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. What would you like to do?
lack nuclear membrane and possess no membrane-bound organelles
Have a nuclear membrane and membrane-bound organeles
What is the fundamental unit of heredity?
Principle of segregation
Diploid's alleles separate in equal proportions
What is a testcross?
Crossing an individual of unknown genotype with a homozygous recessive individual to reveal the unknown.
When a het has a phenotype intermediate between the phenotypes of two homo, the trait is said to be _______
When two alleles separate, their separation is independent of the separation of alleles at other loci. What principle is this?
What kind of crosses reveal the principle of independent assortment?
How do you determine the chi-square value for inheritance of crosses?
chi-square = sum((obs_i - exp_i)/exp_i), with degrees of freedom one less than the expected number of phenotypes
What type of sex determination has males with only one X?
XX-XO sex determination
What type of sex determination has males with two of the same chromosome?
ZZ-ZW sex determination
What determines phenotype in Drosophila?
X:A ratio, A is number of autosomes
In humans, what does the combination of XO chromosomes lead to?
In humans, what does the combination of XXY chromosomes lead to?
In humans, what does the combination of XXX chromosomes lead to?
What is a reciprocal cross?
It is a breeding experiment designed to test the role of parental sex on a given inheritance pattern. In one cross, a male expressing the trait of interest will be crossed with a female not expressing the trait. In the other, a female expressing the trait of interest will be crossed with a male not expressing the trait.
If only one copy of a chromosomal region is present, such as X-linked genes in humans, this is called ______
__________ equalizes the amount of protein produced by X-linked genes in the two sexes.
What is the Lyon hypothesis?
Darkly staining bodies in the nuclei of female cats (known as Barr bodies) were inactive X chromosomes
_______ is when the phenotype of the het is intermediate between the two homozygotes.
The MN blood group is an example of _________.
__________ is the percentage of individuals with a particular genotype that express the expected phenotype.
_______ describes the degree to which a genotype is phenotypically expressed.
A ____________ causes death at an early stage of development, often before birth
lethal allele. Note that this affects the phenotypic ratio outcome
An allele is said to be ______ when it has more than one phenotype.
_______ occurs when genes at multiple loci affect a single phenotype.
The mallard example with duck feather pattern highlighted _________
multiple alleles at a single locus.
______ is a phenomenon in which a one gene masks (hides) the effect of another gene at a different locus
The gene that does the masking is called the _______
The gene that is masked is called the ______ gene.
The presence of either of two recessive alleles at two different loci resulting in a recessive phenotype is called ______
duplicate recessive epistasis
When a single copy of an allele is sufficient to mask the phenotype of alleles at a second locus, this is ______
Interpreting dihybrid cross ratios is often easier if the amount is ...
multiplied by 16 and then divided by the total
______ traits are present in males and females, but are inherited only from the mother and can result in phenotypic variation.
__________ is a phenomenon in which the phenotype of the offspring is determined by the genotype of the mother.
Genetic maternal effect. Note that the offspring's phenotype is determined by mother's genotype, not her phenotype.
_________ is the differential expression of genetic material depending on whether it is inherited from the male of female parent.
At the ______________, the mutant protein functions normally.
At the ____________, mutant protein is inactivated and endocytosis is halted, resulting in paralysis.
Characteristics that have a few easily distinguished characteristics, such as seed color or shape, are called _____.
_________ are characteristics, such as height, for which a continuous distribution of phenotypes is observed. Also called _________.
Continuous characteristics, quantitative characteristics
Continuous characteristics frequently arise because genes at many loci interact to produce the phenotype: ________.
What is the number of genotypes encoded by n loci with two alleles?
Name useful characteristics of genetic model organisms.
- short generation time
- large number of progeny
- adaptable to lab environment
- housed and propagated relatively inexpensively
- genomes sequenced
What model organism has a homolog whose mutant in a human eye disease?
What model organism taught us about differences in skin color?
Zebrafish (Danio rerio)
What is the most widely studied prokaryote?
Escherichia coli, regulation of expression of genes
What is the simplest eukaryotic genetic model organism?
Baker's yeast (Saccharomyces cerevisiae), DNA damage/repair
What is the most basic multicellular animal that is a genetic model organism?
roundworm (Caenorhabditis elegans), neurotransmitters
What is the mammalian genetic model organism?
House mouse (Mus musculus), white blood cells
What is the primary plant model organism?
Thale cress plant (Arabidopsis thaliana); genome structure, plant evolution
_______, the person from whom the pedigree is initiated.
Characteristics of autosomal recessive traits in pedigrees
- Normal parents have affected children
- Equal number of males and females
- 1/4 of offspring of two carriers are affected
- Normal children with affected siblings have a 2/3 chance of being a carrier
- Consanguineous marriages are sometimes involved
- Example: tay-sachs
Characteristics of autosomal dominant traits in pedigrees
- Does not skip generations (unless incompletely penetrant/express)
- Approx equal number males and females
- Most affected individuals are heterozygous
- 1/2 offspring of an affected individual are affected
- These alleles tend to be very rare, even more so for homozygous
- Example: elevated blood cholesterol
Characteristics of X-linked recessive traits in pedigrees
- Males usually affected
- Usual transmission from carrier woman to son
- Daughters of carrier mothers have 1/2 chance of being carriers
- Not passed from father to son
- Affected woman must have an affected father and either an affected mother or carrier mother
- Example: Hemophilia A
Characteristics of X-linked dominant traits in pedigrees
- Affected males only have affected daughters.
- Affected mothers have 1/2 affected kids, and 1/2 normal
- Trait does not skip generations
- Rare: assume heterozygous
- Example: Hypophosphatemia - vitamin D resistant rickets
Characteristics of Y-linked traits in pedigrees
- Very rare
- Father to son transmissions
- All male offspring are affected
______ genes do not assort indepently.
______ is the soring of alleles into new combinations
Genes on the same chromosome belong to the same ________
If the genes of interest are linked, only _______ progeny are produced.
Genes that exhibit crossing over are ________.
A single crossover produces what ratio of recombinant to nonrecombinant gametes?
_____ is the percentage of progeny produced in a cross.
Wild-type alleles in a _____ configuration are on the same chromosome.
Wild-type alleles in a _____ configuration are on different chromosomes.
What are the two types of recombination?
Interchromosomal and intrachromosomal.
Which type of recombination arises from independent assortment?
Who discovered that Intrachromosomal Recombination Results from Physical Exchange Between Chromosomes?
Harriet Creighton and Barbara McClintock
______ are chromosome maps calculated by using the genetic phenomenon of recombination.
Genetic maps, 1 m.u. = 1% recombination
______ are chromosome maps calculated by using physical distances along the chromosome.
Physical maps, distance in base pairs
Why are genetic maps based on short distances more accurate than those based on long distances?
Double crossovers are not detected
In a 3-point cross, classes with the fewest progeny are ____.
In a 3-point cross, classes with the most progeny are ____.
In a 3-point cross, the recombination frequency is calculated by adding all the crossovers (single and double) between two genes and dividing by the total number of progeny.
See example Lecture 10, Slide 7
The degree to which one crossover interferes with additional crossovers in the same region is termed ________.
The ratio of observed double crossovers to expected double crossovers is called the _______.
coefficient of coincidence
The number of expected double crossovers can be calculated by multiplying the total number of progeny by _____.
the product of the probability of recombination for the single crossovers.
1- coefficient of coincidence
Disease causing genes are mapped by _______ analysis which examines the probability of having linkage at a particular RF compared to the probability of independent assortment.
logarithm of odds
A LOD score of 3 indicates linkage with the specified recombination is _____ times as likely to produce what was observed as independent assortment.
Variable genes with easily observable phenotypes are called ____.
Variations in in DNA sequence detected by cutting the DNA with restriction enzymes are called _____.
Restriction Fragment Length Polymorphisms (RFLPs)
Variable numbers of short DNA sequences repeated in tandem are called ____.
Individual variations in the DNA nucleotides are called ____.
Single Nucleotide Polymorphisms (SNPs)
Describe deletion mapping.
- Individual homozygous for a recessive mutation in the gene of interest is crossed with an individual heterozygous for a deletion.
- If the gene of interest is in the deletion region, half of the progeny will dislay the mutant phenotype.
- If it's not, all the progeny will be wild type.
Describe somatic-cell hybridization
See first 3 slides of Lecture 11. It's used for gene mapping
_____ is a method for determining the chromosomal location of a particular gene through molecular analysis.
in situ hybridization
Wild-type bacteria are called ____.
_____ contains all the nutrients required by prototrophic bacteria.
Mutant strains called _____ lack enzymes necessary for metabolizing nutrients or synthesizing essential molecules.
_____ contains all substances required by bacteria for growth and reproduction.
Most bacteria have __#__ _____ (shape) chromosome(s) several million base pairs long.
Many bacteria also contain small (several thousand base pairs), circular DNA molecules called ____.
_____ are plasmids capable of freely replicating and able to integrate into the bacterial chromosome.
The ______ in E. coli controls mating and gene exchange between E. coli cells.
F (fertility) factor
Plasmids Replicate _____ of the Bacterial Chromosome.
What are three types of gene transfer in bacteria?
Conjugation, transformation, and transduction
In ______, a cytoplasmic bridge forms allowing transfer of part of DNA from one bacteria to another.
In _____, naked DNA is tken up by the recipient cell/bacteria.
In ______, a virus attaches to a bacteria cell to inject DNA.
_____ is usually the only DNA transferred during conjugation.
Conjugation between an F+ cell with an F- cell results in an ____ cell.
____ cells contain an F factor integrated into the bacterial genome.
Hfr (high frequency)
Bacterial genes can be transferred from an ___ cell to an F- cell in conjugation.
An Hfr cell can be converted into an F' cell during ______.
Sexduction produces _______, cells with two copies of some genes.
merozygotes (partial diploids)
The transfer times of genes between bacteria indicate the order and relative distances which can be used to construct a ______.
_______ are small circular plasmids that carry genes that encode antibiotic resistance and can be transferred by conjugation.
Cells that take up DNA through their outer membranes are called _____ cells.
Competence is influenced by _____.
Growth stage, concentration of available DNA, and composition of medium; heat shock, chemical treatment
The cell that receives DNA in a transformation is called the _______.
When genes are transformed together, they are ______.
The rate of cotransformation is _______ proportional to the distances between the genes which is useful for gene mapping.
Sometimes bacteria acquire DNA from eukaryotes in a process called ___________.
horizontal gene transfer
The process of passing passing genetic information through reproduction is called ____________.
vertical gene transfer
A simple replicating structure made up of nucleic acid surrounded by a protein coat is called a _____.
__________ are viruses that infect bacteria, and have been used extensively for genetic studies.
Virulent phages reproduce only through the _____ cycle and always kill their host.
_________ phages can undergo either the lytic or the lysogenic cycle.
Phage DNA integrates into the bacterial chromosome and becomes a ________.
When a bacteria lyses, the adjacent bacteria are infected which also lyse resulting in a clear patch called a _____.
If a phage containing bacterial DNA transfers genes to another bacterium, recombination may take place and produce a _______.
The rate of transduction is ___.
The rate of cotransduction is _____ proportional ot the distances between genes.
_____ transduction can only occur near an att site.
In specialized transduction, a phage called ________ can be produced resulting in either an unstable transductant or a stable transductant with a gal+ allele.
lambda gal defective
_______ used the awesome power of phage genetics to make inferences about gene structure.
The sites of different mutations in the same gene can be mapped, referred to as _______.
A _______ test indicates whether two mutations occur in the same or different genes.
A _______ is a functional gene defined by a complementation test.
_______ occurs when there is at least one wild-type copy of each gene, i.e. the mutations are in different genes.
What are the four basic types of chromosomes?
metacentric, submetacentric, acrocentric, telocentric
A complete set of chromosomes possessed by an organism presented as an ordered image is called a _____.
What are three types of chromosome mutations?
rearrangements, aneuploids, and polyploids
What are four types of chromosomes rearrangements?
duplications, deletions, inversions, and translocations
When a single copy of a gene is not sufficient to produce a wild-type phenotype, it is said to be a ______ gene.
______ inversions do not include the centromere, while ______ do.
______ genes are nonviable in both peri and paracentric inversions.
A _______ involves the movement of genetic material between nonhomologous chromosomes.
In a _______ translocation, there is a two-way exchange of segments between two chromosomes.
In a _______ translocation, two long arms of chromosomes are combined creating a large metacentric chromosome.
In homologous pairing in translocation heterozygotes, the best option is ______ segregation.
________ is an increase of a decrease in the number of individual chromosomes.
Aneuploidy in meiosis ___ (I or II) results in 2 trisomic and two monosomic zygotes.
Aneuploidy in meiosis ___ (I or II) results in 1 trisomic, 1 monosomic, and 2 normal (diploid) zygotes.
In humans, ______ results in 44 chromosomes.
In humans, monosomy results in how many chromosomes?
In humans, trisomy results in how many chromosomes?
In humans, ______ results in 48 chromosomes.
What mechanism for controlling gene dosage could account for viability of XXX females?
What creates a carrier in familial Down syndrome?
____ is the presence of more than two sets of chromosomes.
____ are 5n in chromosome number.
_____ is a type of polyploidy where chromosome sets are from a single species.
_____ is a type of polyploidy where chromosome sets are from different species.
The primary structure of DNA is the _____.
Secondary structure of DNA is the _____.
One type of tertiary structure in DNA is ______, which takes place when the DNA helix is subjected to strain by being overwound or underwound.
Supercoiling is controlled by _______, enzymes that add or remove rotations from the DNA helix.
Two types of eukaryotic chromatin are the more common one, ____, and ____, which is present near centromeres and telomeres and along X-inactivated chromosomes.
Most abundant proteins in chromatin are ____ - small, positively charge proteins of 5 major types.
____ consist of DNA wrapped about 4 pairs of histones.
A _____ consists of a nucleosome plus the ____ histone acting as a clamp.
_____ chromosomes arise when repeated rounds of DNA replication take place without cell divisions in certain tissues in Drosophila.
Acetylation occurs when enzymes called _____ attach acetyl groups to lysine amino acids on the histone tails.
Centromeric sequences Serve as binding sites for _____ proteins that provide anchor sites for spindle fibers.
____ was awarded the nobel prize for the discovery of how chromosomes are protected by telomeres and the enzyme telomerase.
_____ are stabilizing sequences at the ends of chromosomes.
______ proteins bind to the G-rich single-stranded sequence of telomeres.
POT (Protection Of Telomere)
A structure called the _____ also functions in protecting the telomere from degradation.
Artificial chromosomes such as the YAC or BAC contain what there essential elements?
Centromere, pair of telomeres, origin of replication
Sequences present at one or a few times in the genome are called ___.
Groups of related genes arising from duplication of unique-sequence DNA are called ___.
The two types of repititive DNA are called ___ and ___ DNA.
moderately repetitive and highly repetitive
Two types of moderately repetitive DNA are called ____ and ___ .
tandem repeat sequences and interspersed sequences
_____ DNA sequences are sometimes called lite DNA and are often found at ___ and ___.
Highly repetitive, centromeres, telomeres
____ are mobile DNA sequences.
______ result from staggered cuts made in the target DNA when a transposable element is inserted.
Flanking direct repeats
____ are present in many TEs are recognized by enzymes that catalyze the transposition.
Terminal inverted repeats
Terminal inverted repeats are ____ and ____ .
inverted and complementary
What are two classes/mechanisms of transposition?
Class I/DNA transposons, and Class II/Retrotransposons
What are two kinds of Class I/DNA transposons?
non-replicative and replicative
Class II/retrotransposons are always ____.
DNA transposons require an enzyme called ____ which is usually encoded by the TE.
In replicative transposition, two DNA molecules are joined and the TE is replicated, producing a ____.
Retrotransposons use an ____ intermediate used to reverse transcribe back into DNA.
An enzyme called ____ is usually encoded by retrotransposons.
Transposable elements were discovered in eukaryotes by _____.
Ds in maize was able to transpose nonautonomously using transposase from Ac elements. What is a possible reason Sleeping Beauty wasn't able to do this?
The inverted repeats were also mutated. Transposase REQUIRES inverted repeats.
What are three types of TEs in bacteria?
Insertion sequences, composite transposons, and noncomposite transposons
The simplest types of transposons in bacteria are the ___ and have 1-2 genes which encode ___.
insertion sequences, transposes(s)
A segment of DNA flanked by insertion sequences can transpose and is called a ____.
____ transposons do not have insertion sequences.
___ found in fruit flies have both a transposase and a repressor of transposition.
What were three models of proposed DNA replication?
conservative, dispersive, and semiconservative
____ performed an experiment to determine which of three models of DNA replication applied to E. col.
Meselson and Stahl
____ replication of DNA takes place in circular DNA in bacteria.
_____ replication of DNA takes place in some viruses and in the F factor of E. coli.
Eukaryotic genomes require multiple ____.
origins of replication
The process of DNA replicaiton includes many componets including (3):
a template, substrates (dNTPs), and enzymes
DNA synthesis always goes in the ____ direction.
Short fragments of DNA produced by discontinuous synthesis on the _____ strand are called ____.
Which mode/l of DNA replication does not have a lagging strand?
Four steps of DNA Replication
DNA Helicase binds to...
The lagging-strand in the 5' to 3' direction and breaks hydrogen bonds while moving along the replication fork
DNA Gyrase ...
relieves torsional strain that builds up ahead of the replication fork due to unwinding
sysnthesizes short strands of RNA including a 3'OH group for replication to begin
How many primers are required on the leading strand?
DNA polymerase III ...
- 5' to 3': polymerase activity: adds nucleotides (primary)
- 3' to 5': exonuclease activity for error correction
DNA polymerase I ...
- 5' to 3': polymerase activity: adds nucleotides
- 3' to 5': exonuclease activity
- 5' to 3': exonuclease activity - remove RNA primers
- Not as efficient as DNA polymerase III
DNA ligase ...
links together DNA at nicks/Okazaki fragments by forming a phosphodiester bond between adjacent nucleotides
Binds to origin and separates strands of DNA to initiate replication
Attach to single-stranded DNA and prevent secondary structures from forming (stabilizes)
Termination can occur when...
- Replication forks meet
- A termination protein (Tus in E. coli) binds to specific sequences to block helicase
What is an Autonomously Replicating Sequence?
Origin of replication found in yeast
Why do origins of replication typically have numerous A-T base pairs?
Easier to break - only 2 H-bonds compared to 3 for C-G base pairs
What is an Origin-Replication Complex?
In eukaryotes, an ORC binds to origins and unwinds the DNA in this region
What is licensing?
In eukaryotes, it is the regulation of precise replication once per cell cycle from a large number of origins
What is telomerase?
Telomerase is a ribonucleoprotein which maintains telomeres by extending the DNA and filling in the gap due to removal of the RNA primer after replication
When does homologous recombination/crossing over take place?
Prophase I, after DNA replication
What are the two models of recombination?
- Double-strand break(*)
What are three DNA binding motifs?
- zinc fingers
- leucine zipper
lac operon is ...
trp operon is ...
trp operon is also regulated by attenuation - when tryptophan level is high:
region 3 and 4 pair resulting in termination of transcription
Antisense RNA regulates transcription by ...
binding to mRNA to create double-stranded RNA blocking the ribosome-binding site
Riboswitches with a regulatory protein...
blocks the ribosome binding site by conformation change
Ribozymes with a regulatory molecule ...
Histone modification includes:
Methylation, acetylation, and phosphylation
Acetyl groups are added by:
Chromatin remodeling complexes...
bind to sites in DNA and reposition nucleosomes allowing transcription factors to bind to promoters and initiate transcription
Heavily methylated DNA ...
is associated with the repression of transcription in vertebrates and plants
Transcriptional activator proteins...
bind to sites on DNA and stimulate transcription specific to a gene or subset of genes
In eukaryotes, repressors ...
do not directly block RNA polymerase, but instead compete with activators or interfere with the basal transcription apparatus
can operate at distant promoters by way of DNA looping out
block the action of enhancers
An example of coordinated gene regulation is:
Several eukaryotic genes respond via consensus sequences to extreme heat producing heat-shock proteins
Alternative splicing allows pre-mRNA to...
be spliced in multiple ways generating different proteins in different tissue or at different times in development
A female fruit fly has a ratio of:
A male fruit fly has a ratio of:
A ratio of 1.0 in fruitfly embryos activates the Sxl gene to produce a protein that causes ...
tra pre-mRNA to be spliced at a downstream 3' site resulting in tra protein which leads to female fruit flies
RNA is degraded by ...
P bodies are:
specialized complexes in which RNA molecules are degraded or sequestered for later release
RNAi inhibits gene expression through:
- Cleavage of mRNA leading to degradation using Dicer, siRNAs and RISC
- Inhibition of translation using Dicer, miRNAs and RISC
- Transcriptional silencing using RITS, siRNA and methylation
- Degradation of mRNA (not via cleavage) using Dicer and RISC
Posttranslational modifications of proteins includes
- Selective cleavage and trimming of amino acids from the ends
- Addition of phosphate groups
- Addition of carboxyl groups
- Addition of methyl groups
- Addition of carbohydrates
When the tinman gene has a mutation, ...
the transcription factor is not produced and the heart doesn't develop
What are the three major types of gene mutations?
- Base substitution
- Base insertion
- Base deletion
Purine to purine mutations are
The phenotypic effects of base mutations are:
- Silent mutations
A neutral mutation is:
a missense mutation that alters the amino acid, but does not change its function
Examples of spontaneous mutations are
- Unequal crossing over
A base analog is
a chemical with a structure similar to any of the four standard bases
Chemical mutagens include
- EMS: alkylation
- Nitrous acid: deamination
- Hydroxylamine: hydroxylation
molecules which insert into DNA in place of nitrogenous bases causing insertions and deletions, e.g. proflavin and acridine orange
1927: showed mutations in fruit flies inducible by radiation
x-rays, gamma rays, cosmic rays
alter base structure, break phosphodiester bonds, and even cause double-strand breaks
less energy, but still mutagenic
can be caused by UV light; create covalent bonds between bases which block replication
eukaryotic system of eta polymerase that can bypass pyrimidine dimers
Four mechanisms of DNA repair are:
- Mismatch repair
- Direct repair
- Base excision
- Nucleotide excision
Replication errors, including mispaired bases and strand slippage
Pyrimidine dimers; other specific types of alterations
Abnormal bases, modified bases, and pyrimidine dimers
DNA damage that distors the double helix, including abnormal bases, modified bases, and pyrimidine dimers
Mismatch repair Mechanism
- Detects 3D distortion
- cuts out distored part with exonuclease
- fill in using original strand as template with DNA polymerase
- repairs nicks with DNA ligase
- Methylation at GATC sequence on old strand for differentiation
removes nucleotides usually at end of DNA strand
seals nick in sugar-phosphate backbone
Direct Repair Mechanism
- Converts modified nucleotides to original form
- e.g. O6-Methyltransferase removes methyl group restoring base to guanine
- e.g. photolyase uses light to break covalent bonds that link pyrimidine dimers
an enzyme that uses light energy to break covalent bonds in pyrimidine dimers
Base-excision repair mechanism
- Excises modified bases and then replaces entire nucleotide
- DNA glycosylase recognizes and removes damaged base
- AP endonuclease cleaves phosphodiester bond on 5' site and removes sugar
- DNA polymerase adds new nucleotide to 3'
- DNA ligase fixes nick in sugar-phosphate backbone
removes nucleotide usually in middle of DNA strand
Nucleotide-excision repair mechanism
- Enzyme complex recognizes 3D distortion
- DNA strand is separated and stabilized with binding proteins
- An enzyme cleaves the strand on both sides of the damage
- Part of damage strand is removed
- Gap filled by DNA polymerase
- Sealed by DNA ligase
A type of genetic recombination in which nucleotide sequences are exchanged between two similar or identical molecules of DNA. It is most widely used by cells to accurately repair harmful breaks that occur on both strands of DNA, known as double-strand breaks.
Two DNA strands are connected thru covalent bonds. Not much know about this.
Common Mechanisms for Nucleotide removal
Damaged section of DNA is recognized
DNA repair endonucleases nick phosphodiester backbone on one or both sides of the DNA damage and one or more nucleotides are removed
DNA polymerase addes nucleotides to the newly exposed 3'-OH group by using the other strand as a template and replacing the damaged nucleotides
DNA ligase seals the nices in the sugar-phoshate backbone
Differences in Mechanisms for Nucleotide removal
How detection and excision are accomplished
autosomal recessive condition caused by nonfunctional repair mechanism for pyrimidine dimers
1973, Cohen and Boyer at UCSF
Created first recombinant DNA molecule
Recombinant DNA technology
Set of molecular techniques for locating, isolating, altering, and studying DNA segments
Stemps requiring Recombinant DNA techniques
- Find gene
- Separate gene
- Make copies of gene
- Insert gene into plasmid without degredation
- Induce bacteria to take up plasmid
- Select bacteria that take up plasmid
- Enzymes that recognize and make double-strand cuts in DNA at specific nucleotide sequences
- Produced naturally by bacteria to defend against viruses
Type I and III Restriction enzymes
Cut outside recognition sequence
Type II restriction enzyme
- Cuts within recognition sequence
- Used in molecular genetic work
- Names indicate original bacteria
- More than 800 isolated
Characteristics of restriction enzymes
- Palindromic recognition sequence
- Fragment end either cohesive or blunt
Cohesive end restriction enzyme
Staggered cut -> sticky ends
Reaction of mixture of DNA, buffer, restriction enzyme, water heated at about 37 C
Standard technique for separating molecules on basis of size/electrical charge
polysaccharide isolated from seaweed
Viewing DNA fragments using electrophoresis
DNA fragments move to positive pole with smaller fragments moving faster
fluorescent or radioactive DNA or RNA fragment complementary to sequence of interest
Transfer of electrophoresis-separated DNA fragments to a filter membrane and subsequent fragment detection by radioactive probe
- A technique used in molecular biology research to study gene expression by detection of RNA (or isolated mRNA) in a sample.
- Size of mRNA molecule
- Relative abundance of mRNA
- Tissue in which MRNA is transcribed
- Transfer of proteins from gel to a membrane
- Probe is usually an antibody
- Determine size of protein
- Pattern of protein's expression
create identical copies of a piece of DNA
DNA molecule into which a foreign DNA fragment can be inserted for introduction into and replication in a cell
Characteristics of an effective cloning vector
- Origin of replication
- selectable marker
- one or more unique restriction sites where DNA can be inserted
Types of cloning vectors
- Plasmid, e.g. pUC19
- Retroviral vectors
The capacity of bacterial cells to take up DNA from the environment
Plasmids that are packaged into empty viral protein coats and transferred to bacteria by viral infection
Originally contructed from F plasmids
DNA molecule that has a yeast ORI, pair of telomeres, and a centromere
Plasmid that can be used to introduce DNA into plants
Vector that allows the production of protein (i.e. transcription and translation)
- 1) Heat to 90-100 C for denaturation
- 2) Cool to 30-65 C for primers to anneal
- 3) Heat to 60-70 C for DNA synthesis
- Requires knowledge of at least part of sequence for primers
- Taq polymerase is poor at proofreading
- Fragments larger than 50Kb cannot be isolated
PCR as a diagnostic tool
Detects presence of a particular sequence, e.g. HIV
Clone all sequences in an organism into vectors
Collection of clones containing all the DNA fragments from one source
Set of bacterial colonies or phages containing fragments in a DNA library
cDNA library: isolating mRNA
isolation of mRNA using oligo(dT) chains
cDNA library: making cDNA from mRNA
- oligo(dT) act as primers
- reverse transcriptase for DNA strand
- Rnase digests most of RNA strand
- Remaining RNA act as primers for second DNA strand
In situ hybridization
A type of hybridization that uses a labeled complementary DNA or RNA strand (i.e., probe) to localize a specific DNA or RNA sequence in a portion or section of tissue
Restriction Fragment Length Polymorphisms (RFLPs)
Variations in the patterns of fragments produced when DNA is cut with a restriction enzyme typically caused by mutation
No OH groups
Sanger Method of DNA sequencing
Uses ddNTP to terminate synthesis of strands of different length which can be read by electophoresis to sequence
allows sequencing of entire genomes in a couple months
- PCR used to amplify STR loci each with large numbers of alleles which assort independently
- Difference in number of tandem repeats have no phenotypic consequence
- Probability of two randomly selected people having the same DNA profile is less than 1 in 10 billion
DNA fingerprinting procedure
- DNA extracted from tissue samples
- PCR primers for specific STR loci used to amplify fragments
- DNA from sample is compared with reference DNA
- Usually use genomic DNA, but mitochondrial DNA can be used as well
Forward Genetic Approach
- Function -> gene
- Frequently used in less complex organisms to discover new genes
Forward Genetic Procedure
- 1. Isolate mutants that have phenotypic mutation.
- 2. Map the mutations.
- 3. Sequence the gene to find the mutation.
- 4. Clone the gene using molecular techniques.
- 5. Further genetic, molecular genetic, and biochemical experiments can further define a gene's function in that process.
Reverse Genetic Approach
- gene -> function
- Frequently used in mice to see if genes discovered in simpler organisms
- have a similar phenotype in mammals
Reverse Genetic Procedure
- 1. Begin with a gene with known sequence.
- 2. Induce a mutation in that gene.
- 3. Look to see what effect these mutations have on the phenotype of the organism
An organism with an added transgene
non-innate DNA added to an organism
Mouse in which known gene has been disabled via homologous recombination
wildtype gene is replaced with known mutant gene
Knockout mouse procedure
- Target "normal" gene disabled by inserting neo+ gene in the middle and a tk+ gene is added at the end
- Disabled gene is transferred to embryonic mouse stem cells to undergo recombination with normal cells resulting in some neo+ tk- cells
- Cells grown in antibiotic, and only recombinated ones survive
- Surviving cells injected into early mouse embryo resulting in variegated mouse
- Variegated progeny interbred resulting in some homozygous mice for the knocked-out gene
Site-Directed Mutagenesis: Method 1
- Short sequence of nucleotides removed and replaced by synthetic sequence containing mutated bases
- Requires flanking restriction sites that are nowhere else in DNA
Site-Directed Mutagenesis: Method 2
- Oligonucleotide created that differs from target sequence by single nucleotide
- Two sequences pair
- Oligonucleotide used as primer which yields molecule with single mismatched pair
- DNA transferred back to bacteria where about half are repaired
- Bacteria then screened for altered sequence
Oligonucleotide-directed mutagenesis (2)
- Often used for making small changes in DNA sequences already cloned into plasmids
- Can't be used in multicellular organism: long, noncircular DNA, multiple ori, etc.
Silencing with RNAi: RNA Knockdown
Can be delivered to cell by injecting or soaking to turn down expression without inducing mutation
Short hairpin RNA (shRNA)
Can be cloned into vectors and used to make transgenic animals
RNAi for the treatment of disease
- siRNAs could be used against RNA viruses, such as HIV
- siRNAs could be used to treat genetic diseases, high cholesterol and cancer
Stable nucleic-acid-lipid-particles (SNALPs)
Used in delivery of siRNA to lower cholesterol thru silencing in monkeys
Gene therapy targets what kinds of cells?
What is genomics?
It is the field of genetics that attempts to understand the content, organization, function, and evolution of genetic information contained in whole genomes
The first living organism to be sequenced was ?
For the Human Genome Project, what kind of method was used for sequencing?
A map-based method
Craig Venter and Celera Genomics used what method to sequence the human genome?
A whole-genome shotgun technique using computers
What is a single nucleotide polymorphism (SNP)?
A site in the genome at which individual members of a species differ in a single base pair
Why are SNPs more commonly found in non-coding regions?
Because there is no selective pressure to weed out the mutations.
What is a haplotype?
It is the set of SNPs and other genetic variants found on a particular part of a chromosome.
Of Africans, Japanese, Chinese, and Europeans, which group has the greatest diversity of SNPs and why?
Africans, as this is consistent with many other studies that suggest humans first evolved in Africa.
What is a contig?
A continuous stretch of DNA
What is bioinformatics?
A field that fuses molecular genetics and computer science
What are two methods to identify genes?
- ab initio approach: scans the sequences looking for characteristics such as an open reading frame
- comparative approach: Looks for similarities between a new sequence and sequences of all known genes
What is an open reading frame?
A frame which includes a start and stop codon in the same reading frame
What is BLAST?
Basic Local Alignment Search Tool, is a program to determine whether a similar gene sequence has already been found in the same or another species
What are two types of homologs?
- Orthologs are homologous genes found in different species that evolved from the same gene in a common ancestor.
- Paralogs are homologous genes in the same organism that arise by duplication of a single gene
What is a protein domain?
A region in a protein that has a specific function or shape
What is a microarray?
An array of numerous microscopic DNA fragments/probes used to find complementary sequences corresponding to known genes
What is a reporter gene?
A gene that researchers attach to a regulatory sequence of another gene of interest that allows for visual identification (e.g. Green Fluorescent Protein, GFP)
Genomic screening for newborns
- Screening for large number of genetic diseases
- Better/earlier preventative treatement
Genomic screening for personalized medicine
- Able to predict responses to different treatments and fine-tune drug therapy for individual
- Genetic testing of both patients and pathogens will allow faster and more precise diagnosis of many diseases
What is population genetics?
The study of the genetic makeup of groups of individuals and how a group�s genetic composition changes with time
What is a gene pool?
A common set of genes in a population
Population geneticists study:
- The variation in alleles within and between groups
- The evolutionary forces that shape patterns of genetic variation
What causes phenotypic varation within a population?
Genotypic frequency: f(AA)
f(AA) = (number of AA individuals)/N, where N is the number of individuals in the sample
Calculating the frequency of an allele
freq of an allele = (# of copies of allele)/(# of copies of alleles at the locus)
Allele frequency: p = f(A)
f(A) = (2nAA + nAa)/(2N)
Calculate allelic frequencies from individual frequencies
p = f(A) = f(AA) + (1/2)f(Aa)
Calculate allelic frequencies at loci with three alleles
p = f(A1) = (2nA1A1 + nA1A2 + nA1A3)/2N
Calculate allelic frequencies from individual frequencies with three alleles
p = f(A1A1) + (1/2)f(A1A2) + (1/2)f(A1A3)
Calculate allelic frequencies at X-linked loci
p = f(XA) = (2nXAXA + nXAXa + nXAY)/(2nfemales + nmales)
Calculate allelic freq's at X-linked loci from individual frequencies
p = f(XA) = f(XAXA) + (1/2)f(XAXa) + f(XAY)
Study example on slide 15
human MN blood-type antigens
What assumptions does the Hardy-Weinberg law make?
The population is large, randomly mating, and not affected by mutation, migration, or natural selection
The Hardy-Weinberg Law predictions:
- Prediction 1: The allelic frequencies of a population do not change
- Prediction 2: The genotypic frequencies stabilize (do not change) after one generation in the following proportions:
- p^2, 2pq, q^2
- The allelic frequencies determine the frequencies of genotypes
If a population meets the Hardy-Weinberg assumptions, can it evolve?
Yes, but it requires external pressure.
When a population is in Hardy-Weinberg equilibrium, the genotypic frequencies are determined by ...
the allelic frequencies
Hardy-Weinberg Law: Comparing Genotypic exp with freq
- 1. Calculate the allelic frequencies
- 2. Find the expected genotypic frequencies
- 3. Compare the observed and expected genotypic frequencies using a chi-square test
How do you calculate the degrees of freedom for the Chi-Square test in Hardy-Weinberg proportions?
In general, the degrees of freedom for a chi-square test of Hardy-Weinberg equilibrium equal the number of expected
genotypic classes minus the number of associated alleles
Positive assortative mating
Tendency for individuals sharing a particular trait to mate
Negative assortative mating
Tendency for individuals that do not share a particular trait to mate
Preferential mating between related individuals
Avoidance of mating between related individuals
How does inbreeding affect homozygosity and allele frequencies?
Inbreeding leads to an increase in homozygosity at all loci, but no change in allele frequencies
The increased appearance of lethal and deleterious traits with inbreeding
Processes that bring about change in allelic frequency
- Genetic Drift
- Natural selection
Sampling error/random effects due to small population size
What are the overall affects of migration?
- Gene pools of populations become more similar
- Increases genetic variation within the recipient population
Genetic drift results in...
the divergence of populations and often results in one allele becoming fixed
What are two causes of genetic drift?
- Founder Effect
- Genetic bottleneck
Three related effects of genetic drift are:
- 1. Change in allelic frequency
- 2. Reduced genetic variation
- 3. Different populations diverge genetically with time
The differential reproduction of genotypes when individuals with adaptive traits produce a greater number of offspring than that produced by others in the population
The reproductive success of one genotype compared with the reproductive successes of other genotypes in the population
Calculating fitness (W)
Divide the mean number of offspring produced by a genotype by the mean number produced by the most proli?c genotype
Selection coefficient (s)
The relative intensity of selection against a genotype
Calculating the selection coefficient
s = 1 - W
General Selection Model
know table 25.4
Three Different types of selection
- Selection against a dominant allele is very efficient
- Selection against an autosomal recessive allele is inefficient
- Balancing selection where the heterozygous genotype is most fit
Do problem on slide 21
calculate relative fitness and next generation frequency of an allele
Mutation's long-term effect on allelic frequency
Equilibrium reached between forward and reverse mutations
Migration's long-term effect on allelic frequency
Equilibrium reached when allelic frequencies of source and recipient population are equal
Genetic drift's long-term effect on allelic frequency
Fixation of one allele
Natural Selection's long-term effect on allelic frequency
- Directional selection: fixation of one allele
- Overdominant selection: equilibrium reached
What's the first step in discovering a gene and determining its function?
Select an appropriate model organism
What are the steps in conducting a mutagenesis screen? (assume we're studying "touch")
- Mutagenize many animals
- Isolate F_2 animals that fail to sense touch
- Map mutation to a small region of genome
- Sequence genes in region
- Look for loss-of-function mutation
Genes are frequently named for their ____.
What kind of search is performed to see if their are homologs that have been studied?
A BLAST or SMART search
What are the steps in subcloning a gene?
- Use a database to find a cosmid that contains the gene of interest
- Determine what restriction sites can be used to cut out the gene of interest
- Digest the plasmid with the same enzyme
- Use gel electrophoresis to separate the fragments from the cosmid
- Ligate the gene fragment from the cosmid to the plasmid vector
- Transform E. coli with the new recombinant plasmid
How do you determine where and when the gene is expressed
- In situ
- Reporter gene like GFP
How do we determine what genes it interacts with?
Determine the subcellular localization of the protein
How do we determine the subcellular localization
- antibody staining
- fuse GFP to the mec-1 coding sequence and inject into animal
What else could we do now to determine if MEC-1 protein can bind collagen and the extracellular domain of the sensory channel?
Biochemical binding assays
What should we do to test whether the mec-1 homolog is necessary for touch sensation in mammals?
Make knockout mice and see if they have defects in sensing touch
If we find that loss-of-function mutations in the mec-1 homolog plays a role in human disease, how could we cure these diseases?
Use gene therapy to reintroduce a wild-type copy of the gene
What would you like to do?
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