Exam #2 Study Guide
I. DNA replication
A. Why is it called semi-conservative?
1. DNA replication is semi-conservative because each helix that is created contains one strand from the helix from which it was copied. The
replication of one helix results in two daughter helices each of which contains one of the original parental helical strands.
B. Know the stages of replication and the enzymes involved
1. DNA replication begins at a fixed origin of replication (ori) and usually involves both DNA strands at the same time. The replication is initiated by addition of an RNA primer by the enzyme primase.
2. In the newly forming nucleic acid strand, nucleotides are added one at a time to the 3’-OH (the 3rd carbon of deoxyribose) end of the growing chain (in 5’ to 3’ direction) by the enzyme DNA polymerase.
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a) Polymerization reaction is another name for replication
3. The fixed position of the ori sites and the 5’ to 3’ direction of DNA synthesis makes it possible for only one out of two new DNA strands to be synthesized continuously. This strand is called the leading strand. The other strand (the lagging strand) is synthesized in pieces, called the Okazaki fragments.
4. Problems with Synthesis:
a) Need to produce single-stranded DNA (super unstable!)
(1) DNA Helicase is used to unwind the double-stranded DNA
b) Polymerase can only accomplish synthesis in one direction (5’ → 3’)
c) Needs a double-stranded primer to initiate the process of
(1) The primer then needs to be removed after synthesis and
replaced with a DNA sequence
a) Helicase: Uses the hydrolysis of ATP to "unzip" or unwind the DNA helix at the replication fork to allow the resulting single strands to be copied.
b) Primase: Polymerizes nucleotide triphosphates in a 5' to 3' direction. The enzyme synthesizes RNA primers to act as a template for future Okazaki fragments to build on to. If you want to learn more check out What does chromium do to your body?
c) DNA Polymerase III: In charge of synthesizing nucleotides onto the leading end in the classic 5' to 3' direction.
d) DNA Polymerase I: In charge of synthesizing nucleotides onto primers on the lagging strand, forming Okazaki fragments. However, this enzyme cannot completely synthesize all the nucleotides.
e) Ligase: This enzyme oversees "gluing" together Okazaki
fragments, an area that DNA Pol I is unable to synthesize.
f) Telomerase: Catalyzes the lengthening of telomeres; the enzyme includes a molecule of RNA that serves as a template for new telomere segments.
g) Nuclease: This enzyme oversees excising, or cutting out, unwanted or defective segments of nucleotides in a DNA sequence. h) Topoisomerase: This enzyme introduces a single-strand nick in the DNA, enabling it to swivel and thereby relieve the accumulated winding strain generated during unwinding of the double helix. i) Single Strand Binding Proteins: Responsible for holding the replication fork of DNA open while polymerases read the templates and prepare for synthesis. We also discuss several other topics like What is the size range of silt?
C. What are the Okazaki fragments?
1. Okazaki fragments are short, newly synthesized DNA fragments that are formed on the lagging template strand during DNA replication. They are complementary to the lagging template strand, together forming short double-stranded DNA sections
D. What is “the end replication” problem? What does it have to do with telomeres, telomerase and the Hayflick limit?
1. Replication of the ends of DNA molecules
a) The newly replicated lagging strand will generate a 3' overhang when the RNA primer is removed from the final Okazaki fragment, or if the lagging strand replication machinery cannot reach the end of the chromosome.
b) Thus, repeated rounds of replication produce shorter and
shorter DNA molecules in somatic cells.
2. Telomerase (reverse transcriptase)
a) In reproductive cells, embryonic stem cells and cancer cells, We also discuss several other topics like How can a society organize an economy?
telomere sequences are extended by the enzyme telomerase.
b) Telomerase is an enzyme (also called reverse transcriptase) that adds DNA sequence repeats ("TTAGGG" in all vertebrates) to the 3' end of DNA strands in the telomere regions, which are found at the ends of eukaryotic chromosomes.
3. The Hayflick limit or Hayflick phenomenon is the number of times a normal human cell population will divide until cell division stops. Empirical evidence shows that the telomeres associated with each cell's DNA will get slightly shorter with each new cell division until they shorten to a critical length.
II. Cell cycle, mitosis, meiosis
A. Know the stages of the cell cycle, mitosis, and meiosis (review Mitosis and Meiosis from the PPT presentations on BB). Know what happens to chromosomes at each step of mitosis and meiosis. Know the key differences between mitosis and meiosis.
1. Stages of the cell cycle
(1) G1: the period between mitosis and the initiation of nuclear DNA replication
(2) S: the period of nuclear DNA replication in preparation for cell division
(3) G2: the period between the completion of nuclear DNA replication and mitosis If you want to learn more check out What is the role of media in advertising participants?
(Video: https://www.youtube.com/watch?v=TKGcfbyFXsw) (1) Prophase
(1) Interphase: DNA synthesis and chromosome replication phase
(2) Meiosis I: separation of homologous chromosome pairs, and reduction of the chromosome number by half
(Video: https://www.youtube.com/watch?v=XGWL9jfPHJ8) (a) Prophase I
(b) Metaphase I: random alignment of homologous pairs of chromosomes along the metaphase plate
(c) Anaphase I: separation of homologous chromosome pairs, and the random distribution of chromosomes into newly divided cells—second mechanism of generating
genetic variation in newly formed gametes
(d) Telophase I
(3) Meiosis II
(Video: https://www.youtube.com/watch?v=mMCcBsSAlF4) (a) Prophase II We also discuss several other topics like Reappointment happens every how many years?
(b) Metaphase II
(c) Anaphase II
(d) Telophase II
(4) Fertilization: the fusion of haploid gametes
(5) Genetic Variation: consequences of meiosis
d) Comparison of Mitosis and Meiosis
(a) 2 Daughter Cells
(b) Chromosome number in each new cell is the same as
the parent cell
(c) Newly formed cells from mitosis are genetically the
same from one another and the parent cells
(a) 4 Daughter Cells
(b) Chromosome number in each new cell is half the
number in the parent cells
(c) Newly formed cells from meiosis are genetically
different from one another and from the parent cell
B. What are chromatids?
1. Just before cell division (and after DNA synthesis), the chromatin condenses into individual chromosomes. The dividing chromosomes appear as two chromatids.
2. The chromatids appear to be made of coiled loops of 20-30 nm-thick nucleoprotein (chromatin) fibers
C. What is the function of checkpoints in the cell cycle? What is the role of CDKs and cyclin in the cell cycle regulation?
1. Cyclins, cyclin-dependent kinases, and the cell cycle checkpoints (Video: https://www.youtube.com/watch?v=Jmqd9Qj_PTA)
(1) There are specific points in the cell cycle when the cycle can be halted (ex.: the G1 and G2 checkpoints).
(2) As a cell enters the G1 stage, proteins called cyclins and cyclin-dependent kinases (CDKs) are synthesized.
(3) CDKs, which exist at constant concentrations throughout the cell cycle, can only become functional if they form
associations with cyclins.
(4) Dimers: groups of proteins that work together to complete a job
(a) Heterodimers: dimers consistent of proteins that are
(i) Example: CDKs and cyclins
(b) Homodimers: dimers consistent of proteins that are
(5) The cyclin concentration in a cell varies throughout the
cycle, depending on the signals from the cellular environment. If the cyclin concentration at G1 is low, the cell goes into a
nondividing state (G0 phase).
(a) Cyclin levels are very low when the cell does not
need to divide
(6) CDKs signal the cell to go into the next stage of the cell
cycle by phosphorylating (supplying with energy) various other proteins involved in the cell cycle process. CDKs are regulated by changing concentrations of the cyclins.
D. Cell cycle breakdown and cancer:
1. malignant vs. benign tumors
a) Malignant tumors can spread to nearby tissues
b) Benign tumors are localized in an organ/tissue
(1) In Benign tumors, cell division stops at some because the cells do not spread to nearby tissues.
(2) If the breakdown of the cell cycle control system is severe (>5 mutations), the cells start to change too rapidly. They
become amoeba-like and can seep through the tissues and get into the bloodstream
c) The spread of cancer cells beyond their original site is called metastasis
(1) This happens to malignant tumor cells only because they are the only ones that spread to nearby tissues
2. proto-oncogenes and their cellular function
a) Proto-oncogenes promote normal cell division in their normal site
3. tumor suppressor genes and their cellular function
a) Tumor suppressor genes suppress normal cell growth and division
b) Mutations in these genes can release cells from their controlling functions (i.e. DNA repair, cell adhesion, or cell cycle inhibition) (1) Mutations in DNA repair genes impair the ability of the cell to repair any further mutations, thus accelerating the progression of cancer
(a) In cancer cells the G2 phase is extremely shortened, and the cells do not have enough time to scan for
c) Tumor suppressor genes usually behave as recessive alleles (i.e. BRCA1 and BRCA2)
(1) BRCA1 is a MARKER for the precursors to breast cancer, IT DOES NOT mean you will 100% develop the disease or that you currently have the disease
(a) It is an ovarian cancer gene
III. Genetic code, transcription, translation
A. Understand the relationship between the DNA and the inheritance of proteins.
B. What is the structure of the genetic code? What makes it redundant?
C. What is a gene? Know the molecular composition of a gene. 1. A gene is a sequence of nucleotide triplets of a DNA molecule bound by a start codon (ATG) and a stop codon (TGA, TAA, or TAG) that specifies a cellular product.
2. Most of the time, the cellular product “coded” in DNA is a protein, in which case the nucleotide triplets in a gene will specify a specific sequence of amino acids on a polypeptide chain.
3. A gene’s final product can, sometimes, be an RNA molecule (rRNA, tRNA).
4. A eukaryotic gene consists of exons (the actual nucleotide sequences that carry a message delivered to the site of polypeptide assembly) and introns (non-coding “spacer” sequences that take part in regulation of genetic expression at the mRNA level).
D. Know the components of the transcription machinery. Study the paper on the eukaryotic transcriptional machinery (promoters, activators, repressors, enhancers, silencers, can tell which ones are proteins and which ones are gene sequences to which respective proteins bind).
1. Promoters, enhancers, silencers, insulators (Nikitin’s Notes) a) The core promoter is the region at the start of a gene that serves as the docking site for the transcriptional machinery
b) The TATA box, found 25-30 base pairs upstream from the transcription start site (TSS), is the binding site for a transcription factor known as TATA-binding protein (TBP), which is itself a subunit of another transcription factor, called Transcription Factor II D (TFIID).
c) Enhancers and silencers function to activate and repress transcription, respectively
d) Proteins called activators and repressors can bind to the enhancers and silencers, thus affecting polymerase binding to the promoters
e) Insulators function to block genes from being affected by the transcriptional regulatory elements of neighboring gene
E. What happens to mRNA during post-transcriptional modification of the primary RNA transcript?
1. Post-transcriptional modification or Co-transcriptional modification is the process in eukaryotic cells where primary transcript RNA is converted into mature RNA.
a) A notable example is the conversion of precursor messenger RNA into mature messenger RNA (mRNA) that occurs prior to protein translation.
b) The process includes three major steps:
(1) Addition of a 5' cap
(2) Addition of a 3' poly-adenylation tail
c) This process is vital for the correct translation of the genomes of eukaryotes because the initial precursor mRNA produced during transcription contains both exons (coding or important sequences involved in translation), and introns (non-coding sequences)
F. Signal transduction pathway:
1. Cell Cycle and the signal transduction pathway
(Video: https://www.youtube.com/watch?v=89W6uACEb7M&feature=related) a) Cells are stimulated to divide by a variety of mechanisms, both internal and external
b) External signals are processed through a signal transduction pathway (STP)
(1) Cells do not normally have a means to respond to an
environmental event directly, they all need a pathway to create a response
(1) Every STP works the same way
(1) Insulin and its control in the sugar pathway
(a) Insulin: universal molecule and hormone (stimulates
growth during the infant phase) in all Mesozoic organisms
(2) Sex Hormones
(a) Steroid lipids, they are not proteins
(b) Harder to destroy, making a longer half life
2. What are transcription factors?
a) A transcription factor is a protein that binds to specific DNA sequences, thereby controlling the transcription of genetic information from DNA to mRNA.
b) Transcription factors perform this function either by promoting (as an activator), or blocking (as a repressor) the recruitment of RNA polymerase to specific genes.
c) A defining feature of transcription factors is that they contain DNA-binding domains (DBDs), which attach to specific sequences of DNA adjacent to the genes they regulate.
d) Transcription factors perform this function alone or with other proteins in a complex, by promoting (as an activator), or blocking (as a repressor) the recruitment of RNA polymerase (the enzyme that performs the transcription of genetic information from DNA to RNA) to specific genes
3. What are the components of the signal transduction pathway? a) There are four main components of an STP:
(1) Growth (stimulating) factors: a naturally occurring
substance capable of stimulating cellular growth, proliferation, and cellular differentiation. It is usually a protein or steroid hormone:
(a) Platelet-derived growth factor (PDGF)
(b) Insulin-like growth factor (IGF)
(2) Growth factor receptors
(a) Every type of stimulus has a receptor molecule
(b) The contact of the receptor molecule with the
stimuli changes the shape of the molecule and induces
(3) Relay molecules
(a) Dedicated group of kinases are used to relay and
amplify the signal to where it needs to go
(i) The kinases that are used in the relaying of
the message are phosphorylated as well and send an
“explosion” throughout the cell
(ii) Phosphorylated kinases can get through the
nuclear envelope and get the signals into the nucleus
(a) The proteins that are in the nucleus have
DNA binding abilities, and after the
phosphorylated kinases get into the cell, they
signal the transcription factors
(4) Transcription factors
(a) DNA-binding domain
(b) Bind to DNA and look for the genes that do
whatever the signal was sent for
G. Know the DNA translation details to the extent covered in lecture and in the PPT presentation.