DNA • DNA is the molecule that carries all of the inherited information in the cell. • DNA was discovered as “nucleic acid”—an acidic material in the nucleus in the later 1800’s. • Its importance was discovered until later. For a long time, DNA was considered too simple to carry genetic information. An Experiment • • • • • • How it was learned that DNA was the hereditary material: experiments by Griffith and then by Avery, Macleod, and McCarthy in the 1920’s through 1940’s. They used bacteria called Streptococcus pneumoniae, one cause of pneumonia. They had 2 strains: R (formed “rough” colonies) and S (formed “smooth” colonies due to a polysaccharide coat). When injected into mice, S bacteria caused pneumonia and killed them. R bacteria didn’t hurt the mice. When he killed the S bacteria by heating them, they no longer caused pneumonia. Same for R bacteria. Here’s the important result: if he injected live R along with the heat-killed S bacteria, the mice developed pneumonia and died. And, they contained live S bacteria. What happened: the hereditary material from the S bacteria survived the death of the bacteria themselves, and it “transformed” the live R bacteria into S bacteria.. Demonstrated that the hereditary material is separate from the property of being alive. More Experiment • • • • • Later work showed that the “hereditary material’ was DNA. Crude extracts were made from the S cells: breaking them apart. The extracts transformed live R cells into S cells and killed the mice, just like heatkilled S cells. The extracts were treated with various enzymes known to digest different cellular components: protein, RNA, DNA, etc. DNAase, the enzyme that digested DNA, stopped the transformation effect, but none of the other enzymes did. This demonstrated that DNA was the active material in transformation, the hereditary material. Numerous other experiments, using different organisms and procedures, continued to show that DNA, and not protein or some other type of molecule, was responsible for inheritance. Structure of DNA • DNA is a macromolecule, a large molecule composed of many subunits. The subunits of DNA are nucleotides. • Each nucleotide is composed of 3 parts: a nitrogenous base, a sugar (called deoxyribose), and a phosphate group (which is a phosphorus atom bonded to 4 oxygen atoms). • There are 4 kinds of nitrogenous bases in DNA: adenine (A), guanine (G), cytosine (C), and thymine (T). More DNA Structure • • • • • The nucleotides are joined into long chains that connect the phosphate of one nucleotide to the sugar of the next nucleotide. The nitrogenous bases of 2 different chains pair with each other, giving a DNA molecule that has 2 sugarphosphate chains on the outside, with bases paired in the center. Base pairing occurs by hydrogen bonds: partial positive and negative charges attract each other. Hydrogen bonds are weak, but there are lots of them in a DNA molecule. The 2 chains are “anti-parallel”—they run in opposite directions. They are twisted together into a corkscrew shape: a double helix. Base pairing is very specific: A pairs with T, and G pairs with C. DNA Replication • • • • • • How DNA makes copies of itself. Occurs during the S phase of the cell cycle, when each chromosome starts with 1 chromatid and ends with 2 identical chromatids. Each chromatid is 1 molecule of DNA. Involves an enzyme: DNA polymerase. The DNA double helix unwinds into 2 separate strands, and a new strand is build on each old one. Thus, each new DNA molecule consists of 1 old strand plus 1 new strand. This is called “semi-conservative” replication. DNA polymerase makes the new strands, using the old strands as a template, with normal base pairing: A with T, and G with C. The energy for this comes from the nucleotide precursors. They all have 3 phosphates on them, like ATP, and 2 of the phosphates are removed to make the DNA. Gene Expression • • • • • Each gene is a short section of a chromosome’s DNA that codes for a polypeptide. Recall that polypeptides are linear chains of amino acids, and that proteins are composed of one or more polypeptides, sometimes with additional small molecules attached. The proteins then act as enzymes or structures to do the work of the cell. All cells have the same genes. What makes one type of cell different from another is which genes are expressed or not expressed in the cell. For example, the genes for hemoglobin are on in red blood cells, but off in muscle and nerve cells. “Expressed” = making the protein product. Genes are expressed by first making an RNA copy of the gene (transcription) and then using the information on the the RNA copy to make a protein (translation). This process: DNA transcribed into RNA, then RNA translated into protein, is called the “Central Dogma of Molecular Biology”. RNA • RNA is a nucleic acid, like DNA, with a few small differences: • RNA is single stranded, not double stranded like DNA • RNA is short, only 1 gene long, where DNA is very long and contains many genes • RNA uses the sugar ribose instead of deoxyribose in DNA • RNA uses the base uracil (U) instead of thymine (T) in DNA. • There are 3 main types of RNA in the cell: • 1. messenger RNA: copies of the individual genes • 2. ribosomal RNA: part of the ribosome, the machine that translates messenger RNA into protein. • 3. transfer RNA, which is an adapter between the messenger RNA and the amino acids it codes for. Transcription • • • • Transcription is the process of making an RNA copy of a single DNA gene. The copying is done by an enzyme: RNA polymerase. Recall that in replication, a DNA copy of DNA is made by the enzyme DNA polymerase. The bases of RNA pair with the bases of DNA: A with T (or U in RNA), and G with C. The RNA copy of a gene is just a complementary copy of the DNA strand. RNA polymerase attaches to a signal at the beginning of the gene, the promoter. Then RNA polymerase moves down the gene, adding new bases to the RNA copy, until it reaches a termination signal at the end of the gene. More Transcription RNA processing • • • • • • Oddly, most genes in eukaryotes are not continuous. They are interrupted by long regions of DNA that don’t code for protein, called “introns”. Introns have no known function. The useful parts of the gene, the parts that code for proteins, are called “exons”. Some genes are more than 99% introns, with only 1% of the gene useful: the cystic fibrosis gene is like this. The entire gene, introns and exons, is transcribed into an RNA copy, but the introns need to be removed before it can be converted to protein. After transcription, snips out the introns, leaving only the protein coding portion of the gene in the RNA. Also, the cell adds a protective cap to one end, and a tail of A’s to the other end. These both function to protect the RNA from enzymes that would degrade it starting on an end and moving inward. Thus, an RNA copy of a gene is converted into messenger RNA by doing 2 things: 1. cut out the introns. 2. add protective bases to the ends. Transcription of RNA processing occur in the nucleus. After this, the messenger RNA moves to the cytoplasm for translation. Genetic Code • • • • • • There are only 4 bases in DNA and RNA, but there are 20 different amino acids that go into proteins. How can DNA code for the amino acid sequence of a protein? Each amino acid is coded for by a group of 3 bases, a codon. 3 bases of DNA or RNA = 1 codon. Since there are 4 bases and 3 positions in each codon, there are 4 x 4 x 4 = 64 possible codons. This is far more than is necessary, so most amino acids use more than 1 codon. 3 of the 64 codons are used as STOP signals; they are found at the end of every gene and mark the end of the protein. One codon is used as a START signal: it is at the start of every protein. Transfer RNA • • • • • • Transfer RNA molecules act as adapters between the codons on messenger RNA and the amino acids. Transfer RNA is the physical manifestation of the genetic code. Each transfer RNA molecule is twisted into a knot that has 2 ends. At one end is the “anticodon”, 3 RNA bases that matches the 3 bases of the codon. This is the end that attaches to messenger RNA. At the other end is an attachment site for the proper amino acid. A special group of enzymes pairs up the proper transfer RNA molecules with their corresponding amino acids. Transfer RNA brings the amino acids to the ribosomes, which are RNA/protein hybrids that move along the messenger RNA, translating the codons into the amino acid sequence of the polypeptide. Translation • Three main players here: messenger RNA, the ribosome, and the transfer RNAs with attached amino acids. • First step: initiation. The messenger RNA binds to a ribosome, and the transfer RNA corresponding to the START codon binds to this complex. Ribosomes are composed of 2 subunits (large and small), which come together when the messenger RNA attaches during the initiation process. More Translation • • • • Step 2 is elongation: the ribosome moves down the messenger RNA, adding new amino acids to the growing polypeptide chain. The ribosome has 2 sites for binding transfer RNA. The first RNA with its attached amino acid binds to the first site, and then the transfer RNA corresponding to the second codon bind to the second site. The ribosome then removes the amino acid from the first transfer RNA and attaches it to the second amino acid. At this point, the first transfer RNA is empty: no attached amino acid, and the second transfer RNA has a chain of 2 amino acids attached to it. Translation, part 3 • The ribosome then slides down the messenger RNA 1 codon (3 bases). • The first transfer RNA is pushed off, and the second transfer RNA, with 2 attached amino acids, moves to the first position on the ribosome. Translation, part 4 • The elongation cycle repeats as the ribosome moves down the messenger RNA, translating it one codon and one amino acid at a time. • Repeat until a STOP codon is reached. Translation, end • The final step in translation is termination. When the ribosome reaches a STOP codon, there is no corresponding transfer RNA. • Instead, a small protein called a “release factor” attaches to the stop codon. • The release factor causes the whole complex to fall apart: messenger RNA, the two ribosome subunits, the new polypeptide. • The messenger RNA can be translated many times, to produce many protein copies. Summary of translation Post-translation • The new polypeptide is now floating loose in the cytoplasm. It might also be inserted into a membrane, if the ribosome it was translated on was attached to the rough endoplasmic reticulum. • Polypeptides fold spontaneously into their active configuration, and they spontaneously join with other polypeptides to form the final proteins. • Sometimes other molecules are also attached to the polypeptides: sugars, lipids, phosphates, etc. All of these have special purposes for protein function. Mutation • • • • • • • • • • Any change in the base sequence of a DNA molecule is a mutation. Mutation is a completely random process: any DNA base can be mutated, whether it is in a gene or not. Basic types: 1. base substitutions: convert one base into another, such as changing an A into a G. 2. Insertions or deletions of large pieces of DNA. 3. Combining parts of 2 different genes together. Mutations are very common: every cell contains multiple mutations. Also, everyone is genetically different from every other person due to the accumulation of mutations. Genetic load: on average, each person has 3 recessive lethal mutations in all cells. We survive because the dominant normal alleles cover up the recessive lethals. Inbreeding—mating with close blood relatives—often causes defective children because the recessive lethals inherited from the common ancestor become homozygous. Many mutations occur in regions where they have no effect: between the genes, or in the introns that are spliced out of the messenger RNA. Only mutations within genes can affect the organism. Base substitution mutations within a gene can alter or destroy the gene’s protein product. The protein may not function at all, or it might be less efficient, or it might have an altered pH optimum or temperature optimum. Many of these changes have little or no effect on the organism: these are called “neutral” mutations, because they are neither good nor bad for the organism. The larger changes that occur with insertions, deletions, and rearrangements are usually harmful, because they usually destroy at least one gene. However, new useful genes sometimes arise from these rearrangements. One event in particular: attaching the control regions of one gene onto the protein-coding part of another gene. This causes the protein to be synthesized in a new time and place within the organism. Mutation Causes and Rate • Rate: for typical genes, base substitutions occur about once in every 10,000 to 1,000,000 cells. Since we have about 6 billion bases of DNA in each cell, this implies that virtually every cell in your body contains several mutations. Clearly, most mutations are neutral: have no effect. • Only mutations in the germ line cells: cells that become sperm or eggs—are passed on to future generations. Mutations in other body cells only cause trouble when they cause cancer or related diseases. • Causes: The natural replication of DNA produces occasional errors. DNA polymerase has an editing mechanism that decreases the rate, but it still exists. • Radiation and certain chemical compounds also cause mutations. Chemicals that cause cancer—carcinogens—almost all work by causing mutations. Gene Control • Most regulation of genes works by controlling transcription, the process of making an RNA copy of a single gene. Thus, a gene is “on” when it is being transcribed, and “off” when it is not being transcribed. • There are many ways to regulate genes—a lot of contemporary biology research is devoted to discovering these mechanisms. • I will describe a few simple mechanisms. Lac Operon • The common gut bacterium Escherichia coli (E. coli) has been studied by scientists for a long time and much is known about it. • E. coli, like most organisms, used glucose as its primary food. However, in the absence of glucose, it can used lactose (milk sugar). • Lactose is a disaccharide. E. coli cells produce an enzyme that breaks it down into glucose. This enzyme is called beta-galactosidase, and it is made by a gene called the lac operon. • The lac operon also makes other proteins that help in the process. Lac Operon Basics • • • • The operon itself consists of 3 regions that code for protein, called Z, Y, and A. The Z gene codes for the important enzyme, beta-galactosidase. A single messenger RNA is made from the entire lac operon. Transcription of the RNA starts at the promoter at the left end of the gene. The operator is a region of DNA between the promoter and the Z, Y, and Z genes. It is an important part of the regulatory system. Another gene makes the “lac repressor”, the protein involved in gene regulation. Lac Regulation • • • • • To conserve its resources, the E. coli need to have the lac operon ON when lactose is present and OFF when lactose is absent. To accomplish this, the repressor protein can be in two different states: the repressor can either bind to lactose, or it can bind to the operator DNA sequence. When lactose is present, the repressor binds to lactose and not to the operator. This allows RNA polymerase to transcribe the gene: it is ON. When lactose is absent, the repressor binds to the operator. This blocks RNA polymerase from transcription, and the gene is OFF. If lactose is added, the repressor falls off the operator and binds the lactose, which allows transcription to start. Positive Control • • • • • • • The lac operon is an example of negative regulation: the regulatory protein (the lac repressor) causes transcription to stop. Positive control, where the regulatory protein causes transcription to start, is more common. The lac operon of E. coli also has an example of positive control. When the cell’s glucose level is high, it doesn’t need to use lactose at all. So, only when the glucose level drops is it necessary to try using lactose. The positive control mechanism turns the lac operon ON when the glucose level drops. This mechanism uses a different protein, called CAP, and the signaling molecule cyclic AMP (cAMP). cAMP is generated when the glucose level is low. cAMP then binds to CAP, and the cAMP-CAP complex attaches itself to the promoter. This complex attracts RNA polymerase and allows transcription to occur at a high rate. The negative regulation system acts on top of the positive system: transcription is allowed whenever glucose is low, but only occurs is lactose is present. X Chromosome Inactivation • • • • Many genes in multicellular organisms are shut down permanently in different tissues. They are never needed because they make products used only in other types of cells. A model for this permanent inactivation is the X chromosome in females. Males have only 1 X and females have 2 X’s. For any other chromosome, this imbalance would be lethal, but it is the normal condition of the X. How can this occur? In every female body cell, only 1 X is active. The other X gets converted into a condensed, inactive blob on the nuclear membrane called a Barr body. This is a simple way to telling male cells from female cells: female cells have Barr bodies and male cells don’t. Only one X is active in every cell: all others are converted to Barr bodies. People with XXY (Klinefelter’s syndrome) are male in appearance, but their cells have Barr bodies. People with Turner’s syndrome (XO, only 1 X) are female but have no Barr bodies. People with 3 X’s (XXX) have 2 Barr bodies in each cell. More X Inactivation • • • • When the embryo has about 200 cells, each cell independently inactivates all but 1 X chromosome. The inactive X stays inactive in all cells descended from the original cell, throughout the individual’s life. The inactivation can lead to interesting effects. Tortoiseshell cats are a mixture of black and orange. They are always female (occasionally Klinefelter’s –XXY – males), because they need 2 X’s. The gene for coat color is on the X and it has a black allele and an orange allele. A heterozygote has one black and one orange allele. But: only 1 X is active in each cell, which means that either the black allele is active or the orange allele is active, but not both. So, as the cells of the embryo develop into patches of skin, the active allele is expressed and you get a black and orange pattern. Calico cats also have white spots. Their black and orange pattern is the result of X inactivation, but the white spots are due to another, autosomal gene. Calico cats are also always female. There is a human condition called anhidrotic ectodermal dysplasia that cause a loss of sweat glands in the skin. In females it leads to patches with and without sweat glands. It is lethal in males. Hormone Signaling • • • Hormones are small molecules that circulate in the blood and alter the expression of genes in many tissues. Hormones are either steroids (lipids with 4 rings of atoms in a characteristic shape) or peptides (small proteins). Steroid hormones can enter the cell directly through the membranes. Once in the cell, they bind to receptor proteins, then move to the nucleus where they stimulate transcription. Peptide hormones bind to receptor proteins on the surface of the cell. The receptor proteins then activate other proteins within the cell, in a cascade that ends up activating “transcription factors” in the nucleus. Transcription factors stimulate RNA transcription of particular genes. Plant Response to Light • • • Many plants will only flower when days are short, while other plants require a long day. Plants are able to determine the length of the dark period. Even a very short –10 second– pulse of light in the middle of the dark period prevents short day/long night plants from flowering. The mechanism for determining day length uses a blue-green pigment called “phytochrome”. Phytochrome is activated by red light, which dominates during the day. The active phytochrome slowly converts back to the inactive form during the night. The amount of active phytochrome left at the end of the night is proportional to the length of the night. When present at the appropriate level, phytochrome stimulates enzymes in the plant cell to start or stop transcription. Different plants have different critical levels of active phytochrome. Genetic Engineering • We have been modifying living things for a long time: domestication of various plants and animals, hybridization of different species (such as horse x donkey = mule), selective breeding for useful traits. • Recently it has become possible to directly modify the DNA of living organisms, in the hope of producing a more useful plant or animal. Also, we can artificially produce natural body proteins that can be used as medicinal drugs. Molecular Cloning • • • • • • Molecular cloning means taking a gene, a piece of DNA, out of the genome and growing it in bacteria. The bacteria (usually E. coli) produce large amounts of this particular gene. The cloned gene can then be used for further research, or to produce large amounts of protein, or (sometimes) to be inserted into cells that lack the gene (people with genetic disease, for example). The basic tools: 1. plasmid vector: small circle of DNA that grows inside the bacteria. It carries the gene being cloned 2. Restriction enzymes: cut the DNA at specific spots, allowing the isolation of specific genes. 3. DNA ligase, an enzyme that attached pieces of DNA together. Plasmid Vectors • • • • • • Bacterial chromosomes are large DNA circles. Bacteria have a single chromosome. Plasmids are small circles of DNA inside bacteria that replicate independently of the bacterial chromosome. They exist naturally, and usually confer some useful but not necessary trait on the bacteria: drug resistance, for example. Each plasmid only has a small number of genes on it. Some plasmids can produce hundreds of copies of themselves inside each bacterial cell. It is possible to insert foreign DNA into the plasmid. This DNA becomes part of the enlarged circle of the plasmid. It replicates along with the plasmid. The plasmid DNA can be manipulated in vitro, outside the cell, then put back into the cell through the process of transformation. Because bacteria can be grown quickly and easily, you can produce large quantities of any DNA inserted into a plasmid Restriction Enzymes • • • • Restriction enzymes are part of the defense system of bacteria: they digest foreign DNA that enters the bacterial cell. Each species of bacteria has its own set of restriction enzymes. Each enzyme cuts DNA at a specific short base sequence. For instance, EcoR1 cuts the DNA at the sequence GAATTC, and BamH1 cuts at GGATCC. There are hundreds of restriction enzymes known. Using properly chosen enzymes, the gene you want can be cut out of the chromosome intact, with very little extra DNA. Many restriction enzymes give a staggered cut across the DNA double helix. This produces short single stranded regions, called “sticky ends”. The ends are sticky because they spontaneously pair with similar ends. DNA Ligase • “ligate” means to tie together. DNA ligase is an enzyme that attaches 2 pieces of DNA together. It forms covalent bonds between the sugars and phosphates of the DNA backbones. • Especially useful: if 2 different DNAs were cut with the same restriction enzyme, they will have matching sticky ends. DNA ligase can then combine these 2 different DNA molecules into a single DNA. This hybrid DNA molecule is called “recombinant DNA”. The Cloning Process • 1. Cut genomic DNA with a restriction enzyme. • 2. Cut plasmid vector with the same restriction enzyme. • 3. Mix the two DNAs together and join them with DNA ligase. • 4. Put the recombinant DNA back into E. coli by transformation. • 5. Grow lots of the E. coli containing your gene. Using Cloned DNA • • • • • • • One major use of cloned genes is to produce large amounts of their protein products to be used as medicinal drugs. As an example, human growth hormone is made in the pituitary gland, a very small organ at the base of the brain. Some people do not produce enough of it, resulting in very short stature and various health-related issues. HGH injections during childhood help. However, pituitary glands must be harvested from human cadavers, and are often contaminated with viruses. Another example: insulin is needed by people with diabetes. In former times it was isolated from pigs or sheep. However, the animal forms had a few amino acid differences from human insulin, and sometimes caused bad immune responses. The solution to these problems is to isolate the human genes using the techniques of recombinant DNA, then cause these genes to express themselves, to produce their protein products. The proteins can then be isolated from the bacteria. The proteins are the normal human forms, despite having been made in bacteria. They are not contaminated with human viruses, and they won’t cause an immune reaction. Getting the genes to express is simple (in principle): RNA copies of genes are made if they have a promoter sequence in front of them. Similarly, proteins are made if the messenger RNA has the appropriate signals on it. So, it is merely a matter of including the proper signal sequences on the plasmid. But of course: complications arise. Gene Therapy • • • • • One way to cure genetic diseases is to insert good (non-mutant) copies of the defective gene into the cells of the affected person. Big problem: how to get the gene into all of the cells. In mice, inject the gene directly into the zygote. The gene incorporates into the chromosomes at some random location, and (usually) functions. Humans usually don’t know about the presence of the disease until the baby is born. In some cases, blood cells can be used. Blood cell precursors are in the bone marrow, which can be extracted, have recombinant genes inserted, then put back into the body. Gene Therapy Problems • • • • Two big problems: 1. The recombinant genes insert into random locations. Sometimes they insert into oncogenes, which causes cancer. Leukemia usually, cancer of the blood cells. 2. There are very few blood stem cells in the body. Stem cells divide repeatedly and never differentiate into the final blood cells. Genes inserted into stem cells are permanently in the body. Genes inserted into cells that have already started differentiating into blood cells will stay in the body for a few weeks, then be lost as the blood cells wear out and die. The bottom line (so far): gene therapy has not been very successful except in a small handful of cases. Genetically Modified Plants • • • • It is fairly easy to insert genes into plants, using a special plasmid vector derived from crown gall tumors. This vector grows in bacteria, but transfers its DNA into the plant genome after infection. One use of this techniques is to insert nutrient genes into plants. For instance, rice is the staple food for a large part of the world’s population. It contains no carotene, the orange pigment that is the precursor for the main visual pigment retinol. People who live on rice alone often develop blindness because they don’t eat enough carotene. Golden rice was developed to solve this problem: genes fro producing carotene were put into rice. This pigment gives the rice its color. Problems: will people eat this oddlycolored food? Will it work well in cooking? Will they accept “Frankenfood”? More Plant Genetic Engineering • • • • In the US, the main uses of genetically modified crop plants are herbicide resistance and insect resistance. If your crop plants are resistant to a herbicide that kills the weeds, you can spray more effectively. Similarly, plants that are given genes for insect resistance don’t need to be sprayed with insecticides. Very effective—farmers find these plants cheaper to grow. However, there is a lot of resistance to eating these plants in Europe. Maybe the genes will somehow leak out and affect people—we digest DNA down to nucleotide subunits before taking it into our bodies, so this shouldn’t happen. Maybe it will affect other plants—probably does happen naturally at a slow rate. Another idea: “pharming” putting genes for useful proteins like insulin into plants, and letting the plant synthesize them in large amounts. Can also be done with animals: proteins secreted into milk. Nuclear Cloning • Why not just take the nucleus from any cell and put it into an egg, producing a new person genetically identical to the original? This is the idea behind nuclear cloning, and it does work on occasion. • Dolly the Sheep was the first example: a nucleus from her parent’s mammary gland was extracted and put into an egg whose own nucleus was removed. The egg was them implanted into the uterus of another sheep, and Dolly was born. She is genetically identical to the donor sheep. More Nuclear Cloning • Although a clone’s nuclear DNA is identical to the donor parent, the parent and offspring are not likely to be exactly identical. Similarly, identical twins are not exactly identical. Events occur after the identical zygotes form: random environmental influences, patterns of development, etc. • It is very difficult to get a nucleus of a cell to regress to the “totipotent” state of an embryonic cell. A totipotent cell can turn into any cell type. After a few cell divisions of the embryo, the cells are restricted: they can no longer become any type. Many genes are permanently inactivated by modifying the DNA molecule. Removing these modifications is, at the moment, more of an art than a science. • The result: the failure rate in cloning is very high: less than 1% success rate. And, of the clones that are born alive, many suffer defects that only become apparent later in life. Dolly, for instance, died at a young age.