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What are the base pairs in DNA? Describe how the nitrogenous bases are linked and explain which pair is easier to separate.
Adenine pairs with thymine using 2 hydrogen bonds, guanine pairs with cytosine using 3 hydrogen bonds. A-T pairs are easier to separate since they are held together by less H-bonds than C-G pairs.
Describe Griffith’s experiment which confirmed DNA as the hereditary molecule.
Griffith injected mice with R-strain and S-strain bacteria. The mice injected with S-strain bacteria developed pneumonia and died, while mice injected with the R-strain stayed alive. Griffith then injected mice with heat-killed S-strain bacteria and a mix of the heat-killed S-strain and live R-strain bacteria. Mice injected with the heat-killed S-strain only stayed alive. while mice injected with the heat-killed S-strain and live R-strain died. Griffith concluded that the R-strain bacteria must have picked up a “transforming factor” from the heat-killed S-strain that made them harmful.
Describe Hershey and Chase’s experiment which confirmed DNA as the hereditary molecule.
Hershey and Chase used a bacteriophage and the bacteria E.coli. They labelled the protein coats on a bacteriophage with the radioisotope S-35, and labelled the phage DNA in another bacteriophage with P-32. Once the bacteria were infected by the bacteriophages, Hershey and Chase found that radioactivity was only found inside the bacteria infected by viruses containing the P-32 in their DNA. They concluded that DNA must be responsible for heredity.
What is a restriction enzyme? What is their purpose in bacterial cells?
A restriction enzyme is an enzyme that recognizes and binds to specific sequences of DNA, called restriction sites. In bacterial cells, their purpose is to remove foreign DNA left by a bacteriophage.
How do scientists use antibiotic resistance genes to confirm the presence of a plasmid in bacteria that have undergone a transformation procedure?
During bacterial transformation, bacteria are mixed with recombinant plasmids that carry antibiotic resistance genes. Some bacteria will transform and contain plasmids with the antibiotic resistance genes, while some will not transform and will instead take up a non-functioning plasmid. To determine which bacteria contain the desired plasmid with the antibiotic resistance genes, all bacteria from the transformation are placed on an antibiotic plate in bacterial selection. Bacteria without the desired plasmid will die, while bacteria that contain the desired plasmid will give rise to a colony.
Briefly describe each biotechnology:
bacterial transformation
gel electrophoresis
polymerase chain reaction.
Bacterial Transformation:
Transformation is the process of inserting a recombinant plasmid into a bacterial cell. First, bacteria are mixed with the recombinant plasmids. Next, the mixture is given a heat shock, which causes the bacterial membranes to become more permeable to the plasmids. Then, some bacteria will transform and contain the desired plasmid, while some will not transform and will take up a non-functioning plasmid.
Gel Electrophoresis:
Fragments of DNA are pulled through a gel matrix by an electric current, separating them by size. Many copies of DNA fragments of the same length form a band on the gel which can be seen by the human eye. The band is compared to a DNA ladder which shows where in the gel different lengths of fragments will appear. Fragment length is measured in base pairs (bp) or kilobase pairs (kbp).
Polymerase Chain Reaction (PCR):
PCR is a common technique used to make millions of copies of a particular region of DNA so it can be cloned or analyzed another way (DNA sequencing, gel electrophoresis). A mixture of template DNA, Taq polymerase, DNA primers that match parts of the target sequence, and DNA nucleotides are inserted into a machine called a thermal cycler. Then, denaturation, annealing, and extension occur.
Denaturation (96 C): Heat denatures the DNA strands, providing single-stranded templates.
Annealing (55-65 C): Cooling allows the primers to form H-bonds with their complimentary sequences on the single-stranded template DNA.
Extension (72 C): Raising the temperature allows Taq polymerase to synthesize new strands of DNA starting from the primers.
Describe the experiment performed by Meselson and Stahl that confirmed DNA replication is a semi-conservative process.
Meselson and Stahl cultured E.coli bacteria in a medium containing the heavy isotope N-15. The bacterial DNA then contained only “heavy” nitrogen. They then transferred the bacteria to a medium containing only N-14, a lighter isotope. They took a sample after the first DNA replication and another after the second replication, extracted the DNA from the bacteria in the samples collected, and centrifuged each DNA sample to separate the DNA molecules based on their mass. Their results showed that each daughter molecule contained a parent strand and a new strand, with molecules containing N-15/N-14 after the first replication, and N-15/N-14 and N-14 only after the second replication, proving DNA replication to be semi-conservative.
Describe the process of DNA replication for:
the leading strand (3’ to 5’)
the lagging strand (5’ to 3’)
The Leading Strand (3’ to 5’):
RNA primase makes an RNA primer at the origin. DNA polymerase adds nucleotides to the primer, building a new 5’ to 3’ strand in the direction of the replication fork until the entire strand is copied.
The Lagging Strand (5’ to 3’):
RNA primase makes an RNA primer at the origin. DNA polymerase adds nucleotides to the primer building a new 3’ to 5’ strand in the direction away from the opening fork. As the fork opens further, another RNA primase makes another RNA primer and DNA polymerase adds nucleotides heading away from the fork until it reaches the first RNA primer that had been added. The strand is built as a series of Okazaki fragments, each with an RNA primer and a segment of DNA.
Describe the role of the enzymes involved with DNA replication:
helicase
topoisomerase
RNA primase
DNA polymerase
DNA ligase
Helicase:
Helicase binds to the open DNA strand and moves into the fork, further unwinding the DNA molecule.
Topoisomerase:
As DNA strands open, the DNA double helix ahead of the replication fork winds very tightly. Topoisomerases function to release the tension by making breaks in the helix, unwinding the molecule, then sealing the breaks.
RNA primase:
RNA primase begins the process of elongation by building an RNA primer, and RNA segment complimentary to DNA at the beginning of the replication fork.
DNA polymerase:
DNA polymerase lll adds nucleotides to RNA primers made by RNA primase.
DNA polymerase l removes the RNA primers and replaces them with DNA nucleotides in termination.
DNA polymerase ll checks for errors in the DNA strand as it is being replicated by analyzing the shape of the DNA molecule.
DNA ligase:
DNA ligase catalyzes the formation of phosphodiester bonds between the newly placed nucleotides that replaced RNA primers and the DNA of the Okazaki fragments.
Describe the process of transcription:
initiation
elongation
termination/processing
Initiation:
RNA polymerase, the main enzyme involved in transcription, binds to the promoter region of DNA, found near the beginning of the gene, and separates the DNA strands to create a single-stranded template.
Elongation:
RNA polymerase “reads” the template strand, one base at a time, and builds an mRNA molecule out of RNA nucleotides that compliment the DNA template.
Termination/Processing:
RNA polymerase transcribes the terminator sequence into mRNA. This sequence causes the mRNA molecule to be released from RNA polymerase. In prokaryotes, the mRNA is ready for translation. In eukaryotes, this pre-mRNA must be modified into mature mRNA before leaving the nucleus for translation.
Describe the process of completing an mRNA from a pre-mRNA. Why does this occur only in eukaryotic cells?
Eukaryotic pre-mRNA needs modifications to become a complete mRNA, including a 5’ cap modification, 3’ tail modification, and splicing.
5’ Cap Modification
A modified G nucleotide is added to the first nucleotide at the 5’ end of the mRNA. This protects mRNA from breaking down and helps it attach to the ribosome.
3’ Tail Modification
An enzyme adds 100-200 A nucleotides to the 3’ end of mRNA. The poly-A-tail makes the mRNA more stable and helps it get exported from the nucleus to the cytosol.
Splicing/Alternative Splicing
Spliceosomes remove introns and paste together exons to make a final, mature mRNA.
More than 1 mRNA can be made from the same gene, it depends on which exons of a gene are pasted back together to make a mature mRNA.
Describe the process of translation:
initiation
elongation
termination
Initiation:
A small ribosomal subunit binds to mRNA. Initiator tRNA carrying the amino acid methionine, with the anticodon UAC pairs with the start codon AUG. The large ribosomal subunit completes the initiation complex with GTP, providing energy for assembly. The initiator tRNA starts in the middle slot of the ribosome, called the P site because it will hold the growing polypeptide.
Elongation:
A tRNA with the anticodon to match the next mRNA codon binds in the A site. A peptide bond forms between the 2 amino acids. The mRNA is pulled through the ribosome so the initiator tRNA is now in the E site, where it will exit the ribosome. The A site is now free for the next tRNA to bind, and the process continues.
Termination:
A stop codon in the mRNA (UUA, UAG, UGA) enters the A site. A protein called a release factor will cause the polypeptide chain to be released from the tRNA. The ribosomal subunits detatch from the mRNA.
How many nucleotides are required to code for each amino acid? How many for a complete polypeptide?
3 nucleotides in a codon are required to code for each amino acid.
What is an anticodon?
An anticodon is a set of 3 nucleotides that bind to a matching mRNA codon.
Which type of cell can undergo the processes of transcription and translation at the same time? Why?
Prokaryotic cells can undergo the processes of transcription and translation at the same time because they lack a membrane-bound nucleus, so transcription and translation occur simultaneously in the cytosol since the ribosomes are able to directly access mRNA as it is being transcribed.
Describe an operon.
An operon is a sequence of DNA containing a cluster of genes under the control of a single promoter. The genes are always expressed together or not at all. There are 3 components to an operon:
Promoter: Upstream sequence to which RNA polymerase binds
Operator: Segment of DNA where a repressor protein binds, blocking RNA polymerase
Structural Genes: Genes regulated by the operon
What is a transcription factor? How is it used to control gene expression?
A transcription factor is a protein that binds to specific DNA sequences to regulate the transcription of genes. It can activate RNA polymerase which causes the gene to be transcribed into mRNA which is then translated to a protein. It can also inhibit RNA polymerase, preventing gene expression.
How does histone methylation affect transcription? acetylation?
Histone Methylation:
Histone methylation involves adding a methyl group to a histone. Histone methylation winds the DNA closer to the histones, making transcription less likely to occur
Histone Acetylation:
Histone acetylation involves adding an acetyl group to a histone, making the histone more negative which pushes the DNA away from the histone, allowing for easier transcription. Deacetylation is the opposite.
What is a frameshift mutation? How do they occur?
A frameshift mutation is a genetic mutation that occurs when there is a deletion or insertion in a DNA sequence that shifts the way the sequence is read
What is a point mutation? How do they occur? Do they always have an affect on the polypeptide?
A point mutation is a genetic mutation that occurs when a single base pair is added, deleted, or changed. Proto-oncogene must mutate into oncogene within the gene to change the protein.
What are proto-oncogenes? What are oncogenes?
Proto-oncogenes are genes that code for proteins that stimulate cell growth and division.
Oncogenes are mutated proto-oncogenes that cause normal cells to become cancerous because of an increase in protein production or activity.
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