7.1 Flashcards
the Hershey & Chase Experiment
In 1952, two researchers, Alfred Hershey and Martha Chase carried out experiments that helped confirm DNA is the genetic material of life.
-They made use of radioisotopes (radioactive forms of elements that decay over time at a predictable rate)
-In their experiment, they grew bacteriophage viruses in two different types of cultures, one included radioactive phosphorus -32 and the viruses produced in culture had DNA in their core, with detectable phosphorus. The other had Sulfur 35,- which was present in the protein outer coat of the virus but since DNA does not include sulfur, S35it was not inside the outer coat
-The two types of bacteriophage with the different radioisotopes were then allowed to infect the bacterium known as Escherichia coli. The E.coli infected with the 32P bacteriophage had radioactivity inside their cell wall. However, the E.coli infected with the 35S had no radioactivity within their cell wall.
Because DNA contains phosphorus and not sulfur, this allowed Hershey and Chase to conclude that DNA, not protein, was the genetic material of the bacteriophage.
The Meselson and Stahl’s 1950 experiment
an experiment to prove that DNA replication was semi conservative and it was first shown in Escherichia coli and subsequently in higher organisms, such as plants and human cells.Bacteria was grown in a medium with heavy nitrogen. DNA contains heavy nitrogen and the bacteria was also placed with light nitrogen. After replication of DNA, the resulting DNA contains both light and heavy nitrogen.
the components of a nucleotide
Nucleotides are composed of three subunit molecules: a nitrogenous base, a five-carbon sugar (ribose or deoxyribose), and a phosphate group consisting of one to three phosphates. The four nitrogenous bases in DNA are guanine, adenine, cytosine and thymine
DNA structure outline including base pairing, bonds, double helix
DNA is a double-stranded molecule formed in the shape of a double helix
Each strand of DNA is composed of a backbone of alternating phosphate and deoxyribose molecules. These two molecules are held together by a covalent bond called a phosphodiester bond or linkage.
In this type of bond, each nucleotide is attached to the previous one. This produces a chain of DNA. The reaction between the phosphate groups on the 5’ carbon and the hydroxyl group on the 3’ carbon is a condensation reaction- where water is released.
Each time a nucleotide is added, it is attached to the 3’ carbon end. A free 5’ carbon end with a phosphate group attached with a free 3’ carbon end with a hydroxyl group forms the alternating sugar-phosphate backbone of each chain.
the antiparallel nature of DNA structure
A DNA molecule is composed of two antiparallel strands. One strand is 5’ to 3’ and the other is 3’ to 5’. DNA strands can only be assembled in the 5’ to 3’ direction because of the action of polymerase III.
- For the 3’ to 5’ template strand, the new DNA strand is formed as described above. The process is continuous and relatively fast, and the stand produced is called the leading strand. The other new strand forms more slowly and is called the lagging strand.
The leading and lagging strands are assembled concurrently. However, there is a slight delay in the synthesis of the lagging strand.
Formation of the lagging strand involves fragments and an additional enzyme called DNA ligase”
The leading strand is assembled continuously towards the progressing replication fork in the 5’ to 3’ direction.
The lagging strand is assembled by fragments being produced moving away from the progressing replication fork in the 5’ to 3’ direction.
Okazaki Fragments
The fragments of the lagging strand are called Okazaki fragments, after the Japanese scientists who discovered them.
Primer, primase, and DNA polymerase III are required to begin the formation of each Okazaki fragment of the lagging strand, and to begin the formation of the continuously produced leading strand.
The primer and primase are only needed once for the leading strand because it is produced continuously.
Once the Okazaki fragments are assembled, an enzyme called DNA ligase attaches the sugar-phosphate backbones of the lagging strand fragments to form a single DNA strand.
Semi conservative DNA replication
begins at special sites called origins of replication. Bacterial DNA is circular and has no histones, and has a single origin. Eukaryotic DNA is linear, has hostones, and has thousands of origins. The presence of multiple replication origins greatly accelerates the copying of large eukaryotic chromosomes.
Replication process follows:
Replication begins at the origin, which appears as a bubble because of the separation of the two strands. The separation or ‘unzipping’ occurs because of the action of the enzyme helicase on the hydrogen bonds between nucleotides.
At each end of a bubble there is a replication fork. This is where the double-stranded DNA opens to provide the two parental DNA strands that are the templates necessary to produce the daughter DNA molecules by semi-conservative replication.
The bubbles enlarge in both directions, showing that the replication process is bidirectional. The bubbles eventually fuse with one another to produce two identical daughter DNA molecules.
Semi-conservative replication means that each parental DNA strand acts as a template to form a complementary strand so eventually two identical daughter DNA molecules are formed.
Eukaryotic DNA molecules are quite long and replication begins at a large number of sites along the molecule. This allows replication to occur at a much faster rate.
Enzymes to know the function of:
DNA Polymerase I: Exonuclease activity, removes RNA primer and replaces with newly synthesized DNA
DNA Polymerase I: Repair function
DNA Polymerase III: Main enzyme that adds nucleotides from the 5’ to 3’ direction
Helicase: Opens the DNA helix by breaking hydrogen bonds between the nitrogenous bases.
Ligase: Seals the gaps between the Okazaki fragments to create on continuous DNA strand (lagging strand)
Primase: Synthesizes RNA primers needed to start replication.
Sliding Clamp- Helps to hold the DNA polymerase in place when nucleotides are being added,
Topoisomerase: Helps relieve the stress on DNA when winding by causing breaks and then realising the DNA.
Single-strand Binding proteins (SSB): Bands to single-stranded DNA to avoid DNA rewinding back.
Leading strand
The strand on the left hand side that goes from 3’ to 5’. DNA helicase unwinds and separates the hydrogen bonds between the complementary base pairs.Next, DNA polymerase adds complementary DNA nucleotides. The newly synthesized strand complementary to the original strand is antiparallel. DNA polymerase is only able to add a nucleotide on the 3’ end of the previous nucleotide. As DNA helicase continues to unwind, the DNA polymerase continues to build the newly synthesized strand in the 5’ to 3’ direction- the same direction in the movement towards the replication fork. The construction of the leading strand takes place continuously.
Lagging strand
DNA Helicase unwinds and breaks the hydrogen bonds between the complementary base pairs. DNA Polymerase also as before adds nucleotides, but because of the antiparallel nature of the original DNA strand, DNA polymerase has to work in the opposite direction on the lagging strand. Because DNA polymerase can only add a new nucleotide to the 3’ end of the previous nucleotide, as DNA Helicase breaks the hydrogen bonds moving upward, DNA polymerase works in the opposite direction, moving down. This results in the completion of the lagging strand in smaller fragments. The lagging strand is completed discontinuously. The fragments created are known as okazaki fragments. In order to finish the replication process, the fragments need to be joined together so Enzyme DNA Ligase joins them together.
Chromatin=condensed DNA
The DNA molecules of eukaryotic cells paired with a type of protein called a histone. Histones help in DNA packaging.
The DNA is attracted to the histones because DNA is negatively charged and histones are positively charged. Between the nucleosomes is a single string of DNA. There is often a fifth type of histone attached to the linking string of DNA near each nucleosome. This fifth histone leads to further wrapping (packaging) of the DNA molecule, and eventually to highly condensed or supercoiled chromosomes.
When DNA is wrapped around the histones and then further wrapped in even more elaborate structures, it is inaccessible to transcription enzymes. Therefore, the wrapping or packaging of DNA brings about a regulation of the transcription process. This allows only certain areas of the DNA molecule to be involved in protein synthesis.
Types of DNA
Regions of DNA that do not code for proteins include areas that act as regulators of gene expression, introns, telomeres, and genes that code for transfer ribonucleic acids (tRNAs).
There may be as many as 100,000 replicates of a certain type per genome. If this repetitive DNA is clustered in discrete areas, it is referred to as satellite DNA.
This repetitive DNA is mostly dispersed throughout the genome. At the present time, as far as we can tell these dispersed regions of DNA do not have any coding functions. They are transposable elements, which means they can move from one genome location to another.
jumping genes are able to change their position within a chromosome, they never actually detach from the DNA molecule they are part of.
frequency of genome
sequence Frequency Protein-encoding genes (exons) 1-2 introns 24 Highly repetitive sequences 45 Structural DNA 20 Inactive genes (pseudogenes) 2 other 7-8
DNA Profiling & STRs
Regions that show specific variation of DNA are known as polymorphisms. To profile Polymorphisms, we often look at a group of 13 very specific loci referred to as short tandem repeats (STRs)
STRs are short, repeating sequences of DNA, normally composed of 2-5 base pairs.
Base pairing
Base pairing: linked by hydrogen bonds:
Thymine and Adenine
Guanine and Cytosine
Adenine and Guanine are double-ring structures known as purines. Cytosine and thymine are single-ring structures known as pyrimidines.
This is complementary base pairing and occurs because of the specific distance that exists between the two sugar-phosphate chains. Two hydrogen bonds link adenine and thymine; three hydrogen bonds link cytosine and guanine.
