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317 Cards in this Set

  • Front
  • Back
Genetics
the study of heredity, genes, and variation of organisms
Levels of genetics
molecules, cells, individuals, populations, species
Why study genetics?
-Basic knowledge about how life works
-Applications that benefit humans
-Evolution, phylogony and taxonomy
-Population and Conservation biology
Classical genetics
-why do offspring resemble parents?
-trait transmission
-patterns of inheritance
-natural and artificial selection
Molecular genetics
-what is the biochemical basis of traits?
-DNA structure and function
-genes to phenotype
-DNA protein interaction
applications that benefit humans
-livestock and crop plant development (agriculture)
-understanding inherited diseases (health)
-understanding cancer
-production of pharmaceuticals
-crime scene analysis
Evolution, Phylogony and Taxonomy
Relationships among species
Population and conservation biology
population dynamics and extinction risk
Genotype
genetic make-up of a cell or individual; refers to specific genes/alleles present
Phenotype
a cell or individual's observable or measurable traits
-may be measured biochemically
Autosomes
all chromosomes in the cell nucleus with the exception of sex chromosomes
Autosomal inheritance
-most common inheritance pattern
-patterns apply equally to males and females
P
parental generation
F1
first filial generation
F2
second filial generation
Dominant
-one trait is expressed over another even though both are present
-determined by whether or not the phenotype can exist with only one copy of the allele
Recessive
trait not expressed when present with dominant trait
"particles" carry contrasting phenotypes
genes are carried on chromosomes
"Particles" are paired, can be same or different
alleles on homologues
"Particles" are split during gamete formation
meiosis (diploid -> haploid)
Monohybrid cross
P: A/A x a/a
F1: A/a x A/a
F2: 1A/A:2A/a:1a/a = 3dominant:1recessive phenotype
Testcross
breed individual of unknown genotype to known homozygous recessive individual (tester)
-useful for determining unknown genotypes
Mendel's First Law: Law of equal segregation
The two members of a gene pair segregate from each other into the gametes; half the gametes carry one member of the pair and half carry the other
-pair = alleles
-segregation into gametes = meiosis
Gene
locus on a chromosome
Allele
all different variations of that gene
What do genes do?
-Make RNA as a final product (e.g. ribosomal RNA)
-Make RNA followed by protein (most genes)
dihybrid cross
-2 traits
-P: y/y.R/R x Y/Y.r/r
-F1: Y/y.R/r
-F2: 9 dom,dom: 3 dom,rec: 3 rec,dom: 1 rec,rec
Mendel's second law: Law of independent assortment
-different gene pairs are inherited (assorted) independently of one another in gamete formation
-NOW: genes on different (non-homologous) chromosomes are inherited independently
Propositus
the first affected member of a family who comes to the attention of a doctor/geneticist
Human Autosomal Recessive Disorders
-Acromesomelic dysplasia - form of dwarfism
-cystic fibrosis
-albinism
-A/A = normal
-A/a = carrier
-a/a = affected
-don't see them often in the wild because it's selected against
Pedigree Analysis: Autosomal Recessive Disorder
-rare - many unaffected
-parents unaffected
-male and female progeny affected
-clustered phenotype (not seen, then 2 affected in same generation)
Consanguineous mating
close relatives mating
-represented in pedigree as a double line
Haplosufficiency
heterozygote has enough normal gene product to be phenotypically normal
-recessive mutations
Human Autosomal Dominant disorders
-Pseudoachondroplasia - more common form of dwarfism
-progeria - accelerated aging, lethal, heavily studied
-Huntington's disease - brain atrophy
-a/a = normal
-A/a = affected
-A/A = lethal
Pedigree analysis: Autosomal Dominant
-affected in every generation (common)
-Parent affected in each case
-Male and female progeny affected
Autosomal polymorphisms
variation for traits within populations
Haploinsufficiency
heterozygotes do not have enough normal gene product to be phenotypically normal
Hemizygous
genes effectively present in single copy in males
X-linked inheritance
-males get one copy of gene in differential region of X chromosome
-Males express whatever single allele they inherit from female parent
-differences in phenotypic ratios by sex, differences in reciprocal crosses
X-linked human disorders
-both dominant and recessive
-fathers can only transmit conditions through daughters
-mothers can affect both sexes - greater probability of affecting sons
-often females unaffected or carrier, males unaffected or affected
*Female parents unaffected having affected sons and unaffected daughters
Y-linked inheritance
-should be possible but few know traits associated with Y linked genes
-would be found in males only, and passed to 100% of male offspring
-females cannot be carriers
-possible e.g. aspects of infertility, external hair on ears
Cytoplasmic inheritance
-mitochondria and chloroplasts have own DNA
-organelles replicate own DNA in cells
-organelles are passed to progeny by single parent, usually female
-female organelle phenotype is passed to 100% of progeny regardless of male genotype/phenotype
Mitochondrial diseases
-nervous system (seizures)
-myopathy (cardiomyopathy, etc)
-diabetes
Chromosomal theory of heredity
genes that encode for traits are located on chromosomes
Important features of chromosomes
-chromosome number is identical from cell to cell within an individual
-chromosome number is identical among individuals of the same species
Characteristics of Chromosomes
-Dynamic structures
-single DNA molecule
Genetics
the study of heredity, genes, and variation of organisms
Levels of genetics
molecules, cells, individuals, populations, species
Why study genetics?
-basic knowledge about how life works
-Applications that benefit humans
-Evolution, phylogony, taxonomy
-Population and conservation biology
Classical genetics
why do offspring resemble parents?
-trait transmission
-patterns of inheritance
-natural and artificial selection
Molecular genetis
what is the biochemical basis of traits?
-DMA structure and function
-genes to phenotype
-DNAprotein interaction
Applications that benefit humans
-livestock and crop plant development (agriculture)
-Understanding inherited diseases (health)
-understanding cancer
-production of pharmaceuticals
-crime scene analysis
Genotype
genetic make-up of a cell or individual; refers to specific genes/alleles present
Phenotype
a cell or individual's observable or measurable traits (can be measured biochemically)
Autosomes
all chromosomes in the cell nucleus with the exception of sex chromosomes
Autosomal inheritance
-most common pattern of inheritance
-patterns apply equally to males and females
Dominant
-one trait expressed over another even though both are present
-determined by whether or not the phenotype can exist with only one copy of the allele
recessive
trait not expressed when present with dominant trait
Testcross
breed individual of unknown genotype to known homozygous recessive individual (tester)
Mendel's First law: law of equal segregation
-the two members of a gene pair segregate from each other into the gametes; half the gametes carry one member of the pair and half carry the other
-gene pair - alleles
-segregation into gametes - meiosis
gene
locus on a chromosome
allele
all different variations of that gene
What do genes do
-Make RNA as a final product (e.g. ribosomal RNA)
-Make RMA followed by protein (most genes)
Mendel's second law: law of independent assortment
-different gene pairs are inherited independently of one another in gamete formation
-NOW: genes on different (non-homologous) chromosomes are inherited independently
Pedigree analysis
examining family records to trace the inheritance of a trait
Propositus
the first affected member of a family who comes to the attention of a doctor/geneticist
Human Autosomal Recessive Disroders
-Acromesomelic dysplasia - form of dwarfism
-cystic fibrosis
-Albinism
-A/A = normal
-A/a = carrier
-a/a = affected
-don't often see in wild because they are selected against
Pedigree analysis: Autosomal recessive
-rare - many unaffected
-parents unaffected
-male and female progeny affected
-clustered phenotype (not seen, then 2 affected in same generation)
consanguineous mating
close relatives mating
-in pedigree represented by double line
haplosufficiency
heterozygote have enough normal gene product to be phenotypically normal
Human Autosomal Dominnt Disorders
-pseudoachondroplasia - more common dwarfism
-progeria - accelerated aging
-Huntington's diseas - brain atrophy
-a/a = normal
-A/a = affected
-A/A = lethal
Pedigree analysis: Autosomal Dominant
- affected in every generation
-parent affected in each case
-Male and female progeny affected
Autosomal polymorphisms
variation for traits within populations
Hemizygous
genes effectively present in single copy in males
X-linked inheritance
-males get one copy of gene in differential region of X chromosome
-males express whatever single allele they inherit from female parent
-differences in phenotypic ratios by sex, differences in reciprocal crosses
X-linked human disorders
-both dominant and recessive, fathers can only transmit conditions through daughters
-mothers can affect both sexes - greater probability of affecting sons
-pedigree: often females unaffected or carrier, males unaffected or affected
*Female parents unaffected having affected sons and unaffected daughters
Y-linked inheritance
-should be possible but few known traits associated with Y linked genes
-Y-linked traits would be found in males only, and passed to 100% of male offspring
-Possible examples: aspects of infertility, external hair on ears
Cytoplasmic inheritance
-mitochondria and chloroplasts have own DNA
-Organelles replicate ownDNA in cells
-organelles are passed to progeny by single parent, usually female
-female organelle phenotype is passed to 100% of progeny regardless of male genotype/phenotype
Mitochondrial diseases
-nervous system(seizures)
-myopathy
-diabetes
Chromosomal theory of heredity
genes that encode for traits are located on chromosomes
Important features of chromosomes
-chromosome number is identical from cell to cell within an individual
-chromosome number is identical among individuals of the same species
Dynamic structures of chromosomes
-diffuse - occupy space in nucleus during normal cell function
-condensed - special form taken during cell division
Single DNA molecule of chromosome
-one densely folded DNA molecule per chromosome
-Genes = functional sequences along DNA molecule
5 levels of DNA folding
-DNA in double helix
-DNA winds onto histone proteins
-DNA-histone complexes coil into solenoid
-Solenoid forms loops attached to central scaffold
-Scaffold and loops arranged in giant super-coil
p-arm
short arm
q-arm
long arm
telomere
ends, work as a clock- reduces telomeres in length with each cell division
heterochromatin
black bands - dense, dark stain
euchromatin
light bands - not as densely packed as heterochromatin
-genes found mostly here
satellite DNA (tandem repeats)
units of DNA repeated over and over again, doesn't code for anything, highly concentrated around centromere and somewhat telomer
Exon
protein coding region, has unique DNA
Intron
in coding region, removed from RNA
Non-coding region
repetitive DNA, promotor regions, 98% of the genome
Mitosis
single nuclear division, asexual cell division
- chromosome number, genotype of daughter cells constant
Meiosis
two successive nuclear divisions in germ cells
- chromosome number halved; daughter cells (gametes) haploid genotype
-ploidy change
Ploidy
the number of sets of homologous chromosomes in cells
-does not change during S phases or during mitosis; reduced by half in meiosis I
DNA content
the number of copies of the haploid genome in cells
-content doubled during S phase; reduced by half in mitosis and in both meiosis I and II
Hayflick limit
the number of times a cell will divide before it stops and becomes senescent
-presumably governed by telomere length shortened each cycle
-ranges but 40-60 divisions is general guideline
-cancer cells do not become senescent; telomere length can be restored
Interphases
DNA replication during S phase
-chromosomes diffuse
Prophase
chromosomes condense; sister chromatids visible, adhesion
Metaphase
chromosomes line up; sister chromatids on equatorial plane of cell
Anaphase
chromatid separation; sister chromatids pulled apart by spindle
kinetochore
point of spindle attachment
Telophase
Nuclear division - chromosomes arrive at poles
-nuclear membrane re-forms
-cell divides into 2
Meiosis in humans occurs?
-gonads, makes eggs and sperm
Meiosis in flowering plants occurs?
-anthers and ovaries, makes meiospores
Prophase I
Chromosomes pair
-sister chromatids visible (dyads)
-homologus dyads pair (tetrads)
-crossing over occurs (recombination)
Metaphase I
chromosomes line up - tetrads migrate to equatorial plane of cell
Telophase I
cell divides into 2 haploid cells
Reductional division
Meiosis I - reduces ploidy of cells by half
Prophase II - Telophase II
similar to mitosis
-no crossing over (homologous chromosomes separated in telophase I)
-4 daughter cells
sister chromatids
identical copies of same chromosome; share a centromere
-same genes and alleles
homologous chromosomes
different copies of the same cromosome; 1 from each parent
-same genes, but different or same alleles
Medical Cryogenics
study of chromosomes; abnormalities
Constitutional abnormalities
present from birth - inherited or introduced early in utero
Acquired abnormalities
occur later in life, often associated with cancer
Deletion
loss of material from a single chromosome
Inversion
2 breaks in a chromosome followed by 180 degree flip and re-attachment
Translocation
exchange of material between 2 or more chromosomes
Nondisjunction
failure to separate; results in extra chromosomes or absent chromosomes
Prenatal screening
-amniocentesis
-transcervical chronic villus sampling
-maternal blood test
-pre-implantation screening
Amniocentesis
-15-20 weeks post conception
-collection of fetal cells in amniotic fluid
-karyotype assembled and chromosome integrity is checked
Transcervical chronic villus sampling
-10-13 weeks post conception
-fetal cells scraped from placenta
-karyotype assembled and chromosome integrity checked
Maternal blood test
-week 10
Pre-implantation screening
-test-tube fertilization
-many 8 cell embryos
-test single cell from each
-implant healthy embryo
Recombination
the production of new allele combinations that differ from that of parental types
Independent assortment
unlinked genes recombine randomly to form new allele combinations
-50% progeny non-parental
Meiotic recombination
crossing over produces new haploid combinations of alleles
-<50% progeny non-parental
Testcross recombination
-if 50% parental types, 50% recombinants due to independent assortment
-if less that 50% frequency of recombinants, genes linked
Linkage
association of genes on the same chromosome
-inherited together
-excess of parental type gametes
Cis conformation
both dominant or wildtype alleles on one homologue
Trans conformation
dominant or wildtype alleles on different homologues
Chiasmata
points of intersection where 2 chromosomes exchange genetic material during crossing over
Linkage maps
conceptual diagram of relative distance between linked loci based on recombination frequency
Linkage maps important because
- understanding gene function
-genome evolution
-facilitate strain building
linkage maps to understand gen function
correlating phenotype with chromosome position (relationship to promoters, other genes)
Neighbourhood effect
clustered genes often transcribed as one unit; proximity to heterochromatin can affect expression (the closer an active gene is to heterocromatin, te less likely it is to be fully expressed)
Linkage maps for genome evolution
comparing relative position of same genes in different organisms
Linkage maps that facilitate strain building
catalogue of markers aids crosses to produce high probability of recombination
4 major interactions from gene to phenotype
-transcription - turned on or off by regulatory proteins
-protein interactions - several proteins from different genes may be required to form molecular machine
-protein activation - protein from gene 1 may need modification by protein from gene 2
-environment - enzymes require substrates, gene expression requires raw materials
Signal transduction
environmental cue triggers consecutive gene-controlled steps in a pathway
Auxotrophic mutants
individuals that grow in culture only when supplemented with substance not required by wildtype
Complete dominance
dominant phenotype expressed when only 1 copy of allele present
-A/A indistinguishable from A/a
-e.g. phnylketonuria (PKU) caused by deficiency in phenylalanine hydroxylase
PKU
phenylketonuria
-absence of PAH (phenylalanine hydroxylase) results in build up of phenylalanine, causes disorder in homozygous recessives
-one copy or dose of wildtype alle is enough to breakdown phenylalanine
-wildtype allele is completely dominant, haplosufficient
Incomplete dominance
heterozygous intermediate between two homozygous phenotype
-e.g. pigmentation in 4:00 flowers
-A/A = red
-A/a = pink
-a/a = white
Gene dosage
each allele yields a certain amount of protein product - finite amount towards phenotype
Codominance
expression in heterozygote of both phenotypes normally shown by either allele
-E.g. human ABO blood typing
Dominance relative to level of measurement
-E.g. individual heterozygote - complete dominance; cellular level of that individual - codominance
-e.g. sickle cell anemia:
--HbA/HbA = normal cell shape and hemoglobin
--HbA/HbS = normal cells unless O2 very limited (complete dominance normally; some sickle cells can be partly induced- incomplete dominance in low O2)
--HbA/HbS - both normal and mutant hemoglobin types, so codominant at molecular level
What is a microsatellite?
A type of genetic marker
-a DNA sequence with a known location on a chromsome that can be used to identify individuals or species
-A short tandem repeat DNA sequence (usually 6-8 bp long)
-Found in many places across genome
Why use microsatellites?
-been found in the genomes of every organism analyzed so far
-show a high level of polymorphism
-neutral genetic markers
-codominant
-small amount of DNA needed
How to use microsatellites
-identify the microsatellite
-design PCR primers
-Collect samples and extract DNA
-amplify DNA using PCR
-separate DNA fragments according to size using capillary electrophoresis
-repeat process for all samples at multiple loci
Identify microsatellite
search DNA sequence for microsatellite repeats
Primer
strand of nucleic acid starting DNA synthesis
Design PCR primers
amplify specific microsatellite repeat in a PCR
-work for every individual in the species
-produces different sized products for each of the different alleles
Pleiotropy
allele of one gene afffects multiple phenotypic traits
Suppressor
allele of one gene reverses effect of mutant allele at other gene; restores wild type
P53
tumour suppressor in humans
-on short arm of chromosome 17
-senses DNA damage; activates DNA repair, regulates cell cycle, can induce apoptosis
Modifiers
mutation in one gene changes expression level at another
Effects of environment of expressivity of gene
conditions for expression of phenotype may not be present
-e.g. individuals with the same genotypes may ave different phenotypes until environment changes
Effects of other genes on expressivity
combinations of alleles at other genes may affect expression of phenotype
Subtlety of phenotype
may be difficult to detect absence of particular traits
Expressivity
degree of expression of particular allele; intensity of phenotype
BRCA 1
causes excess cancer risk; many with genotype get cancer, many don't
-variable penetrance
Penetrance
% of individuals with a given allele who exhibit the phenotype associated with that allele
Marfan Syndrome
all have mutation in FBN1 gene; range of phenotypes
-variable expressivity
Detecting gene interactions
-generate mutants, screen for variants in pathway of interest
-establish multiple independent mutant lines for same phenotype
-conduct select crosses to create double mutants
-examine double mutants for several important types of events
Complementation test
can wildtype be restored by crossing 2 different true-breeding mutants?
-NO- mutants are variants in same gene
-YES- mutations are in different genes that affect same pathway- gene interaction identified
Epistatic mutations
disrupt pathway at earlier point that known single mutant
-always dominant in double mutant
-always in reference to a pathway or phenotype affected by multiple steps
-cause particular changes to Mendeliam ratios
-e.g. Arg synthesis pathway:
--Arg-1 mutant epistatic to Arg-3 (Arg-1 mutant can never see Arg-3 phenotype)
Requirements for DNA as hereditary Material
1. Must be faithfully replicated and past on to all daughter cells
2. Must have information content to encode for wide array of traits
3. Must rarely undergo heredity changes to provide raw materials for natural selection
How DNA meets requirements for hereditary material
1. DNA is replicated via a semi-conservative mechanism and passed to all daughter cells
2. DNA has information content encoded in the order of nucleotides
3. DNA replication is not 100% accurate, makes mistakes periodically
DNA is made up of?
-nitrogenous bases
-deoxyribose sugar
-phosphate
2 types of bases
purines
pyrimidines
Purines
double ring structures
-adenine
-guanine
Pyrimidines
single ring structures
-cytosine
-thymine
nucleotide consists of?
assembled sugar, phosphate and nitrogenous base
Chargaff's Rules
-amount of purines = amount of pyrimidines
-amount of T=A, amount of G=C
Complementary bases
Each DNA base only pairs with one other
-A-T
-G-C
Semi-conservative replication
daughter cells contain double helix, one strand is new, the other directly from parent cell
Replication fork
site at which double helix is unwound to expose templates for copying
DNA polymerases
enzyme group which bring nucleotides to replication fork
Nucleotides added to _____ end only
3prime
leading strand
elongated in direction of fork movement, continuous addition of nucleotides
-requires one initial priming to begin
lagging strand
elongated in opposite direction of fork movement, nucleotides added as discontinuous segments
-requires repeated priming between segments
Okazaki Fragments
segments of DNA added to lagging strand
-avg 1000-2000 bp
Fidelity
accuracy of DNA replication, adherence to base-pairing rules
-less than one mistake per 10^10 bp copied
PCR
polymerase chain reaction
-amplify a specific DNA region
-millions of copies of the target region
-makes DNA for further molecular work
Applications of PCR
-identify alleles/genotypes to assess variability in a population
-characterize mutations
-create sequences for phylogenies to determine taxonomic relationships
-conduct forensic investigations
Thermal cycler steps
- denature double stranded DNA (94degrees)
- anneal primers to single-stranded DNA (55degrees)
- extend primers, yielding new double-stranded DNA (72degrees)
-cycling - repeat steps 1-3 (20-40 times)
PCR requirements
-DNA template
-primers
-nucleotides
-buffer
-Taq polymerase
-MgCl2
DNA template in PCR
provides the target site of interest
Primers in PCR
-known sequence; anneal to single-stranded DNA template at target site
-provide initiation site for addition of nucleotides
nucleotides in PCR
-building blocks for new DNA strands (A,G,C,T)
Buffer in PCR
maintains optimal pH and [salt] for polymerase
Taq polymerase
-thermally stable enzyme
-extends growing DNA strand complementary to DNA template
MgCl2
-in buffer
-provides ions needed for enzyme reaction
Steps in path from gene to protein
-Copy info in DNA into RNA intermediate (transcription)
-Process RNA intermediate into final form specifying order of amino acids (splicing in eukaryotes only)
-Use RNA as template to synthesize amino acid chain (translation)
RNA properties
-ribose as 5-carbon sugar (OH group at 2prime position)
-primarily single-stranded, no double helix structure, can self base-pair to form 3-D structures
-has nitrogenous pyrimidine base uracil (U) instead of T (can also weakly pair with G to form 3-D structures)
-can catalyze reactions
2 major types of RNA
-Messenger
-Functional
messenger RNA
transfers info from DNA-based genes to cellular machinery for protein production (gene expression)
-pro- and eukaryotes
-mRNA is intermediate between DNA and protein
functional RNA
RNA itself is final product, contributes to process of info transfer
-"assistants" to mRNA
-tRNA
-rRNA
-snRNA
-miRNA
tRNA
transfer RNA
-involved in movement of amino acids in cytoplasm
-pro- and eukaryotes
rRNA
ribosomal RNA
-major components of ribosome
-pro-and eukaryotes
snRNA
small nuclear RNA
-part of spliceosome, molecular machine that processes primary RNA transcript
-eukaryotes only
miRNA
microRNA
-regulate expression of other genes
-eukaryotes only
Transcription
production of TNA strand with sequence that matches that of a DNA gene
-DNA based gene used as template, RNA synthesized is complementary
-RNA sequence matches DNA sequence of non-template strand
coding strand
has information that codes for protein
template strand
has template for coding strand
RNA polymerase II
adds nucleotides to growing RNA strand via base pair rules
Stages of transcription
-initiation
-elongation
-termination
initiation of transcription
recognition of start gene, begin RNA synthesis
-requires recognition of coding sequence
Elongation of transcription
synthesis of RNA strand that includes complete DNA sequence of non-template strand
Termination of transcription
stop RNA synthesis at end of gene
General transcription factors (GTF)
binding proteins that attract RNA polymerases, play critical role in initiating transcription
3 types of RNA polymmerase
- (I) transcribes all rRNA genes
- (II) transcribes all protein coding genes - most abundant
- (III) transcribes all small functional RNAs
Primary transcript
RNA strand produced by transcription
-matches coding strand
RNA splicing
processing of eukaryotic genes to remove introns
Spliceosome
made of several snRNP subunits
-snRNP - small nuclear RNA and set of proteins
Functions of spliceosome
-bind introm sequence, recognize intron/exon boundaries
-hold primary transcript in correct position to join exons
-catalyze reactions that remove introns, join exons
Alternative splicing
mRNA produces different proteins from same primary transcript based on which regions are removed
Measuring gene expression
activity of genes can be quantified based on levels of mRNA in cells
DNA microarray
DNA fragments permanently fixed to solid support (glass) in orderly array
-usually as dots; each has known "address" that can be associated to DNA sequence
rtPCR
reverse transcriptase PCR
-conversion of mRNA into cDNA using reverse transcriptase
Primary protein structure
amino acid sequence, affects more complex features
Colinearity
sequence of nucleotides in gene determines sequence of amino acids in proteins (via mRNA intermediate)
Central dogma
unidirectional information flow from nucleic acids to protein, never reverse
Codon
unit of genetic code
-3 nucleotide sequence
Features of genetic code
-non-overlapping: consecutive amino acids specified by consecutive codons
-three bases make up each codon
-code is read from fixed starting point to end of coding sequence
-code is degenerate: some amino acids specified by more than one codon
AUG
methionine
-start codon
Translation
production of polypeptide with amino acid sequence specified by codon sequence of mRNA
transferRNA
adapter molecule, brings amino acids to ribosome during translation
tRNA structure
amino acid binding site and anticodon region complementary to mRNA
-all have similar structure but differ in amino acid acceptor and anticodon sequence
- 4 double-helical stems, 3 single stranded loops
-anticodon loop pairs with mRNA
amino acids attached to tRNA by aminoacyl-tRNA synthestases
-tRNA and mRNA interact with ribosomes during translation
Ribosome interaction
-mRNA binding site is in the small subunit
-tRNAs bridge subunits - anticodon in small subunit, aminoacyl in large
-3 binding sites for tRNA
A site
aminoacyl
-binds incoming tRNA carrying next amino acid in sequence
P site
peptidyl
-contains growing peptide chain, interacts with central portion of ribosome
E site
exit
-deacylated tRNA ready to be released from ribosome
3 Stages of translation
-initiation
-elongation
-termination
Initiation stage of translation
first amino acid placed at AUG codon (Met) by initiator, a special tRNA
Elongation stage of translation
addition of amino acids to polypeptide chain
Termination stage of translation
when stop codon is present in A binding site of ribosome (UGA, UAA, UAG) caused by release factor
What makes the genetic code degenerate?
-more than one codon specifies an amino acid
-modified base pairing rules in codon-anticodon binding
Wobble
relaxation of base pairing rules in codon-anticodon binding in translation
-usually at third base of codon
Molecular cloning
Isolating defined DNA sequence and making many copies of that sequence
-bacterial chromosome and cellular machinery hijacked to make copies of DNA sequence
-frequently used to amplify copies of a specific gene or gene segment for further study
-PCR doesn't count because no cells are involved
Cellular cloning
-derive population of cells (clones) from a single cell in clulture
-used in culturing bacteria, yeast, animal cells
-inoculate growth medium with one cell, colony of clones develops via mitosis
Reproductive cloning
-generation of an individual that has same nuclear DNA as another individual (donor)
-Donor can be live or dead - preserved viable cells required
Somatic Cell Nuclear Transfer
transplant nucleus from adult cell to enucleated egg. Stimulate division, implant into surrogate mother
Is the individual produced via reproductive cloning genetically identical to the donor? why or why not?
-NO
-mtDNA is not from donor, comes from egg
-mutations and re-arrangements occur during embryogenesis
-gene expression patterns are different
-more similar to identical twins
Therapeutic cloning
-SCNT used to create embryos that are not implanted
-stem cells generated from embryos useful for disease therapy
-new organs could be derived from stem cells
-you could donate organs to yourself!
Genetic Toxicology
DNA damaging agents
Consequences of damaged DNA in somatic vs germ cells
-Somatc - mutation - cancer (exposed show phenotype)
-Germ - mutation - unknown (next generation show phenotype)
Studying germline mutation in natural population
-phenotypic mutations rare
-comparisons lack statistical power
Studying germline mutation in lab studies
-large sample size and treatment doses
-do not reflect ambient conditions (high acute doses vs low chronic doses in the wild)
Tandem repeat DNA
-neutral (no phenotypic change)
-high spontaneous mutation rates
-sensitive markers for mutagen exposure
Conclusion from Great Lakes gull work
-Emissions from steel industry play a prominent role in mutagenesis
-Are other organisms similarly affected?
-What is the predominant route of exposure?
Why use Herring gulls?
-not migration from the Great Lakes
-fairly monogomous
Conclusions from first mouse experiment
-Ambient air at steel site induced heritable germ cell changes in exposed adult mice
-Male germline more susceptible than female.
Mechanisms of spontaneous variation
-Spontaneous mutation occurrence is result of balance between damage and repair
-Spontaneous lesions
-Errors in DNA repair
Spontaneous lesions
physical or chemical damage to molecular components of DNA
-Depurination
-Oxidative damage
Depurination
loss of purine bases via severed deoxyribose-purine bond
-bond severed by reactive metabolites
-loss of purine causes altered DNA structure, failure of template during replication
-lost purine base cannot contribute to codon
Oxidative damage
oxygen containing molecules (free radicals), by-products of aerobic metabolism, cause several types of DNA damage
-damaged bases can cause mispairing, mutation
Errors in DNA replication
mistakes made by cellular machinery as DNA strands are copied
-Base substitutions
-Base insertion/deletion
Base substitutions
-wrong base added to elongating strand, polymerase not 100% accurate
Base insertion/deletion
addition or subtraction of one or more nucleotides by replication machinery
-can result from replication slippage
-can result in frameshift - drastically alters protein or eliminates expression
replication slippage
polymerase slips off template and reattaches at incorrect spot
Polymerase proofreading
-most important DNA repair system
-checks adherence to base pairing rules in DNA replication
Base excision repair
-second most important DNA repair system
-minor base damage recognized, base-sugar bond cleaved to release damaged base
-DNA with excised base removed; correct strand synthesized using other strand as template
-only one damaged base at a time; cannot deal with large distortions to double helix or tracts of damage
Nucleotide excision repair
-major, bulky DNA damage; removes stretch of DNA and replaces it
1. damage recognition
2. multiprotein complex at lesion site
3. cutting out damaged areas in ~30bp pieces
4.synthesis of replacement strand
Bulky DNA adducts
-large molecules covalently bond to DNA
-stall replication fork movement, disrupt DNA replication
Xeroderma pigmentosum
-autosomal recessive
-defective nucleotide excision repair
-extreme UV light sensitivity
-unable to repair UV crosslinks
Mismatch repair
-fixes incorrect base pairs missed by polymerase proofreading
-incorrect bases recognized and removed, new strand synthesized
-increases DNA replication fidelity 100-fold
-repair restricted to newly synthesized strand during or immediately following replication
PKU genetic information
-Phenylalanine hydroxylse gene (PAH)
-enzyme metabolizes essential amino acid
-chromosome 12; DNA = 90kb, 13 exons; mRNA = 2.4kb
-more than 70 different mutations in PAH gene cause PKU
PKU clinical symptoms and diagnosis
-Asymptomatic at birth
-progressive psychomotor impairment
-Severe intellectual disability; seizures, spasticity, autistic behaviour
-Neonatal screening; blood PHE levels checked - treatment at 600um
select PAH mutations
-LEU311PRO
-ARG252TRP
-PRO281LEU
-MET1VAL
-non-synonymous mutations
Progeria phenotype
-accelerated aging
-cranio-facial abnormalities
-stunted growth
-cardiovascular failure
-death by ~13
*affects multiple systems in the body
*pleiotropic
Progeria: Point mutation
-90% caused by point mutation in codon 608 of gene for Lamin protein A (LMNA)
-wildtype codon = GGC; progeria codon = GGT; third position in codon unlikely to change amino acid
-GGC = proline
-GGT = proline
-LMNA makes up nuclear envelope
-LMNA = 664 codons in size, gene produces 3 other splice variants
How does synonymous substitution cause progeria
-DNA point mutation changes splicing enzyme recognition site in primary RNA transcript
-creates alternative splice variant - new splice site created by change to GGT in codon 608
-new splice pattern removes 150 nucleotides from adjacent exon = -50 amino acids
-LMNA proteins are modified post-translation
-mutant splice variation doesn't make functional association with other LMNA proteins
Chromosome mutations
variation in DNA sequence at level of whole chromosome or large fragments - affects many genes simultaneously
-can be detected genetically (via crosses) and microscopically
2 major types of chromosome changes
-change in chromosome number
-change in chromosome structure
Changes in chromosome number
-No structural abnormalities in DNA molecule - purely a change in chromosome and gene number
-Aberrant euploidy
-Aneuploidy
Aberrant euploidy
-Change in chromosome number by multiples of whole set - not common in humans
-produces polyploids
-usually sterile - affects meiosis
-in plants 2 common types:
--autopolyploid
--allopolyploid
Polyploids
organisms that have more than 2 full chromosome sets
Monoploid
individuals that are normally diploid but become haploid aberrantly
Genetic load
accumulation of harmful recessive mutations in the genome of an individual or population
Autopolyploid
multiple chromosome sets derived from within a single species
-e.g. bananas = sterile autotriploids
-e.g. some grapes = autotetraploids
Allopolyploids
multiple chromosome sets in one organism derived for 2 separate species (form of hybridization)
-e.g. spotted salamanders
-e.g. new world cotton and wheat = natural allopolyploids
Aneuploidy
changes in the number of parts of chromosome sets
-caused primarily by parental nondisjunction events during meiosis
Changes in chromosome structure
alterations that result in novel sequences or gene arrangements
-breakage and repair that results in altered chromosomal arrangement
-2 types
--balanced, no net DNA change
--unbalanced, changed DNA dosage
Balanced change in chromosome structure
-Inversion
-Reciprocal translocation
-if DNA breaks occur within genes, disrupts function
-disrupts gen clusters that are transcribe together
-breaks 'fixed' by
--non-homologous end joining
--recombination
Inversion
chromosome segment broken in 2 places, flipped 180 degrees, then rejoined
Reciprocal translocation
-2 non-homologous chromosomes, each broken once; resulting chromosome fragments switch places
Unbalanced chromosome structure change
-Deletion
-Duplication
-Both result in gain/loss of DNA and therefore changes in gene dosage
Deletion
segment of chromosome broken 2x and fragments lost; ends of remaining pieces joined together
Duplication
segment of chromosome arm is repeated
-can be tandem duplication of DNA sequence or in different location
Speciation
chromosome changes cause new phenotype, present barrier to reproduction with ancestral type
Human separation from other great apes
-all other great apes have chromosome count of 2n = 48 or n = 24
-Homo sapiens 2n = 46 or n = 23
-chimp-human gene sequence ~98% similar
4 differences between humans and chimps
-~2% sequence difference
-fusion of two chromosomes (ape 2 and 3) with small deletion
-pericentric inversion in chromosome 5
-small telomeric deletion, chromosome 6
Population
group of individuals of same species in the same place and time; an evolutionary unit with common gene pool
Gene pool
the total extent of genetic variation within a group (population, species)
-all possible alleles
2 types of genetic variation
-discontinuous (qualitative)
-continuous
discontinuous genetic variation
-character found in 2 or more distinct forms, easy to catagorize
Continuous genetic variation
unbroken range of phenotypes, can be measured but difficult to catagorize
-e.g. height, mass
Population genetics
study of frequencies of genotypes in populations and changes to genetic composition through space and time
-relates processes of individual heredity to genetic composition of whole breeding groups
Genetic composition
collective frequencies of different genotypes
6 major factors affecting allele frequencies
-effects of mating patterns
-migration
-mutation
-recombination
-natural selection
-genetic drift
Effects of mating patterns
inbreeding, assortative mating, random
Migration
gene flow among groups
Mutation
introduction of new alleles
Recombination
production of novel allele combinations
Natural selection
changes due to differential survival and reproduction of genotypes
Genetic drift
random changes in allele frequencies due to sampling error
2 major side to population genetics
-measuring genetic variation
-predict changes
Measuring variation
-requires simple relationship between presence of allele and trait
-only a limited number of characteristics can be measured this way
Genotype frequency
observed proportion of individuals with particular allele combination in population
Allele frequency
the proportion of a specific allele present in a population based on all copies of allele present for that gene
Polymorphism
more than one allele and associated phenotypes in a population
-can be characterized at levels of
--visible phenotype
--protein composition
--DNA sequence variation
Hardy-Weinberg Equilibrium
allele and genotype frequency are at equilibrium after one round of random mating and do not change in the absence of other influences
Hardy-Weinberg equation
p^2 + 2pq + q^2 = 1
Assumptions of HWE
-Large, closed population
-Completely random mating
-Equal survival of all genotypes
-Equal fertility of all genotypes
-no mutation