Biol 1103 Test 2

Another word for Epigenetics:

Chemical Modifications of DNA

Relationship between DNA and Proteins:

DNA encodes for proteins, and protein enzymes replicate and maintain DNA.

Genomes

Genome: complete set of chromosomes in a cell or an organism (contains all genes). Knowing the order of genome is just part of the story We also must understand how an organism interprets its genome and uses that info to build itself In the human genome: about 20,000 genes are required. They are encoded by less than 2% of our genome. Sequence of a genome: like a simple map. High traffic areas: places where many genes are turned on by proteins (enzymes) binding to DNA. -these areas call for multiple layers of regulation Basic genome sequence has stop signs and start signs (flags) added, and these additions direct the traffic of proteins that bind to DNA 3D Satellite Map: we can see what proteins bind to what DNA sequences in a single cell of a Genome The diff patterns of "on" and "off" genes help produce the diff types of cells that make you, you (ex. skin cells, blood cells, etc) EPIGENOMIC MODIFICATIONS: some of the stop and start signals that direct our cells to translate the map of our genomes -Changes in chem groups that are added/subtracted from DNA base or proteins that bind to DNA. These modifications change the way genes are read. -These modifications can be passed on as cells divide, even though they are not strictly changes to the DNA itself. -ex. Methylation: as human develops, some early cells start to become brain cells. Specific small chemicals (called methyl groups) are added onto the DNA in front of genes that WONT be needed for brain cells (stop signs). Genes that aren't needed for the brain cell stay turned off. Methyl groups are not changes in the sequence of DNA bases, but modifications like this can ACT like mutations by directing if the bases are read or not. ex. In cancer cells, methyl groups are added at inappropriate places in the genome. Cells grow out of control because stop signs have been added in front off genes that would have kept the cell in check. Some cancer therapies: drugs to stop methyl groups from being added where they shouldn't. ex. Azacitidine: used in blood cancer. Last 15 years: Human Genome Project Dutch Hunger Winter: southing was passing down through generations, but NOT changes in the DNA itself Methylation Patterns in the DNA were altered in these children, changing health outcomes Diet directly affects methylation, because NUTRIENTS IN OUR FOOD provide the methyl groups themselves. So, the diet of either parent can affect their children and grandchildren. Smoking can also change methylation patterns And drinking alcohol during pregnancy can alter Epigenomic patterns. These modifications to our DNA play a central role in how the genome carries its work. But Epigenomics aren't the only controls we've learned about through Human Genome Project. What the rest of the Genome Does: Only 2% of the genome's bases are used to encode proteins. What is the rest doing? Some of the genome's bases help determine its structure. ex. Telomeres: the ends of each chromosome. -They keep the ends of your chromosome from fraying (unraveling). Some other parts of the genome POSITION THE GENES IN THE RIGHT PLACES TO BE READ BY THE CELL'S MACHINERY Dark Matter/Junk DNA: the noncoding 98% of the DNA in a genome that is noncoding! -Some parts called "ultra-conserved elements:" have kept the exactas same epigenomic sequence for millions of years in mammals even though they don't contain genes; because, deleting these elements would cause brain changes, and could lead to neurological diseases like Alzheimer's. Up to 45% of your genome is leftover DNA from viruses that infected mammals over millions of years. -That old viral DNA contributes to the exchange of nutrients between mother and baby through the PLACENTA (part of the uterus that nourishes baby through umbilical cord) The rest of the genome includes: REGULATORY ELEMENTS, including areas that INFLUENCE GENES TO BE EXPRESSED at the same time in response to the environment. TRANSCRIBED SEQUENCES: cell reads these but does not use them for making protein And material which ACTS AS A HOTSPOT FOR YOUR CHROMOSOMES TO EXCHANGE INFO WITH EACH OTHER. Video Notes: Epigenetics: differences in traits that aren't due to changes in DNA sequence Just after conception, an embryo's chemical flags are erased. Cells destined to become sperm and eggs get erased a second time, making them PLURIPOTENT, so they can make all the cell types in a new embryo (blank slate). So how is inheritance of our parents' acquired traits possible? Because SOME flags (on/off, stop/go, epigenomic modifications) sneak through. In Epigenetics, DNA interacts with smaller molecules within cells, which can activate and de-activate genes. Proteins help determine a cell's characteristics and functions. Epigenetic changes can boost or interfere with transcription. -DNA (or proteins that wrap around it) get labeled with chemical tags. AN EPIGENOME: All of the chem tags attached to the genome of a cell -ex. Methyl group (a tag) inhibits gene expression by causing DNA to coil more tightly, so now, the gene is silent. Some tags boost transcription and translation by uncoiling DNA Epigenetic changes can survive cell division, so can stay with someone for life All cell types in our body (about 200. ex. liver, blood) have same genome, but its own distinct epigenome!!!! Epigenome mediates the dialogue between environment and gene -ex. external factors cause tags to turn genes on/off part of why identical twins can grow up to have diff lives. as twins get older, their epigenomes diverge, affecting how they age, susceptibility to disease, etc. Most epigenetic marks/flags are erased when egg and sperm cells are framed -but some imprints survive, pass down. Epigenetic changes are sticky but not always permanent!

The "teeth holding together 2 strands of the double helix" are the:

Hydrogen bonded Nitrogen-containing bases.

One way that researchers are identifying cancerous cells is to detect the degree of methyl groups that are present on several cancer causing genes. One gene, RSSF2, appears to be a tumor suppressor and produces a protein that is involved in stopping the cell from dividing. Doctors would expect to see.......... levels of the RSSF2 protein in these abnormally dividing ovarian tumor cells because DNA for the gene had a(n) ......... in methyl groups.

Lower. Increase

Define regulatory proteins and explain their role in adding tags to the epigenome.

PROTEINS CARRY SIGNALS TO THE DNA:Ex. your body is exposed to stress, so cortisol protein goes and signals to the DNA.Once a signal reaches a cell, proteins carry information inside. Like runners in a relay race, proteins pass info to one another.The information is ultimately passed to a gene regulatory PROTEIN that attaches to a specific sequence of letters on the DNA. Once there, it acts like a switch: activates gene transcription or shuts down (methylates) the gene).Epigenetic Tags Added in Response:Gene regulatory proteins recruit enzymes that add or remove EPIGENETIC TAGS. (ex. Enzymes that tag the gene with Methyl groups).

Later in life... Environmental signals such as diet and stress can trigger changes in gene expression. Epigenetic flexibility is also important for

Shaping new memories

All of the following are true about DNA methylation, EXCEPT:

When it occurs, transcription is more likely to occur

Different cell types, for example a muscle or a blood cell in the same organism retain ___.

a complete set of their genes, and retain the ability to express those genes under certain circumstances into mRNA

Gene Regulation

ability of an organism to control which genes turn on and off (regulates how much protein is made).

Acetyl groups are important in modifying histones that wind up DNA. The following table compares a normal cell to a cell treated with the chemotherapy drug Vorinostat. Normal Cell: low Acetylation tightly wound Cell treated with Voriostat: high Acetylation unwound You would predict that treatment with Vorinostat would ____ from genes like tumor- suppressors that would be useful to stopping cell division.

cause an increase in expression of proteins

Of these nucleotides, which ones resemble each other in shape and size?

A and G. C and T.

Genome vs Epigenome

All cell types in our body (about 200. ex. liver, blood) have same genome, but its own distinct epigenome!!!! Epigenome mediates the dialogue between environment and gene

EPIGENOME

All of the chem tags attached to the genome of a cell

Researchers collected mRNA transcripts from rapidly-dividing cancerous breast cells and from normal breast cells. Based on the function of the proteins made from these genes, which gene would be most likely to have higher levels of mRNA expression in breast cancer cells compared to normal cells?

CDCA3

Cancer and Microarrays Lecture:

Cancer and Microarrays: Muscle Cells have specific genes turned on to produce Actin Protein and Myosin Protein (Muscle Proteins). TURNED ON: TRANSCRIPTION. (ex. Microarrays only look at whether mRNA is produced, NOT whether protein is produced). Each spot on the chip contains unique strands of DNA from a different gene. DNA in each spot only sticks to complement. Each spot originally contains unique single stranded DNA sequences from every different gene expressed in a cell. Detects and attaches/base pairs/hybridizes to complement strands (A to T, C to G), when we pour cDNA onto the microarray. Reverse Transcriptase: enzyme that converts RNA into cDNA

Assume you are trying to use a DNA microarray to compare gene expression patterns between normal to cancerous cells. Place the following steps in the order that you would conduct them.

Collect tissue from both cell types. Isolate mRNA from both the cancerous and normal cells. make a fluorescent-labeled DNA copy of the mRNA from the cancerous cells that is red, and a fluorescent DNA copy of the mRNA from the normal cells that is green. (hybridization base pairing between cDNA and probe occurs when you make these mixes) apply the fluorescent DNA to the microarray scan the microarray for red, green, and yellow spots

DNA Structure Reading

DNA made up of nucleotides 3 parts of a nucleotide: pentose 5 C sugar, phosphate group, nitrogenous base -4 types of nitrogenous bases in DNA - A and G are double ringed purines. C and T are smaller, single ringed Pyrimidines. Nucleotide is named according to the base it contains. Phosphate group of one nucleotide bonds covalently with the sugar molecule of the next nucleotide. Forms a long polymer of nucleotide monomers. Sugar-Phosphate groups line up in a backbone for each DNA strand, and the nucleotide bases stick out from this backbone. The C atoms of the 5 C sugar are numbered clockwise from the oxygen 1' to 5' (one prime to five prime). The phosphate group is attached to the 5' C of 1 nucleotide and the 3' C of the next nucleotide. Each DNA molecule: 2 strands held together by H bonds between bases Base pairing happens between a purine and pyrimidine A and T connected by 2 H bonds. C and G by 3 H bonds. Diameter of double helix always uniform throughout. RNA is same, but in these nucleotides, the 5 C sugar is ribose, not deoxyribose. -Ribose has a hydroxyl group at the 2' C, unlike deoxyribose, which just has an H atom. RNA nucleotides contain same nitrogenous bases, except U, instead of T. RNA: single strand There is mRNA, tRNA, rRNA (molecules for protein production) DNA can be about 2 meters/6 feet long Prokaruote chromosomes are much simpler than Eukaryote chromosomes -Most prokaryotes have a single circular chromosome found in the part of the cytoplasm called the "nucleoid." DNA in prokaryotes is twisted beyond the double helix in a SUPERCOILING Eukaryotes: consist of a linear DNA molecule DNA is wrapped around proteins known as Histones to form structures called Nucleosomes. This Nucleosome is linked to the next one by a short strand of DNA free of histones. String and beads: NUCLEOSOMES ARE THE BEADS, AND THE SHORT LENGTHS OF DNA BETWEEN THEM IS THE STRING Nucleosomes stack compactly onto each other to form 30-nm-wide fiber The fiber is coiled into a more complex structure: Chromatin At Mitosis metaphase, when chromatin becomes COMPACT CHROMOSOMES, when chromosomes are lined up in center of cell, chromosomes are most compacted In interphase, chromosomes are decondensed. Have 2 regions: Darkly staining regions usually contain non-active genes, and are found in regions of the centromere and telomeres (ends of chromosome). Light stain regions usually contain active genes, w/ DNA packaged around nucleosomes but not further compacted (so the DNA strings are "loose"). Chromatin: unraveled. Becomes chromosome at mitosis (cell devision) metaphase, when it becomes condensed.

All of the following are true statements about gene expression, EXCEPT:

During the process of cell division, epigenetic tags such as methyl groups are removed from DNA molecule.

What causes what?

Environment causes genes to be turned on/off!!

Epigenetics describes the effect of on gene expression.

Environment, parental care, diet, etc... ALL OF THE ABOVE.

Inheritance traits by mechanisms not directly involving nucleotide sequence is called:

Epigenetic Inheritance

When looking for color patterns in Microarray, when you compare 2 different types of cancer...

First look for which Tumor Samples are similar. (Which ones have similar color patterns, top to bottom). Then, notice horizontal patterns. Then you will see, the SEVERAL PEOPLE (EX. TUMOR 1, 3, 5) have THIS specific GENE TURNED UP A LOT IN TYPE A CANCER CELLS (RED) OR TYPE B CANCER CELLS (GREEN), OR BOTH TYPES OF CELLS (YELLOW). Vertical Groups group together to form types. (ex. Tumor 1, 3, and 5 have same/similar pattern, so they form Type A).

Gene Regulation: Reading Notes

Gene Regulation Turning a gene on: for transcription and translation Gene Expression: process of turning a gene on to produce RNA and protein Whether a single cell or multicellular organism, each cell controls gene expression So, there must be a mechanism to control when each gene is expressed, when to make/stop making RNA and protein. In multicellular organisms, cells are specialized. Cells in diff tissues look very diff and perform diff functions. ex. skin cell vs liver cell. All cells have basic functions: ex. convert energy in sugar to energy in ATP. Each cell has many genes that are NOT EXPRESSED, and expresses genes that are NOT EXPRESSED BY OTHER CELLS, so it can carry out its specialized functions. Due to environment changes (epigenetics) or organism's development, cells will turn genes on/off Demands from environment also cause unicellular organisms (eukaryotic and prokaryotic) to turn on/off genes as well Malfunctions in gene expression can lead to diseases. ex. cancer. Prokaryotes: Gene expression is regulated at Transcription level!! Lack nucleus Transcription and translation happen almost simultaneously When the protein is no longer needed, transcription stops. So, regulation of TRANSCRIPTION is primary method (mechanism) to control how much protein is expressed, in prokaryotic cells. All next steps happen automatically. When more protein is required, more transcription occurs. In Prokaryotic cells, control of gene expression almost entirely at Transcriptional level. ex. "Iac Operon" is a stretch of DNA with 3 adjacent genes that code for proteins (enzymes) that participate in absorption and metabolism of lactose, a food source for E. Coli. -when lactose isn't present, Iac genes are translated in small amounts -when lactose is present, the genes are transcribed, and the bacterium (E. Coli) uses the lactose as a food source. -Iac Operon has promoter for RNA polymerase to bind to for transcription initiation -between the promoter and the 3 genes is the OPERATOR. When there is NO LACTOSE, REPRESSOR PROTEIN binds to OPERATOR, USUALLY PREVENTS RNA POLYMERASE FROM BINDING TO PROMOTER. -So, very little of the protein of the 3 genes is made. -When lactose is present, an END PRODUCT OF LACTOSE METABOLISM binds to REPRESSOR PROTEIN, keeps it from binding to the OPERATOR -So, RNA POLYMERASE can bind to PROMOTER, and transcribe and translate the 3 genes. Protein enzymes are made to metabolize the lactose. Eukaryotic Cells: Have nucleus Have intracellular organelles (within the cell). Much more complex. DNA in nucleus; is transcribed there. Newly synthesized mRNA processed in nucleus and then transported to cytoplasm for translation Transcription and Translation NOT simultaneous Regulation of gene expression can occur at all stages: -May occur when DNA is uncoiled and loosened from nucleosomes to bind Transcription factors (factors like RNA polymerase, promoter, activator, repressor) (Epigenetic level), when RNA is transcribed (Transcriptional level), when RNA is processed and then transported to cytoplasm (post-transcriptional), when RNA is translated into protein (Translational), or after protein is made (Post Translational). -post-processing: 5' cap, poly-A tail, and excision of introns and splicing of exons. Alternative pre-mRNA Splicing: MECHANISM that allows diff protein products to be produced from one gene, when diff combinations of introns (and sometimes exons) are removed from transcript. -Common mechanism of GENE REGULATION in EUKARYOTES!! 70% of genes in humans are expressed as multiple proteins through alternative splicing. -The cause of many genetic diseases is alternative splicing! NOT mutations in sequence. -BUT, alternative splicing DOES create a protein variant without the loss of the original protein. -Similarly, gene duplication allows genes to evolve without eliminated the original functional protein. While all somatic cells (in body) in an organism contain the same DNA, not all cells within that organism express the same proteins! (so not all cells turn on/off the same genes. That's why skin cells diff from liver cells, etc). Prokaryotic Organisms express the entire DNA they encode in every cell, but not necessarily all at the same time. -Proteins expressed only when they are needed (ex. needed as enzymes for lactose metabolism). -and regulation of expression occurs at TRANSCRIPTIONAL LEVEL Eukaryotic Organisms express a subset of DNA that is encoded in any given cell. (So DNA in all cells is same, but diff types of cells turn on certain genes at certain times). In each cell type, the type and amount of protein is regulated by controlling gene expression. (Controlling when to turn on a gene for transcription and translation REGULATES the amount of protein made). Lactose gene codes for Lactase Enzyme. Common in infants. Lactose Tolerant. After weaning, humans stop producing Lactase protein enzyme - Lactose Intolerant Some people carry mutation that keeps lactase gene switched on past infancy A gene is EXPRESSED when it is transcribed, processed, and translated to produce a protein, and then processed again as a protein Person can digest Lactose when the Lactose gene (LCT) is expressed in the cells of the small intestine! Any step can be regulated to reduce gene expression (because eukaryotic). Transcriptional Regulation: Not transcribing in the first place. Most complete and efficient way to turn off gene expression. Suitable to turn off for a long time. Not efficient to change gene expression rapidly! RNA Processing: Not a major way to regulate gene expression. Translational Regulation: regulatory proteins bind to specific mRNA sequences, prevent attachment to ribosome. -OR, RNA Interference: small pieces of RNA bind to mRNA to trigger degradation/block translation. shut down gene expression. Protein Processing: If active proteins (proteins that have already been made) are no longer needed, they are marked for destruction. In most people with lactose intolerance, LCT gene transcription is reduced a lot, resulting in very low lactase levels. -Repressor binds to Operator to prevent Promoter from binding to RNA Polymerase. Turning off LCT gene: block an activator, or turn up a repressor! Populations in Europe and Africa have mutation near the LCT gene, which causes lactase production to continue in adulthood: Lactase Persistence!! A human evolutionary adaptation to drinking animal milk. -This mutation is found in a DNA region that is an ENHANCER for the LCT gene. -Mutation in ENHANCER REGION: causes activator to bind more strongly to Enhancer region, or repressor to no longer bind to the enhanced region. -One Mechanism: Mutation in North Europe: STRENGTHENS THE BINDING OF A TRANSCRIPTION FACTOR CALLED OCT-1 (AN ACTIVATOR FOR LCT) TO THE ENHANCER REGION. INCREASES TRANSCRIPTION. -Africa mutation uses a diff mechanism. Research ongoing. Promoter binds to RNA Polymerase... Unless... Repressor binds to Operator... Unless... Metabolism end unit binds to repressor, keeping it from binding to Operator... Lactase Production strengthened if... Activator binds strongly to Enhancer region of LCT gene, or Repressor binds less strongly to Enhancer region

Largest to smallest:

Genome, chromosome, gene, nucleotide

Green Fluorescence: No Estrogen (hormone) Red Fluorescence: Estrogen (Hormone) In the microarray shown in the figure above, if you were searching for genes whose expression was inhibited by hormone treatment, you would search for sequences spotted on the array that showed ________ fluorescence.

Green

Queen bees are genetically identical to worker bees, however queen bees have a single gene silenced which impacts expression of numerous traits. The two bee types also differ in their diet as larvae, with individuals fed royal honey developing into queen bees. Based on your understanding of epigenetics, you might expect royal honey to be:

High in methyl compounds.

The Confusing Lecture

How does what your parents ate affect you? (methyl groups are inside the food) Gene Turned On: Active (open) chromatin: A gene that is marked for active transcription: loosely wound with many Acetyl groups (Acetylated Histones). NO METHYL GROUPS AT ALL/UNMETHYLATED. Even though both of these mice carry the yellow agouti allele, it is not expressed in the thin mouse, because methyl groups are used to turn off the gene, keep the DNA for the Augouti gene tightly coiled, and inaccessible for transcription. Mothers of 2 mice have the same Agouti gene, but the mother of the fat mouse was supplemented with some other stuff... When it is expressed, the agouti gene in mice causes yellow fur and obesity. It was discovered that certain compounds, like green leafy vegetables, can affect methylation in the developing mouse embryo. If you observe normal (non-agouti gray) baby mice ONLY after feeding the yellow mother leafy greens during pregnancy, you might predict that leafy greens have AN INCREASING EFFECT ON THE LEVEL OF METHYLATION. Familiar nutrients like folic acid, B vitamins, and SAM-e, are key components of methyl-making pathways. Diets HIGH IN THESE METHYL-DONATING NUTRIENTS CAN RAPIDLY ALTER GENE EXPRESSION (turn gene off), ESPECIALLY DURING EARLY DEVELOPMENT WHEN THE EPIGENOME IS FIRST BEING ESTABLISHED. Many vitamins that we take are specifically used to contain these compounds (help methylate DNA for necessary Gene Silencing). Ex. vitamins in pepper, sunflower seeds Other vitamins contain compounds that acylate DNA (turn on gene, open it up). Ex. vitamins in broccoli. How are methyl and acetyl groups added to DNA? The early embryo is made up of stem cells, which can give rise to any type of cell. Early in development, genes are "poised" like runners in the starting blocks, ready to jump into action. The fetus is made up mostly of differentiated cells. In a differentiated cell (specific. Ex. muscle cell, skin cell), only 10 to 20% of the genes are active. Differentiation process: activating certain genes, inactivating other genes IN A CELL. Different sets of active genes make a skin cell different from a brain cell. Later in life... Environmental signals such as diet and stress can trigger changes in gene expression. Epigenetic flexibility is also important for forming new memories. PROTEINS CARRY SIGNALS TO THE DNA: Ex. your body is exposed to stress, so cortisol protein goes and signals to the DNA. Once a signal reaches a cell, proteins carry information inside. Like runners in a relay race, proteins pass info to one another. The information is ultimately passed to a gene regulatory PROTEIN that attaches to a specific sequence of letters on the DNA. Once there, it acts like a switch: activates gene transcription or shuts down (methylates) the gene). Epigenetic Tags Added in Response: Gene regulatory proteins recruit enzymes that add or remove EPIGENETIC TAGS. (ex. Enzymes that tag the gene with Methyl groups). Epigenetic Tags are Heritable to Daughter Cells During DNA Duplication. (When a DNA molecule divides/duplicates into 2, Methyl-Copying enzymes make sure that the New Methyl Tags on the new strands are put in the same places as the original strand. This way, a SKIN CELL duplicates into more SKIN CELLS, to create skin tissue/structure... not into some other types of cells). Epigenetic Changes and Gene Expression: On Gene: LOOSE, LESS METHYL, OR UNMETHYLATED. ACETYL GROUPS. Off Gene: TIGHT, MORE METHYL, METHYLATED, NOT ACETYLATED Cancers and Epigenetics: Tumor Suppressor Gene: Stop Sign: Stop Producing Tissue/Tumor: signal cells to pause the cell cycle, to fix mistakes. -When this gene is unmethylated (on), the stop sign is working, stopping tissue/tumor from being formed. (Healthy Cells). (Expression of Tumor Suppressor genes helps STOP abnormal cells from dividing, prevents cancer). -When this gene is methylated (off), the stop sign is not working, so the cell will make tissue/tumor. (Cancer Cells). (Many cancer cells have stopped making proteins from tumor suppressor genes). -BRCA1 and BRCA2 are TUMOR SUPRESSOR GENES that produce DNA Repair Proteins. -Tumor suppressor genes like p53 produce proteins that can induce apoptosis instead of allowing the cell to progress through the cell cycle. -Some tumor suppressor genes make proteins that suppress the cell cycle when cell division should not happen. SO, TUMOR SUPRESSOR GENES PRODUCE PROTEINS THAT PREVENT TISSUE/TUMOR FORMATION. Proto-Oncogenes: Go Signs: Go ahead and make the tissue/tumor: Signal cells to progress through the cell cycle. -When this gene is unmethylated (on), Go sign is working, so cell will divide/make tissue/tumor. (Cancer Cells). -When this gene is methylated (off), Go sign is not working, so cell will not make tissue/tumor. (Healthy Cells). -Some Proto-Oncogenes like HER2 produce proteins that enable the cell to respond to external signals that tell the cell to divide SO, PROTO-ONGOCOGENES PRODUCE PROTEINS THAT HELP THE TISSUE/TUMOR FORMATION. Remember, when you are making protein, you are not making tumor! And vice versa! Example of Methylation and Cancer: BRCA2: a tumor suppressor gene that is critical in repairing DNA. Some breast cancer patients have hypermethylated BRCA2 genes. The Chemotherapy Drug 5-Azacytidine turns OFF enzymes that add METHYL groups, leading to decreased Methylation. (BRCA2 gene will turn on, and will be able to do its job of repairing DNA, and working as a stop sign). The drug has been approved by the FDA for the treatment of a type of Leukemia. You are running a drug trial testing for 5-Azacytidine as a treatment for breast cancer. You need to determine which, if any, of 3 breast cancer patients would be good candidates for this drug trial. The levels of BRCA2 are measured before treatment here: Healthy Individual: High BRCA2 Patient 1: High BRCA2 Patient 2: Low BRCA2 Patient 3: Low BRCA2 Patient 2 and Patient 3 would benefit from the Chemotherapy 5-Azacytidine drug. The drug will turn off the genes that methylate (turn off) the BRCA2 stop sign. Therefore, the BRCA2 stop sign gene will work/reach higher levels after the drug. When you work out, muscle gets bigger, but muscle cells don't divide/duplicate. Same thing w/ liver, brain, etc. There are stop signs that tell cells not to divide anymore, to maintain set structure. Skin cells need to divide every day. So for these, you NEED "green go" signs. When BRCA2 gene is turned on (loose, not methylated), transcription and translation happens, and the formed protein keeps the cell from dividing further, keeps tumor from growing. When BRCA2 gene is turned off by enzymes adding methyl groups (heavily methylated, tight), transcription and translation not happening, and the cell continues to divide and forms a tumor. Chemotherapy: blocks/turns off the Methyl-adding enzymes, blocking the Methylation so that the gene can be on (loose, opened up, unmethylated) and transcribe, and the cell does not continue to divide and make tumors. Chemotherapy prevents/blocks ALL METHYLATION OF ALL GENES in a cell. Doesn't target a specific gene! So ALL genes in the cell the chemo targets will be turned on/loose. That's why it's answer A, not C.

DNA Microarray

Humans have around 20,000 genes. Each cell has those 20,000 genes. But diff cell types have diff ones turned on or off. Genomics: a way to study many/all genes in an organism at one time. One tool for this: DNA Microarray. Turned on: expressed: producing mRNA, and then protein. -ex. Muscle cell's "on" genes make mRNA to turn into MUSCLE PROTEINS. But doesn't make mRNA for other types of proteins! Using DNA Microarrays, you can pinpoint differences in gene expression (which genes are on/off) between 2 cell types. To make a DNA Microarray: 1. Sequence the entire genome of the organism you're studying. (Already been done for humans). 2. Find out where all genes are in your sequence. 3. Computer: design primer pairs, to use PCR (Polymerase Chain Reactions) to make copies of every gene. 4. Make all primers and do a PCR reaction to copy every gene in the genome. 5. Find out which PCR reactions didn't work. 6. Make copies of genes that didn't work. 7. SEPARATE DOUBLE STRNADED DNA from each gene copy into single strands. 8. Place microscopic drops of each single stranded DNA sample ito ordered rows and columns on the microscope slide. This is the Microarray. 9. Keep track of all gene spots on the Microarray. 10. Make sure all spots contain equal amounts of DNA. Most research labs buy Microarrays from biotechnology companies. Microarrays go by many names, like DNA chip, genome chip, expression chip, gene array. One type: GeneChip. One spot on Microarray represents one gene. There are thousands of spots. Each spot contains multiple copies of a unique DNA sequence, which corresponds to a single gene. Handy for determining the difference between 2 cell types. We will see the difference between a healthy cell and a cancer cell. You can look at difference in appearance between healthy and cancer cells. But sometimes, they look same. And appearance doesn't tell us why they are different. Cancer: disease of "genes gone bad." Many genes control how cells grow, divide, and die. When they stop working right, CELL GROWTH gets out of control, leads to TUMOR FORMATION. Diff types of cancer are caused by different sets of genes working abnormally. Experiment: 1. Collect Tissue 2. Isolate RNA 3. Isolate mRNA 4. Make labeled DNA copy (cDNA) 5. Apply DNA 6. Scan microarray 7. Analyze data We measure which genes are on/off in regular and cancer cells by measuring the types of mRNA found in both cell types. 1. Collect tissue: collect mRNA from melanoma (skin cancer) cells and healthy cells from same patient. So use scalpel to get some skin. 2. Isolate RNA from both tissues, bu dissolving them in solvents. Put samples in pipettes. Then mix the tissue samples on the VORTEX, so the tissue will dissolve, and RNA will be released. The samples are placed in the micro-centrifuge, which will spin the samples and separate the RNA from the rest of the cell contents. -Strands of DNA are much longer than RNA molecules, so it separates. Pour RNA from each into tubes. 3. Each sample (reg and cancer) contains diff types of RNA: mRNA, tRNA, and rRNA. mRNA always ends in a sequence of adenines: poly-A tail. Only mRNA reflects gene expression, so we'll save the mRNA and get rid of the rest. We wash RNA samples over columns filled with small beads that will only bind to RNA with poly-A tail. Other RNA will wash away. We now have columns with mRNA strands attached to poly-T beads by their poly-A tails. (A and T attract, remember). Now, wash a buffer solution over the columns to detach mRNA from the beads. (Buffer does not allow for hybridization, so they detach). 4. Now we make a DNA copy of each RNA. (cDNA). We give the DNA some color: add some labeling mix (colored) to our 2 RNA samples. Labeling mix will assemble a complementary DNA strand opposite every RNA molecule. (Look at picture in notebook). Poly-T Primer: usually referred to as Oligo-dT Primers: bind to poly-A tail of mRNA. Reverse Transcriptase: an enzyme which will synthesize a strand of complementary DNA (cDNA)!!!! Labeled Nucleotides: Will be incorporated into new cDNA molecule. The nucleotides in this tube (healthy) will have a green flourescent molecule attached to them (for the color of the labeling mix). -ONCE REVERSE TRANSCRIPTASE ASSEMBLES LABELED NUCLEOTIDES INTO CDNA, MRNA IS DEGRADED. OUR TUBES NOW CONTAIN CDNA. HYBRIDIZATION: 2 COMPLEMENTARY DNA STRNADS FROM DIFF SOURCES CAN PAIR WITH EACH OTHER -If 2 complementary DNA strands are mixed together, they will base pair, reforming a double strand DNA molecule. Doesn't matter where they came from. This is key to how DNA microarrays work. -so complementary strands that are mixed together in labeling mix will base pair. so most strands of cDNA on each dot will be base pairing. 5. Apply DNA -Stuck to our microarray are little piles of single stranded DNA molecules. -A SINGLE SPOT CONTAINS MANY IDENTICAL COPIES OF THE SAME GENE (several cDNA strands: copies of same one on the same dot). AND EACH SPOT ON MICROARRAY REPRESENTS A DIFF GENE. Computer database shows which gene is contained in each spot!! For every molecule of cDNA in our tubes (which represent human genes), there is a matching spot of single stranded DNA on the microarray. Most cDNA molecules added to the microarray will hybridize to their complimentary DNA strands on the microarray (base pair, form double helix w complimentary DNA strand on the correct dot)!!!!!! -Red (cancer) cDNAs hybridize on same spots. Green (healthy) cDNAs hybridize on same spots. On some spots, green AND red cDNAs hybridize. -And a few stray cDNA molecules do not hybridize. So we WASH OFF THE EXTRA CDNAS THAT DIDN'T BIND TO THE SLIDE/DIDN'T HYBRIDIZE. 6. Scan Microarray - we scan specific portions of it -First, we'll see WHERE the green (healthy) cDNA from healthy cells BOUND. (which dots/genes are turned on/transcribed). -Many spots are green! But not every spot. Dark spots are genes in the healthy cells that were not transcribed. Remember, not every gene gets expressed in every cell type. Now, we scan microarray to see where the red cDNA (from cancer cells) bound. -Lots of red spots (genes turned on in cancer cell). But diff pattern. Healthy skin cell and cancer cell: same genes, diff pattern (which ones are turned on). -Microarray scanner can merge red image and green image and display the composite picture. 7. Analyze Data Any spot that contains both red and green cDNA (gene is turned on in both the healthy and the cancer cell) shows up yellow. Gene hybridized to both green and red cDNA. GENE IS TURNED ON IN BOTH TYPES OF CELL. Yellow genes are not too interesting to us, because their activity doesn't change much when cell becomes cancerous. Dark spots: genes turned off in both healthy and cancer cells. GENE TURNED ON: TRANSCRIPTION -If a gene is showing up on a microarray as "on," it is turned on for transcription. doesn't necessarily mean it is TRANSLATING... Red Spots: show genes that produce more mRNA (turn on/up more, transcribe more) in cancer cells than in healthy cells. Green Spots: show genes whose expression is turned down in cancer cells. Not all of the red spots are "genes gone bad." Many of the genes are normal, but are red on our microarray because they are CONTROLLED BY A GENE THAT HAS GONE BAD!!!!!! Gene 4263 is a gene turned up in cancer cells (red dot). Gene 4263 produces a PROTEIN PRODUCT whose role is to TURN DOWN THE EXPRESSION OF THE GREEN GENES (genes that are expressed/turned up in healthy cells). Gene 6219 is normally turned on in healthy skin cells. In our cancer sample, the gene is defective. Gene transcribes into mRNA, but mRNA can't translate into protein. GENES LIKE THIS ARE YELLOW, BECAUSE THEY ARE TURNED ON/TRANSCRIBED IN BOTH HEALTHY AND CANCER CELLS (just not translated into protein in cancer cells). -Because it shows up as yellow (not black), defect cannot be detected using microarray analysis. Major technology limitation. So how do researchers know if a gene is producing A PROTEIN/TRANSLATING? -Scientists rely on PROTEIN EXPRESSION ANALYSIS, to tell them if mRNA is being translated into protein. DNA Microanalysis: power tool that can identify genes that are expressed differently in 2 diff cell types, by comparing every single gene in a single experiment. Limitations: IT CANNOT: Cure a disease Tell you which gene/genes "went bad" to cause a disease. Identify every gene that is behaving inappropriately. (ex. some genes may show up yellow, because in cancer cells, are turned up/on for transcription, but not functioning for translation).

The GR gene, which codes for a protein that results in anxious behavior in rats, can be inactivated by:

Injecting a drug that results in tightly winding the section of DNA that contains the GR gene.

Each of the thousands of different genes in a human cell can be cut apart from the chromosome, isolated one at a time, and then stuck onto a unique spot on a thin microarray chip. This chip provides a miniature map of all the genes that can then be searched to see if transcripts from these genes are being produced in a cell. For example, if you then wished to know if a particular cell was producing mRNA transcripts for the gene at a specific spot, all you need to do is collect mRNA transcripts from that cell, make them fluorescent with a green dye, and then add them to the chip. Because mRNA is complementary to the gene it was transcribed from, it will stick to only that specific DNA sequence wherever it finds it, but it will not stick to different sequences. So it should just stick to that single spot on the microarray and glow under fluorescent light, thus indicating that that gene is being transcribed into mRNA in the sample tissue. In this figure, you can see that a small section of a microarray was created with single stranded DNA from the following four genes expressed in breast cells: PFK: phosphofructokinase gene produces an enzyme used in all cells to break down glucose for energy Actin: gene that codes for an important cytoskeletal protein used in all cells to maintain cell shape CDCA3 gene that produces a protein growth factor that stimulates cells to divide. KRT8 gene that produces a protein called keratin that forms filaments of protein in structures such as the secretory epithelial cells that line the ducts of mammary glands. mRNA transcripts were collected from breast cells from a woman, made to glow with a green fluorescent dye, and added to the chip. Which of the following gene expression patterns would you expect to see in breast cells from a woman?

Just expression of CDCA3 gene.

REGULATION OF EXPRESSION OF GENES IN EUKARYOTIC CELLS OCCURS AT:

May occur when DNA is uncoiled and loosened from nucleosomes to bind Transcription factors (factors like RNA polymerase, promoter, activator, repressor) (Epigenetic level), when RNA is transcribed (Transcriptional level), when RNA is processed and then transported to cytoplasm (post-transcriptional), when RNA is translated into protein (Translational), or after protein is made (Post Translational).

Genomic Imprinting

Most epigenetic marks/flags are erased when egg and sperm cells are framed -but some imprints survive, pass down. (ex. your mother is exposed to a specific stress/diet that affects how some of her genes are expressed/turned on or methylated/turned off. when most flags are erased at conception, your mother passes down this "genomic imprint" to you). Genomic Imprinting is reversible. STICKY BUT NOT PERMANENT. (ex. then you may experience a non-stressful environment that causes diff genes to be turned on/expressed or turned off/methylated, and this is passed down to your kids.

1/31/20 Reading

Nazis stopped transport of food into Netherlands. 20,000 people starved to death. Pregnant women were uniquely vulnerable, and the children they gave birth to were impacted by famine throughout their lives. As adults, they were heavier than avg. High triglyceride and LDL cholesterol. Higher obesity, diabetes, schizophrenia. Died at a higher rate than people born before/after The hunger winter silenced certain genes in unborn children All cells in a body share the same genes but diff ones are active/silent in diff cells. Program locked in place before birth. Later experiences (like exposure to virus) can cause cells to quiet or to boost gene activity, sometimes permanently. Epigenetics: study of long term gene control. At millions of spots across our DNA, genes may carry a METHYL GROUP. They seem to silence genes, putting fetal programming (prenatal conditions affecting someone's lifetime health) into action. Researchers found methyl groups that were related to famine AND health conditions later in life. These methyl groups disrupt how cells normally use genes. -One methyl group linked to high body mass may quiet the gene PIM3, which is involved in burning body fuel. Theory: Ducth Hunger winter added a methyl group to fetuses born to starving mothers, making PIM3 gene less active, and continued to do so for life. -could be the other way around: gaining weight as you age could trigger change in PIM3 -or, maybe the famine made some cells more common, rather than affecting epigenetics

Which of the following has the "beads on a string" structure?

Nucleosomes Nucleosomes are the beads. DNA is the string.

Sources

Primary Sources: research studies submitted to a scientific source for peer review. Builds upon and includes references to previous research studies. Also original data as graphs/data tables. Sometimes hard to understand the scientific language. Secondary Sources: professional REVIEWS, summaries. In academic or gov journals. Provides references. Often, gives you more info than you can understand. Tertiary Sources: articles in news/magazines. Info thrice removed from original source. Usually report the names of primary researcher and publisher. Evaluating Sources: CRAP Testing: Currency- how recent? Reliability- based on evidence? multiple reliable sources give similar info? how well does it explain info? Authority- does the author have credentials? Purpose- Is the site using a specific agenda? selling something? Primary sources describe a single research study. Secondary sources prevent several research studies about that same phenomenon.

Diff sources

Primary sources These sources consist of research studies performed, written, and then submitted for peer-review to a scientific journal. These studies build upon and include references to previous research studies. They also include original data in the form of graphs or data tables. Example of a Primary Source: Miller, AJ, Sturgis, AL, McHorney, MR, Sinz, MJ. Differences in Body Composition Between Users and Non-users of Hydroxycut. J. Undergrad. Kin. Res. 2006; 1(2): 31-36. Secondary sources Very often, media articles will mention something called reviews. These reviews, called secondary sources, are often written by experts in the field and are designed to provide a summary of the state of current research studies about the topic. As such, they contain descriptions of all the original research conducted. They also provide all references in complete form so that the reader can identify who did the research as well as when, where, and how the research was conducted. These review articles can appear in academic journals or they can also appear in online journals or from government agencies. But, in all cases they will have been critiqued by experts in the field for accuracy (called peer-review). Imagine these review articles are an opportunity to sit down with an expert in the field and ask them which studies they find most important and why. They will give you all the details you want, but often they give you more than you can understand. Example of a Secondary Source: Foreyt, J. P., and Goodrick, G. K., Dieting and Weight Loss: The Energy Perspective. Nutrition Reviews, Volume 59, Issue 1, pages S25-S26, January 2001 Tertiary Sources Articles in the news or from popular magazine are known as tertiary sources because their information is thrice removed from the original source. Instead of being written by the researcher, these media reports are written by identified journalists - not posted anonymously. These media reports rarely contain complete citations or references with names of all researchers and exact source of publication but will usually report the names of primary researchers and often refer to the publisher. Tertiary sources can also include Government warnings such as consumer updates often posted to U.S. Department of Health and Human Services Websites. Encyclopedia entries such as Wikipedia that contain some citations and references are another example of tertiary sources. It is important to remember with Encyclopedic entries that the content of the article may or may not have been fact checked. Primary sources describe a single research study about a phenomenon or question, secondary sources present several research studies about that same phenomenon

Which of the following is the main difference between a primary and secondary source?

Primary sources describe a single research study about a phenomenon or question, secondary sources present several research studies about that same phenomenon

The term "Gene Expression" refers to the:

Process by which genetic information flows from genes to proteins (remember that when genes are flagged/expressed/turned on, that is causing transcription and translation).

If researchers added red-labeled copies of mRNA from rapidly-dividing cancerous breast cells, and green-labeled copies of mRNA from normal cells from and from normal breast cells, what color spots would indicate a gene that had increased expression in cancer cells compared to normal cells?

Red

The KRT8 gene contains an estrogen response element and produces the protein keratin in secretory epithelia in the breast and uterus. Since the KRT8 gene is present in all cells and estrogen travels throughout the body, why is this keratin protein only produced in epithelial breast cells but not in other cells in the breast, for example, in skin cells?

Skin cells do not produce the estrogen receptor protein necessary to stimulate transcription. (Skin cells' genes do not produce profound newestary to stimulate transcription: TRANSCRIPTION FACTORS) THE PROTEIN STIMULATES TRANSCRIPTION. (remember, gene produces protein, and in turn, protein stimulates transcription). remember, proteins affect everything! they act as runners and recruit enzymes to add methyl groups (turn off a gene for transcription), or acetyl groups (turn on a gene for transcription), etc.

REGULATION OF EXPRESSION OF GENES IN PROKARYOTIC CELLS OCCURS AT:

TRANSCRIPTIONAL LEVEL ONLY

FOOD/NUTRIENTS CONTAIN METHYL GROUPS!

TRUE!

If a cell were unable to produce histone proteins, which of the following would be expected to occur?

The cell's DNA could not be packed into its nucleus.

Diff between Heterochromatin and Euchromatin

The major difference between heterochromatin and euchromatin is that heterochromatin is such part of the chromosomes, which is a firmly packed form (tight, darkly stained) and are genetically inactive (off, methylated), while euchromatin is an uncoiled (loosely) packed form of chromatin (loose, light stained, unmethylated, on) and are genetically active.

Which of the following statements concerning the eukaryotic chromosome (found in animals, plants, fungi, and protists) is FALSE?

The number of genes on each chromosome is different in different cell types.

Some limitations of a Tertiary Source:

The source may not be easy to reference because the author may not be given. Not written by a professional in the field. The source may not report any limitations of the original studies it describes

One strand of DNA is the template strand for the synthesis of another strand (replication). What does this mean?

The template specifies the nitrogenous bases on the nucleotides of the strand being made.

How does Chromatin Structure and Epigenetics (chemical modifications of DNA) affect gene expression?

When the Chromatin is Methylated, Tightly Strung Together (Nucleosomes are tightly packaged), and Darkly Stained, gene is OFF. When the Chromatin is Unmethylated, Acetylated, Loosely Strung, and Light Stained, gene is ON.

Studies examining epigenetic tags in twins have been useful in identifying variation in gene expression in humans. Which of the following patterns would you expect to be true based on epigenetics.

Younger identical twins have more similar epigenetic tags on their DNA because they have a more common life experiences.

Genomics

a way to study many/all genes in an organism at one time. One tool for this: DNA Microarray.

DNA microarrays have made a huge impact on genomic studies because they

allow the expression of many or even all of the genes in the genome to be compared at once. Allow us to compare the gene expressions of all genes in a cell at one time (allowing us to see the difference between gene expression in 2 cell types).

If you were able to look very closely at a portion of DNA and find methylated histones (histones with methyl groups), you would:

be looking at a region of inactive DNA

Genome

complete set of chromosomes in a cell or an organism (contains all genes).

Epigenetics

differences in traits that aren't due to changes in DNA sequence Changes in environment/situation can affect which genes are expressed/turned on and off with chemical flags in a genome.

If you were interested in finding out if a specific gene (4361 with DNA sequence that reads ATTCTGCGCTAGCTAATTC) on a microarray was expressed by a cancer cell, a normal cell, or both. You would use which of the following as a probe?

fluorescently-labeled DNA of sequence TAAGACGCGATCGATTAAG you make a fluorescent-labeled DNA copy (cDNA) of the mRNA from the cancerous cells that is red, and a fluorescent DNA copy of the mRNA from the normal cells that is green. (hybridization base pairing occurs when you make these mixes) The PROBE is used to HYBRIDIZE (base pair) with the COMPLEMENTARY (opposite letters) CDNA THAT YOU WILL THEN ADD TO THE MICROARRAY. -so, cDNA is the actual copy of mRNA. -probe is what base pairs with each cDNA single strand to make double helixes. -cDNA BASE PAIRS are what ends up on the microarray.

A densely staining region of highly compacted chromatin is known as _____ and is generally____.

heterochromatin; not transcribed

Which of the following are true about DNA methylation?

it leads to genomic imprinting, which is reversible

Comparing DNA sequences for the KRT8 gene in a breast and a skin cell would show:

no differences

2/3 Notes

· All of our cells have 46 chromosomes, so all our cells have 2 of each gene. But some cells have diff genes turned on/off (if it is turned on it can transcribe and translate). -ex. A digestive cell and a skeletal cell both have lactase gene. But only the digestive cells can produce/have the mRNA and lactase protein enzyme. -There are exceptions to this "gene is there but the mRNA and protein isn't" rule, but we wont talk about that much. · Epigenome: series of chem tags that lie on top of your genes, telling your body which genes to read/activate/turn on or off/ transcribe and translate, and how often. · Promoter of Transcription AND Chromatin Structure affect when, where, and how much RNA is made in transcription 8 Histone proteins wound together (most genes are loosely wound around these histones, making them easy for cell to read) to make a structure called a Nucleosome Nucleosomes are beads, and DNA between them is the string Packed Nucleosomes are known as Chromatin This folding allows 6 feet of DNA to fit into the body. Tightly wound DNA (harder to read/ transcribe and translate) has more METHYL GROUPS on the DNA and proteins (the DNA and attached protein Histones are highly Methylated, turning off the genes). Loosely wound DNA (easier to read) has less Methyl Groups attached to the DNA and protein histones. Less Methylated. Acetylate does the opposite: Acetylate groups added on to the Histones (Histones are Acetylated) will occur when DNA is loosely wound (easier to read). Epigenetic Modifications: the addition of reversible changes to DNA and histone proteins (chemical flags being added to them). -can cause genes to turn on and off. (can sometimes be bad, turning off genes that shouldn't be turned off. Ex. genes that shouldn't be turned off are turned off, causing cancer). Histones: proteins that wind DNA into Chromatin

Biol Mice notes

· It was discovered that SOME people/organisms had identical genotypes (identical nucleotide sequences) but different phenotypes. · The environment can affect the way our genes are expressed, and pass down generation to generation · Ex. Mice with the same genotype can have diff colors of fur, because diff environmental explanations (ex. their mothers being fed diff diets) affected the way their genes would be expressed Separate the litter. High licking mothers were matched with the children of the low licking mothers, and vice versa. This way, genes would have zero effect. They found that the genes were not what mattered; the environmental explanation (the licking) which caused the genes to become expressed is what caused the rats to exhibit different types of behavior.