Define transcription, RNA processing, translation and replication
Copying of DNA to RNA
Modification and transport of RNA
Synthesis of protein from mRNA
Copying of DNA to DNA
What makes DNA more stable than RNA?
DNA lacks an OH group on the 2' position
Describe the chemical structure of the DNA strand
5' end has a phosphate and an OH- on the 3' end
Adenine and Guanine are purines made up of 2 rings and 6' carbon group
Uracil, Thymine, Cytosine are pyrimidines with 4' carbon group
Describe the physical structure of the DNA double helix
Strands are antiparallel
B form shown here is the most common (width = 2 nm)
Length of DNA per turn = 3.6 nm, ~10.5 bp per turn
What makes DNA more stable?
More C-G bonds that give 3 H-bonds
TATA box-binding protein
It binds to a specific DNA promoter sequence just upstream of the start site of many eukaryotic genes.
Why is RNA less stable than DNA? What happens when it is under neutral pH and alkaline conditions?
2' hydroxyl group on ribose allows hydrolysis of the phosphodiester bond under alkaline conditions.
RNA hydrolysis is slow at neutral pH but rapid under alkaline conditions
How much UV light can a single-stranded DNA absorb and why?
They can absorb 260 nm of UV light more than double stranded DNA because single-stranded DNA absorb more because energy is not shared. The ring structures of double stranded DNA absorb UV light when bases are stacked and energy shared thus less absorbtion.
Describe what happens during replication of circular DNA and what it does to reduce stress
Replication of circular DNA induces torsional stress as the two strands unwind to be copied, which leads to super coiling. Cleavage of one phosphodiester bond on one DNA strand (nicking) by topoisomerase enzyme is sufficient to relieve the stress (strands are now free to rotate at the breakpoint).
What kinda of RNA structure are found the most?
Pseudoknot tertiary structure
An enzyme that transcribes the template DNA strand of genes into function RNAs (mRNAs, rRNAs, tRNAs, etc.)
Describe the growth of the RNA chain
It is always 5' to 3'; the α phosphate of the nucleoside triphosphate precursor becomes part of the phosphodiester bond
What are the 5 stages of transcription?
Initiation: Polymerase binds to promoter sequence in duplex DNA. "closed complex"
Polymerase melts duplex DNA (12-14 bp melted) near transcription start site, forming a transcription bubble. "open complex"
Polymerase catalyzes phosphodiester linkage of two initial rNTPs
Elongation: Polymerase advances 3'→5' down template strand melting duplex DNA and adding rNTPs
Termination: At stop site, polymerase releases RNA and dissociates from DNA
Describe the overall gene organization in prokaryotes
Genes that function together are commonly found in clusters called operons and transcribed as a single mRNA. Transcribed mRNAs are translated into proteins as soon as they are made (no nucleus, no processing). mRNAs have separate start sites for translation of each gene.
Describe the overall gene organization in eukaryotes
Single genes are generally dispersed in the genome even if they function in the same way. Primary transcripts made in the nucleus must be processed and transported to the cytoplasm. Mature mRNAs encode a single gene flanked by 5' and 3' UTR (usually short)
Describe mRNA processing in eukaryotes
5' end of primary transcript is modified by addition of a cap structure while it is synthesized. 3' end is generated by cleavage of a longer precursor and addition of a poly(A) tail (100-250 bases). Splicing process removes non-coding introns and ligates coding exons.
What do introns have to do with eukaryotes?
Most genes of higher eukaryotes contain numerous introns; differential removal of introns (alternative splicing) produces multiple transcripts that can encode different proteins from a single gene.
Describe the eukaryotic mRNA cap structure
7-methylguanylate residue is added to nascent (growing) RNA chain using a 5'-5' bond rather than the usual 5'-3' bond. Methyl residues are also added to ribose of first and second base of mRNA transcript by specialized methylase enzymes. Cap structures assists export to cytoplasm, is required for mRNA translation and protects RNA from degradation.
What modifications are found at the ends of eukaryotic mRNAs?
The 5' end has a 7-methylguanylate cap structure and the 3' end has a poly(A) tail of 100-250 bases.
Describe the process of translation
mRNA is translated using ribosome with its associated rRNAs and tRNAs.
What are tRNAs and how many types of RNA do they carry?
tRNAs carry an anticodon sequence that can base pair with triplet codon on mRNA and a matched amino acid at the 3' end. One specific amino acid per 20 different types of tRNAs
What is the role of aminoacyl-tRNA synthetases?
It covalently links a specific amino acid to the 3' end of tRNAs with a matching anticodon. Once tRNA is charged, a ribosome can transfer the amino acid from the tRNA and onto a growing peptide chain.
What are cognate tRNAs?
Amino acids that carry two or three different tRNAs and are modified by the same aminoacyl-tRNA synthetase. Overall the structure is different but the function remains the same.
What are some specialized codons?
AUG = start (met), UAA = stop, UGA = stop, UAG = stop
Describe reading frames in mRNAs
Any mRNA sequence can potentially be translated into three different protein sequences depending on the start site. The starting codon is usually the first AUG from the 5' end and sets the reading frame. In some instances, mRNA are translated into two proteins by starting at a second AUG in a different reading frame
Describe the structure of tRNAs
tRNAs are 70-80 nt long and differ in sequence but all have a conserved overall "cloverleaf" structure.
Many bases in tRNAs are modified using specialized enzymes which are required for function.
3' end contain CCA which isn't included in DNA and is exposed for easy access
Contains inosine which is similar to guanine and has 3H bonds
Describe the general structure of ribosomes for prokaryotes and eukaryotes
Prokaryotes: large subunit contains rRNA (23S,5S + 31 proteins=50S) + small subunit (16S + 21 proteins=30s) = 70S
Eukaryotes: large subunit contains rRNA (28S + 50 proteins=60s) + small subunit (18S + 33 proteins=40s) = 80S
What are the 4 translation initiation steps in Eukaryotes?
Ternary complex assembles with 40S subunit to form a preinitation complex
Preinitiation complex binds to mRNA cap structure to form initiation complex
40S subunit scans mRNA from 5' end until it reaches the AUG translation start codon
60S subunit joins 40S subunit to form 80S ribosome with initiator tRNA in peptidyl (P site)
* ATP and GTP hydrolysis are necessary to drive the reactions forward
What are the peptide chain elongation steps in Eukaryotes?
"Charged" tRNA (carrying the amino acid for the next codon) bound to EF1α and GTP enters ribosome at the A site
GTP hydrolysis promotes conformational change that positions the two tRNAs for peptide bond formation
Peptide bond formation is catalyzed by large ribosomal RNA
GTP hydrolysis promotes ribosome translocation to the next codon on the mRNA (tRNAs move to E site and P sites, leaving A site available for next tRNA)
What are two elongation factors required for peptide chain elongation in eukaryotes?
EF1α and EF2
What are two release factors required for the termination of translation?
eRF1 and eRF3
What are the steps to the termination of translation?
Translation terminates when the ribosome reaches stop codons (UAA, UGA, or UAG)
eRF1 and eRF3-GTP, instead of charged tRNA, enter at or near A site
GTP hydrolysis then promotes release of polypeptide and tRNAs as well as dissociation of 80S ribosome
How do eukaryotic mRNAs form circular complexes? What about prokaryotes?
In cells mRNA caps and poly(A) tails are bound to proteins that interact with each other to form circular structures.
Prokaryotic mRNAs have no cap or poly(A) tails and do not circularize; translation starts internally at the beginning of each reading frame; otherwise individual steps in translation are similar.
Which mRNA feature does the translation preinitiation complex bind to?
5' end cap structure
Describe DNA replication in contrast to RNA replication
DNA polymerases, like RNA polymerases, can only synthesize chains in 5' to 3' direction.
In contrast to RNA polymerases, DNA polymerases cannot initiate chain synthesis de novo
Synthesis by DNA polymerases requires a primer, a pre-existing RNA or DNA strand base-paired to a template strand (a short RNA serves as a primer in cell)
How can cells synthesize DNA in two opposite directions at replication forks?
There are the leading and lagging strands at the replication forks
Leading strand synthesis occurs continuously from a single RNA primer in the direction of fork movement
Lagging strand synthesis occurs discontinuously in a direction opposite from the replicating fork movement
Describe the process of DNA replication at the replication fork
Primase enzyme synthesizes RNA primers ~15 nt long, every few hundred bases apart on the lagging strand
DNA polymerase elongates primers
RNA primers are removed by an exonuclease enzyme associated with DNA polymerase
Short gaps are filled by DNA polymerase
Resulting Ozaki fragments ( a few hundred bases long) are joined by DNA ligase
Describe how DNA is replicated bidirectionally
DNA replication initiates from specific origins in both directions.
Helicases are required for unwinding of DNA at origins and for replication fork movement
DNA polymerases are responsible for replication are DNA pol III in prokaryotes and DNA pol δ in eukaryotes
What are some possible causes for DNA damage? What is the estimated no. of DNA damage per day?
Spontaneous mutations are due to inherent DNA instability (depurination)
Cellular reactions produce chemicals that react and damage DNA (hydroxyl radicals and superoxides)
Radiation and chemicals from the environment can also damage DNA
104 to 106
What are 6 possible DNA repair systems?
DNA polymerase proofreading (enzyme associated)
Base excision repair (due to deamination)
Mismatch excision repair (replication errors)
Nucleotide excision repair (distorted DNA)
Nonhomologous end-joining (dsDNA breaks)
Homologous recombination (dsDNA breaks)
Describe the overall process of proofreading by DNA polymerases
Replicating DNA polymerases have and associated 3' to 5' exonuclease activity. Misincorporated bases cause polymerase pausing and transfer of the growing 3' end to the exonuclease site. After removal of the incorrect base, the growing chain is transferred back to the polymerase active site and synthesis resumes.
What causes deamination? What can repair it?
Deamination of 5-methyl cytosine to thymine due to chemical instability can cause T•G mismatches unless repaired by base excision. The excision process creates the removal of T base from the sugar-phosphate backbone, cuts phosphodiester bond, and removes the remaining deoxyribose phosphate.
What does DNA pol β specialize in?
It is a specialized repair enzyme.
How are replication errors repaired? What does it also repair?
By mismatch excision repair systems. Complex of MSH2 and MSH6 proteins recognize and bind to mismatch region in the newly replicated strand. Several enzymes (endonuclease, exonuclease, and helicase) then work in concert to remove a portion of the DNA strand containing the mismatch (produces a gap). The gap is then filled by DNA pol δ
It also repairs small deletions and insertions introduced during replication
Damage causing distortion of the DNA duplex is repaired by what? What is the best known damage in its class?
Nucleotide excision repair system, repair involves endonucleases that cut a 24-32 base-long segment containing the damage; gap is filled by DNA pol δ. The best known DNA damage in this class is thymine dimers caused by UV radiation (covalent linkage of adjacent thymines).
Describe the two systems for recombination repair of dsDNA breakage
Nonhomologous end joining: Broken ends are ligated by several base pairs at the joint are loss (error prone)
Homologous recombination: Damage sequence is replaced by a segment copied from the undamaged sequence on the homologous chromosome.
What are the cause of cancer predisposition? Define 2
Caused by hereditary defects in DNA repair systems.
Hereditary nonpolyposis colorectal cancer: Due to loss of function in one copy of either MLH1 or MSH 2 genes involved in mismatch excision repair
Hereditary xeroderma pigmentosum: (sensitivity to sunlight and UV) is often due to mutations in endonucleases required for nucleotide excision repair.
Which activity associated with DNA polymerases is responsible for proofreading?
3' to 5' exonuclease
What are the two distinct systems used to repair dsDNA breaks?
Nonhomologous end-joining and homologous recombination
What are the differences between 3'→5' and 5'→3' exonuclease activity?
Removal of nucleotides (hydrolysis of DNA) takes place in the 3'-->5' direction.
Hydrolysis begins only at the 3'-OH terminus
Has a 'proofreading' and 'editing' function in the initial stages of replication. Polymerase I removes mismatched nucleotide pairs and follows this up by resynthesis.
All the three polymerases (I, II and III) show 3'--> 5' exonuclease activity
Hydrolysis is in the 5'-->3' direction
Has a role in the excision of pyrimidine dimers during repair replication.
What are two general strategies in characterizing gene function?
Classical genetic strategy starts with the mutant
Reverse genetic strategy starts with the wild type (more common and makes you in charge of making the mutation)
When do gain-of-function and loss-of-function occur?
Dominant mutations often cause gain-of-function (increase of activity of encoded protein, confer new function)
Recessive mutations usually cause loss-of-function (remove part of the gene, alter protein structure)
What are two types of loss-of-function mutations?
Haplo-insufficient genes: Both wild-type alleles are required for function (loss of function in one allele is a dominant mutation)
Dominant negative mutation: Loss-of-function allele interferes with function of wild-type allele (mutant protein impairs assembly of a structure composed of multiple identical subunits)
What are 5 DNA alterations that cause mutations in the coding region
Silent point mutation: No change in the aa sequence or protein activity (a change in the third base of a codon for aa with multiple codons)
Missense mutation: Substitution of one aa for another (No longer codes the same aa)
Nonsense mutation: aa codon change to a stop codon causing premature termination
Frameshift mutation: Change in reading frame due to small insertions or deletions one or two bases at a time
Lager alterations: Large deletions, insertions, duplications, rearrangements, etc.
What are some of the main advantages of yeast being a model as eukaryotic organism for genetic studies?
Single yeast cells grow rapidly to form colonies on nutrient agar plates (like bacteria)
Yeast can be grown as haploid or diploid cells, which facilitates genetic analysis
What is the ability to perform replica plating crucial?
It allows testing of single cell progeny under different conditions to define phenotype
Describe the process of segregation of mutations in haploid yeast
Haploid cells contain a mixture of two different mating types, α and a cells. Under appropriate experimental conditions, a and α cells will mate (fuse) to produce diploid cells. Mating of cells carrying different alleles of a gene readily determines dominance. Under starvation conditions, diploid cells sporulate (undergo meiosis) and give rise to four haploid spores.
Why are genetic screens essential? What 2 kinds of mutations is it used on?
Mutants are present at low frequency in wild-type population: genetic screens are used to identify and isolate mutants (most easily done with haploid yeast).
Lethal mutations do not allow characterization of the affected function (no haploid cells survive) and only reveal that the affected gene is essential.
Conditional mutations allow characterization of any mutation, lethal or not -mutant phenotype is observed at high but not low temperature or in the abs. but not the presence of a specific unt.
Describe the process of screening for temperature-sensitive cell division cycle (Cdc) in mutants in yeast
Spontaneous mutants for any given gene are present at low frequency in a large population of wild types. A chemical mutagen is generally added to increase mutation frequency before screening. Incubate at 23º C by plating and create a replica-plate and incubate (one at 23ºC and other at 36ºC) Thousands of independent mutations that block colony formation are noticed at only 36ºC can then be isolated.
What is another example of yeast Cdc mutants and its process?
Saccharomyces cerevisiae (budding yeast), cell division initiates with small "buds" that eventually grow to full size cells. Cdc mutants grown in suspension stop growing when shifted from permissive to non-permissive temperature. For the Cdc28 mutant growth, it stops before budding while Cdc7 growth stops after budding but before separation from the mother cell.
Shows that the outcome is the same, but different on different genes due to the different growth stages
Why is genetic screening in higher eukaryotes more complex? List 4
Generation time is much longer than in yeast
Spontaneous or induced mutations in diploid organisms typically change only one allele
Effect of recessive mutations cannot be tested in haploid cells (sperm and eggs)
Inbreeding is required to identify recessive lethal mutations (homozygous progeny does not survive)
Describe complementation analysis in yeast and how it is used in identifying mutations in the same gene
It determines how many genes are represented in a collection of independent recessive mutations affecting the same process (cell division, synthesis of a particular metabolite). Each mutant isolate is mated to every other isolate to produce diploid cells. The phenotype of the diploid cells reveals whether the two mutant isolates carry a mutation in the same gene or two different genes.
Describe the process of complementation analysis in yeast. What does it reveal?
Mate haploids of opposite mating type and carrying different recessive temperature-sensitive cdc mutations
Test resulting diploids for a temperature sensitive cdc phenotype first in permissive temp. then the replica-plates at nonpermissive temp.
Growth/absence of growth should indicate the mutations to be either complementing or non-complementing
How does complementation analysis reveal the number of genes governing a process?
All mutant pairs that fail to complement each other affect the same gene and are assigned to one/same complementation group. Mutants that complement each other are assigned to different complementation group. With a sufficiently large collection of mutants, the number of genes affecting a particular process (ex. cell division) is equal to the number of complementation groups.
Normal functioning of most cellular processes depend on what? Give an example
Protein complexes that require specific binding interactions between proteins encoded by different genes. (enzyme subunits)
Describe what happens in genetic suppression
A single mutants in each of the two genes produce the same defect but the double mutant remains normal, the two gene products are then more likely to interact.
What is synthetic lethality?
It reveals gene product reaction. It occurs when mutation in one of two interacting proteins lead to a partial defect but a mutation in both causes a severe defect.
Describe redundant pathways
Some cellular functions are provided by redundant pathways (either pathway provides the same function). However, in this case mutations in two different genes (one in each pathway) are required for the lethal phenotype (synthetic lethality)
What is frequency of recombination?
The measure of distance between two given genes by convention one recombinant individual per 100 total progeny = one genetic map unit or 1 centimorgan (cM)
What is recombinant DNA methodology and DNA cloning?
A detailed study of the structure and function of a gene at the molecular level requires large quantities of individual genes in pure form.
DNA cloning makes it possible to prepare large numbers of identical DNA molecules of any sort. The basic key to cloning a DNA fragment is to link it to a vector DNA molecule that can replicate rapidly in a host cell such as E. coli
What is the basic scheme to DNA cloning?
vector DNA + DNA fragment → recombinant DNA → replication of recombinant DNA within host cells → isolation, sequencing, and manipulation of purified DNA fragment
How are restriction enzymes the key to cloning?
They are endonucleases found in bacteria that can cleave both strands of DNA duplex but only within a specific sequence or recognition site. The sites are (4-8 bp long) are pallindromic sequences (same sequence of both strands read 5' to 3'). The staggered cuts leave "sticky" single-stranded tail ends four bases long. Under annealing conditions sticky ends can transiently base pair and be religated with DNA ligase
Why do each bacterial species encode its own restriction enzyme and specific methylase enzyme?
Each restriction enzyme has a specific recognition site; these serve to protect a bacterial species against invasion by foreign nucleic acids.
It adds methyl groups to the recognition site to prevent cleavage by its own restriction enzyme.
How does restriction enzyme digests produce a unique set of fragments for a given DNA? How can they be separated and visualized?
DNA molecules contain restriction sites that can be cleaved by one or more restriction enzymes. The size and number of fragments produced by a restriction enzyme depends on the location of the cleavage sites and the resulting pattern is unique for a given DNA. DNA fragments can be separated by size and visualized by gel electrophoresis.
What are DNA cloning vectors?
DNA molecules that can replicate in host cells. There are many different types, but most commonly used are plasmid vectors (2.5 to 10 kbp) that replicate at extrachromosomal circular DNA in bacteria or in some lower eukaryotes like yeast.
How do you insert DNA fragments into vectors?
A genomic DNA sample cut with three different restriction enzymes produces three classes of fragments with different sticky ends. When mixed with a vector DNA cut with one of these enzymes, only genomic fragments cut with the same enzyme can base-pair at the sticky ends. Addition of DNA ligase covalently links the compatible genomic fragments to the vector
What are three components of a typical cloning vector?
Polylinker: Enzyme sites in the region that are unique in the vector (occur only once and can be used for cloning a.k.a restriction sites)
Ori: Replication origin where DNA replication is initiated by host enzymes
Ampr: β-lactamase enzyme which confers resistance to ampicillin (selectable gene)
What is ampicillin
An antibiotic derived from penicillin that inhibits bacterial cell wall formation
What are DNA libraries and genomic libraries? What makes it easier to identify and manipulate protein encoding genes from eukaryotes?
The entire genome of an organism that is too big to clone in a single vector molecule.
Overlapping smaller fragments that are first cloned into vector "libraries" such that all parts of genome are represented at least once.
cDNA libraries are made from mature mRNAs that lack non-coding introns
What are the 10 steps to constructing cDNA libraries?
Hybrizise mRNA with oligo-dT primer
Transcribe RNA into cDNA
Remove RNA with alkali and add poly(dG) tail with transferase enzyme
Hybridize oligo-dC primer
Synthesize complementary strand (Reverse transcritase or DNA polymerase can produce DNA:DNA duplex)
protect cDNA by methylation at EcoR1 sites (EcoR1 methylase modifies all EcoR1 sites in the entire population)
ligate cDNA to restriction site linkers
Cleave with EcoR1 (All EcoR1 are protected)
ligate to plasmid,
transformed E.coli select for ampr
Describe the methods of screening DNA libraries by hybridization
Colonies resulting from transformation with a library are transferred to a nitocellulose filter paper (replica). The filter is incubated in alkali to lyse cells and denature DNA (single-stranded DNA binds to nitrocellulose). It is then incubated under hybridizing conditions with a DNA probe (P32) encoding one specific gene sequence. After washing away the probe, autoradiography (defection of radioactive signal on film) identifies colonies that contain DNA complementary to the probe.
Describe yeast's genomic libraries and why is it better. Also define shuttle vector
The genome is smaller than that of higher eukaryotes and its genes contain fewer introns. It is therefore possible to use genomic rather than cDNA libraries to clone yeast genes. Specialized shuttle vectors that replicate as plasmids both in E.coli and yeast cells are used for this purpose. Shuttle vectors are also engineered to express the clone genes (make corresponding proteins) in yeast cells)
Describe the method of constructing yeast genomic libraries
Genomic DNA is digested with restriction enzyme (Sau3A) under partial conditions (incomplete digestion) to yield overlapping fragments of 10 bp. The fragments ensures that all regions of the yeast are cloned. Ampicillin resistant bacterial colonies are then pooled together and specific yeast genes are isolated by functional complementation in yeast
Describe the method for screening yeast genomic libraries by functional complementation
When the shuttle plasmids from a yeast genomic library are introduced into yeast cells, the promoter sequences are recognized by yeast RNA polymerase II. Plasmids from a wild-type yeast genomic library thus synthesize functional wild-type proteins in transformed cells. When haploid mutant yeast cells are transformed with the plasmid library, a few cells will acquire an extrachromosomal wild-type copy of the recessive mutant gene and show complementation
What are 6 essential methods for characterizing DNA and RNA?
Gel electrophoresis (separating DNA fragments by size)
DNA sequencing (dideoxy chain-termination method)
PCR (polymerase chain reaction)
Southern and Northern blotting (hybridization methods)
Microarray analysis (global gene expression)
Expression Plasmid Transfection (gene function analysis)
How does gel electrophoresis separate DNA fragments by size?
Any fragment of DNA (gene insert in a plasmid) can be purified using restriction enzymes and gel electrophoresis. A sample of DNA layered on top of a gel migrate towards the positive electrode when a current is applied (DNA is negatively charged at neutral pH). DNA moves through pores in the gel according to size; polyacrylamide gels resolve DNA fragments 10-2000 bases and agarose gels (larger pores) separate fragments 200 to 20 kb. DNA bands are detected by autoradiography or ethidium bromide.
What is the difference between ddNTP and dNTP
Absence of 3' OH end on the dideoxyribonucleotide precluding chain elongation (cause termination) on ddNTP
What machine determines which fluorescence wavelength is emitted for each fragment size?
What is polymerase chain reaction? Where is it useful (list 4)?
It enables one to make as many copies of a gene or DNA fragment as needed without cloning. It can amplify a single DNA molecule ans is useful for many applications such as:
direct isolation of a specific segment of genomic DNA
preparation of DNA probes
introducing specific mutations in genes
amplifying trace amounts in DNA in forensic applications
Describe the process of polymerase chain reaction
Oligonucleotide primer sequences that base pair to either side of a region to be amplified are first synthesized. The template DNA is first denatured at high temp. and then annealed at lower temp. to an excess of two primers. A heat-stable DNA polymerase from thermophilic bacteria is included in the reaction to elongate primers. After 20-30 cycles of denaturation and DNA synthesis carried out in thermal cyclers, large amounts of the amplified DNA accumulate; 20 cycles = (220)
Describe the 3 process of detecting specific DNA fragments by southern blotting
Separate restriction fragments by size using gel electrophoresis
Transfer DNA fragments from gel into nitrocellulose paper (blotting)
hybridize DNA bound to paper with a specific labeled DNA or RNA
What is the difference between Northern and Southern blotting?
Different to southern blotting by using gel electrophoresis to separate mRNAs by size.
What are DNA microarrays? What makes them a global analysis of gene expression?
Consists of thousands of individual gene-specific sequences attached to the surface of a glass microscopic slid. Gene-specific DNAs commonly used are chemically synthesized oligonucleotides or PCR products. Oligonucleotide arrays are often called DNA chips (deposition of samples in tightly packed arrays resembles integrated circuits on computers). Hybridization of cDNAs to DNA microarrays monitors the expression of thousands of genes simultaneously.
How are the function of cloned genes analyzed using plasmid expression vectors? What are two types of transfection methods?
Analyzing the function of eukaryotic genes requires synthesis of the gene product in eukaryotic cells. For this purpose, genes are moved from a cloning plasmid into an expression plasmid (cloned directly) taht contain sequence elements required for transcription of the gene. Expression plasmids are introduced into animal cells by transfection, which results in the uptake of an aggregate of several hundred plasmid DNA molecules in each cell.
Transient and stable
Describe the process of transient transfection of expression plasmids in animal cells
Plasmid vectors have the required elements for replication in E.coli (ORI, ampr, polylinker) so it can be prepared in large amounts. The plasmid also contains a viral origin of replication (allows some limited replication in animal cells and a eukaryotic promoter sequence for transcription of the gene). Transfected cells express the cloned gene efficiently but cell division over the course of several days leads to loss of plasmid and expression.
Describe the process of stable transfection of expression plasmids in animal cells
The plasmid vector now includes a selectable gene (neor) and no viral origin of replication (rest is same as for transient transfection). Following transfection, a few rare cells integrate the neor gene into their genome through a non-homologous recombination event. The "transformed" cells are resistant to G-418 antibiotic (neomycin derivative) and grow as colonies over the course of two weeks. Stably transfected cells continue to express the gene of interest as long as the culture is maintaine
What can we learn about gene function from transfection experiments? List 5
The effects of adding new genes (not present in the recipient cells)
Effect of adding mutated versions of "endogenous genes"
Effect of inhibiting endogenous genes with dominant-negative mutants
Determine cellular localization of a given protein using fusion tags or epitope tags (short aa sequence recognized by a specific antibody)
determine promoter sequence elements of a specific genes by linking them to reporter genes (fluorescent protein, firefly luciferase, b-galactosidase)
What are 3 types of DNA polymorphisms used for disease gene linkage mapping? What two are detected by PCR and DNA sequencing?
Restriction fragment length polymorphism (RFLPs): absence or presence of specific restriction enzyme sites
Single nucleotide polymorphism (SNPs): single base pair differences (most abundant type)
Single sequence repeats polymorphism (SSRs): variable number of repetitions of one-, two-, or three- base sequence (also called microsatellite)
SNPs and SSRs
DNA polymorphism does what to restriction enzyme sites? What does it give rise to?
DNA polymorphisms occasionally destroy or create restriction enzyme recognition sites in particular regions of an individual genome. It gives rise to variations among individuals in the length of restriction fragments derived from a given region of their genome.
RFLP analysis is commonly used to map out what?
RFLP analysis is commonly used to map human disease genes relative to known positions of specific RFLPs.
Describe the process of RFLP analysis
Restriction fragments are analyzed by Southern blotting with a probe that hybridizes to a specific DNA fragment at a known position on a given chromosome. The two homologous chromosome of this individual show RFLP when cutting with restriction enxyme A but not enzyme B. Three different alleles are detected in a chromosomal region detected by a labeled probe. If a disease allele is linked to the RFLP (on the same chromosome), then it will segregate along with restriction pattern
What works well with inherited human diseases? Define genetic heterogeneity and polygenic
Linkage mapping works well with inherited human disease that are monogenetic traits (single gene defect) and most of these have already been mapped using DNA-based markers.
Mutations that show complicated patterns of inheritance where mutations in any one of multiple genes can cause the same disease.
Several different genes acting together contribute to the occurrence and severity of disease (diabetes, heart disease, obesity, predisposition cancer, some mental disorders)
What are 3 types of gene-inactivation techniques employed in eukaryotic organisms?
Gene replacement or knockout (replace normal gene with other sequences)
Introducing a mutant allele that inhibits functioning of the wild-type allele (dominant negative)
Using RNA interference to promote the destruction of a specific mRNA or prevent its translation into protein
What are the two stage procedure for the gene disruption (gene knockout) in mice?
Introduce a disrupted allele into embryonic stem cells (ES cells obtained from early embryos can be grown in culture and retain their ability to produce all tissues)
Inject ES cells heterozygous for the disrupted gene into a wild-type blastocyst (very early embryo) which is then implanted in a foster mother).
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