MOLECULAR GENETICS: Experiments Griffith's Experiment Griffith determined that there was a transforming factor or a chemical responsible for

change in the genetic makeup of an organism. In his experiment he had a strain of smooth, virulent bacteria and a strain of rough, non-virulent bacteria. As his control, he injected a mouse with the virulent strain -- it died. He injected another mouse with the non-virulent strain -- it lived. Then he took the virulent strain and used heat to kill it. He then injected a mouse with the killed virulent strain -- it lived. He injected another mouse with a killed virulent strain that had been mixed with a non-virulent strain -- it died. He concluded that something caused the genetic makeup of the non-virulent bacteria to change into something virulent. Hershey-Chase Experiment Hershey and Chase determined that it was DNA that controlled the cell's activities. In their experiment, they labeled a bacteriophage with radioactive sulfur (in the protein coat) and radioactive phosphorus (in the DNA). They then mixed the phage in with other bacteria and waited for infection to occur. Then, they scanned the bacteria for radioactive elements. Hershey and Chase discovered that the radioactive sulfur was discarded by the phages outside of the bacteria cells while the radioactive phosphorus was inside the infected bacterial cells. This led them to conclude that DNA that was the genetic material.

DNA Replication:

during interphase, a second chromatid containing a copy of the DNA molecule is assembled. ---Involves separating (unzipping) the DNA molecule into two strands, which serve as templates to assemble a new, complementary strand. **DNA replication is Semi-conservative (split DNA and complimentsingle strand of old, single strand of new) *DNA is ANTIPARALLEL. 35, 53 strands run in opposite directions. Helicase unwinds DNA at the origins of replication, producing a replication fork. Single-strand binding proteins prevent the single strands of DNA from recombining. Topiosomerase removes twists and knots that form in the double stranded template as a result of the unwinding (tension) induced by helicase. Primase initiates DNA replication at origins of replication with RNA primers. DNA polymerase attaches to the primers and begins elongation. The leading complementary strand is assembled continuously as the double-helix DNA uncoils. The lagging complementary strand is assembled in short Okazaki fragments. The Okazaki fragments are joined by DNA ligase. Proofreading then occurs and if any mistakes are found, they are taken out through excision and the primers are also taken out and replaced by DNA nucleotides. To prevent loss of DNA in the replicated strand, the enzyme telomerase stops elongating the template strand, and ultimately DNA polymerase will be unable to replicate the new, extended portion template.

Protein Synthesis There are three kinds of RNA. Messenger RNA (mRNA) carries a copy of the DNA code to the ribosomes during protein sythesis. Transfer RNA (tRNA) transports amino acids (used to form proteins) to their proper place on the mRNA template. Ribosomal RNA (rRNA) are the building blocks of ribosomes. They are created in the nucleolus.

Protein synthesis is divided into two phases: transcription and translation. In transcription, RNA polymerase unzips DNA and reads it in the 3' to 5' direction. Free RNA nucleotides are base paired with the exposed DNA nucleotides and assembled into mRNA. The mRNA then leaves the nucleus and moves to the ribosomes in the cytoplasm. Transcription ends and translation begins. Translation has four stages. In the amino acid activation stage amino acids are attached to the appropriate tRNA molecules. A molecule of ATP is used to activate the tRNA. In the initiation stage the ribosome moves along the mRNA strand until it reaches the code for formyl methione (AUG). In the elongation stage the tRNA matches its anti-codon (three nucleotides) with the mRNA codon (three nucleotides). The amino acid on the previous tRNA is transferred to the newly arrived tRNA and the empty tRNA leaves. This is repeated over and over as the ribosome moves along the mRNA strand. In the termination stage a nonsense code is reached and the protein is released.

Mutations Mistakes in base-pairing are corrected by DNA polymerase and other mismatch repair enzymes. Radiation and various reactive chemicals can cause thymine dimers where adjacent nucleotides bind to each other instead of to the complimentary strand. Such errors are usually fixed by splicing out the affected nucleotides and replacing them (excision repair). A mutation is a DNA error that is not repaired. Some mutations include using an incorrect nucleotide (substitution), deleting a nucleotide (deletion), and adding a nucleotide (insertion). When insertion occurs, all the subsequent nucleotides are misplaced causing a frameshift mutation.

The Operon Theory All enzymes are made of protein, all proteins are made of amino acids, and all amino acids have a DNA codon. Basically, DNA controls RNA which controls the production of proteins. In 1961, Franois Jacob and Jacques Monod proposed the Operon Theory to explain how cells control the production of enzymes.

The following is a list of the components of the operon. The regulatory gene codes for the repressor protein. The promoter site is the attachment site for RNA polymerates. The operator site is the attachment site for the repressor protein. The structural genes code for the proteins. The repressor protein is different for each operon and is custom fit to the regulatory metabolite. Whether or not the repressor protein can bind to the operator site is determined by the type of operon. The regulatory metabolite is either the product of the reaction or the reactant depending on the type of operon. The messenger RNA. The final enzyme. There are two kinds of operons: repressible and inducible. In the repressible operon, the product is the regulatory metabolite. When the concentration of the product increases, the product binds to the repressor protein allowing the repressor protein to bind to the operator site -- shutting the operon down. In the inducible operon, the reactant is the regulatory metabolite. When the concentration of the reactant increases, the reactant binds to the repressor protein removing the repressor protein from the operator site -- turning the system on.

DNA Organization There are several steps involved to get DNA into the form of a chromosome. First, DNA is twisted into a double helix. Second, the DNA double helix is wrapped around small proteins called histones to form nucleosomes. Third, the nucleosomes are arranged into looped domains. Finally, the looped domains are condensed into chromosomes. Recombinant DNA Recombinant DNA is DNA that contains segments or genes from different sources. These segments are spliced together to form the recombinant DNA. Recombinant DNA technology uses restriction enzymes that cut the DNA after a certain sequence of nucleotides. The staggered cut is referred to as a sticky end. The sticky ends are then reintegrated into a DNA strand by the enzyme ligase. Recombinant DNA technology is the manipulation and combination of DNA molecules from different sources. Recombinant DNA technology uses the techniques of sequencing, rejoining, amplifying, and locating DNA fragments, all of which use complementary base pairing of A with T (or U) and G with C. Crossing over during prophase of meiosis produces recombinant chromosomes. To make rDNA a technician often begins by selecting a vector, by which the recombinant DNA is introduced into a host cell. The most commonly used vector is the plasmid which is a small accessory ring of DNA. The plasmid is not part of the bacterial chromosome and can be replicated independently. Recombinant DNA technology uses restriction enzymes to cut up DNA. They are obtained from bacteria that manufacture these enzymes to combat invading viruses. These enzymes are very specific in that they cut DNA at specific recognition sequences of nucleotides. The cut that restriction enzymes produce across a double-stranded DNA is usually staggered leaving fragments that have one strand extending beyond the complementary- the sticky end. Restriction enzymes also cut the plasmid leaving the same sticky ends in both foreign DNA and plasmid segments. The foreign DNA can now be attached to the cut plasmid by base-pairing at the sticky ends. Foreign DNA can only be inserted into a cut plasmid by utilizing DNA ligase. This enzyme seals the two separate pieces together at the sticky ends resulting in a recombinant plasmid. The recombinant plasmid is then introduced into a bacterium by transformation. Bacterial cells take up recombined plasmids more easily if they are treated with calcium chloride which makes them more permeable. As the host cell reproduces each new cell will

have at least one plasmid therefore each containing the gene of interest. This is known as cloning. Gel electrophoresis is used to separate restriction fragments. DNA fragments are separated as they move through the gelatinous material under the influence of an electric field. Because DNA is negatively charged, the DNA fragments move toward the positive electrode on the other end. The shorter pieces move further than the longer pieces. This process is used to compare DNA between different organisms and determine evolutionary relationships. The fragments of DNA from separate organisms differ in length because of polymorphism- slight differences in DNA sequences. These are known as restriction fragment length polymorphisms or RFLPs for short. RFLPs are used in DNA fingerprinting when comparing the DNA left at the scene of a crime to possible suspects. A genome is the full set of genes of an individual. A genomic library is a collection of bacterial or bacteriophage clones each containing a particular segment of DNA from the source cell. The process of making a genomic library involves the splicing of an organisms DNA and putting those pieces into vectors which are taken up by host bacteria which will then multiply. For human gene expression to occur in a bacterium it must be accompanied by the proper regulatory regions and should not contain introns. This is due to the fact that bacterial cells do not have the necessary enzymes to process primary mRNA. However, human genes may lack introns. Reverse transcriptase, and enzyme, can be used to make a DNA copy of all the mature mRNA molecules from a cell. The result is complementary DNA (cDNA) which does not contain introns. Probes, a single-stranded nucleotide sequence, are utilized to find a particular gene in a genomic library. The probe may be radioactive or fluorescent. Bacterial cells carrying the DNA fragment are placed onto agar in a petri dish. The probe can then hybridize with the gene of interest isolating it from the fragment. It can now be cloned or analyzed. The polymerase chain reaction (PCR) is a technique that uses the enzyme DNA polymerase to produce millions of copies of a particular piece of DNA. Before carrying out this process, primers (sequences of about 20 bases that are complementary to either side of the target DNA) must be available. They are needed because DNA polymerase does not start the replication process; it only continues or extends the process. After the primers bind by complementary base pairing to the DNA strand, DNA polymerase copies the target DNA. Bacteria, plants, and animals are genetically engineered to produce biotechnology products. Transgenic organisms are organisms that have had a foreign gene inserted into them. Organs for transplant from nonhuman sources are also examples of biotechnology products. Biotechnology products are produced by bacteria which create substances such as insulin, human growth hormone, t-PA (tissue plasminogen activator), and the hepatitis B vaccine. Transgenic bacteria have been produced to promote the health of plants. Some bacteria contain genes that code for a toxin which protects the roots of a plant from insects.

Gene pharming is the use of genetically engineered animals to produce pharmaceutical in milk.