Chapter 2 Genes.pdf

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University of Florida
Biology - Biological Sciences
BSC 3096

Chapter 2 Genes Sunday, June 30, 2013 5:12 PM 1. The gene a. Eukaryotes have more than one copy of some genes b. Prokaryotes have only one copy of each gene c. Transcriptionally active chromatin is structurally different from inactive chromatin i. Euchromatin - eukaryotic genes being transcribed ii. Heterochromatin - eukaryotic genes not being transcribed 1) Where repetitive sequence DNA is mainly found 2. DNA a. b. 5' to 3' directionality i. ii. 5' end is attached to phosphate group iii. 3' end Is attached to -OH group c. A forms 2 h-bonds with T d. C forms 3 h-bonds with G e. CANNOT move through nuclear pores 3. Replication a. One time in each life cycle a cell replicates its DNA b. DNA replication is semiconservative i. When a new double strand is created, it contains one strand from the original and one new strand c. Replisome i. Group of proteins that govern replication 1) d. Replication beings towards the middle of a chromosome at the origin of replication i. A single eukaryotic chromosome contains multiple origins of replication e. From the origin, two replisomes proceed in the opposite direction f. Replication fork - the point where a replisome is attached to the chromosome g. Replicons/replication units - discrete segments of DNA replication on the same chromosome h. Helicase unwinds the double helix i. DNA Pol cannot initiate a strand from two nucleotides, it needs a free -OH group to attach on to i. Primase, an RNA polymerase, creates an RNA primer (around 10 nucleotides long) to initiate the strand j. DNA Pol reads parental strand in the 3' to 5' direction, creating the new strand in the 5' to 3' direction i. Each nucleotide added requires the removal of a pyrophosphate group (two phosphates) from the dNTP ii. k. Some of the energy that is released from the hydrolysis of pyrophosphate is used to drive replication l. DNA replication is extremely accurate m. One of the subunits in DNA Pol is an exonuclease, which proofreads the strand as it is synthesized and makes the proper adjustments n. Fast due to multiple ORC o. Telomeres are repeated six nucleotide units from 100-1000 bp long that protect the chromosome from being eroded 4. RNA a. Identical to DNA except RNA is single stranded, carbon # 2 on the pentose sugar is not deoxygenated, and contains uracil instead of thymine i. b. Can move through nuclear pores c. Exists in three forms i. mRNA 1) Delivers the DNA code for AA to the cytosol ii. rRNA 1) Combines with PROTEINS to from ribosomes 2) Synthesized in the nucleololus iii. tRNA 1) Collects AA in the cytosol and transfers them to the ribosome 5. Transcription a. RNA polymerase i. Prokaryotes have one type ii. Eukaryotes have three types b. Promoter i. Sequence of DNA nucleotides that designates a beginning point for transcription ii. THE TRANSCRIPTION START POINT IS PART OF THE PROMOTER ii. THE TRANSCRIPTION START POINT IS PART OF THE PROMOTER iii. The most commonly found nucleotide sequences of a promoter recognized by RNA pol of a give species is called the consensus sequence 1) Variations from the consensus sequence causes RNA Pol to bond less tightly and less often, which leads to those genes being transcribed less frequently c. Transcription factor i. Protein that binds to specific DNA sequences, thereby controlling the transcription of DNA to mRNA d. Initiation i. A group of proteins called initiation factors find a promoter on the DNA strand and assemble a transcription initiation complex 1) TIC includes RNA pol and various transcription factors ii. After binding to the promoter, RNA pol unzips the DNA double helix creating a transcription bubble e. Elongation i. RNA Pol transcribes one strand of the DNA nucleotide sequence into a complementary RNA nucleotide sequence ii. The strand being transcribed is called the template strand or (-) antisense strand iii. The other strand is called the coding strand or (+) sense strand and protects its partner against degradation 1) The result of transcription yields an mRNA strand identical to the coding strand except for U instead of T iv. Transcription is much slower than replication f. RNA Pol DOES NOT contain a proofreading mechanism and thus has a higher rate of error i. Errors in RNA are not called mutations g. Termination i. The end of transcription requires a special termination sequence and special proteins to dissociate RNA pol from DNA h. Activators and repressors i. Bind to DNA close to the promoter and either activate or repress the activity of RNA pol ii. iii. Often allosterically regulated by small molecules such as cAMP i. The amount of a given type of protein within a cell is likely to be related to how much of its mRNA is transcribed i. mRNA has a short half-life in the cytosol, so soon after its transcription is over, the mRNA is degraded and its protein is no longer translated ii. Many proteins can be transcribed from a single mRNA, resulting in an amplifying effect j. Polycistronic i. Several genes in a single transcript ii. Prokaryote k. Monocistronic i. One gene per transcript ii. Eukaryote l. Operon i. Genetic unit usually consisting of operator, promoter, and genes m. Lac operon i. Codes for enzymes that allow e. coli to import and metabolize lactose when glucose is not present in sufficient quantities ii. iii. Low glucose levels lead to high [cAMP] 1) cAMP binds to catabolite activator protein, CAP 2) Activated CAP binds to CAP site found upstream and adjacent to the lac operon promoter 3) The promoter is now activated iv. The operator is found adjacent and downstream of the promoter 1) The operator provides a binding site for a lac repressor protein 2) The lac repressor protein is inactivated by the presence of lactose in the cell a) The lac repressor protein will bind to the operator unless lactose is present 3) If the lac repressor does bind to the operator, indicating there is no lactose present, the transcription of the lac genes is prevented 4) Lactose can therefore induce the transcription of the lac operon only when glucose is not present n. Gene regulation in eukaryotes i. Much more complicated than prokaryotes involving the interaction of many genes ii. Enhancers are regulatory proteins commonly used by eukaryotes 1) Their function is similar to activators and repressors but act at a much greater distance from the promoter 6. Post-transcriptional processing a. Occurs in both eukaryotic and procaryotic cells i. In prokaryotes, rRNA and tRNA go through posttranscriptional processing, mRNA does not (gets directly translated to protein) ii. In eukaryotes, each type of RNA undergoes posttranscriptional processing b. Posttranscriptional processing allows for additional gene regulation c. Initial mRNA nucleotide sequence is called primary transcript/pre-mRNA/heterogenous nuclear RNA(hnRNA) d. Primary transcript is processed in three ways i. ii. Addition of nucleotides iii. Deletion of nucleotides 1) Before leaving the nucleus, the primary transcript is cleaved into introns and exons 2) Enzyme-RNA complex called snRNP's recognize nucleotide sequences at the ends of the 2) Enzyme-RNA complex called snRNP's recognize nucleotide sequences at the ends of the introns and form a spliceosome to remove them 3) Exons of some genes may be spliced together in different order allowing them to code for different polypeptides a) There are only 27,000 to 38,000 genes b) 120,000 proteins made possible by differential splicing of exons iv. Modification of nitrogenous bases e. Before the eukaryotic mRNA is even finished being transcribed, a 5' end cap is added using GTP f. 5' cap i. Serves as an attachment site in protein synthesis and as a protection against degradation by exonucleases g. h. Many more introns (24%) in the genome than exons (1.1%) 7. DNA Technology a. When heated or immersed in high concentration salt solution or high pH, the H-bonds connecting the two strands in DNA denature i. G-C bond has three h-bonds, so it takes more energy to denature ii. Denatured DNA is less viscous, denser, and more able to absorb UV light b. Separated strands will spontaneously associate with their original partner OR other complementary strand i. Nucleic acid hybridization: DNA-DNA, DNA-RNA, RNA-RNA ii. Enables scientists to identify nucleotide sequence by binding a known sequence with an unknown sequence c. Bacteria can produce restriction enzymes to protect themselves from viruses i. Bacteria protect their own DNA from these enzymes by methylation ii. Typically, restriction sites will be palindromic, read the same way both forwards and backwards iii. Most restriction enzymes cleave DNA strand unevenly, leaving complementary single stranded ends iv. These ends can reconnect through hybridization and are termed sticky ends and joined by ligase v. d. Recombinant DNA can be made long enough for bacteria to replicate and then placed within the bacteria using a vector, typically a plasmid or sometimes a virus i. Vector - DNA molecule used as a vehicle to artificially carry foreign genetic materia into another cell where it can be replicated and/or expressed another cell where it can be replicated and/or expressed e. To make a DNA library, take your DNA fragment, use a vector to insert it into a bacterium, and reproduce that bacterium like crazy. i. Now you have a clone of bacteria with your DNA fragment f. Not all bacteria take up vectors and not all vectors take up DNA fragments i. Therefore, a library may contain some clones that do not contain vectors or contain vectors that do not contain the recombinant DNA fragment ii. To screen for the appropriate clones, the original vector has an antibiotic resistance gene and a lac z gene 1) Once the vector is inserted into bacteria and the bacteria grow, if the bacteria did not take the vector, it will die due to no resistance gene. 2) In addition, if the donor DNA has been properly inserted, it will insert and disrupt the lacZ gene (by using a lacZ endonuclese) a) When grown, the bacteria will not be able to metabolize the x-gal, leaving it white iii. g. In order to find the proper DNA sequence to be inserted into the vector a probe is used i. A probe is a radioactively labeled complementary sequence of the desired DNA fragment ii. The radiolabeled clones are identified by laying them over photographic film which they expose and non-radiolabeled clones do not expose and non-radiolabeled clones do not h. Normal eukaryotic DNA cannot be inserted into a vector due to it possessing introns (bacteria have no mechanism for removing introns) i. It must be reversed transcribed from mRNA creating cDNA i. PCR i. Requires the base sequence flanking the ends of the DNA fragment be known, so that the complementary primers can be chosen ii. j. Southern Blotting i. Used to identify target fragments of known DNA sequence in a large population of DNA ii. DNA to be identified is cleaved into restriction fragments iii. Fragments are run through gel electrophoresis and then denatured by turning the gel alkaline iv. Nitrocellulose is used to blot the gel which transfers the resolved single stranded DNA fragments onto the membrane v. A radio-labeled probe with a nucleotide sequence complementary to the target fragment is added to the membrane k. Northern Blotting i. Same as southern blotting but identifies RNA fragments l. Western Blotting i. Detects a protein with antibodies m. Restriction fragment length polymorphisms identifies individuals as opposed to identifying specific genes i. DNA of different individuals possesses different restriction sites and varying distances between sites (polymorphic) ii. After fragmenting the DNA sample with endonucleases, a band pattern unique to an individual is revealed via Southern Blotting iii. DNA fingerprints used to identify criminals in court cases 8. The genetic code a. Start Codon: AUG i. Also codes for methionine b. Stop codon: UAA, UAG, UGA c. The
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