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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
|
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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
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- (I) transcribes all rRNA genes
- (II) transcribes all protein coding genes - most abundant - (III) transcribes all small functional RNAs |
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Primary transcript
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RNA strand produced by transcription
-matches coding strand |
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RNA splicing
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processing of eukaryotic genes to remove introns
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Spliceosome
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made of several snRNP subunits
-snRNP - small nuclear RNA and set of proteins |
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Functions of spliceosome
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-bind introm sequence, recognize intron/exon boundaries
-hold primary transcript in correct position to join exons -catalyze reactions that remove introns, join exons |
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Alternative splicing
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mRNA produces different proteins from same primary transcript based on which regions are removed
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Measuring gene expression
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activity of genes can be quantified based on levels of mRNA in cells
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DNA microarray
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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 |
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rtPCR
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reverse transcriptase PCR
-conversion of mRNA into cDNA using reverse transcriptase |
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Primary protein structure
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amino acid sequence, affects more complex features
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Colinearity
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sequence of nucleotides in gene determines sequence of amino acids in proteins (via mRNA intermediate)
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Central dogma
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unidirectional information flow from nucleic acids to protein, never reverse
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Codon
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unit of genetic code
-3 nucleotide sequence |
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Features of genetic code
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-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 |
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AUG
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methionine
-start codon |
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Translation
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production of polypeptide with amino acid sequence specified by codon sequence of mRNA
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transferRNA
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adapter molecule, brings amino acids to ribosome during translation
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tRNA structure
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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 |
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Ribosome interaction
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-mRNA binding site is in the small subunit
-tRNAs bridge subunits - anticodon in small subunit, aminoacyl in large -3 binding sites for tRNA |
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A site
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aminoacyl
-binds incoming tRNA carrying next amino acid in sequence |
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P site
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peptidyl
-contains growing peptide chain, interacts with central portion of ribosome |
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E site
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exit
-deacylated tRNA ready to be released from ribosome |
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3 Stages of translation
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-initiation
-elongation -termination |
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Initiation stage of translation
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first amino acid placed at AUG codon (Met) by initiator, a special tRNA
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Elongation stage of translation
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addition of amino acids to polypeptide chain
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Termination stage of translation
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when stop codon is present in A binding site of ribosome (UGA, UAA, UAG) caused by release factor
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What makes the genetic code degenerate?
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-more than one codon specifies an amino acid
-modified base pairing rules in codon-anticodon binding |
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Wobble
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relaxation of base pairing rules in codon-anticodon binding in translation
-usually at third base of codon |
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Molecular cloning
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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 |
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Cellular cloning
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-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 |
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Reproductive cloning
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-generation of an individual that has same nuclear DNA as another individual (donor)
-Donor can be live or dead - preserved viable cells required |
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Somatic Cell Nuclear Transfer
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transplant nucleus from adult cell to enucleated egg. Stimulate division, implant into surrogate mother
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Is the individual produced via reproductive cloning genetically identical to the donor? why or why not?
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-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 |
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Therapeutic cloning
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-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! |
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Genetic Toxicology
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DNA damaging agents
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Consequences of damaged DNA in somatic vs germ cells
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-Somatc - mutation - cancer (exposed show phenotype)
-Germ - mutation - unknown (next generation show phenotype) |
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Studying germline mutation in natural population
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-phenotypic mutations rare
-comparisons lack statistical power |
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Studying germline mutation in lab studies
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-large sample size and treatment doses
-do not reflect ambient conditions (high acute doses vs low chronic doses in the wild) |
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Tandem repeat DNA
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-neutral (no phenotypic change)
-high spontaneous mutation rates -sensitive markers for mutagen exposure |
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Conclusion from Great Lakes gull work
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-Emissions from steel industry play a prominent role in mutagenesis
-Are other organisms similarly affected? -What is the predominant route of exposure? |
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Why use Herring gulls?
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-not migration from the Great Lakes
-fairly monogomous |
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Conclusions from first mouse experiment
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-Ambient air at steel site induced heritable germ cell changes in exposed adult mice
-Male germline more susceptible than female. |
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Mechanisms of spontaneous variation
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-Spontaneous mutation occurrence is result of balance between damage and repair
-Spontaneous lesions -Errors in DNA repair |
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Spontaneous lesions
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physical or chemical damage to molecular components of DNA
-Depurination -Oxidative damage |
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Depurination
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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 |
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Oxidative damage
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oxygen containing molecules (free radicals), by-products of aerobic metabolism, cause several types of DNA damage
-damaged bases can cause mispairing, mutation |
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Errors in DNA replication
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mistakes made by cellular machinery as DNA strands are copied
-Base substitutions -Base insertion/deletion |
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Base substitutions
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-wrong base added to elongating strand, polymerase not 100% accurate
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Base insertion/deletion
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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 |
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replication slippage
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polymerase slips off template and reattaches at incorrect spot
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Polymerase proofreading
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-most important DNA repair system
-checks adherence to base pairing rules in DNA replication |
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Base excision repair
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-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 |
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Nucleotide excision repair
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-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 |
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Bulky DNA adducts
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-large molecules covalently bond to DNA
-stall replication fork movement, disrupt DNA replication |
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Xeroderma pigmentosum
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-autosomal recessive
-defective nucleotide excision repair -extreme UV light sensitivity -unable to repair UV crosslinks |
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Mismatch repair
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-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 |
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PKU genetic information
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-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 |
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PKU clinical symptoms and diagnosis
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-Asymptomatic at birth
-progressive psychomotor impairment -Severe intellectual disability; seizures, spasticity, autistic behaviour -Neonatal screening; blood PHE levels checked - treatment at 600um |
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select PAH mutations
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-LEU311PRO
-ARG252TRP -PRO281LEU -MET1VAL -non-synonymous mutations |
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Progeria phenotype
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-accelerated aging
-cranio-facial abnormalities -stunted growth -cardiovascular failure -death by ~13 *affects multiple systems in the body *pleiotropic |
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Progeria: Point mutation
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-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 |
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How does synonymous substitution cause progeria
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-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 |
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Chromosome mutations
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variation in DNA sequence at level of whole chromosome or large fragments - affects many genes simultaneously
-can be detected genetically (via crosses) and microscopically |
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2 major types of chromosome changes
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-change in chromosome number
-change in chromosome structure |
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Changes in chromosome number
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-No structural abnormalities in DNA molecule - purely a change in chromosome and gene number
-Aberrant euploidy -Aneuploidy |
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Aberrant euploidy
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-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 |
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Polyploids
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organisms that have more than 2 full chromosome sets
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Monoploid
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individuals that are normally diploid but become haploid aberrantly
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Genetic load
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accumulation of harmful recessive mutations in the genome of an individual or population
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Autopolyploid
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multiple chromosome sets derived from within a single species
-e.g. bananas = sterile autotriploids -e.g. some grapes = autotetraploids |
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Allopolyploids
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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 |
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Aneuploidy
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changes in the number of parts of chromosome sets
-caused primarily by parental nondisjunction events during meiosis |
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Changes in chromosome structure
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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 |
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Balanced change in chromosome structure
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-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 |
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Inversion
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chromosome segment broken in 2 places, flipped 180 degrees, then rejoined
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Reciprocal translocation
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-2 non-homologous chromosomes, each broken once; resulting chromosome fragments switch places
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Unbalanced chromosome structure change
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-Deletion
-Duplication -Both result in gain/loss of DNA and therefore changes in gene dosage |
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Deletion
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segment of chromosome broken 2x and fragments lost; ends of remaining pieces joined together
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Duplication
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segment of chromosome arm is repeated
-can be tandem duplication of DNA sequence or in different location |
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Speciation
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chromosome changes cause new phenotype, present barrier to reproduction with ancestral type
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Human separation from other great apes
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-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 |
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4 differences between humans and chimps
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-~2% sequence difference
-fusion of two chromosomes (ape 2 and 3) with small deletion -pericentric inversion in chromosome 5 -small telomeric deletion, chromosome 6 |
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Population
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group of individuals of same species in the same place and time; an evolutionary unit with common gene pool
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Gene pool
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the total extent of genetic variation within a group (population, species)
-all possible alleles |
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2 types of genetic variation
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-discontinuous (qualitative)
-continuous |
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discontinuous genetic variation
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-character found in 2 or more distinct forms, easy to catagorize
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Continuous genetic variation
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unbroken range of phenotypes, can be measured but difficult to catagorize
-e.g. height, mass |
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Population genetics
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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 |
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Genetic composition
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collective frequencies of different genotypes
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6 major factors affecting allele frequencies
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-effects of mating patterns
-migration -mutation -recombination -natural selection -genetic drift |
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Effects of mating patterns
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inbreeding, assortative mating, random
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Migration
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gene flow among groups
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Mutation
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introduction of new alleles
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Recombination
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production of novel allele combinations
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Natural selection
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changes due to differential survival and reproduction of genotypes
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Genetic drift
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random changes in allele frequencies due to sampling error
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2 major side to population genetics
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-measuring genetic variation
-predict changes |
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Measuring variation
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-requires simple relationship between presence of allele and trait
-only a limited number of characteristics can be measured this way |
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Genotype frequency
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observed proportion of individuals with particular allele combination in population
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Allele frequency
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the proportion of a specific allele present in a population based on all copies of allele present for that gene
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Polymorphism
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more than one allele and associated phenotypes in a population
-can be characterized at levels of --visible phenotype --protein composition --DNA sequence variation |
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Hardy-Weinberg Equilibrium
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allele and genotype frequency are at equilibrium after one round of random mating and do not change in the absence of other influences
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Hardy-Weinberg equation
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p^2 + 2pq + q^2 = 1
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Assumptions of HWE
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-Large, closed population
-Completely random mating -Equal survival of all genotypes -Equal fertility of all genotypes -no mutation |