Lecture 23, 11/5 (Maybruck 8); Operons + Bacterial Genetics

You can get the audio for lecture on 11/5 here.

• • Review of last class - check out the animations on blackboard.
  
• • Slide 1 (handout from 10/26) Lactose operon: inducible operon
• Operons (structures found in bacteria (prokaryotes)) – grouping of genes that are adjacent to each other and are involved in coding for one particular phenotype
• Their transcription will be regulated together
• Lac operon (inducible operon) – the only way that transcription is going to occur is if lactose is present.
• Promoter region – where RNA polymerase is binding. Once it binds there it will start transcribing (3’-5’ direction). This is what operons are regulating – with the help of an operater region. This is found “downstream” of the promotor. (downstream indicates that it is in the direction that transcription occurs – 3’-5’ . . . upstream is the opposite.)
• If you want transcription to happen in lac operon you must get rid of repressor protein. The repressor protein has two binding sites one binds to the operating region. Once bound it prevents RNA polymerase from transcribing. The other binding site will bind to lactose. Lactose is the inducer of this operon. Lactose binds to repressor protein and causes it to change protein form. This protein can no longer bind. This allows rna polymerase to begin transcribing. The only time this operon will transcribe is when lactose is present.
• What is being transcribed in this operon?
• • Beta-galactosidase : this enzyme is responsible for breaking down lactose into galactose and glucose.
• • Slide 2 arg operon: repressible operon
• Arg operon – responsible for a group of genes that are responsible for building the amino acid argenine. RNA polymerase comes along and transcribes Arg operon. Argenine acts as a repressor. It associates with a repressor protein (which has two binding sites like the other one). One site is for argenine the other is for the operator region of that operon. When argenine is no longer available to bind to the repressor protein the repressor protein will move away from operater region and allows transcription to occur once more.
• • Slide 1 Application of bacterial genetics (handout11/5/07)
• Biotechnology – manipulating the biochemical processes of an organism to benefit humanity.
• • Example: insulin → there are bacteria that make our human insulin
• • It can also help us identify different bacteria
• • Slide 2 restriction endonucleases
• Restriction endonucleases - Internal cutting of DNA
• The restriction endonucleases are bacteria’s immune system. They try to stifle viruses attempt to inject their DNA into the bacteria.
• They always cut palindrome sequences.
• Sticky ends and blunt edges
• • Sticky ends created by restriction endonucleases. They occur when the DNA is split. These sticky ends go on and bind to other complimentary pairs.
• • Blunt ends are cut clean
• Sticky ends are used in a process called recombinant DNA. Recombinant DNA splices foreign DNA.
• Restriction fragment length polymorphism (RFLP)– a piece of DNA that has been cut by a restriction enzyme. We can use the RFLP to distinguish between different organisms.
• • Slide 3 analysis of DNA + getl electrophoresis
• Polymorphisms – the subtle differences that are occurring between the different nucleotide sequences of the organisms.
• EcorI cuts up certain base pair sequences consistently for different organisms. The cuts leave certain lengths which cause it to be identifiable.
• Gel electrophoresis helps identify cut pieces
• • Add the samples (cut pieces) to gel wells.
• • The gel is exposed to an electrical current which creates a negative charge at one end and a positive charge at the other end. The positive charge is always away from the wells. DNA has an overall negative charge to it. This means that it will want to move away from the negatively charged wells.
• • The fragments then spread out through the gel. Smaller fragments migrate faster. Large fragments move slower.
• • From there you can compare electrophoresis tests to compare and classify different organisms.