeat until you have your full strand.
- Site-directed mutagenesis: If you want an A instead of a T at a certain point in a plasmid, synthesize a complementary chain with the replacement you're looking for. Allow the synthesized chain to bind to the original one, then use "tricks" of some kind to make the base mismatch repair processes resolve the discrepancy in favor of the new mutation.
What are the main conformations of double helical DNA?
B-DNA (Watson and Crick) is the most common.

A-DNA
Z-DNA
Why didn't all of the organeller genes get transferred to the nuclear genome
some proteins need to be translated in the organelle due to hydrophobic interactions that would be difficult in the cytoplasm
How is the automated method set up?
Same thing as before, just you have a laser that emits a fluorescent light that is detected by a detector and then analyzed by a computer to give you nice sharp peaks
What does the large ribosomal unit do?
It binds to the small one, creating a complete ribosome with the met-tRNA complex sitting in the P site
What is the 1st step in cDNA cloning? and what is needed to do this?
Make DNA copy of RNA with Reverse Transcriptase
HMT
histone methyl transferase
What is dNTP ?
DNA
deoxyribonucleic acid, responsible for processes of hereditary in all plants and animals.
transcription factors
-can help initiate transcription-modify histones, bend DNA, bind the preinitiation complex-have an activation domain
Is transformation successful with linear DNA?
NO
RNA
ribonucleic acid, a natural polymer that is present in all living cells and that plays a role in protein synthesis
A-Adenine
U-Uracin
C-Cytosine
G-Guanine
Histone code
actively transcribed genes are associated with hyperacetylation of histones H3 and H4 and methylation of H3 lys 4, while the condensed heterochromatic and non-transcribed region are hypoactelylated and methylated at h3 lys 9
Histones
basic proteins found in nucleosomes; DNA-binding proteins
TBP
-TATA binding protein-binds via a beta-sheet, not an alpha helix-binds the minor groove
What is hyperchromicity?
 
At which wavelength of UV important for detection?
 
Effect depends on what?
 
Tm?
See script
What are exons?
They are coding sequences
Quorum Sensing
- Prokaryotic cell-cell ocmmunication that allows behavior (expression) modification as a function of cell density.
- Underlies biofilm formation, virulence, etc.
- Staph A: At low concentrations, bacteria express proteins for adherence and concentration. At high levels, they express proteins for toxicity and proteases.
- Utilizes a 2-component signaling pathway with a sensor histidine kinase autophosphorylating and then passing messages along to a DNA-binding/transcriptional activator domain.
Meselon, Stahl
1953- discovered DNA replicated semi- conservatively
Bromodomains
bind to acetylated histone tails and help prevent inappropriate spreading of silencing. At HMR, mutation of BDF1 or YTA7 genes lead to spread of silencing
30nm fiber
form of the interphase chromosomes; more condensed version of the complex
transposons
-mobile element which have a DNA intermediate as they transpose from one location (donor) to the other (recipient) in the genome-if a copy is left behind it is "replicative"; if not then "conservative"
constitutive expression
expression that results in continuous expression of a gene or a set of genes that is normally subject to regulatory control
RNAs associated with splicing
-snRNAs-small nuclear RNAs-whole complex is called "snurp"
Dipliod
refers to twice the number of chromosomes in a gamete. from 23 to 46 chromosomes.
What is important in footprinting?
1.DNAse2.DMS-DNA methylating agent3.Hydroxyl Radical
What occurs?
The ribosome advances 3 nucleotides along the mRNA in the 5’ ‡ 3’ direction
Imprinting
- A specific form of epigenetic modification where only one of two parental genes is expressed.
- The parent whose gene is expressed is maintained, but the gene silenced is reset each generation. Thus, a mutant allele could be passed down the paternal line without ever being expressed if that particular gene was always silenced in the chromosome inherited from mom.
- This can lead to trouble especially in uniparental disomy.
Which direction do newly synthesized strands made out of dNTP's by poylmerase run?
5'-3' direction
codon
a triplet of adjacent nucleotides in the messenger RNA chain that codes for a specific amino acid in the synthesis of a protein molecule.
exons
expressed sequence of DNA; codes for a protein
Intragenic suppression
second site mutation is within the same gene as the first mutation.
transposon tagging
-placing a lacZ reporter ORF (lacking its promoter) in frame in a transposon-if transposon lands in a gene lac z will be expressed yielding blue colonies-problem is screening all of the mutants
prions
-coded for by one of our own genes-mutated for PrPsc predisposes one to Creutzfeldt-Jakob disease-power to induce other PrPsc or even PrPc proteins to take one that same perverted, pathogenic structure
activating transcription factors
-must help the general transcription factors-without these transcription will not proceed
inverted palindrome
-RNA sequence folds into a stable hairpin, this RNA-RNA base pairing being favored over the DNA-RNA pairing that normally occurs within the transcription bubble
What is transformation in prokaryotes?
introduction of foreign DNA
Explain the sigma cycle
Upon promoter clearance, sigma dissocaites from RNA polymerase and joins new core to initiate another RNA chain.
Otherwise?
A portion of viral RNA functions as mRNA, which is translated into RNA replicase immediately after entering the hose cell
Genotype/Phenotype Correlations
- Modifier genes alter the phenotype associated with mutations in another gene.
- An allelic series is a set of mutant alleles at a given locus that give different phenotypes.
- Poor correlation between genotype at the locus and phenotypic severity indicates that other variables (environmental, modifier genes) play a major role in the disease.
F and R Factors
- Conjugation involves uni-directional transfer of information.
- F-factor is a plasmid that carries genes that allow it to be transferred via conjugation (sexual reproduction). It codes for the f-pilus, which attaches to specific receptors on F- cells.
- F' occurs when some bacterial DNA is incorporated into the F-loop, and then the areas right around it are sometimes transmitted. Only the flanking regions are transferred.
- High Frequency Recombination occurs when F loop is incorporated into bacterial DNA.
* During conjugation, DNA transfer begins in the middle of the f-factor (at the orit marker) and proceeds in one direction. Because it doesn't make it all the way around, some host genes are transferred INSTEAD of the F-factor.
* Recipient bacteria no longer become F+, because they don't get the whole F+ genes.
- Using independently derived HFR strains allows for chromosome mapping by the frequency of genes transferred.
adenine
a purine base, C5H5N5, one of the fundamental components of nucleic acids, as DNA, in which it forms a base pair with thymine, and RNA, in which it pairs with uracil.
List the following steps in order
 
1. spindel fibers contract
2. centrioles move to opposite poles
3. Chromosomes unwind into chromatin threads
4. nuclear membrane disolves
 
 
2, 4, 1, 3
nucleoid structure
40 to 50 supercoils around the protein core
aminoacyl-tRNA synthetase
-20 of these-the enzyme which charge a tRNA with an amino acid-one amino-acyl synthetase per amino acid
Define Result and Conclusion
Result—product of an experiment or other—raw data—observations Conclusion—results make up the conclusion, this is a general statement about data, interpret results
What are 3 common molecular separation techniques?
1.Chromatography 2.Electrophoresis3. Ultracentrifugation
Describe the sigma cycle
During initiation sigma can be recycled for additional use in a process called the sigma cycle.Core enzyme can release sigma which then associates with another core enzyme
Do any DNA viruses replicate and transcribe in the cytoplasm?
A few do
What happens with initiation?
Synthesis begins when the small ribosomal subunit binds to the mRNA near its 5’ end in the presence of proteins called initiation factors
Cri du Chat syndrome is a deletion on ....
5p
Reverse Transcription and Telomeres
- Retroviruses, which are like transposons that have gained the ability to leave the cell, encode a reverse transcriptase that copies RNA into 2-stranded DNA.
- Telomerase is a special form of reverse transcriptase.
* Telomeres appear at the end of Euk chromosomes and are short sequences of NT's repeated ~2000 times.
* Because Okazaki fragments require an RNA primer, the final one begins before the end of the linear template, and some of the telomere is lost.
* Telomerase uses a bound RNA template to form new DNA telomere repeats, which are then matched on the opposite side by DNA polymerase.
Outline the steps in transcription
Initiation: RNA polymerase binds the sense strand to the dna which opens the helix.
Elongation: rna polymerase starts building single stranded mRNA. uracil is used instead of thymine
Termination: rna polymerase reaches end of gene and recognizes this because of a specific termination sequence (stop signal). mRNA detaches from the DNA strand and the DNA reforms
over expression of a gene
lean to an unnatural phenotypepowerful promoters are expressed only in the correct tissue type
tyrptophan synthesis operon
-if trp is present RNA pol terminates its transcription at this point without reaching ORF-if trp is limiting, ribosome stalls and the hairpin loop is able to form
What else do expression vectors have? Why?
Inducible promoters Inducible promoters--one that can be controlled, turn on or off, you control when it is expressed or not expressed
What is Gel Filtration Chromatography?
Separate substances based on physical dimensions (size)
What are teh binding sites for the Fis protein?
Transcriptional activator
Where does translation occur?
In the cytoplasm and involves tRNA, ribosomes, mRNA, amino acids, enzymes, and other proteins
What does it consist of?
Either double stranded or single stranded DNA or RNA
Polymerase Chain Reaction
- Requires a DNA template that you want to multiply, 2 primers complementary to each end, taq or another heat-resistant DNA polymerase, and a bunch of all 4 dNTPs.
- As you raise and then lower the temperature, your resistant polymerase synthesizes complement chains to each of the two strands of your original DNA template, they "melt" and denature, and the cycle repeats.
- You can use mRNA and reverse transcriptase for RNA PCR.
What are some of the physical characteristics of fragile X syndrome?
Elongated face.
Large ears.
Autistic behaviour.

Often maternally carried.
Triplet repeat disease.
What is the HapMap project?
An international effort to catalog genetic variation in multiple ethnic groups. Provides a reference for correlation between SNP's.

Can be used to select SNP's for genetic association studies.

Correlated SNPs are said to be in ”linkage disequilibrium” (LD). This actually means that they are highly correlated with each other despite the name.
Regulation in Prokaryotes by Specialized Sigma Factors
- Sigma factor initiates transcription by joining RNA polymerase and binding to the Pribnox box and -35 sequence.
- Sigma factors in the RNA polymerase holoenzyme can be replaced sequentially in response to different biological stimuli.
- For example: when sporulation genes need to be expressed, they require the prior translation/expression of specialized sigma factors.
- Different sigma factors recognize different promoter sequences.
Descripe Gal4p activation domain
They are acidic with high levels of aspartic and glutamic acid. They also have key hydrophobic residues. These residues are though to form adhesive surface for protein interaction. The activation domain function is regulated recruitment of proteins important for transcription.
GU-AG mechanism
-2' OH on the A of the branch site attacks the phosphate of the terminal G--TRANSESTERIFICATION-2'-5' a-g phosphodiester bond resulting in a lariat-3' OH of first exon attacks the 5' phosphate of the 2 exon
What results in Recombinant molecules?
Cloning DNA pieces (i.e. cutting from one source and pasting into another molecule)
What are 3 types of Gel electrophoresis?
1. DNA Gel electrophoreis (agarose/acrylamide or pulsed-field)2. Protein Gel electrophoresis3. 2D Gel electrophoresis
How many bonds does T always form with A?
Two hydrogen bonds
What is the inducer?
It is usually the substrate, or a derivative of the substrate, upon which the enzyme normally acts
Outcome of Cellular Infection by Viruses
- A _cytocidal_ infection rapidly kills the cell and produces a burst of new infectious diseases.
* Acute infections are usually cleared w/i weeks because of strong immune responses to the virus.
- A _persistent_ infection infects the cell and produces infectious virus without affecting the cell's viability.
* Persistent infections are not cleared b/c of weak immune response or limited access to the site of infection by the immune system (as in the brain). Virus and viral proteins are continually produced, as in HIV and Hepatitis.
- A latent infection has no detectable viral products and no effect on the cell, but can later become cytocidal.
* Latent virus infections result when the virus is not cleared initially, and the virus exists either integrated or as an extrachromosomal element with occasional reactivation (and symptoms). Herpes, HIV and Hepatitis.
- Slow virus infection includes an initial acute infection followed by a period of low-level viral persistence, with eventual reactivation.
What is a polysome structure?
An assembly line of ribosomes that line up to work cooperatively.
What are the three components of a nucleotide?
Phosphate group, sugar, nitrogen bases.
Topoisomerase?
 
How many types and what is special about them?
 
ATP ?
Topo 1 = breaks single strand
Topo 2 = breaks double strand
 
Topo 1 does not use ATP
Topo 2 uses ATP
 
What does qPCR use to detect the amount of DNA in each cycle?
fluorescent probes attached to quenching probes
What is the shorthand of a W. Blot?
1. Electrophoresis2.Blot proteins from Gel to membrane3. Detect using Antibody4. Labeled Second antibody to bind to 1st one 5. Detect AntibodyW.EBDLD
Is sigma recycled or is a new sigma used in each transcritption event?
It can be recycled
What does this mean?
It means one mRNA strand codes for one polypeptide
Other than RNA POL II, what is needed in the eukaryotic holoenzyme transcription complex?
General transcription factors, mediators, chomatin remodelling complexes and histone acetylases.
Translation Release Factor Example of Self-Regulation in Translation
- If there's sufficient RF floating around, the stop codon in the middle of the gene encoding RF2 ends translation, producing a nonsense fragment.
- If levels of RF2 are low, the ribosome "slips" over the stop codon, and continues on to produce the finished protein.
How do you add sticky ends where there are none?
Use Terminal Transferase + dCTP
How does data come in in UCF?
In a graph with peaks representing where and what fraction number is most concentrated (top or bottom)
What is the big theme in this class?
interaction b/w specific protein and specific dna sequence and how they interact with other proteins (recruit other proteins to regulate proceses)
What does the RNA polymerase, primase, do?
It synthesizes the primer, which binds to a segment of DNA to which it is complementary and serves as the site for nucleotide addition
How many possible codons are there?
There are 4 nucleotides in the mRNA sequence, so there are 4^3 (64) permutations of the 3-letter code.
How would you determine if your cloning was effective using bacteriophages?
You can take foreign DNA and insert into a phage and begin infecting it into bacteria and you would look for absence of growth where the bacteria have been infected and killed
What is the Travers and Burgess experiment in 1969?
They did a SDS-PAGE of RNA polymerase from E. coli and it showed several subunits.
What is needed for transcription to occur?
An inducer must bind to the repressor, forming an inducer-repressor complex
Genetic Contributions to Sporadic Disease: A Recessive Locus at 12q24 Commonly Contributes to Patient Ductus Arteriosis
- PDA: In fetus, a duct (the DA) btw pulmonary artery and aorta allows fetal blood to bypass the lungs. In PDA, this doesn't close at birth as it should.
* One method of treating is to inhibit prostaglandins (which keep arteries open).
- The authors found much higher rates of PDA in Iran than the States, and specifically in families with consanguinity.
- By using areas of short-term tandem repeat markers we can compare the likelihood that two alleles inherited were inherited from a common ancestor. If many people affected with a certain disease have identical haplotype blocks, that section is likely linked to the disease.
- The sequences with the most conservation btw people are most likely to be linked t othe gene of interest. After identifying sequences, use PCR and look for common mutations in the shared LD blocks.
What do we mean when we say DNA replication is semi-discontinuous?
The leading strand is synthesized continuously toward the replication fork.

The lagging strand is synthesized discontinuously in short Okazaki fragments directed away from the fork.
Specifically what kind of primer is needed?
A 3 prime hydroxyl Put down by primase and a primer is needed, built on preexisting nucleotides
What were the four lanes of in the PAGE Gel? (describe the gel)
FIRST LANE: R+S- (first lane) NOT including S1 Nuclease so it is not chopped up at all and it ended in step 4 SECOND LANE: R+S+ (including S1 Nuclease) full expermeint [what we are interested in] you can see the size of the bubble here Goes from -9-->+3. Size is about 12 nucleotides This is about 12-17 nucleotides THIRD LANE : NO polymerase ( you have the first initial piece of DNA because nothing was cut [step 1]) FOURTH LANE: R-S- (nothing was cut or no methyl was added)
Why and How to Create a cDNA Library
- cDNA libraries allow us to analyze mRNA structure and abundance with the more stable and manipulatable DNA copy.
- cDNA allow us to determine the intron/extron positions on DNA by allowing hybridization.
- cDNA is created by using a 5' DNA primer to make a single cDNA strand complementary to the mRNA, then using a second primer (from the other end) to create a DNA version of the original mRNA strand.
- Place each cDNA into a library of vectors, and you have a library.
What did Heil and Zillig want to find out?
which subunit has the resistance to Rifampicin
MeCPs
-Methyl-CpG-binding proteins-bind the off signals that attract HDACs which take the acetyl groups off of the histone
GATA-1
the first erythroid specific transcription factor identified, required for erythroid cell maturation. binds to WGATAR, which is overrepresented in the globin locus.
DNA replication involves coordination of how many proteins/ enzymes?
 
 
>10
Retroviruses
- Retroviruses are positive-strand diploid viruses that replicate through a chromosomally integrated DNA intermediate.
- Retrovirus virions contain a reverse transcriptase and tRNA which prime DNA synthesis, and produce a double-stranded DNA copy of the genome while in the cytoplasm. This migrates to the nucleus, is integrated into the chromosome (with integrase), and then is transcribed and replicated.
- Virus is assembled at the cell membrane and released by shedding (non-cytolytic!)
- Variability is introduced during conversion of viral RNA to DNA (infidelity of reverse transcriptase) and transcription of DNa into RNA (infidelity of RNA polymerase II).
- Retroviruses also undergo recombination during reverse transcription, adding to variability.
Nondisjunction
Occurs when two homologous chromosomes fail to seperate during mitosis or meiosis.
Rap1p and Abf1P. multifuntion proteins can act as transcriptional activators at some promoters, but are repressors at the HM loci
plasmids
-not absolutely essential under normal conditions-can be ultimately dispensed; but have much of their DNA in plasmid form
self-splicing
-tetrahymena needs no snRNAs-uses G nucleotide to launch the attack via its 3' OH on the 5' PO4 of the 5' end of the intron-no lariat is produced-ribozyme
What is FRET?
Flurescnence Resonance Energy Transfer
What is ORMDL3?
An asthma susceptibility gene.
transcription
process in which part of the nucleotide sequence of DNA is copied into a complementary sequence in RNA
 
 
 
Crossing over
the exchange of genetic material between two homologous chromosomes.
Site directed mutagenesis
-synthesize the oligonucleotide in order to create a new ssDNA circle-50% of the clones wont have the mutation
virus genomes
-have dsDNA, ssDNA, dsRNA, and ssRNA-can be linear or circular-some are even segmented with one segment per one or two genes-nonsegmented the whole viral genome serves as an operon
Nuclear Matrix
-protein like network that permeate the cell
RNA turnover
-bacterial mRNA half life: few minutes-yeast mRNA half life: 20 minutes-human mRNA half life: many hours
Kozak sequence
-consensus sequence around the AUG start codon
What does HindII recognize and CUT?
5'GTPyPuAC3' 3'CAPuPyTG5'
What is needed for transcription?
1.RNA polymerase2.DNA template3.Ribonucleotides (rNTPs)4.Regulatory sequences (promoters and terminators to control gene expression)
Cancer-Causing Genes
- Oncogenes: Normally stimulate normal growth.
- Suppressor Genes: Normally inhibit growth or stimulate apoptosis.
- Repair Genes: Normally limit mutations.
What is the trinucleotide repeat in fragile x syndrome?
CGG
transformation
the act or process of transforming; change in form, appearance, nature, or character
transfer RNA
type of RNA molecule that transfers amino acids to ribosomes during protein synthesis.
Continuity of Life
succession of offspring that share structural similarities with those of their parents
sir1p
required for full silencing of HMR and HML. Sir1 mutants consist of 80% silenenced cells and 20% derepressed
human genome
-low genetic density, lots of genome wide repeats microsatellites-1.5% actually genic-11 genes per mb meaning 30,000-40,000 genes
Structural Domains
-loops of DNA between the nuclear matrix attachment points
housekeeping genes
-a gene that is continually expressed in all or at least most cells of a multicellular organism
DNA conformations?
 
Right /left?
A= turns right
B =turns right
Z =turns left
How does Automated DNA sequencing help?
Adds Colors!
How is DNA synthesized?
By the enzyme reverse transcriptase
Transmission-Disequilibrium Test
- Similar to linkage analysis, TDT looks at trios of parents and their affected child(ren). If a particular marker is transmitted more than the predicted 50% of the time to _affected_ children, it is likely either a susceptibility allele, or in disequilibrium with one.
Which eukaryotic DNA polymerases have have 5’ to 3’ polymerase activity.
All of them.
gene
the basic physical unit of heredity; a linear sequence of nucleotides along a segment of DNA that provides the coded instructions for synthesis of RNA, which, when translated into protein, leads to the expression of hereditary character.
messenger RNA
RNA that copies the coded message from DNA in the nucleus and carries the message into the cytoplasm
minisatellites
form cluster up to 20 kb in length, with repeat units up to 25 bp in length
bacterial vs eukaryotic mRNAs
-bacterial mRNA do not need maturation; eukaryotic do-both have to lay down certain number of nucleotides before elongation-in bacteria the sigma factor then falls off-in eukaryotes, after about 30 bp, the capping begins of the 5' end
What is sigma factor?


A sigma factor (σ factor) is a prokaryotic transcription initiation factor that enables specific binding of RNA polymerase to gene promoters
How do you purify a fusion protein (simplified version)?
TransformLyseExtractMixture ColumnElution agent ProteaseMixture Column TLEMEPM
How big is the "moving" transcription bubble where active initiation/elongation occurs?
about 17 BP
What is transcription?
It is the process whereby information coded in the base sequence of DNA is transcribed into a strand of mRNA
Viral Envelopes
- Many animal viruses have a lipid bilayer envelope.
- The envelope of the influenza virus, for example, contains two integral glycoproteins, HA and NA. These proteins have internal and external domains that help create the envelope, and the external domains are involved in attachment to host cells and penetration.
- The external glycoproteins are the most sensitive targets of antibodies, and are species-specific (as far as viruses have species).
Which prokaryotic DNA polymerases have 5' to 3' exonuclease activity?
Only DNA POL I.
Translation Initiation in Prokaryotes and Eukaryotes
-Prokaryotes: SD interaction is engaged, 3 Initiation Factors help bring the tRNA(met) and ribsomal small subunit over to the mRNA.
*tRNA(met) is placed at the P site and GTPase brings over the large subunit.
* Multiple ribosomes can bind to various SD sites on the mRNA, translating the multiple proteins on one polycystronic mRNA at once, or one protein multiple times.
-Eukaryotes: Complex of proteins binds the cap and polyA tail to form a loop.
* IF2 binds and helps locate AUG by scanning the loop 5'-3' from the cap, tRNA(met) is deposited, and large subunit joins.
Chargalf's Rule
the percent of adenine is close to the percent of thymine, and the percent of cytosine is close to the percent of guanine.
Cromosome conformation capture analysis
used to detect loops. Use formaldehyde to seal the bonds between DNA and protein. Then use restriction enzymes. then use proximity ligation and analyze with PCR. Sequences that were thousands of base pairs apart in the chromosome are now ligated together only a few hundred base pairs apart
prokaryotic transcription initiation
-use sigma factor which binds to the RNA polymerase and allows it to bind specifically to the promoter
What is an expression vector?
specialized vectors for expression of foreign genes
What is an isoschizomer?
Recognize the same sequence and make the same cut
In S. blots what must the Target DNA be in order for the DNA to bind on the probe?
Single Stranded
What is polycistronic?
It is what a strand of prokaryotic mRNA may be
Using Plasmids to Grow Protein
- Plasmids are generally several kilobases long, contain an origin of DNA replication, a resistance gene, and a useless section into which we can stick foreign DNA.
- Plasmids are usually capable of propogating on their own.
- Human DNA, DNA ligase, and a restriction enzyme are inserted, bacteria are plated on a selective plate, and only those bacteria that incorporated the plasmids with our DNA of interest (as well as the resistance gene) will survive.
- The surviving bacteria can then be used as a factory for growing our protein of interest.
What are subtelomeric FISH probes?
A powerful new tool
in detecting
cryptic telomeric
chromosomal rearrangements that often lead to mental retardation.
What are the different types of polymorphisms that are useful in complex disease studies?
Microsatellites (simple sequence repeats, SSRs):
Usually 10-20 copies per repeat unit of 2-6 nucleotides (di-, tri-, tetra-, etc.)
Detectable by PCR.
Microsattelites are used mostly in genetic mapping.

Single nucleotide Polymorphisms (SNPs):
Single base changes
Penicillin, Penicillin Resistance, and Penicillin-Derivatives
- Transpeptidatse catalyzes release of 1 ala, then attack by the amine from DAP or the glycine bridge on the remaining Ala carboxy terminal.
- Penicillin's B-Lactam rings mimic the D-ala-D-ala and inhibit transpeptidase by permanently binding to the enzyme.
- Because the cell wall cannot be reinforced with the peptidase bridges, the cell lyses.
- Penicillin resistance is usually a B-lactamase that cleaves penicillin. Gram- bacteria are innately resistant to penicillin because of LPS.
- We can fight resistance by changing the side chains to form, say, methicillin, which also works against Gram- bacteria because it passes through pores in the outer membrane.
What are the differences between somatic mutations and gametic mutations?
somatic mutations affect somatic cells and will not be passed on to offspring.
Gamatic mutations, however, affect sex cells and will be passed on to offspring.
try synthesis operon
-turns on and off the operon of the gene based on tryptophan concentrations
Why is that important?
To protect your DNA of interest from being digested by the cell
What is a DNA Library?
collection of clones that covers an entire source (like a chromosomes in fragments) collection of entire starting material except it is in fragments and cloned into plasmids
What are Northern Blots?
similar to S. blots but you are looking for RNA, most likely mRNA
How is Viral RNA replicated and transcribed?
In the host cell’s cytoplasm
Types of Tumors
- A tumor is any growth, period. This includes hernias and cysts (non-neoplastic tumors).
- Neoplastic tumors can be benign (polyps, adenomas) which means they can't move, or malignant (leukemias, carcinomas, sarcomas, etc.)
- A tumor is fundamentally a group of cells that have lost their 1:1 ratio of cell death to birth to something higher.
What is Hereditary Nonpolyposis Colorectal Cancer (HNPCC)?
It is a defect in mismatch repair

Mutations in Mut protein homologs are found in the majority of HNPPC patients.

Mutation rate sin these patients are elevated by 100- to 1000 fold.

Accumulation of mutations eventually leads to tumor formation.
Attenuation Mediation of Transcription (in prokaryotes)
- Only possible b/c translation occurs simultaneously.
- trp operon example: Consists of a leader region and structural genes for synthesizing trp.
* In a high-trp environment, the leader region (consisting of two codons for trp) is transcribed, and the terminator region of mRNA following forms a hairpin loop for rho-independent termination of transcription.
* In a low-trp environment, the ribosome stalls at the leader region (b/c it has no trp), newly synthesized mRNA forms a _different_ "antiterminator" loop, the RNApolymerase is able to continue onto the body of the trp operon (which codes for the production of trp), and the ribosome is free to follow along and continue translating.
DNA binding proteins "reading"
-can read wheter or not they attach to a specific sequence in the major groove-use hydrogen bonding and van der walls forces to make the perfect fit
How can you remove a nick?
use DNA polymerase in the 5-->3' direction
Why don't RE cut host DNA?
RE are paired with methylase in "restriction-modification system" protecting it from digestion/cutting
What are the steps for transcription initiation?
1.formation of closed complex at promoter2.formation of open complex (unwinding/melting)3. polymerization of first 9-10 nucleotides are polymerized while polymerase remains at promoter4. Promoter clearance: a. Transcript forms stable hybrid with template b. Polymerase adopts "elongation" conformation and moves away from the promoter (sigma is release)
Dosage Control, X-Inactivation, and Y Chromosome Function
- Aneuploidy for any autosome disrupts development. To deal with the XY/XX conundrum, then, we have X-inactivation.
- In females, at about the 1000 cell mark of development, each cell randomly inactivates one of the two X's.
- Partial explanation for mechanism: Inactivation starts at the Xic location on one chromosome and spreads along it, probably by Xist RNA coating the entire inactive chromosome.
- Relatively high frequencies of X-aneuploidy survival, b/c of inactivation mechanism.
- Functions of Y chromosome are primarily male sex determination and spermatogenesis.
- Clonal inheritance from father to son implies that sequence changes simply accumulate with time (no recombination).
What is a nonsense mutation?
One which causes an amino acid codon to change to a STOP codon.
What is the function of UAS binding
to bring the activation domain into proximity of the promoter
What does the plasmid need before replication can occur?
Like chromosomes, it will need a single origin of replication (ORi)
Look at figure 20.4 what happens if one of the components is missing?
something will go wrong in transcription
What dos each daughter helix contain?
It contains an intact strand from the parent helix and a newly synthesized strand
Tissue culture and production of monoclonal antibodies
- Tissue culturing can be used to study viruses and their life cycles.
- You can transfect DNA into cultured cells, integrating added DNA into the host genome. Difficult and unreliable.
- Can use cultured cells as factories to produce useful proteins, including specific antibodies.
- Production of monoclonal antibodies involves injecting antigen into mouse, isolating b-cells, fusing them with tumor cells to increase production, allowing them to grow and then testing for the presence of antibodies against the antigen of interest.
Why do some cells differentiate into epithelial cells, hepatic cells, neurons, etc.?
Genes are regulated in different cell types to alter expression levels.
Where does the Phosphates bind in DNA?
 
Where does the base bind to the sugar in DNA?
 
 
1. 5 prime and 3 prime positions 
2. The 1 prime position
 

 
 
With DNA gel Electrophoresis, why must one load standards?
fragments of a known size help you have a reference for your fragments
What has the resistance to Rif?
The core has the resistance to Rif so when an old sigma can attach to a resitant core in +Rif transcription still occurs
What happens when viral DNA becomes integrated into host DNA?
It is called a provirus or prophage after that
What is a chromatin fiber or solenoid?
The beads on a string braided together by the interaction between the H1 proteins associated with the chromatosomes.
DNA Replication (Including Replicons, Fidelity, and Initiation, Elongation and Termination Steps)
- A unit of DNA replicated in one go is called a replicon. In Proks it's a circle, in Euks there are multiple replicons per genome.
- Fidelity in replication comes from b.p. specificity, a 3'-5' exonuclease, and DNA repair mechanisms.
- Initiation: Protein machinery is assembled at the replicon origin.
* Helicase unwinds, topoisomerase removes supercoils, binding proteins maintain the DNA in its unwound state, and primase creates RNA primers for DNA polymerase.
* In Euks, the Origin Recognition Complex binds the origin and opens the fork.
Elongation: Polymerase sees the 3' -OH group on the primer and begins synthesizing in the 5' to 3' direction.
* The lagging strand is also synthesized in the 5' to 3' direction, "doubling back" to remove the RNA primers in front of each Okazaki fragment and ligate together individual DNA fragments.
Termination: Bacteria have 1 point of origin and 1 termination region. When the forks going opposite directions around the circle hit, the 2 daughter genomes are decatenated by topoisomerase (the interlocking circles are cut).
* In Euks, the replication forks hit randomly and combine.
How do we determine probability of cut?
1/4 ^ x x being the number of nucleotides per sequence
What are 2 pieces of information we can gain from the Recycling Sigma experiment?
1. Sigma is recycled because it is released2. Core has antibiotic resistance.
What is a breakapart probe for FISH?
A probe that binds to an entire gene and fluoresces if the gene breaks apart.
What do we mean when we say unbalanced translocation?
Loss or gain or chromosome material is associated with unblanced translocations.

Abnormal phenotypes are likely.
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