Sollars - Combined Flashcards
What can be said on the distribution of mitochondrial genomes?
2 - 10 copiesCytoplasmic segregationHeteroplasmy (uneven distribution)
What pathology is common in mitochondrial DNA disease?
Liver pathology
For MtDNA disease, what is the effect of Heteroplasmic vs Homoplasmic?
Heteroplasmic is rare, and strong (multisystem, death)Homoplasmic is more common, and weaker (associated w/disease)
Why would someone being treated for HIV-1 or Hepatitis B be affected by Mitochondrial DNA Depletion?
gamma-Pol resembles HIV polymerase (which we attack)
What do MtDNA diseases in the exonuclease domain deal with?
Proofreading—fidelity
What do MtDNA diseases in the polymerase domain deal with?
Efficiency
What is the role of y-Pol (gamma)?
Replicates MtDNA
What can occur when checks in the cell cycle to not properly occur to ensure fidelity of genome?
Cancer
What is faster, prokaryotic or eukaryotic DNA replication?
Prokaryotic (50 bp/s vs 1000 bp/s)
If you see a DNA Pol + Greek symbol, what are you dealing with?What about a Roman numeral?
Eukaryote = GreekProkaryote = Roman
What enzyme acts on the unrprotected end of linear chromosomes? What other problems can occur
ExonucleaseChromosomal fusion events
Cellular Senescence
Phenomenon which normal cells cease to divide, possibly triggered by gradual loss of telomere
Cellular Crisis
When Cellular Senescence does not occur, and cells continue to divide–can lead to mutation events and cancer
Telomerase
Enzyme to replicate end of chromosomes
When will a cell enter Senescence?
When it reaches Telomere Associated Repeats (TAR)
What is the mechanism for Telomere building?
After removal of original RNA primer, will have stretch of ssDNA1. Telomerase contains RNA template matching telomeric repeat element2. ssDNA above is extended farther3. Primase synthesized RNA primer4. 3’ ends of primer accepts DNA polymerase5. RNA primer removed from 5’ end
Fragile Sites
Non-staining gaps, observed in characteristic sites on several chromosomes.
How are fragile sites inherited?
Mendelian codominant fashion
When would chromosomes be visible in the classic fashion?
After S-Phase
What is the purpose of the centromere?
Attachment of chromosomes to cytoskeleton
What 3 things do you need for chromosomes?
CentromereTelomereOrigin of Replication
What are three morphological classifictions of chromosomes?
MetacentricSubmetacentricAcrocentric
Satellite DNA
Any fraction of the DNA that differs sufficiently in its base composition from that of the majority of the DNA fragments to separate as one or more bands distinct from the bands containing the majority of the DNA during centrifugation
When does each chromosome have an identical nucleoprotein copy joined by a centromere (sister)?
After S-phase and DNA replication
Replication of Origin
Nucleotide sequence at which DNA synthesis begins
Type of Chromosome w/a centrally placed centromere =Type of Chromosome that appears J-shaped at anaphase because the centromere is nearer one end than the other =Type of Chromosome with a nearly terminal centromere =
MetacentricSubmetacentricAcrocentric
Complex of nucelic acids (DNA/RNA) and proteins (histones/nonhistones) comprising eukaryotic chromosomes
Chromatin
What type of chromatin is highly condensed and shows no evidence of active gene expression?What type contains transcriptionally active DNA and is extended?
HeterochromatinEuchromatin
How many chromosomes does a haploid cell contain?
23
What is the structure of mitochondrial chromosomes?
Circular
What is the difference between Unique Sequence DNA and Repetitive DNA?
Unique = single copy per haploid genome, low conservation, protein coding, intronic, intergenicRepetitive DNA = Repeated sequences, >50% of genome
Satellite DNA
Any fraction of the DNA that differs substantially in base comp (either A+T rich, or G+C rich); formsbands
Short Interspersed Nucleotime Elements (SINES)andLong Interspersed Nucleotide Elements (LINES)
SINES:AlurepeatsLINES: L1 (Line-1)Both are Dispersed Repetitive DNA
What are major differences between mitochondrial DNA and Nuclear Genome?
Mitochondrial genome is 10,000x smaller; each cell contains thousands of copies; is highly conserved for coding; and displays maternal inheritanceNuclear genome is much larger but only contains two copies and displays traditional maternal/paternal inheritance
What is the most common cell used for chromosome preparation?
Circulating Lymphocytes
What cell cycle would you stop the cell at in order to best study the chromosomes?
Metaphase
Define each number for the following gene:10p23
10(chromosome) p(arm) 2(region) 3(band)Hint: Remeber C.A.R.B
What type of study is treatment with trypsin following by staining with Wright’s (Giemsa)?What is characteristic of this study?
G-bandingCharacteristic, and reproducible patterns of light and dark bands
What type of study features fluorescent dye binding to A-T rich regions preferentially?
Q-banding
What technique freezes cells earlier in their cycle, and is useful in detecting subtl structural abnormalities?
High-resolution banding (prophase or metaphase banding)
What is the difference between R and G bands?
R = early replication, light bands, genes are mostly concentrated hereG = A-T rich dark bands, few expressed genes, rich in L1 sequences
What type of band is light, G-C rich, contains expressed genes, and is rich in Alu?
R-band
What type of band is A-T rich, has few expressed genes, and is rich in L1 repeated sequences?
G-bands
Fluorescentin situHybridization (FISH)Commonly used for what?
Analysis method to us a labeled,chromosome specificDNA segment to hybridized with metaphase, prophase, or interphase chromosomes and visualize under a fluorescent microscopeConformation of Down’s syndrome
Spectral Karyotyping (SKY)
Using multiple fluorescent probes specific for each chromosome to generate specific colors along most of its length.Greatly incrased accuracy and efficiency of detecting abnormalities.
Comparative Genomic Hybridization (CGH)
DNA from test source is labeled with a green gluoropore and control normal DNA is labeled with red fluorophores. Two mixed, hybridized to normal chromosomes. Deletions or additions will alter the green:red ratio.Widely used in cancer genetics.
Individualized Medicine
Treating patient based upon biochemistry and genetic factors. “Tailored” medicine
Pharmacogenomics
Study of the role of inherited and acquired genetic variation in drug response
Cytogenetics
Study of chromosome structure and identification of microscopically visible abnormalities. Examination of the ordered array of chromosome or karyotype
Molecular Genetics
Study of structure and function of genes, includes gene mapping and recombinant DNA based tech
Biochemical Genetics
How genetic defects disrupt normal metabolism/cell control–need this to design treatment
Population Genetics
Study of how allele frequencies present in a population.
Clinical Genetics
Diagnosis/detection of inherited disorders; treatment/management; risk assessment; genetic counseling; and pharmacogenomics
Pharmacogenomics
Study of the role of inherited ad acquired genetics variation in drug response
Law of Segregation (1st Law)
Each individual possesses two genes for a particular characteristic. Only one of these genes is transmitted to the offspring
Which law deals with unaffected carriers of disease?
Law of Segregation
Monohybrid cross
Mating cross designed to produce an individual that has parent contributions that are different for one trait
What is the ratio of progreny in the F2 generation?
3:1
What are characteristics that appear in the F2 generation?
Recessive
Dominant
Forms of gene that fully manifest their effects on a trait when present in the heterozygous state
Recessive
Forms of a gene whose effects on a trait are masked by dominant allelles
Codominance
Alleles are codominant if each is expressing independently of the presence of the otherEx: A/B alleles in blood typing
Genotype vs Phenotype
Genotype: Genetic (allelic) composition of individual (Xx, etc)Phenotype: Observable physical, biochemical, etc feature (color, height, etc)
Law of Independent Assortment (2nd Law)
Members of different pairs of factors assort independently.or..Members of different pairs of alleles are assorted independently into gametes during gametogenesis, and that subsequent pairing of male and femail gametes is random.
What is the molecular basis for the law of Independent Assortment?
Genes are present on independent chromosomes, and chromosomes undergo recombination along their length fairly frequently
Epistasis
when one gene masks the expression of anotherEx. Blue eyes
Linkage
greater association in inheritance of two or more nonallelic genes than is to be expected from independent assortment
Genomic Imprinting
– degree of which a gene expresses itself depends upon parent transmitting
Mitochondrial Inheritance
inheritance of a trait encoded in the mitochondrial genome
Germline Mosaicism
when an individual is composed of two or more cell lines in their germ cell population, where these cell lines are of different genetic or chromosomal constitutionex. Seen in schizophrenia
Single Gene Defect
Stem frompolymorphism or mutationsin one gene; easy to recognize modes of inheritanceExamples: Cystic fibrosis, sickle cell, Marfan
Chromosome Disorder
Defect is due not to a single mistake in genetic blueprint, but to an excess or deficiency of genes contained in whole chromosome or chromosome segmentsEx: Down’s, Patau
Multifactorial Inheritance
Most common defect, can include environmental and genetic factors.These can recur in families, but do not show any particular pedigree pattern
What can be useful in determining if disorder is inherited?
Comprehensive family historyMedical literatureTwin studies/segregation analysis
Congenital
Present at birth,ex. Congenital AIDS
Familial
Trait or disorder tends to cluster in families
Sporadic
Indicates no evidence for vertical transmission or familial clustering exists
Inherited vs Genetic
Inherited - vertical transmission (e.g. father to daught) of the diseaseGenetic - a gene or chromosome is involved in the susceptibility to or cause of the disease
Types of repair involving excision and replacement
MismatchNucleotide excisionBase exchange
What does Mismatch Repair preserve?
Fidelity
When does Mismatch repair occur?How is the correct strand recognized?What can result when it’s broken?
Repairs errorsafter replicationDNA methylationmakes the correct strandHNPCC (Lynch Syndrome)can result when broken
What does Nucleotide Excision Repair fix?What can a defect in Excinuclease result it?
Repairs damage resulting from large distortions in the DNA double helix.Defect in excinuclease can result inXeroderma Pigmentosum
What can UV light do to DNA?What repair system corrects this?
Can cause pyrimidine dimers–adjacent thymines dimerize.Nucleotide Excision Repair fixes this.
What does DNA contain thymine, not uracil?
Cytosine can spontaneously convert to Uracil, and you would have C-G, A-U. Would throw off repair systems with no way to dope the scope.
What creats an abasic site in DNA, and much slower in ribonucleotides and RNA?
Depurination
How does Ricin work?
Depurination of RNA
Base-Excision RepairWhat comes through and fixes the last step?
Repairs altered bases resulting from spontaneous changes and environmental stress (deamination, depurination)Produces an apyrimdinic site that needs to be repaired (gap in the chain)Deoxyribose Phosphate Lyaseremoves the base free phosphate, andDNA Polymerase and DNA Ligasefill in the gap
What is the most common single base mutation in human cancer?What can remove the incorrect methyl markers?
C to T or G to ABase Excision Repair
Direct Repair
Specific enzymes are dedicated to repairing specific DNA damage; following use will be “inactive” and degraded.
Double Stranded Break RepairCause?NHEJ?HR?Experimental Use?
Cause: High energy radiation and oxidative free radicalsNHEJ = Nonhomologous end-joining (NHEJ) repair; uses different DNA strand to repair;more error prone!HR = Homologous Recombination (HR);BRCA1/BRCA2;may signal breast cancer risk if abnormal BRCAUse:CRISPR
DNA Recombination
Rearrangement or exhange of genetic information b/t two DNA molecules of b/t two different parts of the same DNA molecule
Legitimate DNA RecombinationWhat type of junction is formed?When is it most commonly seen?
Fidelity is preserved, event occurs at aligned points on genetic map,no information is lost,regions of high homologyHolliday JunctionMeiosis
illegitimate Recombination
Fidelity isnotpreserved; recombination occurs between segmentsnotaligned on the map.Ex:Unequal Crossing Over
Viral Integration
Example of illegitimate DNA recombination; some are site specific, others not.HIV is not site specific
Movement of Transposons
Example of illegitimate DNA recombination, over 1-million Alu sequences through genome,some disruptions can cause disease if transposon is inserted where it would disrupt another gene
What are the three rules of DNA Replication?
- DNA Replication is semiconservative2. Replication begins at an Orgin, proceeds bidirectionally 3. DNA synthesis proceeds in a 5’ - 3’ direction and is semi continuous
What does each daughter cell duplex consist of?
One parental strand and one newly synthesized strand
Where are origins usually found–why?
Around A-T base pairs due to weaker hydrogen bonding compared to G-C (2 vs 3)
Which end are deoxynucleotides added to growing chains?
3’ end
What initiates strands during DNA synthesis?
RNA primers
Synthesis of the leading strand is ______?Synthesis of the lagging strand is ______?
Continuous Discontinuous
What are the three steps of initiation?
- DNA-A binds origin of replication2. Helicase dissociates helix3. ssDNA binding proteins keep DNA from reannealing
What does elongation require?What enzyme grows leading strand?What is the orientation compared to the parent strand?
RNA primersDNA Polymerase III;works in 5’-3’ direction; beginning at the 3’ end of each RNA primerComplimentary and antiparallel to the parent template
How is the lagging strand synthesized in DNA synthesis?
SmallOkazaki Fragments
What initiates Okazaki Fragments?What completes them?
RNA Primer Synthesis by PrimaseCompleted byDNA Polymerase III, made in a 5’ - 3’ direction
What molecule class alleviates supercoiling strain via nicks and repairing nicks?
Topoisomerases
When does proofreading occur?What does it ensure?
ElongationEnsures fidelity
If DNA Polymerase mismatches a nucleotide–how does proofreading occur?
3’-5’ Exonuclease activity to excise the mismatched nucleotide;occurs bia enzymebacking up
DNA Polymerase I
Removes RNA primersSpecial 5’-3’ Exonuclease ActivityProofreadingDNA Repair
DNA Polymerase II
DNA repair
DNA Polymerase III
Latches on, stays onSynthesizes most DNA during leading and lagging strandsProofreading
DNA Polymerase IV, V
DNA Repair
What three methods is genetic fidelity preserved?
- Geometry - Active site of DNA Polymerase; must have 3 rings… tautomerization causes problems2. Proofreading; 3’-5’ Exonuclease Activity3. Mismatch Repair and DNA Methylation - Parent strandmethylated,copied not. Helps identify correction version to repair.
What does proofreading’s 3’-5’ Exonuclease activity preserve?
Fidelity
Why is the parent strand methylated and copied not?
Helps identify correct version to repair
What is the 1st rule of DNA replication?
DNA replication is semiconservative; each parental strand is used as a template for the synthesis of a new complementary strand
What does the first rule state a daughter cell will receive?
One of the two identical DNA molecules
What does each daughter duplex consist of?
One parental strand, and one newly synthesized strand
What is conservative replication?
Both templates go on to the same cell
Where are origins of replication found and why?
Sites rich in A-T, due to weaker hydrogen bonding (2vs3) than G-C pairing
What direction do replication forks move from the origin of replication?
Bidirectional
What is the 2nd law of DNA replication?
Replication begins at an origin, and proceeds bidirectionally
What is the 3rd rule of DNA replication?
DNA synthesis proceeds in a 5’-3’ direction, and is semi-discontinuous
Where are new deoxynucleotides added to the growing chain?
3’ end
What initiates each new DNA strand during replication?
RNA primer
How is synthesis of the leading vs lagging strand?
Leading = continuousLagging = discontinuous
AAA+ ATPases
DnaA binds to the origin of replication and disassociates the helical strands; energy of ATP cleavage is used to produce conformational change in DnaA, which forces the strands apart
DNA Ligase
Creates phosphodiester bonds by using the energy of ATP cleavage, to seal “nicks” in the DNA strand
DNA Polymerase
“Workhorse” enzyme; responsible for strand elongation, require ssDNA template AND RNA primer
Helicase
Cause disassociation of the two strands of the double helix, unwinding the structure using energy released from ATP cleavage
Nucleases
Sever phosphodiester bonds of the DNA backbones; two types–exo/endo nuclease
Primase
Responsible for synthesizing short stretches of RNA complementary to the template DNA strand that serve as primer
TopoisomeraseWhat additional functions do they contain?What is difference in Type I and Type II
Adjusts supercoiling of DNA double helices, both alleviating stress and introducing negative supercoiling. Endonuclease and Ligase (cut and seal) Type I = cleave one strand of double helixType II = cleave both strands of double helix
What occurs at Initiation?
- DnaA binds the origin of replication2. Helicase breaks double helix3. ssDNA binding proteins maintain disassociation
Elongation is accompanied by what function to ensure proper base pairing?
Proofreading
What molecule is instrumental in alleviating supercoiling strain?
Topoisomerases
What initiates the Okazaki fragment?
Synthesis of an RNA primer by primase
What powers DNA synthesis?
dNTP to dNMP (2 Pi)
What is DNA gyrase?
Type II Topoisomerase
Topoisomerase II Poisons
Stabilize covalent DNA topoisomerase II complex
Catalytic Inhibitors
Topoisomerase II inhibitors (w/out stabilizing covalent complex)
When does proofreading occur and what does it ensure?
Elongation, fidelity
What enzyme excises the mismatched pair in proofreading?
3’-5’ Exonuclease
When does termination occur?
When replication forks join up
What is the central dogma?
DNA - Transcription - RNA - Translation - Protein
What is the general structure of DNA?
Polynucleotide sequence joined through 3’-5’ phosphodiester bonds. Polar primary structure, strands run antiparallel, exterior is hydrophilic, interior hydrophobic, helix has major and minor grooves
What is often found at the 5’ and 3’ ends of DNA?
5’ - Phosphate 3’ - Hydroxyl
What is the role of Actomycin D?
Anticancer drug which intercalates b/t the planar bases of DNA
What are Chargaff’s Rules?
A=T, G=C, Purines = Pyrimidines (A+G=T+C)
What is a major exception to Chargaff’s Rules?
Base composition in an organism can be affected by environment, age, and/or nutrition
What are the hydrogen bonding rules in DNA? What does this result in?
A-T = 2G-C = 3Sequences rich in A-T pairings will be weaker than those rich in G-C pairings
What is the most common form of DNA?What is Z-DNA? A-DNA?
B-DNALeft handed double helix rich in G-CA-Form made by dehydrating B-DNA, found in RNA hybrids or RNA-RNA double strands
What has an impact on viscosity and UV-absorption? What is this called, and how is it used?
Denaturation Hyperchromic Effect, used as standard to measure purity of DNA in preparations
Is melting point uniform for strands of DNA?
No, elevation/depression depends on differences in A/T , G/C rich regions (2 vs 3 hydrogen bonds)
What is Chromatin?
Complex of nucleic acids (DNA/RNA) and proteins (histones/nonhistones) comprising eukaryotic chromosomes
What is the role of histones in DNA secondary structure?What is their electrochemical makeup?What is the role of Histone H1?
DNA wraps around histone complexes to form nucleosomesLarge amounts of basic amino acids (H, K, R) leading to a positive charge which attracts negatively charged DNAHistone H1 associated with linker DNA b/t nucleosomes, inducespositive supercoilingin the linker region
What are the two types of Chromatin?
Euchromatin - loosely packed,transcriptionally activeHeterochromatin - tightly packed,transcriptionally inactive
What occurs when the axis of the DNA helix is coiled on itself?What types of this are there?What must cells do as a result?
SupercoilingPositive - Winds in same direction as helixNegative - Winds in opposite direction as helixCells must actively maintain an unwound statenecessary to facilitate access to DNA
What class of molecules relax supercoiling stress?What type of supercoiling to they maintain? How?
TopoisomerasesMaintain Negative Supercoiling by breaking DNA then resealing the breaks
What does Ciprofloxacin target?
Bacteria topoisomerase
What does doxorubicin target?
Human type II topoisomerase inhibitor
What drives the differences in packaging in viruses, bacteria, and higher organisms?
Genome size.In humans, DNA is organized in chromosomes; in mitochondira and backteria they arecircular(which are also loosely termed chromosomes)
CentromereWhat is different about metaphase?
Region to which spindle traction fibers attach during mitosis/meiosis.Joining point for two replicated chromatidsDuring metaphase, centromere is narrower than distal regions, termedprimary chromosomal constriction
Telomere
Repeated DN sequence w/specialized proteinsfound and ends of eukaryotic chromosomes; chromosomes lose 100 nucleotides from ends per cell division (can be repaired by some cells)
Origin of Replication
Nucleotide sequence at which DNA syntheis begins, multiple sites in humansBacteriaonly haveonedue to circular chromosome
Rett Syndrome
X-linked genetic disorder observed in females; 99% of cases are mutations–lack of reproductive function.Cause:Defect in methyl-CpG-binding protein 2,involved in chromatin formation—DNA packaging error
What is Royal Jelly an example of?
Epigenetics–also a deviation from Chargaff’s rules
DNA MethylationWhere does it usually occur?What is the usual result?
Acts to sterically inhibit interaction with DNA molecules and further serve as abinding site for proteinsUsually occurs in thepromoter regionUsuallysilences genes–but may also activate
DNA Methyltransferases (DNMTs)
Catalyze DNA methylation using S-adenosyl methionine (SAMe) as donor
Ten-Eleven Translocation (TET) Enzymes
Remove methylation
Thymine-DNA Glycolase (TDG)
Removes thymine moieties from G/T mismatches by hydrolyzing the carbon-nitrogen bond
What modification is occuring in Histone Modification?How does it occur?What is the result?What major enzymes cause this to occur?
AcetylationTransfer of acetyl group fromAcetyl Co-AtoE-amino group of lysinePartial neutralization of the (+) charge on histon, decreasing affinity to DNAHAT (+ acetyl)HDAC (- acetyl)
HATs / HDACs
Histone Acetylransferases (HATs) - add acetyl to histoneHistone Deacetylase (HDACs) - remove acetyl from histone
How are hypomethylating agents used?What does it do?
Treatment of some medically fit patients with adverse features of AMLBlocks DNA Methyltransferae (Inhibits DNA methylation)
HDAC Inhibitors?
Used in some individuals with lymphoid malignancies, such as multiple myeloma and Sezary Syndrome
What is the basic formula for a nucleotide?
Nitrogeneous Base (information) + Pentose (structure) + Phosphate (energy)
What are the purines?What are the pyrimidines?What is the general structure of each?
Purines: Adenine (A), Guanine (G)Pyrimidines: Cytosine (C), Thymine (T), Uracil (U)Purines are two rings, Pyrimidines are single ringHint:Purines areAlwaysGiganticPyrimidines areCertainlyUsuallyTiny
What is the major weakness of the bond structure of all nitrogeneous bases?
Tautomerism alters the hydrogen bonding side of the molecule
What is the structural difference of Ribose (RNA) and Deoxyribose (DNA)?
Ribose has an additional hydroxyl group at pos. 2 of the pentose ring
What is the formula for a nucleoside?What is the predominant form of nucleosides?
Purine base + ribose sugarAnti (syn = minor)
What is the general structure for a nucleotide?How are these used to store energy, what is an exampe?
Nucleoside + Phosphate GroupThe high E bonds of the phosphate groups; example is Adenosine Triphosphate (ATP)
How do nucleotides function as energy sources in the cell?
Phosphate groups give method to carry energy in bonds
How are do nucleotides function as building blocks in the cell?
Nucleotides can bind to glucose, or similar molecules to donate in various reactions; can also serve asmethyl donorin certain pathways
How are do nucleotides function as Regulatory Signal in the cell?
Substances like Cyclic AMP (CAMP) can act as second messengers mediated the response from certain hormones
How are do nucleotides function as Coenzymes in the cell?
Give energy and participate in oxidation-reducation reactions; can carryacyl groups (fatty acids)in metabolism
What is the structure and role of 5-Fluorouracil?
Anti Cancer Agent; nitrogeneous base analogue which causes DNA to break
What is the structure and role of AZT?What is one known side-effect?
Anti HIV drug, activated by polymerase which transcribes viral genomeMitochondrial issues .. remember where bacteria came from bro
