78 terms

cell bio ch 10

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cDNA library
Collection of cloned DNA fragments synthesized using all of the mRNAs present in a particular type of cell as a template.

- all mRNAs extracted, dsDNA copies produced by reverse transcriptase and DNA polymerase
- introduced into bacteria and amplified
- gene of interest (without introns) can be isolated using a probe that hybridizes to DNA sequence
genomic library
represents entire genome of organism
*used for chromosome specific libraries/ complete Chromosome Painting methods
1. Take DNA, cleave w restriction nuclease- Generates millions of genomic DNA fragments of varying sizes
2. Fragments inserted into plasmids using DNA ligase =plasmids with recombinant DNA
3. Introduction of plasmids into bacterial cells (E. coli) at a conc. that ensures only 1 taken up
transformation
controls bacterial uptake of DNA molecules in surroundings - "transform" one bacterial strain into another. DNA purified from pathogenic strain used to transform a harmless bacterium into a deadly one. E. coli that take up recombinant DNA suspended in nutrient-rich broth and allowed to proliferate. Each time bacterial pop doubles, # copies of recombinant DNA molecule also doubles, producing many plasmids along with DNA fragment. Bacteria can then be split open and plasmid DNA purified from rest of cell contents. Cut it out with same restriction nuclease used to insert it, then separate from plasmid DNA by gel electrophoresis--> Allows amplification/purification of any DNA segment from genome of any organism
DNA ligase
reseals nicks in DNA backbone during DNA replication and repair in cells
- can join together any 2 DNA fragments in vitro to produce recombinant DNA molecules using ATP to reseal backbone
- of either same or different restriction nuclease - if different before fragments undergo ligation, DNA pol + mix of dNTPs are used to fill in staggered cut
plasmids
vectors used for gene cloning- small circular DNA molecules
- contains origin of replication
southern blot/ gel transfer hybridization
Used to verify presence/absence of specific nucleotide sequence in DNA from different sources and identify restriction fragment that contains the sequence
1. DNA from each source digested with specific restriction enzyme
2. DNA restriction fragments (or dsDNA fragment) separated by gel electrophoresis
- can then put nitrocellulose filter paper
3. Fragments visible with ethidium bromide
4. ss fragments from gel and blotted onto nitrocellulose paper/nylon filter
- buffer, drawn toward paper towels, carries alkali-denatured DNA fragments from gel to paper
5. Paper removed
6. Radioactively labeled nucleic acid probe hybridized to complementary nitrocellulose-bound DNA bands
- probe certain sequences of DNA after remove from gel -- probe also denatured (bc double stranded)
7.Bands visualized by autoradiography/ nylon membrane visualized with X-ray
- reanneal based on specificity to DNA sequence
DNA probes
used to determine in which tissues and at what stages of development a gene is transcribed
DNA cloning
Production of many identical copies of a DNA sequence
- amplification - makes possible
- cell extract containing plasmid isolated from bacterial cells
- insert into vector with 3 elements: 1. cloning site (where foreign DNA inserted)
2. drug resistance gene (destroys Abs to allow selective growth of host cell (ex: AmpR))
3. Replication origin- allow plasmid to replicate in host cell
biotechnology
use of biological organisms and processes to create useful products in industry & medicine
genomics
study of genomes - from the structure & alignment of genes to how a chromosome is packaged with proteins & how the DNA works in a biological system
HGP goals
1. Mapping the human genome
(& eventually determining the sequence of all 3.2 billion letters in it); 2. Mapping and sequencing the genomes of other
organisms important to the study of biology;
3. Developing technology for analyzing DNA;
4. Studying the ethical, legal and social implications
of genome research.
1990: ELSI Founded
BAC
bacterial artificial chromosome = a piece of human DNA fitted into a bacterial "vector", a carrier of DNA
- Researchers began to study how to efficiently produce stable carriers of large DNA inserts in bacteria, so-called BACs
- Typically 100,000 to 300,000 base pairs long - is more stably inherited than in YACs
- Many copies of the human insert are made when the bacteria multiply. They can be used as the pieces of the puzzle when making a physical map of the genome and can be chopped up further for sequencing
- used to manufacture DNA for both the public genome consortium and the private sequencing effort of Celera
-They are generally preferred to YACs
(yeast artificial chromosomes) as DNA microfactories because they are easier to separate from host cell chromosomal DNA, and they don't have a predilection, as do YACs, for incorporating large pieces of DNA from multiple sites in a genome
--less background to hybridize
EST
expressed-sequence tag = a stretch of DNA sequence made by copying a portion of an mRNA molecule
- all ESTs replicate sequences from genes
- A relatively small portion (approximately one-tenth) of the human genome is thought to be transcribed, or "expressed"
- Looking at ESTs is a way to home in on the expressed, clearly functional sequences in the genome that the nucleus sends out to the rest of the cell
genetic marker
always inherited along with a certain disease gene - indicates that the gene is located near the marker
RFLPs
restriction fragment length polymorphisms - used as markers in early genetic maps
- The first genetic map that spanned the entire human genome with microsatellites was an improvement because they are more easily discovered and distinguished
genetic mapping
if a particular genetic marker is
inherited with a disease gene, the gene likely resides near the genetic marker
- want to generate many markers
- critical early step in the hunt for disease genes - allowing researchers to find out which chromosome a gene lies within and approximately where in that chromosome
- human genome had nearly 6,000 markers, with markers spaced an average of 700,000 base pairs apart
MANIPULATING AND ANALYZING DNA MOLECULES
• Restriction Nucleases Cut DNA Molecules at Specific Sites
• Gel Electrophoresis Separates DNA Fragments of Different Sizes
• Bands of DNA in a Gel Can Be Visualized Using Fluorescent Dyes or Radioisotopes
• Hybridization Provides a Sensitive Way to Detect Specific Nucleotide Sequences
restriction nucleases
Enzymes discovered within bacteria which serve to defend host from foreign DNA, cleaving DNA at highly specific sites.
- restrict DNA transfer between certain bacterial strains and own bacterial DNA (protected by chemical modification of these sequences)
- Cuts btwn BOTH DNA strands- palindromic sequence
can generate staggered (EcoRI, HindIII) or blunt (HaeIII) ends!
Gel electrophoresis
Cleaved DNA molecules separated by size - smallest fragments migrating furthest into gel.
Fragment sample sizes are determined by reference- compare sample unknown DNA to known size DNA and look @ sequences!
DNA ladder of known fragment sizes
- based on taking dsDNA, cutting with diff. restriction endonucleases, load onto gel (polyacrilamide, agarose)
- DNA neg. charged- runs to pos. electrode
- heavier/larger DNA @ top, lighter/smaller @ bottom - based on weight
bands of DNA in gel
Can Be Visualized With Fluorescent Dyes or Radioisotopes
- devel of nontoxic fluor probes
Hybridization
2 complementary nucleic acid strands come together and form H bonds to produce a double helix; used to detect specific nucleotide sequences in either DNA or RNA.
Provides a Sensitive Way to Detect Specific Nucleotide Sequences
1. dsDNA denatured by heating to ss (H bonds btwn nt pairs broken)
2. Single strands slowly cooled to renature/restore double helices (nt pairs re-formed)
- DNA can be denatured by heating or alkali treatment-
- 1961 discovery-very powerful technique- launchpad for biotechnological techniques used for DNA/RNA
DNA CLONING IN BACTERIA
• DNA Cloning Begins with Genome Fragmentation and Production of Recombinant DNAs
- fragmenting DNA - mass produce a lot to analyze
• Recombinant DNA Can Be Inserted Into Plasmid Vectors, then THEY can be expanded and ?turned into recombinant DNA? also
• Recombinant DNA Can Be Copied Inside Bacterial Cells
• Genes Can Be Isolated from a DNA Library
• cDNA Libraries Represent the mRNAs Produced by Particular Cells
- vs. genomic: all mRNAs made by certain cell
2 ways to make recombinant DNA
1. joining two fragments cut by the same restriction nuclease
staggered end
- cut by same restriction enzyme
- w/ addition of ATP + ligase, get a unified molecule

2. joining two fragments cut by different restriction nucleases
blunt end (blunting)
addition of DNA pol - join blunt ends together
- addition of dNTPs
DNA Fragments
can be inserted into a bacterial plasmid via DNA Ligase and the fragment can be cloned/expanded
1. circular double-stranded plasmid DNA (cloning vector)
- cleavage with restriction nuclease-
2. open double-stranded plasmid DNA
- add in DNA fragment to be cloned, and form covalent linkage by DNA ligase-
3. recombinant DNA formed
Recombinant DNA
Can Be Copied Inside Bacterial Cells
- transfect into bacterial cells w/ recombinant plasmid
- copy and multiply millions of these specific recombinant DNAs to study
1. double-stranded recombinant plasmid DNA introduced into bacterial cell
2. cell culture produces hundreds of millions of new bacteria
3. many copies of purified plasmids isolated from lysed bacteria
Identifying bacterial colony with particular DNA clone via Hybridization
1. Petri dish with colonies of bacteria containing recombinant
2. Peel paper from dish to produce replica of colonies
3. Lyse bacteria and denature DNA with alkali
- radiolabeled DNA probe added in
4. Incubate with probe and wash
5. Expose paper to photographic film- radioactive
- position of desired colonies detected by autoradiography
Preparation of Complementary DNA (cDNA) from mRNA of Specific Cell
1. lyse cells in culture and purify mRNA
- take cDNA from mRNA of cell
- cells growing in culture
- lyse- look @ fibroblasts
- purify all mRNA made from particular cell type
2. hybridize mRNA with poly T primer
- mRNA has 5' and polyA 3' tail
3. make DNA copy with reverse transcriptase to form RNA/DNA double helix - mRNA and cDNA hybrid
4. partially degrade RNA with RNAse
- residual RNA primer of 5'->3' strand left on 3'->5' strand
5. synthesize a complementary DNA strand using DNA polymerase
- rest of other half of DNA by adding DNA pol
-- *double-stranded cDNA molecule of ONLY expressed genes of that specific cell type made!
How are Genomic DNA Clones and cDNA Clones Different?
Prep of Genomic Library:
- Restriction nuclease digestion to produce DNA fragments
- both introns and non transcribed DNA are included in clones. and most have either no coding sequence or only part of coding sequence of a gene; the DNA that regulate the expression of each gene are also included
- genes A and B are transcribed and thus represented equally in the genomic library
GENOMIC- ALSO HAS INTRON SEQUENCES!

Prep of cDNA library:
- Treatment with reverse transcriptase and DNA polymerase to produce cDNA copies of mRNAs
- intron sequences have been removed by RNA splicing during formation of mRNA, and a continuous coding sequence is present in each clone
- gene B is transcribed more than A in cells from which cDNA library was made, so it will be represented much more often than A in the cDNA library
CDNA- ONLY GENES THAT ARE TRANSCRIBED
DNA CLONING BY PCR
• PCR Uses a DNA Polymerase to Amplify Selected DNA Sequences in a Test Tube
• Multiple Cycles of Amplification In Vitro Generate Billions of Copies of the Desired Nucleotide Sequence
- Used to Obtain either Genomic or cDNA Clones
1. Isolate total DNA/mRNA from cells
2. DNA/mRNA sequence to be cloned
3.
DNA:
1. Separate strands (by heating) and add primers (cool to anneal)
2. PCR Amplification
3. Genomic Clones

mRNA:
1. Add First Primer, Reverse Transcriptase, and Deoxyribonucleoside Triphosphates
2. Separate strands (heat) and add second primer (cool)
3. PCR Amplification with Both Primers Present
4. cDNA Clones

• PCR is Also Used for Diagnostic and Forensic Applications
A. ex: determine if infected with HIV
1. blood sample from person
- Remove CElls by Centrifugation
2. Rare HIV particle in plasma of infected person
- Extract RNA
3. Reverse transcription and PCR Amplification of HIV cDNA
4. Gel Electrophoresis with infected sample and control (using blood from noninfected person)

B. Forensic Science - distinguish individuals
- DNA sequences analyzed are short tandem repeats (STRs) composed of repeating 2 letter sequences - number of repeats in population is highly variable - hereditary
A) PCR using primers that recognize unique sequences on either side of one particular STR locus produces a pair of bands of amplified DNA from each individual, one band representing maternal and other paternal STR variant
- different people can have several bands in common, but overall pattern differs - band can serve as DNA fingerprint!
- more loci = more confidence - decreasing likelihood of sharing same fingerprint by chance -- also used in paternity test
PCR Primers
Direct the Amplification of a Desired DNA Segmment In Vitro - start w specif region of dsDNA you want amplified that you isolated

1. Heat to 94-96 C to separate target dsDNA into single strands
2. Cool to 50-65 C to Anneal Primers (bracket DNA region to be amplified) to complementary sequences
3. Heat to 72 C to allow Taq polymerase to attach at each priming site and extend (synthesize) new DNA strand - DNA Synthesis
- when do this, get products of 1st cycle of amplification
- keep doing using Taq DNA polymerase (isolated from thermophilic bacterium- stable at higher temps than euk DNA pols, so not denatured and doesn't have to be added again after each cycle!)- get strand denaturation, anneal primers and synthesis
- specif amts generated quickly
- all you need is primers! to be able to use PCR
PCR steps
Identify region of dsDNA to be amplified
1. Heat to separate strands
2. Cool to anneal primers
- DNA exposed to large excess of a pair of specific primers- designed to bracket the region of DNA to be amplified- and the sample is cooled to allow primers to hybridize to complementary sequences in the 2 DNA strands
3. DNA synthesis - add in DNA pol and dNTPs (dATP, dCTP, dGTP, dTTP)
- Mixture incubated with DNA pol and 4 dNTPs so DNA can be synthesized starting from 2 primers
-- First cycle of amplification
Repeated rounds of strand denaturation, hybridization, and synthesis amplify DNA in PCR - repeat by reheating sample to separate newly synthesized DNA strands
- Second cycle produces 4 dsDNA molecules
- Third cycle produces 8
dideoxy/Sanger sequencing
The standard method of determining the nucleotide sequence of DNA; utilizes DNA polymerase and a set of chain-terminating nucleotides.
- relies upon chain termination via dideoxynucleoside triphosphates
- Normal deoxyribonucleoside triphosphate has 3'OH - allows strand extension at 3' end - 5' C of 'incoming' dNTP jointed to 3' C at end of chain
- hydroxyl groups in each position form ester linkages with central phosphate- chain elongates
- Chain-terminating deoxyribonucleoside triphosphate
DNA microarray
A surface on which a large number of short DNA fragments (typically in the tens of thousands) immobilized in an orderly pattern. Each DNA fragment acts as probe for mRNA produced by a specific gene, allowing the exp of every gene in a genome to be monitored
1. mRNA collected from different samples - can be used to look at diff genes, species, tissue types, or cancer cell vs normal cell if you are searching for cancer disease genes up-regulated/down regulated, etc.
2. each is reverse transcribed to cDNA complementary to mRNA, with different color labeled fluorochrome
3. cDNA hybridizes to DNA samples in microarray (incubate)
4. Wash, scan for fluorescent color signals and combine images
- cDNAs complementary to type A mRNAs hybridize with DNA (in array) complementary to type A cDNA
- if cells don't exp any mRNAs that correspond to cDNAs, no cDNAs are made that binds to those mRNAs
- typical cell exp's 100s-1000s of mRNAs - 100s-1000s of spots ID'd - study many genes simultaneously from a particular type of cell, or cells exposed to environmental condition
- Automated fluorescence microscope determines which mRNAs were present in the original sample based on the array positions to which the cDNAs are bound
- 1 drawback: sequences of mRNA samples to be analyzed must be known in advance and represented by a corresponding probe on the array
- color shown = gene in sample that is expressed at a higher level than the corresponding gene in the other sample
- mix of 2 colors - equal level of expression of genes in both cell samples
- intensity = how much RNA present from a gene
- dark spot = little/no expression of gene whose fragment is located at that position in the array
- represents lots of genes - allows for direct comparison of specific genes expressed under both conditions
- 1 dot = 100,000 bp of human genome
EXPLORING AND EXPLOITING GENE FUNCTION
• Whole Genomes Can Be Sequenced Rapidly
• Next-Generation Sequencing Techniques
Make Genome Sequencing Faster and
Cheaper
• Comparative Genome Analyses Can Identify
Genes and Predict Their Function
• Analysis of mRNAs By Microarray or RNA-Seq Provides a Snapshot of Gene Expression
- ex: embryonic vs adult
• In Situ Hybridization Can Reveal When and
Where a Gene Is Expressed
• ReporterGenesAllowSpecificProteinstobe
Tracked in Living Cells
Sanger Method
Produces 4 Sets of Labeled DNA Molecules
To determine complete sequence of a single-stranded DNA fragment:
1. DNA hybridized with short DNA primer labeled with fluorescent dye or radioisotope
2. DNA polymerase and excess of all 4 normal deoxyriobonucleoside triphosphates (A, C, T, G) are added to primed DNA, which is then divided into 4 reaction tubes
3. Each tube receives a small amount of a single chain-terminating dideoxyribonucleoside triphosphate (A, C, T, G)
4. Because chain-terminating ddNTPs will be incorporated only occasionally, each reaction produces a set of DNA copies that terminate at different points in the sequence
5. Products of these 4 reactions are separated by gel electrophoresis in four parallel lanes of a polyacrylamide gel (A, T, C, G)
6. In each lane, bands represent fragments that have terminated at a given nucleotide, but at different positions in the DNA
7. By reading off the bands in order, starting at the bottom of the gel and reading across all lanes, the DNA sequence of the newly synthesized strand can be determined
8. The sequence, which reads 5' -> 3' bottom to top, is complementary to the sequence of the original single-stranded DNA fragment sequenced
5' GCAT (primer) ATGTCAGTCCAG 3'
3' CGTA TACAGTCAGGTC 5' (sequence of original DNA strand)
Fully automated machines run Sanger sequencing reactions
Uses excess amount of normal dNTPs + mixture of four different chain-terminating ddNTPs, each of which is labeled with a fluorescent tag of a different color
- reaction products loaded onto a long, thin capillary gel and separated by electrophoresis
- camera reads color of each band on gel and feeds data to computer that assembles sequence (size-separated products)
- each colored peak = a nucleotide in DNA sequence
Shotgun Sequencing
Preferred method for smaller genomes
Most straightforward approach to sequencing a genome
- 2-3 genomic libraries (short, medium, and long fragments) made by shearing DNA from entire genome into fragments
- clones selected at random from each library and sequenced (at ends of fragments)
- software used to assemble long stretches of sequence from overlapping short fragments, using sequences from larger clones as a framework
Clone-by-clone approach to shotgun sequencing
1. Break genome into overlapping 100-200 kb pair fragments
2. Plug segments into BACs and insert them into E. coli (BACs are similar to bacterial plasmids but carry larger pieces of DNA)
3. As bacteria divide, they copy BACs, producing a collection of overlapping cloned fragments
4. Determine where each DNA fragment fits into existing map of genome by using different restriction nucleases to cut each clone to generate a unique restriction-site "signature"
5. Locations of restriction sites in each fragment allow to map each BAC clone onto a restriction map of a whole genome generated previously using same set of restriction nucleases
6. Select 30,000 BACs knowing relative positions of cloned fragments, shear each BAC into smaller fragments, and determine nucleotide sequence of each BAC separately using shotgun method
7. Assemble whole genome by stitching together sequences of thousands of individual BACs that span the length of the genome
- Reduced likelihood that regions containing repetitive sequences would be assembled incorrectly
- Eliminated possibility that sequences from different chromosomes will be mistakenly joined
second-generation sequencing methods
allow multiple genomes to be sequenced in parallel
- most rely on PCR amplification of a random collection of DNA fragments attached to a solid support (glass slide or microwell plate)
- for each fragment, amplification generates a 'cluster' with 1000 copies of individual DNA fragment
- tens of millions on a single slide/plate are sequenced simultaneously
how do 2nd gen seq methods work?

PCR-amplified DNA!

- DNA allocated to slide/plate
- molecule you want sequenced in excess copies
- add primer, polymerase - terminator NTPs, nucleotide - for marker- ex: Adenine (A) fluorescently labeled
- do over and over and look @ snapshots on place
- use computer to see where they are
third-generation sequencing methods
permit sequencing of just a single molecule of DNA
- each DNA molecule pulled through tiny channel
- bc each of 4 nucleotides has a different characteristic shape, the way a nucleotide obstructs the pore as it passes through reveals its identity
- info then used to compile sequence of DNA molecule
- requires NO AMPLIFICATION OR CHEMICAL LABELING
- obtain complete human genome in hours!
RNA-Seq
- RNAs are converted to cDNAs, which are then sequenced by second-generation sequencing methods
- more direct approach for cataloging RNAs produced by a cell
- provides a more quantitative analysis of the transcriptome
- determines number of times a particular sequence appears in a sample
- detects rare mRNAs, RNA transcripts that are alternatively spliced, mRNAs that harbor sequence variations, and noncoding RNAs
- replacing microarrays as the method of choice for analyzing the transcriptome
transcriptome
complete collection of RNAs produced by a cell under certain set of condtions
In Situ Hybridization
Allows a specific nucleic acid sequence - either DNA or RNA - to be visualized in its normal location to see where a particular RNA is made
- reveals exactly where in the cell or tissue mRNAs are produced
- Uses single-stranded DNA or RNA probes, labeled with either fluorescent dyes or radioactive isotopes, to detect complementary nucleic acid sequences within a tissue, cell, or isolated chromosome
- used to study expression patterns of a gene/collection of genes in an adult or developing tissue
GFP fusion
standard strategy for tracking location and movement of specific proteins in living cells
- use of multiple GFP variants that fluoresce at different wavelengths can provide insights into how different cells interact in a living tissue
reporter gene
Map the location of specific proteins in unfixed, living cells through recombinant DNA techniques fusing the gene of the reporter molecule (GFP) to the gene of the protein of interest (i.e. Drosophila protein in neurons)
- Use regulatory DNA sequences of protein-coding gene to drive expression of reporter gene that encodes a protein easily monitored by fluorescence/ enzymatic activity-- mimics expression of gene of introduced, producing reporter protein when, where, and in same amt as normal protein made
- same approach used to study regulatory DNA sequences that control gene's expression
- location monitored by fluorescence microscopy
- alternate to using Ab to visualize a protein
- for a gene that encodes a protein, location of protein within cell/tissue/organism gives clues to gene's function
BAC Clones
Get Ordered on the Physical Map on the Basis of Their Restriction Site Signatures
- cleavage sites for restriction nucleases A-E
- restriction pattern for individual BAC clones - aligned- important for gene mapping via chromosome walking!
Visualizing A Portion of the Human Genome
Macro Metaphase Chromosome Level vs Micro DNA Base Sequence Level
Comparative Genomic Hybridization
Huge advance in looking @ tumor DNA!
- label each w diff flurochrome
- hybridization with excess Cot 1 DNA to prevent binding repetitive sequences - mix w Cot 1 DNA - binds all rep DNA seq's
- conventional CGH-> metaphase chromosome spread-> position on chromsome
- vs. cDNA microarray->fluorescence image->position on sequence
- exons from normal DNA- compare to genes of tumor DNA- see how quantitatively different!
- in conventional genomic hybridized, hybridized to 2 metaphase chroms
- look @ intensity of seq's as they hybridize!
- normal = green, tumor = red
see where they are different!
microarrays - array plates
- done w/ exons
- can test/look for down/upregulated, or under/overexpression
CGH vs. SKY
CGH detects only unbalanced chromosomal changes
SKY can detect complex rearrangements, translocations etc.
- comparative genomic hybridization-
- good for seeing large gains or losses of genomic material!
good @ detecting unbalanced chrom changes
- generate chromosome profile
SKY
label 24 flow-sorted chroms using flow cytometer- isolate diff chroms by size
- clone and make DNA libs
- chrom painting probes
- Cot DNA- eliminates repetitive seq's
- unique chrom painting probes
- through img analysis, asign a color to each chrom
FISH Use in Nuclear Organization Studies
chrom organization in interphase nucleus
-ex: human fibroblast nucleus

- 24 color 3D-FISH Representation and Classification of Chromosomes in a Human G0 Fibroblast Nucleus
- through confocal imaging- 3D reconstruction
Comparing GENOMES: DNA TO DNA!
FISH
ISH can be used to locate genes on isolated chromosomes
- mark an entire library of genes as in chromosome painting or locate individual gene loci with both chromosome 5 homologues showing two gene signals:
- 6 different DNA probes used to mark locations of their nucleotide sequences on chromosome isolated from mitotic cell in metaphase
- Probes labeled with different chemical groups, and detected using fluorescent Abs specific for those groups
- Maternal/paternal copies aligned
- Each probe produces 2 dots on each chromosome because chromosomes undergoing mitosis already replicated their DNA - each chromosome has 2 identical DNA helices (FISH)
- ex: Simultaneous detection of expression of 5 genes in Drosophila embryo by FISH with 5 RNA probes, FISH of HPV DNA Sequences Detected in Infected Epithelial Cells, FISH generated gene expression map of mouse brain - GFPs that fluoresce at different wavelengths help reveal connections that individual neurons make in the brain- neurons randomly express diff combos of diff colored GFPs, so can distinguish/trace individual neurons
2 common reporter genes
1. B-galactosidase
2. GFP
How to Use Reporter Genes to Investigate Gene Expression
Constructing a Reporter Gene:
1. Using recombinant DNA techniques, replace coding sequence for protein X (of certain cell type) with that of reporter gene Y (easily monitored visually)
2. Expression of reporter gene Y will now be controlled by the regulatory DNA sequences that determine expression of X
- coding sequence for protein X shows expression pattern of gene X on normal gene, and same for Y on recombinant reporter gene (both express B, E, F)
Using a Reporter Gene to Study Gene X Regulatory Sequences:
3. To determine which regulatory sequences normally control expression of X in particular cell types, reporters with various combinations of regulatory regions associated with gene X can be constructed
- these recombinant DNA molecules are then tested for expression after their introduction into different cell types
Conclusions:
- regulatory sequence 3 turns on gene X in cell B, 2 turns on gene X in cells D, E, F, 1 turns OFF gene X in cell D (why it isn't expressed in normal or recombinant reporter genes) - and turns off all after that, so NOTHING is expressed!
mutant organisms
differ in phenotype
- accelerate process by radiation/chemical mutagens - randomly disrupt gene activity
- generates large numbers of mutants - each can be studied individually - "classical genetic approach"
- most applicable to organisms that reproduce rapidly and can be analyzed genetically (bacteria, yeast, nematodes, fruit flies (zebrafish, mice))
RNA interference (RNAi)
silencing of gene expression after transcription, triggered by double-stranded RNA homologous to portions of the gene. exploits natural mechanism used in plants/animals to protect selves against viruses and proliferation of mobile genetic elements
- Due to antisense RNA- complementary to specific mRNA (bind)
- gene silencing results from cleavage and degradation of a target gene's mRNA, but can also result from blocking translation of intact mRNA - potential for treating disease
- RNA molecules that silence genes are short (20 bp)
- inhibit activity of specific genes- important for regulating gene expression - guides embryo development by turning down specif genes at critical times
- injecting dsRNA turns down exp of genes with matching nucleotide sequence - critical for gene silencing
reverse genetics
gene of known sequence inactivated and effects on cell or organism's phenotype observed
forward genetics
begin with randomly generated mutant and identify responsible gene
Green Fluorescent Protein (GFP)
Use Allows Genes to Be Tracked and Located in Living Cells
RNA Silencing by Small Noncoding RNAs
Utilize short 21-23 nucleotide RNAs that can target an mRNA for translational silencing or mRNA decay
Limitations of RNAi
Non-target genes sometimes inhibited along with gene of interest
- certain cell types resistant to RNAi
- often temporary - "gene knockdown"
Two classes of small noncoding RNAs that can silence gene expression
microRNAs (miRNA) and small interfering RNAs (siRNA)
miRNA silences gene expression
short RNA sequences not perfectly complementary to target mRNA - result in the translational silencing / decay of mRNA
siRNA silences gene expression
perfectly complementary to the target mRNA - triggers cleavage of the mRNA target
How are siRNA and miRNA are processed?
dsRNA is cleaved into 21-23 nt fragments by Dicer
The RNA is assembled into a complex referred to as RISC
(RNA- Induced Silencing Complex)
Depending on the complementarity/homology of the short RNA to the target mRNA, it either results in the degradation of the mRNA or translational silencing of the sequence/gene (mRNA)
Net outcome:
protein/gene from the target mRNA is not produced.
There may be small residual activity, so this procedure can also be called GENE KNOCKDOWN (vs Knockout)
RNA Silencing by Small Noncoding RNAs Process
1. precursor miRNA in nucleus (with polyA tail) processed and exported to cytoplasm
2. formation of RISC on ss-miRNA (3'->5') by RISC proteins
3. Search for complementary target mRNA
a. extensive match: RISC released and mRNA rapidly degraded
b. less extensive match: translation reduced, mRNA sequestered and eventually degraded
GMOs/transgenic organisms
- coding sequence of a cloned gene mutated to change functional properties of its protein product, or regulatory region changed so amount of protein made altered or gene expressed in a different type of cell or at a different time during development
- altered gene is re-introduced back into original organism to produce mutant - studied to determine gene's function - inserted into genome of reproductive cells so it can be stably inherited by subsequent generations
- ideal: normal gene replaced by altered one - analyze function of mutant in absence of normal protein
how to find particular gene within DNA/genomic library
Use labeled DNA probe to bind specifically to part of the gene's DNA sequence
- identify rare bacterial clones in DNA library that contain the gene (or portion of it) by hybdirization
how to design a probe to detect a gene
determine part of protein's AA sequence
- use to deduce corresponding gene sequence
- generate DNA probe
DNA Cloning Steps
1. restriction endonuclease cleaves plasmid DNA @ specific sites - produces sticky ends- hybridize w any DNA also cut w that nuclease
2. Foreign DNA with sequence want to clone is digested with nuclease and then mixed with cleaved vector
3. Sticky ends of foreign/plasmid DNA hybridize, sealed with DNA ligase - forms recombinant plasmid - each has fragment, ampR, and ori
4. E. coli added to recombinant plasmids
- treated w calcium chloride - a few uptake recombinant plasmid by transformation (most don't)
5. On agar, only cells resistant can grow
6. At 37C, transformed grow and multiply - each form separate colony. Nontransformed die
7. Rep origin allows plasmid to repilcate using host cell's enzymes
- plasmid rep indep of host cell division, but plasmid distributed to each daughter cell when host divides
8. as plasmids rep and host multiply, # of copies increases
- daughter cells form colony/clone
- all contain copies of same recomb plasmid w fragment of foreign DNA
- assay used to determine which has DNA seq want to isolate
sanger sequencing steps
1. DNA separated into two strands.
2. The strand to be sequenced is copied using chemically altered bases.
3. These altered bases cause the copying process to stop each time one particular letter is incorporated into the growing DNA chain.
4. This process is carried out for all four bases, and then the fragments are put together like a jigsaw to reveal the sequence of the original piece of DNA.
2 ways to introduce RNAi
Technique:
1. introduce dsRNA with nucleotide sequence that matches gene to be inactivated
2. dsRNA cleaved/processed by special RNAi machinery to produce shorter, ds fragments and direct their degradation - sometimes same fragments can direct production of more siRNAs allowing continued inactivation of target mRNAs
- frequently used to inactivate genes in cultured mammalian cell lines, Drosophila, and nematode C. elegans
- test gene function - dsRNA introduced into C. elegans by:
1. feeding worms E. coli that express dsRNAs that trigger RNAi - these RNAs get converted into siRNAs, which get distributed throughout body to inhibit expression of the target gene in various tissue
2. injecting dsRNA into gut
- in an embryo in which a gene has been silenced by RNAi, the pronuclei (egg and sperm that fuse after fertilization) fail to migrate and come together - showed function of gene in embryonic development
- for organisms whose genomes have been completely sequenced, RNAi can be used to explore function of any gene
(large collections of DNA vectors that produce these dsRNA are available)
RNA-dependent RNA polymerase
produces dsRNA when triggered by high levels of mRNA
- creates dsRNA that shut down gene expression when acting on abundant mRNA
how RNAi works
- post-transcriptional but pre-translational
- presence of dsRNA in cell is rare
- RNAi process begins with presence of long double-stranded RNA molecule
- Dicer enzyme recognizes and cuts long dsRNA into short 21-25bp molecules (siRNAs) using ATP hydrolysis - acts as endonuclease
- siRNAs bind to several proteins and form RNA-induced silencing complex (RISC)- activated when ds-siRNA is unzipped (requires ATP hydrolysis)
- Once activated, RISC can recognize and then bind to target mRNA- Once bound, subunits of RISC cleave mRNA - shuts off gene. Other proteins further degrade mRNA, preventing protein production
1. longer fragment of RNA in nucleus processed by Drosha enzyme
2. miRNA fragment brought into cytoplasm
3. goes thru Dicer enzyme
4. inhibition of protein formation from mRNA
map-based sequencing
1. Chromosomes are fragmented into large pieces (100,000-200,000 bp).
2. Fragments are cloned into vectors that can accomodate large DNA sizes (BACs)
3. Fragments that can hybridize to the DNA markers in the genetic map are ID'd
4. Other fragments ID'd from their ability to hybridize to DNA isolated from the ends of the previously identified fragments
5. One after another, additional overlapping fragments are identified until a contiguous sequence of clones ("contig") is built
7. Direct analysis of contig provides physical map of region - shows size of DNA region, location of restriction sites, and location of new DNA markers within the contig
8. Smaller fragments small enough to be sequenced (0.5-1 kb) are produced from larger, and sequenced individually
9. Computer finds areas of overlap
shotgun sequencing steps
1. Break multiple copies of genome into smaller, overlapping random fragments
2. Sequence each fragment and assemble genome based on overlapping sequences
- Issue: Reassembly process can be derailed by repetitive nucleotide sequences (rare in bacteria, but a large fraction of vertebrate genomes)
-- when sequence assembled incorrectly, intervening info lost
--- To avoid: combined with clone-by-clone approach
3 ways RNAi is made
1. dsRNA might've been product of virus/jumping gene as part of life cycle
2. introduced into cell by researcher
3. integration of gene into chrom next to endogenous gene can cause antisense RNA synthesis
DNA Hybridization/Probe Steps
1. dsDNA denatured to ssDNA @ high temp, anneal at lower temp (bc of base pairing of complementary strands)
3. If similar nucleotide sequences, complementary strands from different sources will ALSO anneal
* Hybridization = each DNA from different source!!*
4. Regions of dsDNA in which ssDNA strands hybridize to one another are homologous (have similar identical nt sequences)
5. High specificity of base pairing can be used to locate a specific nt sequence in a sample
6. If DNA from one source is immobilized by attachment to a solid surface (i.e. nitrocellulose), homologous DNA from another source will hybridize
- Basis for DNA probe techniques
7. Non-homologous sequences do not bind