Gene Expression

Gene expression All cells in one organism have the same DNA. But different cells have very different functions. In each cell at certain times only some genes are expressed. Which genes are expressed at which times?

Cells muscle nerve

Double-stranded DNA

DNA Structure

DNA matching Every A forms two weak hydrogen bonds with T. Every T forms two hydrogen bonds with A. Every C forms three weak hydrogen bonds with G. Every G forms three hydrogen bonds with C.

RNA RNA is also a sequence of nucleotides. RNA means “ribonucleic acid”. DNA means “deoxyribonucleic acid”.

Nucleotides

RNA

DNA Structure

DNA vs RNA Both are strings of nucleotides. DNA is usually double-stranded; RNA is single-stranded. RNA is usually much shorter than DNA. RNA replaces each T by U (uracil). DNA contains deoxyribose while RNA contains ribose. This makes DNA more stable chemically than RNA.

DNA and RNA DNA in your cells is in the nucleus; RNA can be anywhere in the cell. Proteins are made directly using RNA, not DNA.

Central Dogma A protein-coding region of DNA is copied to messenger RNA (mRNA) by transcription. The mRNA leaves the nucleus and goes to a ribosome. The ribosome uses the mRNA to make a protein by translation.

Central Dogma

Translating codons Ala/AGCT, GCC, GCA, GCGLeu/LTTA, TTG, CTT, CTC, CTA, CTG Arg/RCGT, CGC, CGA, CGG, AGA, AGGLys/KAAA, AAG Asn/NAAT, AACMet/MATG Asp/DGAT, GACPhe/FTTT, TTC Cys/CTGT, TGCPro/PCCT, CCC, CCA, CCG Gln/QCAA, CAGSer/STCT, TCC, TCA, TCG, AGT, AGC Glu/EGAA, GAGThr/TACT, ACC, ACA, ACG Gly/GGGT, GGC, GGA, GGGTrp/WTGG His/HCAT, CACTyr/YTAT, TAC Ile/IATT, ATC, ATAVal/VGTT, GTC, GTA, GTG STARTATGSTOPTAG, TGA, TAA

Protein primary structure

3D views of proteins

DNA for beta hemoglobin ATGGTGCATCTGACTCCTGAGGAGAAGTCTGCCGTTACTGCCCTG TGGGGCAAGGTGAACGTGGATGAAGTTGGTGGTGAGGCCCTGGG CAGGCTGCTGGTGGTCTACCCTTGGACCCAGAGGTTCTTTGAGTC CTTTGGGGATCTGTCCACTCCTGATGCTGTTATGGGCAACCCTAA GGTGAAGGCTCATGGCAAGAAAGTGCTCGGTGCCTTTAGTGATGG CCTGGCTCACCTGGACAACCTCAAGGGCACCTTTGCCACACTGAG TGAGCTGCACTGTGACAAGCTGCACGTGGATCCTGAGAACTTCAG GCTCCTGGGCAACGTGCTGGTCTGTGTGCTGGCCCATCACTTTGG CAAAGAATTCACCCCACCAGTGCAGGCTGCCTATCAGAAAGTGGT GGCTGGTGTGGCTAATGCCCTGGCCCACAAGTATCACTAA

Primary structure for beta hemoglobin MVHLTPEEKSAVTALWGKVNVDEVGG EALGRLLVVYWTQRFFESFGDLSTPD AVMGNPKVKAHGKKVLGAFSDGLAHL DNLKGTFATLSELHCDKLHVDPENFRL LGNVLVCVLAHHFGKEFTPPVQAAYQ KVVAGVANALAHKYH

Part of the two strands for beta hemoglobin ATGGTGCATCTGACTCCT… TACCACGTAGACTGAGGA… The top is the sense or template; the bottom is the antisense or coding strand.

Transcription: Make mRNA ATGGTGCATCTGACTCCT… sense TACCACGTAGACTGAGGA… coding AUGGUGCAUCUGACUCCU… mRNA

Structure of mRNA

mRNA goes to a ribosome, outside the nucleus AUGGUGCAUCUGACUCCU… mRNA

Eukaryotic cell (1) nucleolus (2) nucleus (3) ribosomes (little dots) (5) rough endoplasmic reticulum (ER) (9) mitochondria (10) vacuole (11) cytoplasm

Ribosomes The ribosome functions as a factory to make proteins. It uses two kinds of input: (a) mRNA (b) tRNA It outputs a protein.

Ribosome translates mRNA Ribosome (2) straddles mRNA (1) It makes the protein (3). It starts at AUG and ends at UAG

Ribosome large subunit

Transfer RNA (tRNA) Each tRNA molecule has on one side a conformation that binds to the specific codon and on the other side a conformation that binds to the corresponding amino acid.

tRNA CCA tail in orange, Acceptor stem in purple, D arm in red, Anticodon arm in blue with Anticodon in black, T arm in green.

tRNA carries the amino acid matched to the codon UAC … M tRNA will bind with the codon AUG in the mRNA. CAC … V tRNA will bind with the codon GUG in the mRNA.

mRNA in a ribosome has the genetic information AUGGUGCAUCUGACUCCU… UAC … M tRNA will bind with the codon AUG. CAC … V tRNA will bind with the codon GUG.

Translating codons Ala/AGCT, GCC, GCA, GCGLeu/LTTA, TTG, CTT, CTC, CTA, CTG Arg/RCGT, CGC, CGA, CGG, AGA, AGGLys/KAAA, AAG Asn/NAAT, AACMet/MATG Asp/DGAT, GACPhe/FTTT, TTC Cys/CTGT, TGCPro/PCCT, CCC, CCA, CCG Gln/QCAA, CAGSer/STCT, TCC, TCA, TCG, AGT, AGC Glu/EGAA, GAGThr/TACT, ACC, ACA, ACG Gly/GGGT, GGC, GGA, GGGTrp/WTGG His/HCAT, CACTyr/YTAT, TAC Ile/IATT, ATC, ATAVal/VGTT, GTC, GTA, GTG STARTATGSTOPTAG, TGA, TAA

mRNA goes to a ribosome AUGGUGCAUCUGACUCCU… mRNA UAC …. M tRNA CAC … V tRNA The ribosome matches UAC on tRNA with AUG on mRNA, then uses the M on the other end in the protein.

mRNA goes to a ribosome AUGGUGCAUCUGACUCCU… mRNA UAC …. M tRNA CAC … V tRNA The ribosome matches CAC on tRNA with GUG on mRNA, then uses the V on the other end to extend the protein.

Ribosome In this manner, the ribosome continues to make the protein until it reaches a STOP codon.

When is a given gene being expressed? A given protein is being made when its mRNA is present in the cell. The DNA is always present.

When is a given gene being expressed? To tell what is being expressed at a given time in a given cell, find out which mRNAs are present. For each kind of mRNA, measure the quantity present.

A microarray

Microarrays A microarray consists of a pattern of thousands of features. Each feature has some DNA that will probe and possibly bind with an mRNA sample. Typically the feature is made to fluoresce under the presence of binding mRNA. The brightness of the dot corresponds to the quantity of mRNA of the given sort that is present.

Two gene chips

Microarrays Typically the probe is attached to a solid surface which is a glass or silicon chip. It is then called a gene chip or Affymetrix microarray.

Introns Introns are inserts in the DNA within portions that code for one protein. The parts that code are exons.

Introns must be removed to make the mature mRNA

cDNA Complementary DNA (cDNA) is DNA synthesized from mature mRNA using reverse transcriptase. AUGGUGCAUCUG mRNA TACCACGTAGAC cDNA

cDNA cDNA is more stable than RNA. cDNA corresponds with the part of the genome from which introns have been removed. cDNA does not correspond exactly to nuclear DNA.

The mature mRNA

The probes Each dot can contain DNA, cDNA, or an oligonucleotide (oligo). An oligonucleotide is a short fragment of single-stranded DNA, typically 5 to 50 nucleotides long.

Gene expression profiling In an mRNA or gene expression profiling experiment the expression levels of thousands of genes are monitored simultaneously in parallel. This can be used to distinguish (a) the effects of certain treatments (b) the effects of diseases (c) the effects of different stages of development.

Gene expression profiling For example, microarrays can identify genes whose expression is changed in response to pathogens by comparing gene expression in infected cells to that in uninfected cells.

A microarray experiment Suppose there are two cells--type 1, healthy, and type 2, diseased. Both have four genes A, B, C, D. We want to compare the expression of these genes in the two types of cell.

Procedure 1. Prepare the DNA chip using the chosen target DNAs. 2. From the cells, isolate the mRNA. 3. Use the mRNA as templates to generate cDNA with a fluorescent tag attached. Typically a green fluorescent tag is used for mRNA from healthy cells, while a red tag is used for mRNA from diseased cells.

Procedure 4. Prepare a hybridization solution with a mixture of the fluorescently labeled cDNAs. 5. Incubate the hybridization solution with the DNA chip. 6. Detect bound cDNA using laser technology. 7. Analyze the data.

Appearance afterwards

Interpreting colors A spot with just healthy cDNA is green. A spot with just diseased cDNA is red. A spot with both is yellow. A spot with neither is black.

Comparison of cells Microarrays are used to compare the genome content in different cells for the same organism.

Single Nucleotide Polymorphisms A single nucleotide polymorphism is a single substitution in the genome. Example: AUGGUGCAUCUGACUCCU standard AUGGUGUAUCUGACUCCU SNP

Detecting SNPs Microarrays can be used to detect SNPs between or within populations. This can measure predisposition to diseases or identify appropriate drugs.

How are chips made? In spotted microarrays the probes may be small fragments of DNA. An array of fine needles is controlled by a robotic arm that is dipped into wells containing the DNA probes. Each needle then deposits a probe at the desired location on the surface. The probes are fixed to the surface. Then the chip is ready to be washed in a solution containing the targets.

DNA microarray being printed by a robot

Flexibility of microarrays Thus scientists can produce arrays from their own labs, customized to an experiment.

Bioinformatics problems 1. How long should the probes be (i.e., how many nucleotides long)? If too short, you get false signals. If too long, it is expensive.

Bioinformatics problems 2. Which parts of a sequence should be cloned in the probe?

DNA for beta hemoglobin ATGGTGCATCTGACTCCTGAGGAGAAGTCTGCCGTTACTGCCCTG TGGGGCAAGGTGAACGTGGATGAAGTTGGTGGTGAGGCCCTGGG CAGGCTGCTGGTGGTCTACCCTTGGACCCAGAGGTTCTTTGAGTC CTTTGGGGATCTGTCCACTCCTGATGCTGTTATGGGCAACCCTAA GGTGAAGGCTCATGGCAAGAAAGTGCTCGGTGCCTTTAGTGATGG CCTGGCTCACCTGGACAACCTCAAGGGCACCTTTGCCACACTGAG TGAGCTGCACTGTGACAAGCTGCACGTGGATCCTGAGAACTTCAG GCTCCTGGGCAACGTGCTGGTCTGTGTGCTGGCCCATCACTTTGG CAAAGAATTCACCCCACCAGTGCAGGCTGCCTATCAGAAAGTGGT GGCTGGTGTGGCTAATGCCCTGGCCCACAAGTATCACTAA

Statistical issues in microarrays 1. There is variability in how well each probe in the microarray was made. 2. There is variability in how uniformly the target got washed across the chip. 3. There is variability in how accurately the probe binds with the target.

Statistical questions What level of expression is statistically significant? If there are 20,000 probes, a 95% confidence means there are ? events with probability less than 5%.

Statistical questions What level of expression is statistically significant? If there are 20,000 probes, a 95% confidence means there are 1000 events with probability less than 5%.

Statistical issues How can the data be normalized (ie, compared with known probability distributions, like the normal curve)? P values: there will be false positives and false negatives.

Experimental design issues Replication of biological samples Replication of RNA samples from each experiment Replicate each spot on the microarray

Data warehousing The data bases are huge hence hard to understand.