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I will be away next week at a conference in Europe. My colleague Bettina Francis, with whom I alternate teaching this course bi-annually, will give the lectures. Her PowerPoint style is more the classic bulletpoint style, so you might want to make a point of attending lecture. On the next slide is a histogram of the raw scores out of 50 for the first exam last Wednesday. Hopefully you will be able to find them online soon, otherwise you will get your exams back in lab this week. I indicate rough grade cutoffs. These are intended to give you an indication of how you are doing. Half of you are doing okay (As and Bs), but there a quite a few struggling (D and below). The latter need to reevaluate how they are approaching this class, for example, if your other classes are going well, then make this your focus for three evenings each week from now on for reading the relevant chapter and reviewing the lecture file. Also consider joining a student study group, consult with your TA during lab, and please feel free to come to me with unresolved questions. IB104 - Lecture 15 - Gene Regulation Reading - Chapter 15 (not flower development) 1. All organisms need ways to regulate when genes are expressed and how much protein is produced, and eukaryotes also need regulation of where, that is, in which cells, genes are expressed. That is, temporal and spatial regulation of gene expression is essential. 2. In bacteria the major form of control involves operons, complexes of multiple genes that are co-regulated. The most famous is the Lac operon. A B median C D F The Lac repressor gene is constitutively expressed at a low level so a small amount of the Lac repressor protein is always present in the E. coli cell. In the absence of lactose, this repressor binds the operator regions that overlap the promoter of the Lac operon, preventing transcription. repressor protein lactose operon regulatory gene transcription, translation repressor protein promoter gene 1 gene 2 gene 3 operator operator in DNA operator in DNA 1 Lactose in the environment enters the cell and binds the Lac repressor, changing its shape, and thereby freeing the operon to be transcribed. No lactose present allolactose lactose One context for this gene regulation is when E. coli bacteria find themselves in the guts of young mammals. Then they don’t need to express their Lac operon. mRNA operator promoter operator RNA polymerase gene 1 3. The Lac operon is an inducible operon, that is, in the presence of lactose transcription is induced and the enzymes thus encoded metabolize the lactose, upon which the operon is shutdown. This is also another neat example of negative feedback regulation. The basics were worked out by two Frenchmen, Jacques Monod and Francois Jacob (Nobel prize in Physiology or Medicine 1965). Bacterial cells have many of their genes organized in operons, and some of them are instead repressible operons. These are ones where the genes encode proteins involved in synthetic pathways, such as for amino acid synthesis. When the amino acid is available in the environment, and the bacteria no longer need to make their own, the bacteria can shut down these operons. Here the repressor protein only binds the operator when the end-product amino acid is present. For example, the His operon has eleven genes encoding all the enzymes needed to make the amino acid histidine from scratch, and is only transcribed if there is no available histidine in the cell’s environment. Lactose present 4. Multicelled eukaryotes: Several levels of control are used to build a complicated organism like ourselves where only bone cells must make bone, only blood cells must make hemoglobin, only cells in islets of Langerhans must make insulin, only hair follicle cells make that particular kind of keratin, only muscle cells make large amounts of myosin and actin, etc. There can be controls at all the levels from DNA to protein that we have examined in the past week. A. TRANSCRIPTIONAL - usually having to do with promoters and interactions at the DNA level. This is perhaps the most important level of gene regulation and the only one we will consider. B. TRANSCRIPT PROCESSING - having to do with the splicing or other modifications of pre-mRNAs. C. TRANSLATIONAL - having to do with the behavior of ribosomes on the transcript and the rapidity of degradation of the transcript. D. POST-TRANSLATIONAL - modification of proteins by cutting or phosphorylating or adding sugars, or complexing with other proteins, and the rate of degradation of proteins. 5. Eukaryotic gene regulation at transcription - five levels. A. Similar kinds of regulation to bacteria, but without the operon structure, that is, one gene at a time, occur in eukaryotes to regulate expression of various enzymes. For example, those involved in detoxifying particular toxins are only produced when the relevant toxin is encountered, which means they must also have receptors for relevant toxins, and a mechanism for signalling need for the detoxification enzyme. (Amazingly, nematodes like C. elegans independently evolved operons, another remarkable case of convergent evolution.) B. In multicelled organisms like animals, a lot of gene regulation involves differential expression in various tissues. This means that there is lots of specialization of tissues, meaning that while every cell still contains all the DNA, only some genes are expressed. The complexity of how this is accomplished is only starting to be un-ravelled, for example, in Drosophila flies. Here the controls primarily involve proteins called transcription factors that bind upstream of the promoters and control the binding of RNA polymerase and transcription. 2 Early development in Drosophila takes only 24 hours, and a series of transcription factors progressively divides the embryo into segments. Egg to larva in 24 hours Worker honey bees result from methylation of unknown genes. Upon sequencing the bee genome, an Australian colleague and I searched it for the genes known to perform DNA methylation in vertebrates (we already knew that Drosophila flies do not have these genes - they must have been lost by large deletions). We found them, and then my colleague asked whether they and DNA methylation might be involved in regulating whether females bees become sterile workers or fertile queens. Normally queens result from female larvae fed especially rich food, called Royal Jelly, but when he inactivated the DNA methylation genes, most female bees developed as queens. Queens, or reproductive individuals, are obviously the ancestral state, so the derived worker developmental pathway is apparently mediated by epigenetic CpG methylation of some genes in their genome. He is now working to identify those genes, and understand how they work. C. DNA methylation and Epigenetics. This generally involves methylation of cytosine when it occurs before a guanosine. That is, a methyl group (-CH3), is attached to the single-ring base of the cytosine when it occurs as a CpG (not a base pair, but a sequential pair of nucleotides along a strand – p means phosphate). This “mark” is present on the DNA of many different kinds of organisms, and has several diverse roles, but the best studied is involvement in gene expression. Because this mark is not inherited in the regular fashion of DNA replication, but must be added anew after each DNA replication, it is called “epigenetic”. We’re only beginning to understand the impacts of epigenetic changes, but they are cause for concern. For example, bisphenol-A (BPA) is an environmental toxin found in plastic bottles, with effects via its similarity to steroid hormones like oestrogen, and because it modifies DNA methylation. D. Changes can also occur in the organization of DNA. Histones are the fundamental proteins involved in binding DNA into nucleosomes. And several additional levels of packing are required to fit all the DNA into a nucleus. The level of packing affects whether genes are expressed, and is mediated by histone modifications, specifically, addition of acetyl groups to the tails of the histones. Histone tail 3 E. A truly major form of regulation at the chromosome level is the inactivation of the X chromosome in female mammals. This occurs in a mosaic pattern, seen clearly in calico cats, which are females with one copy of a gene on the X chromosome for orange fur, and one for black fur. They are a patchwork of orange and black splotches according to which X chromosome is inactivated (white fur is differently controlled). The condensed X chromosome appears cytologically as a dark spot attached to the nuclear membrane, called a Barr body. The best understood example is colon cancer, but today similar progressions of gene mutations and losses are being shown for others. 6. Cancer is a somatic disease in which control of cell division is lost. We now believe that cancer is largely a genetic disease in which genes have been mutated in various ways. Regulation of cell division in normal cells involves a balance of positive and negative controls, all involving various proteins encoded by various genes. One of the early breakthroughs was the discovery of oncogenes, that is, genes that foster cancer development. They were discovered as genes in cancer-causing viruses, and Harold Varmus and Michael Bishop got the Nobel prize in medicine in 1989 for the discovery in 1975 that our cells contain normal copies of these genes, called proto-oncogenes. These are genes that normally promote cell division, so when overexpressed by an infecting virus, can convert cells to cancerous fates. We now know that for a cell lineage to become a cancer it has to undergo a series of mutations and genetic insults, even loss of chromosomes. This process also removes at some point genes that impede cancer, for example, by causing cell suicide, so-called suppressor genes. There are seemingly endless varieties of cancer, broadly classified by the major tissue or organs they affect (breast, skin, lung, brain cancers), but within each are many subtle subdivisions, depending on precisely which kind of cell started dividing out of control. The details are crucial to improve both treatment and research. Cancers seldom kill when restricted to one place, however, after additional changes in a process called metastasis, the cells lose adhesion to each other and spread via the blood circulation, eventually sticking in capillaries and forming new tumors all over the body. This can happen very rapidly for some cancers like melanomas of the skin, which makes these particularly dangerous and hence even youngsters like yourselves should have annual checkups that include a complete skin exam. 4