Regulation of enzyme synthesis

Transcription

1 Regulation of enzyme synthesis The lac operon is an example of an inducible operon - it is normally off, but when a molecule called an inducer is present, the operon turns on. The trp operon is an example of a repressible operon - it is normally on but when a molecule called a repressor is present the operon turns off. Enzyme repression Is the regulatory mechanism that inhibits gene expression and decreases the synthesis of enzymes. Is a response to the overabundance of an end-product of a metabolic pathway. Is mediated by regulatory protein repressors, which block the ability of RNA polymerase to initiate transcription. Repressor: is an allosteric protein that binds to the operator (after it binds to the corepressor) region of the DNA and blocks transcription Operator: A specific region of the DNA located at the beginning of the gene, where the repressor protein binds Corepressor is a small molecule that combines with a repressor protein and alters its conformation. Only the altered repressor can bind to the operator Enzyme Induction Turns on transcription of gene(s). An inducer is a small molecule that initiates enzyme induction by combining with a repressor protein and altering its conformation. The altered repressor is released Inducible enzymes are those synthesized in the presence of inducer.

2 Genetic Engineering or Recombinant DNA technology Applications of molecular biology have allowed scientists to modify the genetic characteristics of organisms at the DNA level -- the most widely used of these techniques is called recombinant DNA technology -- this technology is revolutionizing agriculture, medicine and forensics. Recombinant DNA technology -- procedures by which DNA from different species can be isolated, cut and spliced together -- new "recombinant " molecules are then multiplied in quantity in populations of rapidly dividing cells (e.g. bacteria, yeast). Hepatitis B vaccine is being made by yeast carrying a gene for part of the hepatitis virus (viral coat protein), thus eliminating the need to use the whole virus. New recombinant DNA techniques can be also used to amplify DNA. Since each person s DNA is unique (except identical twins), the procedure is useful in the field of criminology. This technology is based on a number of important things: 1. bacteria contain extrachromosomal molecules of DNA called plasmids. They are the cloning vectors (small, independently replicating genetic elements used to replicate genes, and most are derived from plasmids or viruses) 2. bacteria also produce enzymes called restriction endonucleases that cut DNA molecules at specific places -- DNA cut into many smaller pieces called restriction fragments. Restriction Enzymes There are many different kinds of restriction endonucleases -- each cuts DNA at a specific site defined by a sequence of bases in the DNA called a recognition site -- several hundred endonucleases have been extracted from bacteria and many are used in recombinant DNA research. Example: endonuclease called EcoRI cuts DNA where the sequence GAATTC occurs -- GAATTC is the recognition site for this endonuclease -- cut occurs between the G and the A so that the cut is staggered and each fragment has 2 sticky ends. Typical restriction enzymes recognize 4-, 6- or 8-base sequences.

3 The two strands have the same sequence if both are read 5 3 or both read 3 5. Restriction enzymes make staggered cuts in the 2 strands of DNA (cuts are not directly opposite to each other), leaving stretches of single-stranded DNA at the end of DNA fragment called sticky end which is characteristic to that enzyme. Sticky ends join to complementary stretches of single-stranded DNA by base pairing (by hydrogen bonding). Then DNA ligase enzyme is used to covalently link the backbone of the DNA pieces and thus producing recombinant DNA molecule. Cloning Vectors Cloning vectors are small, independently replicating genetic elements used to replicate genes, and most are derived from plasmids or viruses (bacteriophage). Cloning: isolation & incorporation of certain gene or DNA fragment in a vector to be replicated To clone a DNA sample, the same restriction enzyme must be used to cut both the vector and the DNA sample. Cloning vectors share four common properties: 1.Ability to promote autonomous replication. 2. Contain a genetic marker (usually dominant) for selection. 3. Unique restriction sites to facilitate cloning of insert DNA. 4. Minimum amount of nonessential DNA to optimize cloning.

4 Gene Libraries: A genomic library, also clone bank or gene bank, is a collection of DNA from a single organism, ideally though not necessarily containing its entire genomic DNA sequence. The DNA from the source organism of interest is divided into multiple fragments and packaged within cloning vectors such that each carries a portion of it. The vector DNA can then be inserted into host organisms - commonly a population of bacteria - for amplification and retrieval. Plasmid vector

5 Each fragment of DNA containing about one gene is carried by a vector, either a plasmid within a bacterial cell or a phage. Gene Library Creating a Genomic Library 1. DNA molecules of an organism of interest are isolated. 2. The DNA molecules are then partially digested by endonuclease restriction enzymes, splitting the helix into small workable portions. Several different restriction enzymes may be used at once, and the DNA molecules may be digested for different lengths of time in order to ensure that the DNA has been digested to manageable sizes. 3. DNA molecules are separated by size using agarose electrophoresis. Individual DNA pieces are ligated into host vectors. 4. The hosts are kept in liquid media and can be frozen at -808C for a long period of time for later experimental use.

6 Gel Electrophoresis The process in which molecules (such as proteins, DNA, or RNA fragments) can be separated according to size and electrical charge by applying an electric current to them while they are in a gel. The current forces the molecules through pores in a thin layer of gel, a firm jelly-like substance. The gel can be made so that its pores are just the right dimensions for separating molecules within a specific range of sizes and shapes. Smaller fragments usually travel further than large ones. After certain time the gel is stained with ethidium bromide which will fluoresce under UV light. In Lane A a restriction enzyme digestion of a standard DNA sample where the size of each band is known Marker or Ladder Other lanes have purified sources of DNA cut by one or more restriction enzymes. The banding pattern of a given DNA is reproducible since a given restriction enzyme always cut at the same site. By comparison with the standard, the fragment size can thus be determined.

7 The bright bands on this gel are DNA fragments produced by restriction enzyme digestion. The bands to the far sides are DNA size markers. Uses of Gel electrophoresis: Comparative studies of 2 or more DNAs. Studies in the classification of microorganisms and their genetic relationship. To determine DNA fragment sizes after cutting the DNA with restriction enzymes (a), or it might be necessary to check DNA that has been isolated and purified MUTATION A mutation is a change in the base sequence of DNA. Such a change in the base sequence of a gene will sometimes cause a change in the product encoded by that gene. For example, when the gene for an enzyme mutates, the enzyme encoded by the gene may become inactive or less active because its amino acid sequence has changed. Such a change in genotype may be disadvantageous, or even lethal, if the cell loses a phenotypic trait it needs. However, a mutation can be beneficial if, for instance, the altered enzyme encoded by the mutant gene has a new or enhanced activity that benefits the cell. Many simple mutations are silent (neutral); the change in DNA base sequence causes no change in the activity of the product encoded by the gene. Types of mutations The most common type of mutation involving single base pairs is base substitution (or point mutation), in which a single base at one point in the DNA sequence is replaced with a different base. If the base substitution results in an amino acid substitution in the synthesized protein, this change in the DNA is known as a missense mutation. By creating a nonsense (stop) codon in the middle of an mrna molecule, some base substitutions effectively prevent the synthesis of a complete functional protein; only a fragment is

8 synthesized. A base substitution resulting in a nonsense codon is thus called a nonsense mutation. Frameshift mutations are mutations in which one or a few nucleotide pairs are deleted or inserted in the DNA. This mutation can shift the "translational reading frame-that is, the three-by-three grouping of nucleotides recognized as codons by the trnas during translation, which results in the production of an inactive protein. Spontaneous mutations occur in the absence of any mutation-causing agents. Agents in the environment, such as certain chemicals and radiation that directly or indirectly bring about mutations are called mutagens. A wide variety of chemicals, many of which are common in nature or in households, are known to be mutagens. Many forms of radiation, including X rays and ultraviolet light, are also mutagenic. In the microbial world, certain mutations result in resistance to antibiotics or altered pathogenicity. Mutagens Chemical Mutagens One of the many chemicals known to be a mutagen is nitrous acid. Nitrous acid can convert the base adenine (A) to a form that no longer pairs with thymine (T) but instead pairs with cytosine (C). Eventually, some AT base pairs of the parent will have been changed to GC base pairs in a granddaughter cell. Like all mutagens, it alters DNA at random locations. Another type of chemical mutagen is the nucleoside analog. These molecules are structurally similar to normal nitrogenous bases, but they have slightly altered basepairing properties. Examples, 2-aminopurine and 5-bromouracil. When nucleoside analogs are given to growing cells, the analogs are randomly incorporated into cellular DNA in place of the normal bases. Then, during DNA replication, the analogs cause mistakes in base pairing, resulting in base-pair substitutions. Some antiviral and anitumor

9 drugs are nucleoside analogs, induding AZT (azidothymidine), one of the primary drugs used to treat HIV infection. Radiation X rays and gamma rays are forms of radiation that are potent mutagens because of their ability to ionize atoms and molecules. Another form of mutagenic radiation is ultraviolet (UV) light, a nonionizing component of ordinary sunlight. However, the most mutagenic component of UV light (wavelength 260 nm) is screened out by the ozone layer of the atmosphere. The Frequency Of Mutation The mutation rate is the probability that a gene will mutate when a cell divides. Spontaneous mistakes in DNA replication occur at a very low rate, perhaps only once in 10 9 replicated base pairs (a mutation rate of 10-9 ). The occurrence of random mutations at low frequency is an essential aspect of the adaptation of species to their environment, for evolution requires that genetic diversity be generated randomly and at a low rate. For example, a mutation that confers antibiotic resistance is beneficial to a population of bacteria that is regularly exposed to antibiotics. Once such a trait has appeared through mutation, cells carrying the mutated gene are more likely than other cells to survive and reproduce as long as the environment stays the same. Soon most of the cells in the population will have the gene. Identifying Mutants 1. Positive (direct) selection e.g., penicillin resistant mutants can be identified by exposure to penicillin 2. Negative (indirect) selection; detects mutant cells because they do not grow. Auxotroph is a mutant organism or cell that requires growth supplements that could normally be synthesized by wild-type strains.

10 Replica Plating Replica plating is a very effective means of isolating mutants that require one or more new growth factors. Replica Plating

11 Identifying Chemical carcinogens Ames Test Ames Test: The Ames test yields a number of growing bacterial colonies which is a measure of the mutagenic activity (potency) of a treatment chemical. This value is often expressed as the number of revertants per microgram of a pure chemical (mutagen) or per gram of food containing that mutagen. About 909 of the substances found by the Ames test to be mutagenic have also been shown to be carcinogenic in animals.

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