Gregor Mendel – 1840’s Conclusion: Heredity material was packaged in discrete transferable units; came up with law of segregation and law of independent assortment.
Thomas Morgan – early 1900’s Discovered that fruit flies’ genes were associated with chromosome inheritance. Conclusion: Chromosomes were known to be composed of proteins and DNA, so genes must be one of these two macromolecules
FREDRICK GRIFFITH -1928 Experiment If dead (heat-killed) pathogenic bacteria was mixed in a culture with living harmless bacteria, the harmless bacteria would become deadly. Conclusion Transformation in bacteria allows a change in genotype and phenotype due to the assimilation of external DNA by the cell.
AVERY’S EXPERIMENT -1944 He separated the components of the heat-killed, deadly bacteria & divided it into smaller samples (proteins, lipids, carbohydrates, or nucleic acids) & left the other molecules intact. He then mixed each sample of the treated lethal strain with living samples of the non-lethal strain. AVERY’S CONCLUSION Only the DNA extract from the deadly bacteria would allow the live harmless bacteria to become deadly.
HERSHEY AND CHASE - 1952 Conclusion: The DNA molecule entered the bacteria cell & not the protein. This showed that DNA and not protein controls traits that are passed on.
CHARGAFF - 1947 Experiment: Studied the composition of DNA and the concentration of each of the nitrogenous bases. Conclusion: DNA base composition varies from one species to another; bases are not present in equal amounts in any one species but they are found in a predictable ratio; concentration of T=A and concentration of C=G.
Franklin and Wilkins Experiment: Took x-ray diffraction pictures of DNA in its different forms Conclusion: Discovered that the alpha form of DNA was double helix in structure.
Watson and Crick Experiment: Built a 3-D model that reflected the base pairing rules determined by Chargraff & the distance between bases suggested by Franklin’s X-ray photos. Conclusion: DNA was a double helix that made one full turn every 3.4nm with bases 0.34nm apart & sugar/phosphate molecules on the outside of the ladder.
Let’s make some DNA • Red = phosphate • White = deoxyribose • Yellow = adenine • Blue = thymine • Orange = cytosine • Green = guanine • Pink = uracil • Plastic connectors = hydrogen bonds
DNA Molecule Sugar and phosphate backbones are antiparallel (their subunits run in opposite directions) Adenine and guanine are purines (both have 2 organic rings) Cytosine and thymine are pyrimidines (both have 1 organic ring) Adenine forms 2 hydrogen bonds with thymine Cytosine forms 3 hydrogen bonds with guanine
What is the purpose of DNA Replication? • To produce a copy of DNA identical to the original in preparation for mitosis or meiosis.
Meselson and Stahl experiment 1st replication in the 14 N medium produced a band of hybrid. This eliminated the conservative model. 2nd replication produced both light and hybrid DNA this eliminated the dispersive model & supported the semiconservative model. Conclusion: DNA replication follows the semiconservative model.
E. coli vs Human DNA Replication E. coli Human 46 DNA molecules; each in a chromosome 6 billion nucleotide pairs Can replicate all chromosomes in a few hours • Has a single chromosome • 4.6 million nucleotide pairs • Can replicate its chromosome in less than an hour. Replication process is similar in prokaryotes and eukaryotes.
Some of the “players” involved… • DNA Polymerase- adds nucleotides to a preexisting chain. • Ligase- joins the sugar phosphate backbones of all Okazaki fragments. • Primase- synthesizes the primer that’s 5-10 nucleotides long. • Helicase- unzips the DNA • Topoisomerase-relieves the strain of overtwisting DNA by braking, swiveling, & rejoining DNA strands.
In order for replication to begin a primer is needed & it is an RNA primer The primer is about 5-10 nucleotides long The new DNA strand starts from the 3’ end of the RNA primer DNA Polymerase adds nucleotides to the preexisting strand
Replication occurs from 5’ to 3’ Leading strand- is made going towards the replication fork and is continuous Lagging strand- is made going away from the replication fork and is synthesized discontinuously, as a series of segments called Okazaki fragments. In eukaryotes Okazaki fragments are 100-200 nucleotides long. Leading strand needs only one primer & lagging strand needs a primer for each Okazaki fragment.
DNA polymerase I – removes the primer and replaces it with DNA nucleotides; one by one Ligase- joins sugar-phosphate backbone.
How is replication of one side of each double strand different than the other? • Because bases can only be added in the 5’ to 3’ direction, the 3’ to 5’ strand must be assembled in fragments that are later annealed together by a ligase protein.
Proofreading and Repairing DNA Errors amount to 1 in 10 billion nucleotides in the final DNA product Initial pairing error amount to 1 in 100,000 more common.
How is the new strand ensured to be identical? • The bases are matched in a consistent pattern • The daughter strands are half new, half old • There is a proofreading mechanism that checks for errors in both strands.
Why are mistakes made? • Spontaneous chemical changes under normal conditions. • Exposure to mutagens • EX: cigarette smoke and X-rays There are many different DNA repair enzymes. -E. coli has 100 known repair enzymes -Humans have 130 identified repair enzymes
Teams of enzymes detect & repair damaged DNA, such as this thymine dimer (often caused by UV radiation), which distorts the DNA molecule. A nuclease enzyme cuts the damaged DNA strand at 2 points, & the damaged section is removed. Repair synthesis by a DNA polymerase fills in the missing nucleotides. DNA ligase seals the free end of the new DNA to the old DNA, making the strand complete.
Replicating the ends of DNA molecules This kind of thing does not occur prokaryotes with a circular chromosome The primer on the end is removed but can’t be replaced with DNA because DNA polymerase can only add nucleotides to the 3’ end of a preexisting polynucleotide
Because of the 5’ to 3’ constraint & the need for each replication event to by initiated with a primer, one side of the replication will be inhibited at the end. What will happen to the length of the chromosome after numerous replications? It will get shorter
What is done to compensate for this problem? Telomeres (located at the ends of DNA molecules) are made of repeated units that are non-coding so that, as they get shorter, no genes are lost. The enzyme telomerase lengthens telomeres in germ cells Cells can only go through a limited number of replications before they are put to death. plus also
Let’s Replicate!!! with narrative
How Does a Gene Become a Protein? With a lot of help, I’ll tell you that!!!
Let’s start with the 2 nucleic acids involved DNA and RNA These molecules have structural similarities and differences that define function.
Comparison of DNA to RNA DNA Made of nucleotides Connected by covalent bonds to form a linear molecule from 5’ to 3’ Contains ribose Nitrogen bases A,U,C, & G Single stranded Able to travel out of the nucleus (eukaryotes) RNA • Made of nucleotides • Connected by covalent bonds to form a linear molecule from 5’ to 3’ • Contains deoxyribose • Nitrogen bases A,T,G, & C • Double stranded • Restricted to the nucleus (eukaryotes)
Is Uracil a purine or a pyrimidine? • Since thymine is a pyrimidine and in essence uracil replaces thymine in RNA it would make sence that uracil is also a pyrimidine. Wouldn’t it?
The sequence of the RNA bases, together with the structure of the RNA molecule, determines RNA function. • mRNA: carries information from the DNA to the ribosome. • tRNA: are molecules that bind specific amino acids and allow information in the mRNA to be translated to a linear peptide sequence. • rRNA: are molecules that are functional building blocks of ribosomes. • RNAi: plays a role in regulation of gene expression at the level of mRNA transcription. • To be discussed later
Genetic information flows from a sequence of nucleotides in a gene to a sequence of amino acids in a protein This occurs in two parts
Transcription • Is the synthesis of RNA using one side of a segment of a DNA strand. • The DNA segment serves as a template. • The RNA made is mRNA. • It is antiparallel to the DNA template • mRNA is made from 5’ to 3’ (reading the DNA in a 3’ to 5’ direction). • This is all completed in the nucleus.
RNA Polymerase opens the DNA strands & joins the RNA nucleotides that are complimentary to the DNA template. • Needs no primer to begin • Bacteria have a single type of RNA polymerase that synthesizes all types of RNA. • Eukaryotes have at least 3 types of RNA polymerase.
Termination of Transcription • Differs between bacteria and eukaryotes • In bacteria • Go through a terminator sequence in DNA. • Once RNA polymerase hits the terminator signal it releases from the DNA and the mRNA that was being made. • In eukaryotes • RNA Polymerase II transcribes a sequence on the DNA which codes for a polyadenylation signal (AAUAAA) in the pre-mRNA. • About 10-35 nucleotides later proteins cut the pre-mRNA from the polymerase & undergoes processing…
Modifying of the pre-mRNA in Eukaryotes • Both ends of the mRNA transcript are altered. • In most cases, certain interior sections are cut out and the remaining pieces are spliced together. • These actions produce a mRNA molecule that is ready for action!!!
RNA Processing For ribosome binding • The cap and tail: • help facilitate the mRNA leaving the nucleus & help protect the • mRNA strand from degradation by hydrolytic enzymes. • help the ribosomes attach to the 5’ end of the mRNA
More RNA Processing Average length of transcription unit = 8000 nucleotides, but average size protein is 400 amino acids so only about 1,200 nucleotides long. This indicates long noncoding stretches of nucleotides which happen to be interspersed in the coding segments. • RNA splicing: • Removing portions of the RNA molecule & putting the other ends together. • The parts to be “edited out” are interspersed between the coding segments. • The segments that intervene with the coding segments are called: introns • The segments that will eventually be expressed & exit the nucleus are called: exons
HOW IS PRE-mRNA SPLICING CARRIED OUT? A small nuclear ribonucleoproteins (snRNPs) recognize the splice sites. These are located at the end of introns. Composed of RNA & protein Several snRNPs and additional proteins form a larger assembly: spliceosome These molecules release introns & join the exons
Why have introns? • One idea is that introns play regulatory roles in the cell. • Splicing process is necessary for mRNA to leave the nucleus. • Consequence for having introns & exons • Genes are known to give rise to 2 or more different polypeptides, depending on which segments are treated as exons during RNA processing = alternative RNA splicing.