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Forward genetics
o Phenotype to genotype
Reverse genetics
o Genotype to phenotype
Cell is the basic component of organisms
o Nucleus contains the genes
o Mitochondria have their own genome
o Prokaryotic cells differ
Genetic material in a nucleoid region
Cell is organized but has no organelles
Almost everything is encoded in the DNA
o DNA karyotype-lay out chromosomes
Centromere
o Helps the chromosomes migrate from the middle of cell to poles
o Metacentric=middle
o Submetacentric=below the middle
o Telocentic=at the end
Cell division is essential to life
o Mitosis-division (exact copy)
o Meiosis-gametes (not an exact copy due to crossing over)
Spermato/oogenesis
2 separations to get haploid cells
o Cell must condense into chromatin
o Spindle attaches to kinetochore via the centromere
DNA replication can induce errors
o Mutations or other changes
o If it was perfect there would be no variation
Source for variation
o DNA replication and repair
o Crossing over and chromosome segregation
Cell cycle is monitored by checkpoints
o G, S, and M
o G0= nondividing cell
o Interphase is G and S
o The checkpoints can let mistakes through
They check for DNA damage or a failure to replicate
Something is wrong=apoptosis
Phenotype
o Appearance
o What is expressed
o Could be complex
Genotype
o What do the genes say
o Homo/heterozygous
o Dominant vs recessive
WT vs Mutant
o Wildtype is the normal that is defined
o Mutant is any changes
o Only 1 WT, but many mutants
Mendel
o Found that in the F1 generation only one gene/phenotype dominated
o But if F2 it was a 3:1 phenotypic ratio
o Chose phenotypes coded for by 1 gene
Monohybrid cross
o Only one gene being crossed
o Start with homozygous parental strains
Recessive alleles
o Only expressed when two copies of the gene are present
o In most cases the WT is dominant, but WT can also be recessive
Homozygous
o Two alleles the same
o Can be dominant or recessive
Heterozygous
o Two alleles are different
o Dominant will be expressed in most cases
Hemizygous
o Only one allele present
Dihybrid cross
o Two genes cross to see effect
o 9:3:3:1 outcome in the F2 generation
o Independent assortment
Independent assortment
o Combine the probability of one trait w/ probability of getting another
o Multiply
Test cross
o Can determine genotype if unknown but have a known phenotype
o Difference between homo and heterozygous
o Cross unknown with homo recessive
If homo- get all dominant expression
If hetero-get some recessive expression (1/2)
Human crosses
Dominant mutations
o Why is simple genetic dominance most often observes for geno/phenotype?
Only need one allele present to function completely
Mutations
o Missense
Point mutation where the codon and changes the AA and protein
o Neutral
Changes the codon and AA but not the protein
o Silent
Changes the codon but not the AA or protein
o Nonsense
Premature stop codon
Complete dominance
o Homo/heterozygous express the same phenotype
Incomplete dominance
o Heterozygotes have an intermediate phenotype
Codominance
o Express both alleles in the heterozygote
o Ex: blood type
Recessive lethal mutations
o The homozygous recessive is lethal and will not survive
o Do not factor it into the probabilities since they are unable to pass it on
Mixed modes of inheritance modify the 9331 ratio
Epistasis
o The effect of one gene depends on the presence of one or more modifier genes
Ex: agouti mice-can only get the agouti pattern if colored a certain color
o Recessive or dominant epistasis
Novel phenotypes
o Get something completely unexpected from a cross
Pleiotrophy
o One mutation has a cascade of effects in the body
Complementation
o Helps to determine where in the genome the gene is located
o If in the same place, the cross leads to a mutation
o If in different places the cross leads to a normal phenotype
Sex linked
o Genes located on the x chromosome
o Males have to get their x from the mom and their y from the dad
Pedigrees
o Again help see the expression pattern
Does a genotype always result in the same phenotype
DNA
Functions
o Replication
o Information storage
o Info expression
Variation through mutation
o Allows new characteristics to evolve
Central Dogma
o DNA
Transcription
o RNA
Translation
o Protein
Has to flow in this direction unless a virus goes from RNA to DNA with reverse transcriptase
Ribosome is formed by rRNA
mRNA is loaded into the ribosome
tRNA brings AA to the ribosomes
DNA and genome size
o More genes doesnt mean more complexity
o Such thing as alternative splicing
DNA as the genetic material
o Griffiths transformation
Found that transformation occurred by some molecule
o Avery, Macleod, McCarthy
Only when using DNAse did transformation not occur
o Hershey-Chase
Used bacteriophages and labeled molecules
DNA with phosphate
Protein with Sulfer
Found labeled DNA in the cell
RNA can be the genetic material
o Viruses can have ss/ds DNA or RNA
o Reverse transcriptase
o Integration
Discovery of DNA
DNA Polym READS from 3 to 5 and SYNTHESIZES from 5 to 3 a new DNA strand
DNA polym
o I, II, III
o All can proofread and replace their mistakes
Holoenzymes
o Protein machine made up of multiple proteins and TFs
Bacteria
o Origin of replication is defined by a repeated sequence (9 mer)
o DNAa molecules bind and create an initial bubble of replication
o DNAb/c bind to the bubble and initiate helical unwinding
o Primase adds an RNA primer
o DNA polym starts
Leading vs lagging strand
o All replication proceeds towards the replication fork
o One strand is continuous
o One strand is discontinuous
Needs multiple primers
Multiple okazaki fragments
Ligase sticks together
DNA poly I
o Replaces the RNA primer with DNA
DNA gyrase
o Untangles the DNA helix
Speed
o Euk > Pro because there are multiple origin sites
Euk are not circular
o Have an issue with end of chromosomes
o Some cells have telomerase, which acts as an end primer to avoid losing some of the
telomere
o Most cells dont have telomerase and lose a small portion with each replication
o Telomerase is only very active during large periods of replications, or when the cell is a
stem cell
Replication and recombination
o Need a single stranded break, then a ligation to a different place
o Crosses with its homologous region and allows for recombination because a piece of
DNA switched from one chromosome to another
Transcription
Transcriptome-all transcripts
Proteome-all proteins
Metabolism-all metabolic compounds
Transcription
o Help get an RNA message
o Need a template strand of DNA to get to RNA
o RNA is identical to the coding strand, but is matched up with the template strand
Prokaryotic cell
o Replication, transcription, translation occur in the nucleus (nucleoid region)
o Need an RNA polymerase
o Scans for an RNA binding site
o Need the sigma subunit to recognize the specific initiation sequence
Nascent RNA
o Transcript
Sigma factor dissociates after a few nucleotides of the RNA strand is built up
o Only necessary for binding and recognizing the promotor
o Recognize TATA box upstream
Operons
o Genes often found in a segment together
o Get a polycistronic mRNA
o Only found in prokaryotes
Ribosomes translate as mRNA is being transcribed
o No posttranslation modification
o Occurs faster than in Euk
o Quickly ramp up protein production
Eukaryotes
o Many more regulation of the mRNA
o Separated into compartments
RNA types
o mRNA
o tRNA
o rRNA
o miRNA
o catalytic RNA
Chromatin in Euk
o Densely packed DNA and organized by histones
o Before transcription may need to modify the chromatin
Hetero/Euchromatin
o 3 types of RNA polymerase
I=rRNA
II= mRNA and snRNA (nucleoplasm)
III=ssrRNA, tRNA (nucleoplasm)
o RNA Polym II promotors have a core promotor, and enhancer elements
TATA box
o Not a lot have it, but if a gene has it, it is essential to transcription
o Binds the RNA polymerase after binding TATA Binding Protein
o Allows for a transcription regulation
o Brings other RNA poly to site to increase regulation
CAAT box
o Another example of a TATA like binding element
Enhancers
o Specific sequence that can be located in front of, in, or after the gene
o If located in the gene it keeps the gene from being translated
o Can activate or depress depending on location
TF
o Generalized proteins that bind specific sequences to regulate genes and expression level
Transcript
o Eukaryotes need to mature it
o Add a methyl G cap and poly A tail to stabilize
o Alternative splicing
Exons vs introns
Introns spliced out
Immature RNA s always longer than mature RNA (remove introns)
Complexity
o Think about number of proteins, not the number of genes
o Genes also interact with each other in different ways (regulate)
Splicing
o Group 1
Make rRNA
Need a guanine to bind to an active site within the intron
Expressed hydroxyl, this attacks the donor site at the other end of the intron
and splices it out
o Group 2
mRNA
needs snRNPs
get a complex that forms lariat loops that splice out introns
exons ligated
modify the transcript
o RNA editing
o Substitution editing
Get a change of a nucleotide in a transcript
o Two forms of a protein depending on editing
o Insertion/deletion editing
Can alter the function and shape of protein
Or bring proteins into the proper reading frame to establish function
Translation
Amino Acids
o R Group only thing that changes
o Hydrophilic/hydrophobic
o Polar (charged)
The r group differs in forl/function
o Change the folding by mutations
Protein sequence
o Primary=AA sequence
o Secondary=alpha helix or beta sheet (H-Bond stabilized)
o Tertiary=whole protein folding
o Quaternary=multiple proteins folding
Domain-functional part of protein that has a certain structure
Post protein modifications (post-translational)
o N terminal AA is often modified
o Add carbs to the protein
o Golgi editing
Functions of proteins
o Structural
o Contractile
o Signaling
o Storage
o Transport
o Enzymatic
Roles
o Enzymatic
Lower activation barrier
o Signal sequence-domain that attracts substrate
o Membrane anchoring
Mutations
Germline vs soma
o Much more dangerous in germline, passed onto future generations
Classes= LOF, GOF, null
Transition
o Purine changed into a different purine
Transversion
o Purine changed for pyrimidine
Repeat expansion
o Continue to get repeated sequences
Genetic analysis
o Use mutations to ID mutations and their resultant phenotyoes
Evolutionary Genetics
Darwinian evolution
o Species have a common ancestor
Neodarwinism
o Discovery of genetics
Evolution requires:
o Variation between organisms
o Competition between individuals
o Selection
Descent from common ancestors
o Can use genetics to find these relationships
Two forms
o Micro/macroevolution
o Large and small scale
Phylogenetic tree-shows relationship between species
o Stasic-doesnt change
o Anagenesis-one species evolved into a different one
o Cladogenesis-species diverged into 2 separate ones
Morphology
o Species based on the way they look?
o Not a great model due to different looking organisms being of the same species
DNA organization
Simple chromosomes
o Viral and bacterial chromosomes often consist of single DNA molecules
o Bacteriophage=lambda (lollipop head)
Circular replication
o Cut bu a nuclease
o Copied discontinuously and continuously
Bacterial DNA packaging
o Ecoli supercoils the DNA
o DNA has no tension due to turns
Eukaryotes
o Organize using histone proteins
o Condensed state get G-bands (dark and light)
o Can alter the packaging to get to genes
DNA loops out of chromosomes when needed
Nucleosome
o Histone octamer
Solenoid
o Group of 6 nucleosomes
Looped domains
Chromatin fiber
Chromatid
Net packing ratio of 500:1
Repetitive DNA
o 98% is repetitive DNA
o Centromeres
Sister chromatid cohesion
Assembly site for kinetochore
CEN
The minimal DNA required for centromere function
o Satellite DNA
Repetitive pieces of 2 or 3 nucleotides that are constantly repeated
o DNA isolation
Satellite DNA has a lower density
Less dense with more A-T bonds
o VNTR
Variable number of tandem repeats
o STR
Short tandem repeats
Very short 5 or less bases
o LINE
Long interspersed nuclear elements (transposon)
o SINE
Short
o Ribosomal genes
Repeated in the DNA
Epigenetics
o Histone modification
o Can be passed on
o Reversible
Epigenators
o Environmental signals (internal or external)
o Signal is transduced to the cell
Histone modification
o Histones have a tail that can be modified
Acetylation
o Opens up
o Deacetylation closes
Methylation
o Opens or closes depending on location
HDAC
o Histone deacetylation complex
o Closes the DNA up
HAT
o Histone acetylation complex
o Opens dna up
CPG islands
o Sites where the DNA is methylated
Imprinting
o
o
o
Karyotype
o Group chromosomes and banding patterns
Aneuploidy
o 2nx chromosomes
Euploidy
o Multiples of n chromosomes
Polyploidy
o Multiples of the same gene
o Auto/allopolyploidy
Auto=duplication of whole genome
Allo=duplication of 2 diff species
Nondisjunction
o Doesnt separate
o Leads to trisomy
o Trisomy 21=downs
o Trisomy 13=patau
o Trisomy 18=Edwards
Chromosomal rearrangements
o Need breakage of a chromosome
o Terminal deletion (lose piece at origin)
o Intecalary deletion
Form a deletion look and it ejects a gene out of a chromosome
o Deletion loop
Duplication
o Unequal crossover between 2 sets of homologous chromosomes
o rRNA present in many copies
CNV (copy # variants)
o Chunks of repeated DNA in chromosomes due to duplication
o Can be present within promotor regions
o Can cause an increase in the replication of the gene
Inversion
o May express new genes
o Can happen due to loop
o Forms a 4 part breakage
Paracentric
o Doesnt change arm length
Pericentric
o Changes the length of the arms
Consequences during chromosomal inversion
o Inversion heterozygote=one inverted and ore normal chromosome
Crossing over leads to nonfunctional chromosomes
Nonreciprocal translocation
o One chromosomes steals from another
Reciprocal
o They share-just changes chromosomes
o Forms a cruciform tetrad during meiosis
Robertsonian translocation
o Exchange of small arm of one chromosome for the large arm of another
o Can get familial downs
Fragile X
o Pieces of the X can break off at the end
o Its so thin because the DNA isnt as condensed
Microbial genetics
o Exactly the same genes but cant see changes without mutations
GWAS (Genome wide association study)
o The goal is to map phenotypes and where they appear on chromosomes based on maps
Extranuclear inheritance
IS elements (bacteria)
o Insertion sequence
o Defined by inverted terminal repeats
o Flanked on both sides of the gene
o Transposons
Recognizes inverted terminal sequences specific for an IS
Inserts the sequence somewhere else in the genome
o DNA bases transposon elements (tn)
Can be larger
Heteroduplex
o The complementary sequence that helps it bud off
In the presence of Ac, Ds is not transposable
o But Ac alone can transpose
o
o
Recombinant DNA
Genomics
Sanger sequencing
o Able to do short segments of the genome (about 1000)
Next gen sequencing
o Sequences the entire genome
Clone by clone sequencing
o
o
o
Omics
Human Genetics
Females XX (homogametic)
Males XY (heterogametic)
Theory-the embryo can develop into male/female
o At one point important changes due to the presence of the Y chromosome set the male
into action
Y chromosome
o PAR region (pseudo autosomal region)-allows the Y chromosome to pair with the X for
mitosis and meiosis
o MSY region (male specific)-genes that make a male a male
o SRY region (sex determining region)-induces the development of testes
Expressed 6-8 weeks into development
o TDF=testes determining factor
X inactivation
o Due to dosage compensation
o Creates a barr body
Utilizes the Xic region and the T-six gene
Single Nucleotide polymorphisms (SNPs)
o Differences in genomes between organisms, or genetic variation
Genomic variation (types of SNPs)
o RFLP
Operons
o The idea of an operon is that in prokaryotes, many genes that are expressed together
are under the control of the same promoter elements
Inducible operons (also known as adaptive, facultative)
o Only expressed when necessary
o System can be turned on/off depending on environmental stimuli
o Positive control
RISC
RITS
Gene Function
Forward genetics
o Genome wide genetic screens for mutants with specific phenotypes
o Id the genotype that creates the phenotype
Reverse genetics
o Define every gene in the genome based on sequence analyses
o Reduce/eliminate functions of specific genes and assess the phenotypic impacts
Model organisms
o Easy to grow
o Short generation
o Abundant progeny
o Can cross in large numbers
Yeast
o Simplest eukaryote
o Haploid and diploid alternating generations
o Phenotypes are evident in haploid
o Diploid allows for recessive lethal mutations to be studied
Drosophila
o No meiotic crossing over in males
o Diploid
o Recessive lethal mutations are maintained in strains heterozygous for balancer
chromosomes
P-Elements
o DNA transposons that insert into the genome
o Can enable transformation
Wild type or altered copy of the gene to assess transgene function
Reporter gene in which enhancer/promoter drives expression of beta-gal or
other detectible genes
o Need a positive selective marker
o Can use this to destroy genes
Randomly inserts itself into the open reading frame
o P elements either insert or destroy gene
Mice
o Genomic synteny with humans
o Large scale genomic screens difficult
o Creating transgenics and gene knockouts/replacements is more feasible
Mutagenization
o Mutagenize parental strain, then perform crosses to generate progeny that can be
assessed for phenotypes of interest
Types of mutations
o Chemical
EMS, ENU
o Radiation
X-Rays, gamma radiation
Screen mutations
o Genetic screen helps select out the ones which were mutated
o Can look at yeast and determine the stages of cell cycle
See any arrested development
Will not grow if mutated
o Replica plating
Get the same colony and grow under different stressors to see if mutations are
sensitive or if new mutations appear under stress
o Screening mutants (Balancer Chromosomes)
Can screen for recessive mutations in diploids by creating a collection of
mutagenized chromosomes in balanced heterozygotes
Assess the phenotypic impact of homo/hemizygocity
Retain mutations of interest
Untangling paths
o The order of generation is based on epistasis
o The effect of mutation in one gene masks or modifies the mutation in another gene
o Pathways affected by this-because every gene must be present to make a product
o Use epistasis analysis to determine which gene is at the top and which is at the bottom
Can determine pathway order with mutation to genes
o Screens for suppressor mutations can ID additional genes in a pathway not Ided in an
initial screen (second round of mutagenesis)
Second mutation by chance mutates the other one t try and bypass the initial
mutation
Modify the original phenotype
o Suppressor mutants
Diminish/eliminate the phenotype caused by the initial mutation
Gene product sequence
o May reveal gene function
o Presence of domains known to have specific functions
Bioengineering
Body Plan
Developmental genetics
o Genetic and molecular mechanisms underlying cellular and organismal development,
homeostasis, aging and senescence
Development
o Develop tissues
o Death of specific tissues
o Balance between growth and death
Specification
o When genetic and positional cues confer a spatially discrete ID on cells
Determination
o Cells time when a specific developmental state becomes fixed
Differentiation
o Process by which a cell achieves its final form and function
Hypothesis
o Development-attainment of a different state by all somatic cells in an organism
Variable gene activity hypothesis
o Differential expression and action of genes
Controls development
o When and where are genes expressed and active
o How is gene expression regulated
Preformation
o Sperm had little human inside that became bigger
Fertilization occurs when an egg and sperm fuse
o Maternal cytoplasmic components
o mRNA and proteins
first components to trigger development
without these nothing would happen
body plan
o very similar in organisms within the same species
o Pattern of organization-characteristics and recognizable traits
Pattern formation
o Aspects of development of the body plan
o Leads to genesis of patterns or structures that make up the body plan
Number of axes (primary)
o Anterior
o Posterior
o Dorsal
o Ventral
Animal body plans are segmented
o The body plan has 11 segments
o Often has appendages
Drosophila
o Homologies among embryonic, larval, and adult body plans
o Governed by a set of genes
o Different segments develop into different parts
Segmental organization of embryonic and adult tissues is homologous
Segmental disks develop into extremities
o Imaginal discs rise to external structures
Mutations that alter the body plan affect the pattern formation
o 3rd segment develops into a second segment-fly has 2 sets of wings
Embryogenesis over 24 hours get the body plan and imaginal discs
Syncytial blastoderm (multiple nuclei)
o Followed by nuclear migration and cellularization
o The pole cells form at the posterior and are the precursors to a germ cell line
o Maternal functions direct the AP and DV axes
Zygotic genes
o Part of the genome but regulated by maternal effect genes
o Gap genes, pair rule genes, and segmental polarity genes form the body plan
Then homeotic genes (HOX genes) determine the fate of cells and specify the type of
cell they will become
Nuslein-Volhard and Wieschaus
o Determined which genes are important to body plan
Maternal effect genes
o Form the anterior posterior gradiants
o Gap genes are triggered (they are TFs)
Trigger certain genes in gap genes to form the band regions
o Formation of discrete bands triggers pair rule genes
Divide gap gene bands into smaller regions
o Activation of pair rule genes activate segment polarity genes
Even more divided
o Then the hox genes are activated and specify the ID of each segment
Gap genes are zinc finger TFs
o Activate the next set of genes
Pair rule genes
o Often encode helix turn helix TFs
o Overlap of TFs or non overlap specifies specific segments
Mutations
o Runt (mutated RunX2 protein) encodes a TF
o Mouse doesnt have proper muscle/bone development
o Humans get cleidocranial dysplasia
o Autosomal dominant diseases
Two Hox genes clusters in drosophila
o Antennapedia complex and Bithorax complex
Hox genes and TFs with Homeobox
o DNA binding homeodomain
o Different complexes influence the further specification of segments into specific cell
types
Gene organization with Hox genes
o Hox genes have a logical order in the DNA
o Not intermixed
o Collinear with expression patterns in the embryo
Humans
o 4 human hox gene clusters
39 total
o Control A-P patterning in humans (and other vertebrates)
o 5 end genes
o Limb development
o Humans dont get mutated very often (need a double mutation since diploid)
get smaller changes (ex: polydactyly)
o
o
o
o
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Cancer genomics
o
o
o Radiation
o Smoking
o Other factors
Drug design
o Use the exact path to develop drugs
o Many times specific to a certain mutation
o Gleevec
Acts as ATP and binds the site that allows BCR-ABL to stimulate cell division
when bound to ATP
o Trastuzumab
Binds to HER-2 and induces its removal (down regulation)
HER signals less intense and the cell therefore divides less often