Modern Genetics

Aneuploidy

Gain or Loss of one Chromosome, but all else are normal (2n ± x)

Euploidy

An unnatural complete haploid set of chromosomes are present (3n, 4n etc.)

Nondisjunction in MI

Sees two gametes with both chromatids and two gametes without any chromosomes, becoming trisomic/monosomic post-fertilization respectively.

Nondisjunction in MII

Two normal gametes, one trisomic gamete, one monosomic

Monosomy

Loss of Single Chromosome in Diploid Genome (2n - 1)

Trisomy

Gain of single chromosome (2n + 1), e.g. Downs, Patau, Edwards (21, 13, 18). Often lethal in spontaneously aborted fetuses

Haploinsufficiency

When the gene product from only one copy of a gene is not sufficient. Seen in recessive lethal mutations and monosomies (e.g. Turners)

Trisomy 21

Due to nondisjunction not occuring in Anaphase I/II, ovum source 95% of time

Polyploidy

2+ haploid sets of chromosomes, based on the number of sets found. Common in plants.

Autopolyploidy

Addition of 1+ sets of chromosomes identical to haploid complement of same species. Often tetraploids, where chromosomes replicate but fail to divide before interphase

Allopolyploidy

Combination of chromosome sets from different species as consequence of interspecific mating

Allotetraploidy

Often results from hybridization of two closely related species. Often sterile product.

Amphidiploid

If a allopolyploidy hybrid undergoes natural chromosome doubling, produces this fertile offspring.

Deletion

A lost piece of chromosome after a break

Terminal Deletion

A deletion near the end of a chromosome

Intercalary Deletion

A deletion in the interior of a chromosome. During synapsis of chromosome with large intercalary deletion, the homolog loops to not pair with mis-complements of missing region.

Cri du Chat Syndrome

Deletion of terminal part chromosome five, inducing anatomical malformations, cat like cry. Deleted gene CTNND2 is a neuronal protein, a haploinsufficient loss of function dominant allele.

Duplication

Repeated segment of genetic material, due to unequal crossing over during M or replication errors

Inversion

Rearrangement of linear gene sequence. Requires two breaks in chromosomes and subsequent reinsertion of inverted segment, often due to looping.

Paracentric Inversion

An inversion in which the two chromosomal arms remain at the same ratio as before.

Pericentric Inversion

An inversion in which the length ratios of the two chromosomal arms is not the same as beforehand

Translocation

Movement of chromosomal segment to new location in genome

Reciprocal Translocations

Exchange of segments between two nonhomologous chromosomes, where genetic info is lost or gained as deletions/insertions

Nonreciprocal Translocation

Genetic information is rearranged between nonhomologs but not exchanged

Four key characteristics of Genetic Material

Expression of Information (Phenotype), Storage of Complex Information, Capacity for Variation, Ability to Replicate

Griffith’s Experiment I

Showed avirulent strains of streptococcus pneumoniae could be transformed to virulence by some transforming principle.

Avery, Macleod, McCarty Experiment II

Repeated Griffith’s experiment but treated cells were heat killed with proteases, RNase, and DNase. DNase destroyed transforming activity, showing the transforming principle had to be DNA, not protein.

Hershey-Chase Phage Experiment

Noted that T2 Phages are 50/50 Protein/DNA, phage infects bacterial cells by attacking cell wall, and they produce progeny within cell. Labeled DNA with 32P and protein 35S radioisotopes, seeing in infected cells 32P radioactive, 35S not. Progeny phages which lysed from 32P infected cells were also radioactive. Therefore, DNA is is not protein and is genetic material responsible for heredity in bacteriophages

Nucleotide Structure

Consists of pentose sugar, nitrogenous base, phosphate group

Nucleoside Structure

Only contain nitrogenous base and pentose

The -OH in Pentose

At the 2’-Carbon, ribose has an -OH while deoxyribose has an H. Makes RNA less chemically stable and DNA better for long term storage

Uracil and Thymine

Uracil is found only in RNA. Thymine is found only in DNA

Pyrimidines

Single Ringed, including Cytosine, Uracil, and Thymine

Purines

Double Ringed, including Adenine and Guanine

Nucleotides Phosphate Group

Nucleotides can have 0-3 phosphate groups on the 5’ Carbon

Phosphodiester Bonds

Nucleotides are connected by these bonds where 5’ phosphate of one group bonds with 3’-OH of next. Makes all polynucleotide strands polar, due to phosphate and hydroxyl at free ends

Chargaff’s Rule

A = T and C = G

Rosalind Franklin

Used X-Ray Diffraction to produce images of DNA. Found a 3.4 Angstrom periodicity consistent with helical structure

Watson/Crick Model

Used Franklin’s Imaging and Chargaff’s Rule. Two polynucleotide strands wrapped about central axis forming right-handed, clockwise rotation double helix. Contains major and minor grooves alternating every 34 Angstroms or 10 bases. Antiparallel strands

Double Helix Model

Hydrogen bonds pair between base pairs. Bases are flat, perpendicular to axis, giving stability. A-T bonds is a pair of hydrogen bonds, C-G a triplet of hydrogen bonds

DNA Variations

Watson-Crick model is B-DNA, most similar to cellular conditions. A-DNA more compact, C/D/E-DNA are right handed but less compact. Z-DNA is left-handed form for regulatory purposes

RNA Structure

Ribose Sugar, single stranded molecule, can form double-stranded regions by folding into secondary structures

mRNA

Messenger RNA acts as template for protein synthesis

rRNA

Ribosomal RNA acts as a component of ribosomes, making up most % of total RNA.

Nucleic Acids UV Light

254-260 nm light is absorbed by nucleic acids due to interactions of light and bases ring system

Hyperchromic Shift

Denaturation of DNA determines a melting temperature (Tm), causing half of DNA single strand to coil

Hybridization of DNA

Single strands of nucleic acids combine to form duplex structures bound by hydrogen bonds

FISH

DNA is denatured from double strand, fluorescent probes are then renatured and combined with specific DNA sequences to form new hybrids that can be analyzed.

Nucleic Acid Electrophoresis

Separates DNA and RNA Fragments by size. Smaller fragments migrate faster through gel than larger. Uses agarose gel as porous matrix with given properties.

Conservative Replication

Entire double strand is template for new strands, yielding one heavy and one light strand.

Semiconservative Replication

Two nucleotide strands unwind with each serving as template for new strand, yielding two intermediate strands.

Dispersive Replication

Double stranded DNA breaks into pieces, with each piece serving as a template for new strands, yielding noncontinuous intermediate strands

Meselson/Stahl Experiment

Used 14N and 15N nitrogen isotopes to grow E. coli. E. coli in 15N media transferred to 14N media, subjected to equilibrium density gradient centrifugation. DNA extracted after each replication saw (1) two continuous intermediate strands then (2) two intermediate, two light.

Origin of Replication (ORI)

DNA Replication begins here, where helix is unwound and replication fork begins. Since replication is bidirectional, two replication forks form

Replicon

Length of DNA replicated

Bacterial Replication

Originates at OriC, a single circular piece fold creating two forks which eventually merge at termination region (ter)

Euk/Prok Replication Differences

Prokaryote DNA is lesser in information, circular, and devoid of proteins. Eukaryote DNA is full, linear, and complexed of proteins. Eukaryotes have multiple origins of replication at one point.

DNA Polymerization

Elongation from 5’ to 3’ by addition of one nucleotide at a time to 3’ end. During addition, two terminal phosphates are cleaved off. Requires a RNA primer oligonucleotide

5’-3’ Exonuclease Activity

Excises primers and fills in gaps

3’-5’ Exonuclease Activity

Proofreads newly synthesized DNA. Replaces or removes incorrect nucleotides.

DNA Poly I, II, III

Polymerases capable of 5’-3’ polymerization

DNA Poly I, II, III

Polymerases capable of Exonuclease Activity 3’ - 5’

DNA Poly I

Polymerases capable of 5’ to 3’ activity

Unwinding the Helix

DNAA initiator protein binds to ORI to change conformation and destabilize/open helix. DNA Helicase (DNAB), a hexamer of subunits, assembles around ssDNA, recruiting holoenzyme to bind replication fork and initiate replication. Single Stranded binding proteins stabilize open conformation of single strand.

To Reduce Supercoiling

Unwinding yields Supercoiling. DNA Gyrase relieves supercoilling, making single/double stranded cuts to relieve tension, driven by ATP hydrolysis. DNA Gyrase is a DNA Topoisomerase

Replication Primer Synthesis

DNA PolIII requires a free 3’-OH primer to add bases to. RNA Primase, an RNA Polymerase, recruited by helicase synthesizes the primer

Direction of Replication

Synthesis occurs on both strands simultaneously, causing DNAPolIII to move away from origin. DNA is antiparallel, replication occurs 5’-3’. Continuous on leading strand, discontinouos at lagging strand which jumps at each Okazaki Fragment and returns to ORI.

DNA Polymerase I

Removes RNA Primer and fills gaps during synthesis

DNA Ligase

Joins Okazaki Fragments together, catalyzing phosphodiester bonds and sealing nicks.

Replication Proofreading

DNAPolys w/ 3’-5’ exonuclease activity (I/II/III) allow proofreading, where mismatched bases are detected, removed, and synthesis can reoccur 5’-3’

Termination of Replication

When two replication forks meet (ter site), termination proteins bind to sites to block movement of helicase.

DNA Polymerase Variants

alpha, delta, epsilon involved in replication of nuclear DNA. DNA Poly Gamma involved in mitochondrial replication. Others involved in repair

Processivity

Strength of Association between polymerase and template. Low Processivity correlates to shorter chain. High Processivity can stay with the chain for longer, generating a longer continuous chain.

Polymerase Switching

Once a new primer is down, polymerases can switch among themselves. Poly Alpha, Delta, Epsilon for lengthening chain (Delta Lagging, Epsilon Leading)

Telomere

Long stretches of repeating sequences at end of chromosome, protecting intact euk chromosomes from improper fusion, degradation. Repetitive G-rich protrusions at 3’ end of dsDNA forms t-loops and complex of six proteins stabilizing ends.

Reverse Transcription

RNA and RT Primer to cDNA

Telomere Replication

Telomerase enzyme elongates telomere w/ complementary binding at 3’ end, synthesizing complementary DNA oligonucleotide which translocated and repeats of six nts to 3’ end. After complement made, telomerase releases, RNA primase synthesizes primer at new 3’ end, and replication can occur on telomere.

HU and HNS

Small positively charged proteins which bond ionically to phosphate backbone

Virus Chromosomes

Nucleic acid, D/RNA, circular or linear, inert until released by host, able to package long DNA into small volumes

Supercoiling

Compacts closed-circular DNA, allows ready sedimentation. Forms in direction opposite of unwinding. Negative Supercoiling implies DNA is being unwound

Linking Number (L)

L = DNA Length/10.4. Left handed DNA is negative, Right handed positive. Change in L is equal to actual number of turns in plasmid

Topoisomers

Molecules identical but with different linking number. Produced by topoisomerases, which cut one/both DNA strands, wind/unwind helix, then reseal ends.

Topoisomerases

Type I are transient single strand breaks (nicks), while type two are transient double stranded breaks

Histones

Positively charged proteins associated w/ chromosomal Eukaryotic DNA. Lots of Lys/Arg, making electrostatic bonds with phosphates. Five types H1, H2A, H2B, H3, H4

Nucleosomes

Tetramers of Histones, (H2A)2 - (H2B)2 and (H3)2 - (H4)2, which repeat as unit combinations as octomers. H1 histone acts as linker bound to regions between core particles. 200 bp, but can be digested into 174 bp unit, which can be further packed into 30 nm diameter structure.

Acetylation of Histones

Opens up chromatin fibers

Methylation of Histones

Increases gene activity, sometimes decreases

Euchromatin

Uncoiled and active, appears unstained during interphase

Heterochromatin

Condensed, mainly inactive, appears stained during interphase

Pseudogenes

Large number of single-copy noncoding regions, representing evolutionary vestiges, mutations, or regions not transcribed.

Codons

Every three ribonucleotides, a triplet code, each specifying one amino acid unambiguously

Degeneracy of Genetic Code

One amino acid can be specified by more than one triplet code

Universal Nature of Genetic Code

Codon chart is single code dictionary used by viruses, prokaryotes, archaea, eukaryotes

n-let codes

Four nitrogenous bases codes for 20 amino acids proving degeneracy. A code based on singlet/doublet/n-let can encode for 4^n amino acids.

AUG, Methionine

The Start Codon, modified into N-formylmethionine (fmet). Only one codon encodes this codon

UAG, UAA, UGA

The Stop Codons

Reading Frames

Continuous Sequences of nucleotides. Shifted by insertions/deletions

Frameshift Mutations

Shows genetic code is in triplets. Insertions/Deletions can change the groupings of three.