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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)