Genetics Exam 2

Chromosome Maps

relative location of genes on chromosomes

frequency of crossing over = relative distance on map

• ex: 12cM = 12% of cross overs

  • if cross overs based on 12 map units in primary oocyte mult. by 2!! (ex: 12×2 = 24%)

Single cross overs: one independent cross over

Double cross overs: the chance of TWO independent crossing over events happening on a chromosome

Linked genes + meiotic consequences

Genes that are close together and have a tendency to be inherited together

Genes that cannot undergo independent assortment

• Independent assortment = no linkage

• linkage w/o crossing over = complete linkage

• linkage w/ crossing over = recombinant gametes

3 pt chromosome mapping procedure

Map unit: cM = % recombination
(Addition of single and double crossovers/total 100)
Most recombination = parental phenotype

• recombination is the formation of new allele combinations due to crossing over

followed by farther apart (and so on)
total distance if addition of each induvidual distance
***as distance between genes increase, map estimates decrease

a - distance - b - distance - c

Reciprocal classes

compliments have a wt and a mutant of each
(heterozygote derived)

Interference

inhibition of further crossover events
I = 1 - C (negative = MORE DCO than expected, positive = less DCO than expected)

coefficient of confidence: observed double crosses/expected double crossover events

• expected found by mult prov of each single crossover (2 map distances)

Extensively mapped genes

drosophillia

Lod Score Method

• determines linkage from pedigrees

• uses probabilities of traits showing up together

Somatic cell hybridization (called?) + what kind of testing?

• fusing cells into a single hybrid cell - one of the two parental chromosomes are lost

  • allowed assigning of human genes to

    respective chromosomes

• heterokaryon - early hybridized cell state with 2 nuclei

• synkaryon: fused nuclei after heterokaryon (in vivo)

(only genes/chromosomes with certain traits survive at a time)
Syteny testing: testing the presence of a gene product when different number of chromosomes are present

• Presence or absence of each chromosome, with presence or absence of each gene product

• Four gene products tested in relation to eight human chromosomes

DNA markers

• short segments of DNA with known sequence and location

• Landmarks/checkpoints when mapping

Examples of DNA markers:

• RFLPs: Restriction fragment length polymorphisms

  • cuts specific sequences (fragments of different lengths)

• Microsatellites: short repetitive sequences throughout genome

• SNPs: Single neucliotide polymorphisms

  • single base difference from the common sequence

  • linked to some diseases

Polymorphic sites

sites with multiple alleles/versions at the same location/gene (loci)

Exchanges in mitosis

sister chromatid exchanges DO occur during in mitosis, but don’t cause any changes

harlequin chromosomes: sister chromatids involved in mitotic exchanges (have a patch like appearance when stained and viewed)

Types of chromosome mutations

# of chromosomes vary, deletions, duplications, rearrangements

• deletions: a chromosonal mutation where a portion of the chromosome is removed

• inversions: a portion of the chromosome is flipped 180 degrees

• translocation: a portion of the DNA breaks off and attached to another chromosome

• duplication: a mutation where a copy of 1+ dna segments is produced

Aneuploidy (and types of it)

variation in chromosome number (NOT full sets)

• monosomy: 2n-1 (lose a chromosome)

  • unmasks recessive/lethal alleles

  • reveals haploinsufficiency: one copy is not enough for survival

• trisomy: 2n+1 (add a chromosome)

  • usually ore viable

  • lethal for autosomes w/ larger chromosome size (ex: drosophillia)

  • Often found (30%) in spontaneously aborted fetuses & 20 percent of conceptions

• indicate 2n is necessary for development

Euploidy

Having a whole number set of chromosomes (haploid sets of chromsomes) - normal number of chromosomes

• Polyploidy: more than 2 complete sets of chromosomes

  • triploid: having 3 SETS of chromosomes

  • tetraploid: having 4 sets of chromosomes

Diagnostic Testing

• Amniocentesis or chorionic villus sampling (CVS)

  • Fetal cells obtained from the amniotic fluid/chorion of placenta

• NIPGD: Noninvasive prenatal genetic diagnosis

  • Fetal cells and DNA obtained from maternal circulation

Autopolyploidy

Type of polyploidy of the same species
identical set of chromosomes is identical to parent species

• Diploid gamete is produced

• Two sperm fertilize one ovum (rare)

autotetraploids: 4 sets (more common; balanced gametes)

autotriploid: 3 sets

Allopolyploid

type of polyplolidy from hybridizing at least 2 distinct but related species with complete sets

• usually plants

• sterile: cant pair/synapse

• may have characteristics of both parental species

Endopolyploidy

only certain cells are polyploid
Set of chromosomes replicates repeatedly without diving (cancer/stem cells)

Amphidiploid

2 complete diploid sets (4n - allotetraploid) from a hybridized cell

linkage ratio and group

ratio: the phenotypic ratio when genes are linked (non-mendelian), which measures how strongly 2 genes are linked
group: all the genes on a chromosome

Deletions

missing regions of chromosomes (breaks)

• terminal deletion: near the end

• intercalary deletion: in the middle of the chromosome

Inversion

segment of chromosomes turned 180 (may occur when chromosomes loop)

• paracentric inversion: one side (doesnt affect both arms)

• pericentric inversion: centromere part of inversion, affects both arms

(not having a centromere is acentric)

Gene redundancy

multipe genes that have the same pupose (ex: to code rrna)

gene amplification/duplication

multiple copies of a gene made (rapidly sometimes)

• may be a source of new genes

  • supported by multiple gens with common DNA sequence but different products

• play a role in evolution

Gene families

groups of genes who’s products have the same function

CNVs

Copy number variants: number of copies of a gene compared to the standard

• coding and non coding regions

Translocations

rearrangement of chromosome peices
reciprocal translocations: 2 breaks and swaps of chromosomes

Robertsonian translocation: 2 long arms fuses

fragile sites

sites more susceptible to breaking off because of the nucleotide sequence

Extranuclear Inheritance**

genetic info passed through the cytoplasm (usually through one parent)

• 3 types: organelle and infectious heridity and maternal effect

  • Organelle: mother’s PHENO affects childs PHENO

  • Maternal effect: mother’s GENO affects the offprings PHENO

Organelle Heredity (+ difficult to do what?)

inheritance from the michochondia or chloroplast

• determined by PHENOTYPE of ovule source (mom’s phenotype)

• difficult to analyze: nuclear + organelle genes contribute to a function

Heteroplasmy

a mixture of mutant and wt mtDNA in a cell

• variable number of organelles with mutated genomes

• Zygote receives most organelles through egg

• Mutation in one or few will be diluted out by many mitochondria that lack mutation and function normally

*Adult cells have variable mixture of normal and abnormal organelles

Endosymbiotic Theory

organelles were independent then absorbed for cell respiration/photosynthesis/mutual benefit

mtDNA & cpDNA similarities

• double-stranded, circular DNA

• no histones

• multiple copies of the DNA per organelle

mtDNA and cpDNA differences

mtDNA:

• smaller then cpDNA

• few duplications of segments or introns (non coding regions)

cpDNA:

• altered G/C bases content

• long noncoding regions

Human mtDNA (+why more susceptible to mutations)

• 16,500 bp & 37 genes

• 13 genes code protiens for cellular respiration

• more susceptible to mutations

  • no structural protection from histones, little DNA repair mechanism, and impacted by high ROS from cell resp.

  • 10X mutation rate to nuclear DNA

criteria of disease/inheritance to be attributed to mtDNA

• maternal inheritance pattern (mother’s pheno in all children)

  • mitochondrial or chloroplast DNA in egg

• deficiency in bioenergetic function of organelle

• mutation in a mitochondrial gene

mtDNA disorders

• MERRF: myoclonic epilepsy and ragged-red fiber disease

• LHON: Leber’s hereditary optic neuropathy

• KSS: Kearns–Sayre syndrome - eyes and heart/muscles

also associated with

• anemia, blindness, diabetes, autism, infertility, parkinsons/alzheimer’s, malignancies

Prevention of mtDNA disorders (statistic?)

1 in 5000 humans have mtDNA-based disease or are at risk for developing one

• Can be detected by genetic testing

• Mitochondrial replacement therapy (MRT) or three-parent in vitro fertilization

  • nucleus of parent placed into egg w/o nucleus (removed) to create new egg w new DNA and healthy mtDNA

Maternal effect

• offsprings phenotype under control of egg GENO

  • nucleus’ gene products control phenotype regardless of genotype

  • egg’s products accumulate in cytoplasm then distributed to gametes cells = influence over phenotype

Prortroph vs Auxotroph

• prototroph: grown on minimal media - wt: synthesizes all organic compounds compounds (humans cant do this)

• Auxotroph: mutant, need complete media, lost ability to synthesize essential compounds

  • let to recombine in mixed media then plated on minimal to see which recombinants survive

Growth of bacteria

log growth: lag phase → log phase (exponential growth) → stationary phase

Vertical vs Horizontal gene transfer

• vertical gene transfer: parent cell to daughter cell of one species

• horizontal gene transfer: Transfer of genetic information between related but distinct species (can be same species)

  • Transfer in the same ‘generation’

  • Plays significant role in evolution of bacteria

Types of horizontal gene tranfer

• Conjugation: direct transfer of DNA through cell to cell contant

  • uses a bridge to connect the cells called pillus/pilli

• Transformation: taking in stray DNA from an environment

  • cotransformation: several linked genes are transformed at the time time

• Transduction: a virus (plage) transfers genetic material into a cell

F Factor vs Hfr (F+ factor transfer called what?)

type of conjugation!

fertility factor: gives cells the ability to transfer genetic material

• F+ → DNA donor; F- → DNA recipient (F+ + F- —> 2 F+)

• has fertility factor separate from other DNA

• low frequency recombination

• ONLY TRANSFERS F factor NOT chromosomal genes (Hfr do!)

• merezygote: recipient of F factor, partial diploid

Hfr: high frequency recombination - type of F+ cell

• can donate genetic information but DOESNT transfer F factor - has F in chromosoms

• high frequency recombination

  • because it takes more time to transfer the ENTIRE chromosome

• Different strains of e. coli may have different F factors and therefore different initiation points for chromosome transfer revealing different linkage arrangements

R plasmids

resistance plasmids: provides extra DNA - 2 parts

• resistance transfer factor: encodes genetic information for transfer between plasmids (physical transfer component)

• r determinants: antibiotic/metal resistance encodings

Col Plasmids

encode colians (protiens toxic to strains w/o the same plasmid)

• kill neighboring bacteria

• comes from E. coli (colE1)

Transformation steps

1) entry of foreign DNA

2) recombination between foreign DNA + homologous region of recipient chromosome

outcomes:

• only step one completed and DNA is in cytoplasm

• both steps completed and recombination actually happens

Bacteriophages/phages

viruses that have bacteria as hosts

reproduce during bacteria’s genetic recombination

type of transduction (can also happen from defective phages that package host cell DNA)

lytic cycle steps:

• phage DNA absorbed

• host DNA degrades

• new phage parts made + assembled

• cell is lysed

• phages released

Lysogenic cycle:

• phage DNA coexists/recombines w/ host DNA (called prophage)

• replicates with chromosomes into daughter cells

• can trigger lytic cycle later and lyse cell

Types of phages

temperate phages: can lyse or be prophage

virulent phages: can only lyse cell (lytic cycle)

Viral mutations and variants are often categorized by changes in

• host range (range of host cells)

• Plaque morphology: holes in media indicating lysed cells from viruses

Complementation and Complementation groups and cistrons

• complementation: when 2 strains affect a cell and provide the missing wt part causing the wt to be expressed again

• complementation group: fail to complement = same complementation group (have the same defective gene)

• cistrons: the same gene - same as same compliment!

hot spots

areas more prone to mutation

reqs to be considered genetic material

• replicate

• store

• express information

• have variation from mutations

Experiments that prove DNA is genetic material

Griffith’s transformation

• rats killed by virulent strain

• when injected by killed virulent and avirulent strain living virulent found

  • Avery, mcleod, and McCarty: tested combinations of DNA and determined DNA is genetic material

    • found they could convert nonvirulent R-type Diplococcus pneumoniae bacterium to the virulent S-type

Hershey & Chase

• tagged DNA & proteins in plages

  • used SULFER and PHOSPHORUS

    • phosphorus is a part of DNA but not protiens and sulfer is in protiens not DNA

• found only tagged DNA incorporated in the cell

protoplasts

lysed outer portion of cell (cell wall removed)

Transfection

Infection by only viral nucleic acid

• → Proves conclusively that viral DNA alone contains all necessary information for production of mature viruses

Indirect Evidence of DNA as genetic material

• weight: diploid cells were exact double weight of haploid cells (not as variable as proteins etc.)

• mutagenisis: most mutations occurred when when the UV wavelength that DNA absorbed was used

Direct evidence of DNA as genetic material

recombinant DNA technology: eukaryotic DNA spliced into bacterial DNA resulted in eukaryotic products being made and also passed on to daughter cells

RNA(can be genetic material when…) (unique RNAS)

• tobacco mosaic virus (TMV): has RNA as genetic material

• retroviruses: viruses that use RNA as genetic material, then is turned into DNA in the host/cell

*RNA uses ribose sugar, uracine instead of thymine, and is usually single stranded

• goes: rRNA < tRNA < mRNA

  • rRNA: sturctural compnent of ribososmes

  • tRNA: translates mRNA for amino acids (codons)

  • mRNA: copies DNA into RNA for protein synthesis

• DOUBLE STRANDED in ANIMAL VIRUSES (the exeption)

Unique RNA

• Telomerase RNA & RNA primers: assist with DNA replication

• SnRNA (small nuclear RNA): process mRNAs

• antisense RNA, miRNA, siRNA, IncRNA: all involved in gene regulation

Nucleosides/Nucleotides
**(types of bases?)

Nucleotides

• made of bases, sugar, and phosphate

• building blocks of nucleic acids (which make DNA)

• Bases

  • purines: nine member ring, A (2 bonds) & G (3 bonds)

  • pyrimidines: six member ring, T, U, (2 bonds) & C (3 bonds)

• nucleotides called [start of base]ylic acid (deoxy at start if DNA not RNA)

  • ex: adenylic acid, guanylic acid, etc.

Nucleosides

• only base + sugar

• called: [start of base]ine/sine/dine

  • ex: guanosine, cytodine, thymidine (deoxy at start if DNA)

Watson & Crick/DNA

discovered semiconservative model of DNA

• antiparallel double helix

• genetic info stored in based sequences (which unrelated are in equal ratios)

• mutations are to base changes (which are connected w h bonds btw)

*dna is in a double hellix but can have different packing/formation in certain areas

Analytical Techniques of DNA

• Absorption of UV light

  • nucleic acids absorb 254-260 nm strongest

  • used in visualization, isolation, characterization

• De/renaturation of nucleic acids + molecular hybridization

  • forming double strands from single strands of different sources

• Florescence in situ hybridization (FISH)

  • finding a specific chunk of DNA by introducing a probe (part of DNA) that binds to the specific location and can be seen in fluorescent light

• Reassociation kinetics

  • analyzes the rate of reassociation of complementary single stranded DNA

  • reassociated reveals size and complexity of an organism

• Gel Electrophoresis:

  • cuts of DNA made and floated to different poles based on size or electric charge

Meselson-Stahl Experiment

1) DNA tagged in heavy nitrogen (N15)

• spun by gravity to see where it lands (by weight)

• All Gen 0 are heavy

2) Allowed to replicate once in light N (14)

• gravitational force spins and finds hybrid (middle- 1s = 14, 1s=15)

• Gen 1 = rules out CONSERVATIVE replication (2 completely new strands)

3) Second replication in N 14

• half hybrid, half light

• rules out DISPERSIVE replication (patches of original and new) because that would still only be hybrid

4) 3rd gen of N 14

• less hybrid, more light

• confirms semiconservative (using both strands as template) replication

Taylor-Woods-Hughes

Vivica Faba (broad bean) experiment:

• Monitored process of replication with labeled 3H-thymidine and performed autoradiography

• Like meselson-stahl they tracked T and found only one strand had it after replication

  • agreed that it was semiconservative replication

DNA Polymerases + structure

prokaryotes

polymerases add or remove dNTPs (which become nucleotides) to a strand (results in 2 extra Ps left over —> nNMPs)

• DNA Polymerase I: can polymerize and remove (exonuclease actvity) in both direction (5 → 3 too)

• DNA polymerase II: can polymerize (3 → 5) and remove 3 → 5

• DNA polymerase III: MAIN POLYMERASE, same as II can build 3 to 5 and remove 3 to 5

  • proofreading done 3 to 5

• DNA polymerases 1, 2, 4, 5: can repair DNA that is damaged (like by UV light)

Structure:

• sliding clamp:

  • Forms a ring around the DNA

  • Holds DNA polymerase in place

  • Prevents the polymerase from falling off

  • Allows the polymerase to synthesize thousands of nucleotides without stopping

• sliding clamp loader:

  • Uses ATP to open the sliding clamp and secure it again

  • Places the clamp around DNA at the primer–template junction

  • Releases the clamp so polymerase can bind and start synthesis

dnaA & ORI (oriC)

Prokaryotes

DnaA: initiator protein that binds to ORI (oriC in prokaryotes or ARCs in yeast) (origin for replication), causes helix to open up into ssDNA

• Multiple ORIs in eukaryotes (including yeast)

DNA Helicase

prokaryotes

assembles around the exposed ssDNA

uses “holoenzyme” to bind replication fork and initiate replication

uses ATP to break H-bonds and open DNA

SSBPs

prokaryotes

single stranded binding proteins: bind to single stands and stabilize the open conformation of the DNA

DNA gyrase (in what family?)

prokaryotes

• part of a larger enzyme group of DNA topoisomerases (enzymes that relieve coiling + seal nicks in backbone to separate the loops)

• makes loops and double or ss cuts to relieve coiling tension ( negative supercoils)

  • uses ATP

Primase

prokaryotes

(type of RNA polymerase)

builds the RNA primer

Lagging Strand

Okazaki Fragments

• 1000 - 2000 bp used to allow bidirectional synthesis to occur

• needs a primer at the start of each (removed by polymerase I)

DNA Ligase

• joins okazaki fragments

• catalyzes formation of phosphodiester bonds/covalent bonds

Tus/Ter

prokaryotes - bacteria

“a protein that binds to terminator sequences and acts as a counter-helicase when it comes in contact with an advancing helicase.^[2] The bound Tus protein effectively halts DNA polymerase movement” ← ends replication for circular chromosomes

processivity

Prokaryotes

• the productivity of an enzyme and its ability to catalyze reactions before releasing the substrate - in this case to assist in nucleotide synthesis before releasing the substrate

Temperature sensitive mutation

a mutation only expressed at certain temperatures

• conditional mutant

Eukaryotic & Bacterial Replication (similarities + differences)

• double stranded, unwound at ORC/ORI, replication forks, polymerases requre 4 dNTP+ template + primer

Eukaryotic only:

• more complex

• more DNA

• linear DNA

• has nucleosomes: unit of wrapped DNA that makes up chromatin

Yeast

contain 250-400 origins

• ARS: autonomously replicating sequences - acts as the origin of replication

  • 120 bp of repeat to signal replication/consensus sequence

  • consensus sequence: sequence that is the same in all yeast ARS

• Yeast ORI control timing of DNA replication

pre-RC / ORC

eukaryotic

Prereplication complex (pre-RC)

• Assembles at replication ORIs @ early G1 phase of cell cycle:

▪ Origin recognition complex (ORC) recognizes ORI and tags ORI as site of initiation

Eukaryotic Polymerases

DNA Polymerase

• alpha: RNA/DNA primers, initiation of DNA synthesis

• Delta: lagging strand synthesis, repair, recombination, proofreading

• Epsilon: leading strand synthesis, repair, recombination, proofreading

Switching of these allows for greater processivity

*3 → 5 exonuclease activity (can directly move back like poly I)

Telomers

repeating “dead” sequences at the ends of chromosomes

• shorten with each cell division

• In most eukaryotic somatic cells, telomerase is not active

  • Replicative senescence: cells stop dividing because the telomers are too short after a while

• Stem cells and malignant cells maintain telomerase activity—immortalized

• Telomerase activity and telomere length linked to aging, cancer, and other diseases

Telomerase

Eukaryotes - code: TTAGGG

• Ribonucleoprotein: RNA serves as template for synthesis of DNA complement

  • Telomerase RNA component (TERC)

    • reads DNA and lays down an RNA template

  • Telomerase reverse transcriptase (TERT)

    • uses TERC’s RNA to lay down the needed DNA

needed on ends of lagging strands: once RNA primer removed on lagging strand, no free 3′-OH to elongate

Telomerase adds repeats of six-nucleotide sequence to 3′ end to fill gaps

Genetic Recombination (+steps)

through crossing over new combinations of alleles are made

• endonuclease cuts phosphodiester bonds

• strand displacement and pairing

• branch migration

• duplex structure/holiday structure

• endonuclease nick

order of DNA replication

1. Origin recognition

• DnaA binds oriC

• DNA unwinds and opens the helix

2. Helicase loading + unwinding

• DnaB helicase is loaded by DnaC

• Helicase moves along DNA, breaking H‑bonds (Your notes: “Hexamer… assembles around ssDNA… moves ahead of the fork.”)

3. Stabilizing single strands

• SSB proteins bind exposed ssDNA

• Prevent re‑annealing and protect strands (Your notes: “SSBPs stabilize the open conformation.”)

4. Relieving supercoiling

• DNA gyrase (topoisomerase) cuts DNA to relieve tension

5. Primer synthesis

• Primase (dnaG) lays down short RNA primers

6. DNA chain elongation

• DNA Pol III extends primers

• Leading strand: continuous

• Lagging strand: discontinuous (Okazaki fragments)

7. Primer removal

• DNA Pol I removes RNA primers (5'→3' exonuclease)

8. Gap filling

• DNA Pol I fills in DNA where primers were removed

9. Fragment joining

• DNA ligase seals nicks between fragments

10. Proofreading

• DNA Pol III (and Pol I) use 3'→5' exonuclease to correct mismatches

11. Termination

• Replication forks meet at ter sites in bacteria

12. Eukaryotic extras

• Multiple origins (ARSs)

• ORC + pre‑RC assemble in G1

• Polymerase switching (Pol α → Pol δ/ε)

• Telomerase solves end‑replication problem