Gene Expression Whats that How do we regulate

Gene Expression. . . What’s that?

How do we regulate the expression of our genes?

Involved in gene expression • DNA regulatory sequences • Regulatory genes • Small regulatory proteins (RNAs)

Regulatory sequences • Stretches of DNA that interact with regulatory proteins to control transcription. Regulatory genes • A sequence of DNA encoding a regulatory protein or RNA.

Gene Regulation among bacteria • Bacteria cells are able to express the genes whose products are needed by the cell. – EX: need for tryptophan. • Have both positive & negative control mechanisms

-Expression of specific genes can be turned “on” by the presence of an inducer or can be inhibited by the presence of a repressor. -Inducers & repressors are small molecules that interact with regulatory proteins &/or regulatory sequences.

Regulatory proteins inhibit gene expression by binding to DNA and blocking transcription (negative control). Regulatory proteins stimulate gene expression by binding DNA & stimulating transcription (positive control) or binding to repressors to inactivate repressor function. Some genes are continuously expressed; they are always turned “on” EX: ribosomal genes

Operons are one way in which genes are regulated.

The switch is the operator (segment of DNA) -it controls the access of RNA polymerase to the genes - Regulatory proteins stimulate gene expression by binding to DNA & stimulating transcription (positive control) or binding to repressors to inactivate repressor function. Operon= the operator, promoter, & genes they control –the entire stretch of DNA required for enzyme production for the tryptophan pathway.

Two types of Negative Gene Regulation • Repressible Operon: transcription is usually on but can be inhibited (repressed) when a specific small molecule binds to a regulatory protein. – EX: tryptophan • Inducible: usually off but can be stimulated (induced) when a specific small molecule interacts with a regulatory protein. – EX: lac operon (lactose) http: //biology-animations. blogspot. com/2007/11/lac-operon-animation. html

Repressible Operon (tryptphan)

Inducible lac operon

Which do you think is more common for inducible operons? - the gene in its non-repressed state? Or in its repressed state? What about repressible operons? Inducible operons are more commonly found in the repressed state. Repressible operons are more often actively transcribing, thus are not repressed

Which type of operon would be used for anabolic reactions (making new molecules)? Repressible operons are only turned off when there is an excess of gene production

Which type of operon would be used for catabolic reactions (breaking down of molecules)? Inducible operons are only turned on in the presence of the a substance produced by metabolism (metabolite) in order to break nutrients down.

Positive Gene Regulation vs Negative Gene Regulation • Positive: When a regulatory protein interacts directly with the genome to switch transcription on. • Negative: When the operons are switched off by the active form of the repressor protein

Positive Gene Regulation When glucose is in short supply as an energy source, E. coli will use lactose. E. coli will then synthesize high quantities of the enzymes to breakdown the lactose. How does the cell sense a shortage of glucose? c. AMP accumulates when glucose is scarce. c. AMP binds with CAP (the activator & regulatory protein) & stimulates the transcription of a gene c. AMP binds to CAP & CAP assumes its active shape. CAP attaches at the upstream end of the lac promoter which stimulates gene expression.

If the amount of glucose increases the c. AMP concentration falls & therefore CAP detaches from the operon. The lac operon is under negative regulation by the lac repressor & positive regulation by CAP.

Can you hypothesize some other ways that might increase or completely shut down the transcription of a gene? EX: activators that help the RNA polymerase have greater affinity with the promoter region.

What differences in gene regulation might we see in the eukaryotic genes?

When a Gene Turned Off Is a Matter of Life or Death: Epigenetic Influences on Gene Regulation LET’S DO A CASE STUDY!!!

Identical twins share the same DNA but are they exactly identical? How might they be different? WHY? ?

Consider the cells that are in the tissue of your big toe. Which genes are those cells going to need to use? How much DNA will be present in a given cell that won’t be used at any point except when the cell replicates? 95 -97% of the genome of any given cell goes untranscribed When a cell receives a signal to transcribe specific genes, what facilitates its search for the genes? DNA is organized very precisely on a scaffolding of proteins that attach to nuclear lamina & cytoskeleton, thus every part of every strand is in a known location.

Each “bead” is a nucleosome. -the basic unit of DNA packing The looped domains coil & fold forming the characteristic metaphase chromosome

Gene expression in eukaryotes is controlled by a variety of mechanisms that range from those that prevent transcription to those that prevent expression after the protein has been produced. 5 kinds of general mechanisms that can be used. Transcriptional - These mechanisms prevent transcription. Posttranscriptional - These mechanisms control or regulate m. RNA after it has been produced. Translational - These mechanisms prevent translation. They often involve protein factors needed for translation. Posttranslational - These mechanisms act after the protein has been produced.

Gene expression can be regulated at any stage, but the key step is transcription • All organisms – Must regulate which genes are expressed at any given time • During development of a multicellular organism – Its cells undergo a process of specialization in form and function called cell differentiation

ON/OFF SWITCHES Complete loss of genes or chromosomes that occurs in amphibians after each phase of metamorphosis VOLUME CONTROLS Enhancers that bind ~1000 base pairs upstream of a promoter can help RNA Polymerase find & bind to the promoter more often so that more transcriptions are made for say, insulin

Why is it an evolutionary advantage to be able to turn some genes off temporarily or permanently? Having genes that are always turned on when the gene product is not needed would be wasteful & use up the resources within a cell. Why are “volume controls” an advantage? Some gene products are in very high demand & need to have a greater number of transcriptions made so that the cell can function efficiently.

Signal NUCLEUS Chromatin modification: DNA unpacking involving histone acetylation and DNA demethlation DNA Many key stages of gene expression can be regulated in eukaryotic cells Gene available for transcription Gene Transcription RNA Exon Primary transcript Intron RNA processing Tail Cap m. RNA in nucleus Transport to cytoplasm CYTOPLASM m. RNA in cytoplasm Degradation of m. RNA Translation Polypetide Cleavage Chemical modification Transport to cellular destination Active protein Degradation of protein Degraded protein

Regulation of Chromatin Structure & Histone Modifications Can affect the configuration of chromatin and thus gene expression

DNA Methylation The addition of methyl groups to certain bases (usually cytosine) in DNA is associated with reduced transcription in some species. Genes that are not being expressed have a tendency to be heavily methylated Removal of the extra methyl groups can turn on certain genes. Experiments have shown that deficient DNA methylation due to lack of a methylating enzyme leads to abnormal embryotic development. In these cases, DNA methylation is essential for the long-term inactivation of certain genes.

Epigenetic Inheritance Chromatin modifications don’t necessarily involve a change in DNA and yet they may be passed on from parent to offspring The inheritance of traits transmitted by mechanisms not directly involving the nucleotide sequence is called: Epigenetic Inheritance Let’s read an article

• A certain laboratory strain of the fruit fly Drosophila melanogaster has white eyes. • If the surrounding temperature of the embryos, which are normally nurtured at 25 degrees Celsius, is briefly raised to 37 degrees Celsius, the flies later hatch with red eyes. • If these flies are again crossed, the following generations are partly red-eyed – without further temperature treatment – even though only white-eyed flies are expected according to the rules of genetics.

Combinatorial Control of Gene Activation • In Eukaryotes, the control of transcription depends largely on the binding of activators to DNA control elements. – Will be able to activate transcription only when the appropriate activator proteins are present Figure 19. 7 a, b Enhancer Promoter Albumin gene Control elements Crystallin gene Liver cell nucleus Available activators Lens cell nucleus Available activators Albumin gene not expressed Albumin gene expressed Crystallin gene not expressed (a) Liver cell Crystallin gene expressed (b) Lens cell

upstream The rate of gene expression can be increased or decreased by the binding of specific transcription factors, either activators or repressors to the control elements of the enhancers. The combination of transcription factors binding to the regulatory regions at any one time determines how much, if any, of the gene product will be produced.

What determines how much of a gene product will be produced? The combination of transcription factors binding to the regulatory regions at any one time determines how much, if any, of the gene product will be produced.

Gene regulation accounts for some of the phenotypic differences between organisms with similar genes.

Embryonic development • A zygote going through cell division over and over would just produce a ball of all the same type of cells with the same genes. • So…where does the differentiation come in? – A sequential program of gene regulation placed in the egg by the mom is carried out as cells divide.

How do they know this? • In the 1950’s F. C. Steward worked with carrots Conclusion: At least some differentiated (somatic) cells in plants are totipotent, able to reverse their differentiation and then give rise to all the cell types in a mature plant.

Using one or more somatic cells from a multicellular organism to make another genetically identical individual is called: CLONING

Nuclear Transplantation in Animals • Not the same as plants regarding differentiated cells. • Differentiated cells in animals do not develop into multiple cell types. Conclusion: The nucleus from a differentiated frog cell can direct development of a tadpole. However, its ability to do so decreases as the donor cell becomes more differentiated, presumably because of changes in the nucleus.

Reproductive Cloning of Mammals • Clone mammals using fully developed differentiated cell. – Would need to “reprogram” to be totipotent

Problems Associated with Animal Cloning • Only a small % of cloned embryos develop normally. • Many exhibit defects – Prone to obesity, liver failure, premature death • The donor nuclei is seen to have more methyl groups on their DNA which will effect gene expression compared to non-cloned embryos.

Stem Cells • Cells that are undifferentiated & under the right conditions is able to differentiate. • These cells are taken during the blastula stage (or blastocyst)

Embryonic Stem Cells • Cells that start to take different development paths to become specialized cells, such as blood stem cells, which means they can no longer produce any other type of cell. Can give rise to any and all tissues in the body they can differentiate into some, but not all, cell types.

2 sources “tell” a cell which genes to express Cytoplasm of egg Environment around a particular cell

• The egg’s cytoplasm contain cytoplasmic determinants (influence development) – Cytoplasm of the egg is distributed into other cells. • Depending on which portions of the zygotic cytoplasm a cell received determines the cells fate because of the variants of gene expression.

• The environment around the particular cell. – Interactions between embryonic cells help induce differentiation.

Once a cell has undergone determination it is irreversibly committed to being that type of cell. Determination at the molecular level is when the cell is expressing tissue specific proteins.

Pattern Formation In Plants & Animals Development of a spatial organization in which the tissues & organs of an organism are all in their characteristic places.

In Animals • Occurs in embryo stage. – Cytoplasmic determinants & cell inductive signals provide positional information – Cell lineage • genes affect formation

In Plants • Mechanisms for plant development – Cell lineage is less important – Depend more on positional information • Cell signaling & transcriptional regulation • Formation In Flowers – Environmental signals • Day length & temperature

LET’S GO GMO…OR NO WHAT IS GMO? THEN… COME UP WITH A LIST OF BENEFITS AND CONCERNS OF USING GMO’S

Plant Biotechnology • Innovations in the use of plants for human usage. • GMO’s (genetically modified organisms) – DNA/genotype of an organism is artificially changed • Use of GMO’s in agriculture and industry – GMO corn is engineered to produce its own insecticide by transferring the Bt (Bacillus thuringiensis) crystal protein gene into the corn genome. – GMO soy is engineered to resist being sprayed with weed killers.

Why go GMO? Let’s look at the GM corn: • More food is grown • Reduced the need to clear say rainforests to grow crops • Lowers cost of production • Less pesticides/ fertilizers/ chemicals in general

Concerns over GMO’s • Unknown risks to humans & the environment – When drugs are tested & results show concerns it can be stopped. Not so with crops. – Risk of soil contamination over long term • Possible human risks: – Transfer allergens • Effects on non-target organisms – Ex: caterpillar died consuming laboratory milkweed because of the pollen from the GM corn with bt gene • Transgene Escape – EX: GM crop for herbicide resistance & a wild relative have genetic transfer

Besides GMO’s there’s….

Artificial Selection

Types of Genes Associated with Cancer

Cancer results from genetic changes that affect cell cycle control

What types of things influence having cancer? Mutations of genes associated with cell growth such as: random mutation, chemical carcinogens, X-rays, and some viruses.

Types of Genes Associated with Cancer Oncogenes Proto-oncogenes Tumor-Suppressor Genes

Cancer most often results from mutations in genes • Proto-oncogenes: they often code for proteins that stimulate cell division, prevent cell differentiation or regulate programmed cell death (apoptosis). • Tumor suppressor genes- produce proteins that signal cells when they are getting too crowded.

Oncogenes & Proto-Oncogenes Cancer causing genes Genes that stimulate normal cell growth & division • Converting Proto-Oncogenes into Oncogenes Genetic changes that lead to an increase in product or a change in activity of protein

Tumor-Suppressor Genes • These genes encode proteins that prevent uncontrolled cell growth. – Repair damaged DNA – Control the adhesion of cells to each other or to the extracellular matrix – Components of cell signaling pathways that inhibit the cell cycle. • A mutation happens here and cells will divide uncontrollably = cancer.

Cell cycle – stimulating pathway No growth factor even needed with this mutation

Cell cycle – inhibiting pathway This signal is started because of damaged DNA. May be the result of exposure to UV light p 53 gene halts cell cycle until DNA can be repaired or activate genes that are involved in DNA repair. When DNA is irreparable the p 53 gene activates the “suicide” genes that cause cell death (apoptosis).

• Mutations that knock out the p 53 gene or if the p 53 gene is defective or missing… – Can lead to excessive cell growth and cancer (c) Effects of mutations. Increased cell division, possibly leading to cancer, can result if the cell cycle is overstimulated, as in (a), or not inhibited when it normally would be, as in (b). EFFECTS OF MUTATIONS Protein overexpressed Cell cycle overstimulated Figure 19. 12 c Protein absent Increased cell division Cell cycle not inhibited

Multiple steps for the development of cancer. More than one somatic mutation is needed to produce full-fledged cancer cells. (the older we get the more likely we are to develop cancer) At least: 1 active oncogene and mutation or loss of several tumor-suppressor genes are recessive so both alleles must be “knocked out”

And finally…the telomerase gene is usually activated in many tumors Enzyme prevents DNA from shortening and when activated removes a natural limit on the number of times a cell can divide

Genetic Predisposition & other Factors Contributing to Cancer

Risk Factors • Inheriting an oncogene puts you one step closer to accumulating the mutations for cancer. – Breast cancer: a person inheriting one mutant BRCA 1 allele has a 60% probability of developing cancer before the age of 50 compared to someone homozygous for normal (2%). • DNA breakage – Minimize exposure to these agents: • UV radiation, chemicals from cigarette smoke, X-rays • Viruses – Viral integration; can contribute oncogene, alter tumor supressor genes, or convert proto-oncogenes to oncogenes.

How deadly? Cancer Type Estimated New Cases Estimated Deaths Bladder 76, 960 16, 390 21% Breast (Female – Male) 246, 660 – 2, 600 40, 450 – 440 16% - 16% Colon and Rectal (Combined) 134, 490 49, 190 37% Endometrial 60, 050 10, 470 17% Kidney (Renal Cell and Renal Pelvis) Cancer 62, 700 14, 240 23% Leukemia (All Types) 60, 140 24, 400 41% Lung (Including Bronchus) 224, 390 158, 080 70% Melanoma 76, 380 10, 130 13% Non-Hodgkin Lymphoma 72, 580 20, 150 28% Pancreatic 53, 070 41, 780 78% Prostate 180, 890 26, 120 41% Thyroid 64, 300 1, 980 3%