BIO 3010 Midterm I Study Questions

Darwin described the process of natural selection. What is needed for this process to occur?

Natural selection requires variation, fitness differences, and inheritance.

There must be variation among individuals in a population for some trait

Differences in the value of that trait must be consistently associated with difference in fitness, meaning reproductive success through survivorship ro fecundity.

Trait must be heritable meaning there must be a consistent relationship between the trait value in parents and their offspring

How does our modern understanding of inheritance differ from the pre-Mendelian ideas of inheritance?

Modern genetics: Hereditary information is stored in DNA and passed through genes and alleles as discrete units.

Pre-Mendelian ideas:

1. Preformationism

   1. A miniature offspring, called a homunculus, already existed in the sperm or egg.

2. Inheritance of acquired  characteristic

   1. Proposed by Lamarck, in which traits acquired during an individual’s life time could be passed to offspring

3. Blending inheritance

   1. Proposed that offspring were a mixture of their parents

Modern genetics instead explain inheritance through genes and alleles that are transmitted from parents to offspring  (passed between generations s discrete genetic information

What is a gene? What is an allele?

A gene is a specific region of DNA that contributes to a biological function or trait, often by encoding a functional RNA or protein. An allele is a specific variant of a gene found at a particular locus. For example, a gene influencing pea color can have different alleles that contribute to yellow or green phenotypes.

What are Mendel’s “Laws” of Segregation and Independent Assortment and what analyses led him to these laws?

Mendel’s Law of Segregation states that the two alleles of a gene separate, or segregate, during meiosis, so each gamete receives one allele; the alleles then reunite during fertilization. The Law of Independent Assortment states that alleles of different genes generally assort independently during gamete formation, so inheritance of one gene does not affect inheritance of another. Mendel developed these laws by performing controlled crosses with pea plants and analyzing the consistent phenotypic ratios among offspring across generations, leading him to conclude that hereditary factors are discrete and follow predictable patterns of transmission.

What “complications” can cause phenotype numbers and proportions to differ from those expected by the Laws of Segregation and Independent Assortment?

Several complications can cause observed phenotype numbers and proportions to differ from simple Mendelian expectations. First, many traits are continuous rather than discrete, because they are often influenced by multiple genes (polygenic inheritance). Second, alleles are not always transmitted at the expected ratios because some alleles can affect meiosis, gamete survival, or offspring survival, causing transmission distortion. Third, many alleles are neither simply dominant nor recessive but have additive effects, including incomplete dominance and codominance, where heterozygotes have intermediate or combined phenotypes. Fourth, alleles at different genes do not always assort independently because genes that are physically close together on the same chromosome are linked and tend to be inherited together; crossing over can separate them. Finally, heritable material may exist outside the simple nuclear genetic model, such as mitochondrial or chloroplast DNA, which can produce inheritance patterns that differ from Mendelian expectations.

What are some of the major technological adavances that have facilitated genetic and genome analyses since ~1970?

Since ~1970, several major technologies have transformed genetic and genomic analysis. DNA sequencing, particularly Sanger sequencing beginning in 1977, made it possible to determine the nucleotide sequence of specific genes. PCR, developed in 1983, allowed scientists to exponentially amplify very small amounts of DNA so they could be analyzed or used in experiments. The Human Genome Project, conducted from the 1980s through 2000, demonstrated that an entire human genome could be sequenced. More recently, targeted genome editing such as CRISPR has allowed researchers to specifically alter genes. Together, these technologies helped move genetics into the modern era of genomics and made DNA analysis increasingly accessible outside research laboratories, although they also raise ethical concerns.

Explain how PCR works

PCR amplifies a specific DNA sequence through repeated cycles of denaturation, annealing, and extension. During denaturation, heat seperates the two DNA strands. During annealing, the temperature is lowered so primers bind to complementary sequences on the template DNA. During extension, a heat-stable DNA polymerase such as Taq polymerase adds nucleotides to the primers to make new DNA strands. Repeating these three steps in a cycle cases the target DNA to be amplified exponentially, allowing a very small starting amount of DNA to become a large amount that can be analyzed.

Describe the basic structure of nucleotides and DNA or RNA

A nucleotide consists of a sugar, a phosphate group, and a nitrogenous base, while a nucleoside consists only of a sugar and base. DNA contains deoxyribose and the bases A, T, G, and C, wheras RNA contains ribose and uses U instead of T. Nucleotides are connected by phosphodiester bonds to form long chains. DNA usually consists of two antiparallel strands forming a double helix, with sugar-phosphate backbones on the outside and complementary bases held together by non-covalent hydrogen bonds on the inside. RNA is usually single-stranded but can fold into complex 3D structures.

What observations suggested that particular bases pair in DNA?

Chargaff’s observations provided evidence that particular DNA bases pair with one another. When he measured DNA from different organisms, he found that the amount of adenine was approximately equal to thymine and the amount of guanine was approximately equal to cytosine, giving A:T and G:C ratios close to 1. Although the overall base composition differed between organisms, these relationships remained constant. We now understand that A pairs with T through two hydrogen bonds and G pairs with C through three hydrogen bonds. These complementary pairs allow the two antiparallel DNA strands to form a stable double helix.

What are examples of chemicals that interact with specific features of the DNA helix?

The DNA double helix has major and minor grooves that expose chemical features of the bases for interactions with other molecules. DAPI binds to the minor groove, especially in A-T-rich regions, and becomes strongly fluorescent, allowing DNA to be visualized in cell nuclei under a fluorescence microscope. Furanocoumarins interact with DNA and can cause DNA damage, interfering with replication and, with UV light, causing cross-linking between DNA strands. These examples show how DNA structure allows chemicals to specifically recognize and affect DNA.

Define “life” at least according to NASA

According to NASA's 1979 definition, life is “a self-sustaining chemical system capable of Darwinian selection.” This definition emphasizes that life must maintain itself chemically while also having the ability to undergo variation and selection. Therefore, life requires some form of genetic material capable of transmitting information and allowing new variation to arise.According to NASA's 1979 definition, life is “a self-sustaining chemical system capable of Darwinian selection.” This definition emphasizes that life must maintain itself chemically while also having the ability to undergo variation and selection. Therefore, life requires some form of genetic material capable of transmitting information and allowing new variation to arise.

What are the essential features of a genetic material and what observations point to RNA as a particularly good candidate for being the first genetic material; are there any caveats to this model?

A genetic material must be able to store information, express information, replicate, and accommodate new variation so that Darwinian selection and evolution can occur. RNA is considered a plausible first genetic material because its nucleotide sequence can store information, while its ability to fold into complex three-dimensional structures gives it chemical and catalytic functions. RNA can act as a ribozyme; examples include catalytic ribosomal RNAs, RNase P, and self-splicing introns. Ribosomal RNAs are also highly conserved across the tree of life, suggesting very ancient origins. Laboratory experiments have additionally produced RNA molecules with replicase activity and shown that modified RNA bases can help produce small peptides. However, the model has major caveats: modern nucleotides require complex chemistry and activation, phosphates and some bases may have been scarce, ribose is problematic, RNA is unstable under some conditions, and naturally occurring RNA polymerase ribozymes are not known. Therefore, the first genetic material may have been a precursor resembling RNA rather than modern RNA itself.

What observations and experimental evidence suggest that self-replicating ribozymes might have existed in nature?

Evidence for the possibility of self-replicating ribozymes comes from both modern ribozymes and laboratory evolution. Modern RNAs such as ribosomal RNA, RNase P, and self-splicing introns demonstrate that RNA can perform enzyme-like catalytic functions. However, no known modern ribozyme has the complete replicase activity required by the RNA World hypothesis. Importantly, experimental evolution has shown that RNA replicase activity can be selected for in the laboratory. Researchers repeatedly selected RNA molecules that could add nucleotides to a template, introducing variation between rounds. After only seven rounds of selection, they produced a ribozyme capable of adding many nucleotides, especially in triplet units. These experiments show that substantial replicase activity can evolve through selection, making self-replicating ribozymes plausible, although they do not prove that such ribozymes actually existed in nature.

What early evidence showed that genetic material could be transferred between organisms, and that such material is DNA?

Griffith demonstrated transformation using Streptococcus pneumoniae. Live smooth (S) bacteria were virulent and killed mice, whereas live rough (R) bacteria were nonvirulent. Heat-killed S bacteria did not kill mice, but a mixture of heat-killed S and live R bacteria killed mice, and S-type bacteria could subsequently be recovered. This showed that some heritable material from the dead S cells had been transferred to the live R cells, a process called transformation. Griffith did not identify the molecule responsible. However, soon after, Avery et al. then isolated material from heat-killed S cells and selectively destroyed lipids, proteins, RNA, or DNA. Transformation still occurred after destruction of lipids, proteins or RNA, but destruction of DNA with DNase eliminated transformation. Therefore, DNA was identified as the transforming substance and strong evidence was provided that DNA is the genetic material.

Explain the experimental results that led to the “one gene, one enzyme” hypothesis. What modern genetic and genomic data suggest the original hypothesis was framed too narrowly?

Beadle and Tatum used X-ray mutagenesis to generate mutations in haploid yeast and screened for mutants defective in amino acid biosynthesis. Mutants were first grown on complete medium to identify viable cells and then tested on minimal medium lacking amino acids. Mutants that could not grow on minimal medium were supplemented with individual amino acids to determine which pathway was defective. They identified several arginine-deficient mutants, including ARG-E⁻ and ARG-H⁻. By testing whether arginine or pathway intermediates such as ornithine, citrulline, and argininosuccinate could rescue each mutant, they determined that different genes were required for different steps in the arginine biosynthetic pathway. This supported the “one gene, one enzyme” hypothesis: each gene appeared to specify an enzyme required for a particular biochemical reaction. However, modern genetics shows that this was too narrow because not all genes encode enzymes (could encode proteins or functional RNA) and one gene can produce multiple RNA and protein products through processes such as alternative splicing. Thus, the modern view is that genes encode functional products, including RNAs and/or proteins, that contribute to cellular functions and phenotypes.

What are the fundamental properties of the genetic code and how were they “deciphered”?

The genetic code was deciphered through experiments that determined how nucleotide sequences are organized and translated into proteins. Brenner investigated how the code is structured by creating small insertions and deletions in a gene using the mutagen proflavin. When one or two nucleotides were added or removed, the gene stopped functioning because the reading frame was shifted. However, when three nucleotides were added or removed, gene function could be maintained or restored. This demonstrated that the code is read in groups of three nucleotides, called triplet codons, and it also provided evidence for frameshift mutations. A frameshift occurs when an insertion or deletion is not a multiple of three, changing how all the following codons are read. Brenner's experiments also showed that codons are non-overlapping, meaning each nucleotide is read as part of only one codon. Nirenberg and colleagues then worked out how specific nucleotide sequences correspond to specific amino acids. They used synthetic RNA molecules in cell-free translation systems and observed which amino acids were produced when different RNA sequences were translated. By testing RNAs with different repeating nucleotide sequences, they could determine that the RNA was being read three nucleotides at a time and identify which codons corresponded to particular amino acids. From these experiments, the six main properties of the genetic code were established: (1) triplet codons — three nucleotides specify one amino acid; (2) non-overlapping codons — each nucleotide belongs to only one codon; (3) polarities of codons and amino acids correspond — the order of codons corresponds to the order of amino acids, with the 5′ end of the mRNA corresponding to the N-terminus of the protein and the 3′ end corresponding to the C-terminus; (4) degeneracy — more than one codon can specify the same amino acid; (5) three stop codons — UAA, UAG, and UGA terminate translation; and (6) a start codon — AUG marks where the reading frame begins and where translation starts. Finally, mutations can change the genetic message: a missense mutation changes one amino acid to another, a nonsense mutation changes a codon into a stop codon, and a frameshift mutation changes the reading frame because of an insertion or deletion that is not a multiple of three. Brenner's experiments were especially important for discovering this frameshift principle and showing how the structure of the genetic code determines the effect of mutations.

What are the different elements comprising a eukaryotic gene? What differs between eukaryotic and prokaryotic genes?

A typical eukaryotic gene contains a core promoter, transcription start site (TSS), 5′ UTR, exons, introns, coding sequence, 3′ UTR, and transcription termination region. It can also contain cis-regulatory elements located upstream, downstream, or within the gene. The core promoter is where the transcription machinery, including transcription factors and RNA polymerase, is recruited. The TSS is the first nucleotide transcribed into RNA. The 5′ UTR is transcribed but not translated. Exons remain in the mature mRNA, while introns are removed by RNA splicing. The coding sequence contains the information translated into protein, and the 3′ UTR remains in the mature mRNA but is not translated.

A typical prokaryotic gene is simpler and generally contains a promoter, TSS, 5′ leader sequence, coding sequence, stop codon, and 3′ UTR/termination region, with few or no introns. Unlike eukaryotic genes, prokaryotic genes are often organized into operons, allowing multiple genes to be transcribed from one promoter. Eukaryotic genes also commonly have cis-regulatory elements that can act from much farther away, whereas prokaryotic regulatory elements are generally located closer to the genes they regulate.

The region of a gene constituting exons and introns is sometimes referred to as the "gene body." What are differences between exons and introns (composition, function, etc.). Why can it be useful to define the region of a gene body, as opposed to the gene itself?

Exons are regions of DNA that, after transcription and RNA processing, are represented in the final/mature mRNA. Some exons contain coding sequence that contributes to the protein, while others contain untranslated sequence such as the 5′ or 3′ UTRs. Introns are regions between exons that are transcribed into the initial RNA but are removed during RNA processing and therefore do not appear in the mature mRNA. Together, the exons and introns make up the gene body. Defining the gene body is useful because the complete functional definition of a eukaryotic gene can be difficult: cis-regulatory elements can occur far upstream, downstream, or even within introns, sometimes very far from the coding region. Therefore, it can be difficult to determine exactly where a gene begins and ends. The gene body provides a more practical way to refer to the defined region containing the exons and introns, rather than trying to define the entire functional gene based on all of its potentially distant regulatory elements.

What are cis-regulatory element and what do they do? Why do you think they most often need to be detected experimentally, rather then being clearly identifiable in the DNA sequence itself?

Cis-regulatory elements are short DNA sequences that regulate the expression of a gene and are located on the same DNA molecule as the gene they regulate. They are binding sites for transcription factors, which interact with the transcription machinery and influence whether and how much a gene is transcribed. Cis-regulatory elements can be located upstream, downstream, or within introns, and they can sometimes be very far from the gene they regulate. They are often difficult to identify simply by looking at the DNA sequence because they are short and highly variable. Unlike coding sequences, which follow a relatively precise genetic code, cis-regulatory sequences can tolerate variation and may differ in how strongly they function. Therefore, researchers often need to use experiments to determine whether a particular DNA sequence actually regulates a gene and how it affects transcription.

Explain the nature of three major challenges facing the Human Genome Project at its inception (or that face any large-genome sequencing project); how was each challenge overcome with specific methods as sequencing technology evolved?

The Human Genome Project faced three major challenges. First, the genome was enormous, about 3.6 Gb, while early Sanger sequencing could read only a few hundred bases at a time. This was addressed by increasing sequencing throughput, first with improved fluorescent Sanger sequencing and then with second-generation Illumina sequencing, which could generate millions to billions of short reads simultaneously. Second, random sampling meant that some regions would be missed or sequenced too few times, so the genome had to be sequenced at high coverage, meaning each region was sampled many times to increase confidence that the entire genome was represented. Third, the resulting short sequence reads had to be assembled into the correct order, which was especially difficult when the genome contained repetitive or nearly identical sequences. The Human Genome Project addressed this by using bacterial artificial chromosomes (BACs) to divide the genome into manageable ~150–300 kb pieces, sequence each piece, and assemble them into a larger tiling path of overlapping contigs. Later, third-generation long-read sequencing such as PacBio and Nanopore reduced this assembly problem by producing much longer reads that can span repetitive regions.

How does "Sanger sequencing" work and what modifications to the original method contributed to increased efficiency (and safety)?

Sanger sequencing uses DNA replication to determine the sequence of a DNA template. A DNA polymerase extends an oligonucleotide primer using normal dNTPs, but the reaction also contains a small amount of ddNTPs (dideoxynucleotides). When a ddNTP is incorporated, DNA synthesis stops because it cannot be extended further. Because ddNTPs are present at low abundance, different DNA molecules stop at different positions, producing fragments of many different lengths. Originally, the four ddNTP types were placed in separate reactions, and the fragments were separated by gel electrophoresis and detected using a radioactive label, producing an autoradiograph from which the sequence could be read. The method became much more efficient and safer when radioactive labeling was replaced with fluorescently labeled ddNTPs. All four ddNTPs could then be included in one reaction because each had a different fluorescent dye. Capillary electrophoresis separated the fragments by size, and a detector identified the fluorescent color of each fragment, producing an electropherogram that could be read by a computer. These modifications increased automation, throughput, and read length to roughly 250–800 bases per sequence, while eliminating the need for radioactive materials.

Sanger sequencing is sometimes referred to as "first generation" sequencing, whereas several methods of "second generation" (sometimes referred to as "next generation") sequencing have been devised. Currently the most commonly used of these is Illumina Corporation's Solexa sequencing. In what ways does this method differ from Sanger sequencing?

Sanger sequencing produces fewer, longer reads and uses ddNTPs and electrophoresis, while Illumina produces millions to billions of shorter reads using massively parallel sequencing. Sanger sequences DNA fragments individually, whereas Illumina sequences many amplified clusters simultaneously on a flow cell. Illumina therefore produces vastly more sequence data much faster than Sanger.

Second generation sequencing (Illumina/Solexa) sequencing differs from Sanger sequencing in scale, setup, and how sequences are detected. Sanger sequences a small number of DNA templates in a liquid reaction using chain-terminating ddNTPs, then determines the sequence by separating DNA fragments by size. Illumina uses massively parallel sequencing: DNA fragments are attached to a flow cell and amplified into clusters. It uses reversible fluorescent terminators for sequence-by-synthesis, detecting each incorporated nucleotide by fluorescence before the next cycle. Thus, Illumina produces millions to billions of short reads simultaneously, giving it much higher throughput than Sanger.

What is meant by "third generation" sequencing and what approach used in the Human Genome Project did these methods replace? What distinguishes the two third generation methods we went over in class?

Third-generation sequencing refers to long-read sequencing methods that can read much longer DNA molecules than Sanger or Illumina. These methods replace the BAC-based approach used in the Human Genome Project, where large DNA fragments were individually sequenced and then assembled. The two methods are PacBio HiFi and Nanopore sequencing. PacBio uses sequence-by-synthesis: DNA is copied repeatedly by a polymerase, and fluorescent nucleotides are detected as they are incorporated. Repeated copies are combined to produce a highly accurate consensus sequence. Nanopore does not use DNA synthesis; instead, DNA passes through a protein pore, and changes in ionic current are used to determine the sequence. Thus, PacBio is highly accurate through repeated sequencing, while Nanopore can produce much longer reads.

Describe two ways in which the current state of sequencing technology enables the goals of "precision medicine" — which we have not yet discussed but you could easily learn about — ways that would have been impossible when Frederick Sanger first published his sequencing method in 1977.

Modern sequencing enables precision medicine by allowing doctors to use an individual's genetic information to guide disease diagnosis and treatment. First, massively parallel sequencing can rapidly sequence an individual's genome and identify disease-associated genetic variants, allowing doctors to assess genetic risk or explain the cause of disease. Second, sequencing can identify genetic differences that affect how an individual responds to a treatment, allowing therapy to be better matched to that person's genome. These approaches were impossible with Sanger sequencing in 1977 because sequencing an entire human genome was far too slow and expensive.

Early estimates of gene number varied widely. What do whole genome sequences that have been annotated for genes tell us about actual numbers of genes, and what kinds of genes they are? What else makes up the genome and about how much of the genome consists of protein coding genes vs. other types of features?

Early estimates of the number of genes in the human genome ranged from ~40,000 to 2 million. Whole-genome sequencing and gene annotation showed that humans have approximately 20,500 protein-coding genes and 22,000 non-coding genes, plus about 13,000 pseudogenes. Thus, the lower end of the early estimates was close when considering both protein-coding and non-coding genes. However, protein-coding genes make up only about 1–2% of the ~3.2 Gb human genome. Most of the genome consists of other types of DNA, including non-coding and repetitive DNA, with about half of the genome made up of repetitive sequences such as transposable elements and microsatellites.

Define "locus" and "allele." What do these terms mean when applied to non-protein coding DNA (like microsatellites)?

A locus is a specific, identifiable location in the genome, while an allele is an alternative version of that locus. These terms can apply to non-protein-coding DNA as well as genes. For example, a microsatellite may contain repeated CA sequences at a particular locus. Different individuals may have different numbers of CA repeats, producing larger or smaller alleles. An individual with the same repeat length on both homologous chromosomes is homozygous, while an individual with two different repeat lengths is heterozygous. Microsatellites usually have no obvious function, but their variation makes them useful as genetic markers.

Genome browsers like Ensembl, NCBI. or UCSC. provide a wealth of information. What are some of the features that can be readily accessed usingsuch a browser? Do these browsers indicate every functionally important part of a gene?

Genome browsers such as Ensembl, NCBI, and UCSC provide information about the organization and variation of genes. They can show a gene’s name and chromosomal location, direction of transcription, exons, introns, 5′ and 3′ UTRs, transcriptional start site, start and stop codons, coding sequence, protein sequence, and flanking DNA. They can also show mRNAs mapped back to the genome, alternative transcript isoforms, known insertions and deletions, other genetic variants, repeated sequences, transposable elements, chromosome bands, gene density, and reference sequences. However, genome browsers do not necessarily show every functionally important part of a gene. The gene model shown may be incomplete or somewhat arbitrary, and the canonical transcript is only one reference model. A single gene may produce multiple alternative RNA transcripts or isoforms, so the browser’s displayed model may not represent every transcript or every functionally important feature.

Do genes ever overlap in the genome? Why or why not?

Yes, genes can overlap in the genome because DNA has two strands, and genes can be encoded on either strand. A gene on one strand may be transcribed in one direction, while a gene on the opposite strand is transcribed in the other direction. Their gene bodies can therefore overlap even though they produce different RNA transcripts. Most commonly, exons of one gene overlap introns of another, but exonic overlap can also occur. When exons overlap, mutations and evolutionary changes are more constrained because they must preserve the function of both genes.

What are the cellular and phenotypic consequences of prenatal Zika virus infection?

Prenatal Zika virus infection targets neural stem cells and neural progenitor cells, which must rapidly divide during brain development. Zika generally arrests these cells in S phase, allowing the virus to exploit the cell’s replication machinery for its own replication. As a result, DNA damage and other cell-cycle problems accumulate. When infected cells attempt to enter mitosis, they take much longer to progress, and chromosome segregation becomes abnormal. The resulting nuclei may be polyploid, and many cells fail to complete mitosis and die, increasing cell death. The loss of neural progenitor cells reduces the number of cells available to build the developing brain, so the brain does not expand normally and has reduced tissue, producing severe microcephaly.

How do growth factors promote the G1 to S transition in normal cells and what roles are played by cyclins/CDKs, E2F/Rb and the p53 pathway during normal development or in the case of DNA damage?

Growth factors (ligands) promote the G1-to-S transition by binding receptor tyrosine kinases, which activate signaling pathways that increase transcription of cyclin genes, including Cyclin D and Cyclin E. Cyclins bind and activate CDKs, allowing them to phosphorylate target proteins. During G1, Cyclin D–CDK4 and Cyclin E–CDK2 phosphorylate Rb, causing Rb to release the E2F transcription factor. E2F then activates genes required for DNA synthesis and S-phase progression. If DNA damage is detected, the p53 pathway prevents the cell from entering S phase. p53 induces CDK inhibitor proteins such as p21, which inhibit CDKs and prevent Rb phosphorylation. Rb therefore remains bound to E2F, keeping E2F inactive and stopping DNA replication from beginning. If the damage is severe, p53 can also activate genes that cause apoptosis. Loss of functional RB or TP53 can remove these controls and promote uncontrolled cell-cycle progression and cancer.

What is "Peto's paradox," what is the evidence that variation in tumor suppressor copy number may "solve" the paradox, and why haven't humans evolved to have even more tumor suppressor gene copies than we do already?

Peto’s paradox is the observation that body size and lifespan are not strongly correlated with cancer risk across species. Large, long-lived animals such as elephants have many more cells and more time for mutations to occur, so they should have more opportunities to develop cancer, yet they do not show the expected increase in cancer risk. One possible explanation is that elephants have increased tumor-suppressor protection. For example, elephants have approximately 20 copies of TP53, while humans and some smaller mammals have about two copies. Elephants also have duplications of other genes involved in DNA damage recognition and repair, cell-cycle checkpoints, and telomere maintenance. These extra copies may provide greater protection against damaged cells becoming tumors, helping balance the cancer risk associated with having more cells and a longer lifespan.

Evolution of additional copies of tumor suppressor genes may have been limited in humans due to possible trade-offs associated with having extra copies that would hinder the growth, regeneration, and development processes by increasing the probability of halting cell division and initiating apoptosis. Therefore, the existing number of copies may represent a balance between cancer protection and maintaining normal cell growth and function.

What are origins of replication and what does it mean for one to be "licensed"? Are there particular characteristics that make a site likely to be licensed? Are all sites that are licensed actually used?

An origin of replication is a region of DNA where replication begins. Because eukaryotic chromosomes are large and linear, many origins are needed to be activated. A potential origin is first recognized by the origin recognition complex (ORC), which recruits other proteins, like the MCM complex, CDC6, and others to form a pre-replication complex (PreRC). When this complex is assembled, the origin is considered licensed, meaning it is prepared and available for replication. Sites are more likely to be licensed when they are in accessible chromatin that allows the replication machinery to bind and load the necessary proteins. In eukaryotes, there is not one specific DNA sequence that defines every origin; instead, licensing depends on features of the DNA and its surrounding chromatin. However, just because the origin is now licensed does not mean that replication will begin at that site. Not every licensed origin is actually used. Some origins are constitutively used, some are inactive or dormant despite being licensed, and others are used only in particular cell types or circumstances.

Once a licensed origin has been selected as a site for replication, what factors are needed to get the job done and how and why do some differ between strands?

DNA replication is semi-conservative, meaning that each newly formed DNA double helix contains one parental strand and one newly synthesized strand. After a licensed origin is activated, MCM proteins act as helicases to unwind the parental DNA and create a replication fork. RPA, a single-strand binding protein, binds the separated strands to prevent them from re-annealing. Because DNA polymerases cannot begin synthesis on their own, polymerase α first lays down an RNA primer, which provides the starting point for DNA synthesis. DNA polymerases can only add nucleotides in the 5′ to 3′ direction. On the leading strand, polymerase ε follows the replication fork and synthesizes DNA continuously. On the lagging strand, polymerase δ must synthesize DNA away from the replication fork, so it cannot make one continuous strand. Instead, polymerase α repeatedly creates RNA primers and polymerase δ produces short sections of DNA called Okazaki fragments, making the lagging strand discontinuous. The RNA primers are later removed by FEN1, the resulting gaps are filled with DNA, and DNA ligase joins the DNA fragments by completing the phosphate backbone. PCNA, the sliding clamp, helps hold the newly synthesized DNA in place during replication. The leading and lagging strands therefore require different modes of synthesis because the two parental DNA strands run in opposite directions while DNA synthesis can occur only 5′ to 3′.DNA replication is semi-conservative, meaning that each newly formed DNA double helix contains one parental strand and one newly synthesized strand. After a licensed origin is activated, MCM proteins act as helicases to unwind the parental DNA and create a replication fork. RPA, a single-strand binding protein, binds the separated strands to prevent them from re-annealing. Because DNA polymerases cannot begin synthesis on their own, polymerase α first lays down an RNA primer, which provides the starting point for DNA synthesis. DNA polymerases can only add nucleotides in the 5′ to 3′ direction. On the leading strand, polymerase ε follows the replication fork and synthesizes DNA continuously. On the lagging strand, polymerase δ must synthesize DNA away from the replication fork, so it cannot make one continuous strand. Instead, polymerase α repeatedly creates RNA primers and polymerase δ produces short sections of DNA called Okazaki fragments, making the lagging strand discontinuous. The RNA primers are later removed by FEN1, the resulting gaps are filled with DNA, and DNA ligase joins the DNA fragments by completing the phosphate backbone. PCNA, the sliding clamp, helps hold the newly synthesized DNA in place during replication. The leading and lagging strands therefore require different modes of synthesis because the two parental DNA strands run in opposite directions while DNA synthesis can occur only 5′ to 3′.

Why do chromosomes shorten with repeated replication and what are the properties of telomeres, telomerase and other factors that allow cells to deal with this problem? What are the cellular and organismal consequences of telomeres that become too short with time in healthy individuals and why are some cell types more susceptible to these consequences than others?

Chromosomes shorten with repeated replication because DNA polymerase cannot fully copy the ends of linear chromosomes. After the last RNA primer on the lagging strand is removed, there is no DNA upstream to fill the remaining gap. Telomeres are repetitive DNA sequences at the ends of chromosomes that act as protective buffers. Their single-stranded G-rich overhang can fold back to form a T-loop, while the Shelterin complex helps protect the chromosome end from being recognized as damaged DNA. Telomerase can prevent or slow telomere shortening. It contains TERT, a reverse transcriptase, and TERC, an RNA template, and uses these components to add telomeric DNA repeats back onto the chromosome end.

Telomerase is highly active in germ cells, stem cells, and some rapidly dividing cells but is low or absent in most somatic cells. Therefore, telomeres gradually shorten in frequently dividing somatic cells. When telomeres become critically short, cells can enter replicative senescence or undergo apoptosis. This reduces tissue renewal and contributes to age-related loss of tissue function. Cells that divide frequently are more affected because their telomeres shorten faster, while cells with active telomerase can better maintain their telomeres. Telomere shortening also helps prevent cancer by limiting how many times a cell can divide, although cancer cells can bypass this limit, often by reactivating telomerase.

What are the pathological consequences of telomere maintenance disorders within and between generations?

Telomere maintenance disorders occur when mutations impair proteins or RNA required for maintaining telomeres, including components of telomerase. Because somatic cells normally have relatively little telomerase, they can develop problems as their telomeres become critically short. These disorders can also affect the germline, creating consequences between generations. If an affected individual passes on chromosomes whose germline telomeres were not properly maintained, the next generation begins with shorter telomeres, and subsequent generations can begin with progressively shorter telomeres. As a result, disease tends to appear earlier and become more severe in each successive generation. In very severe cases, the disorder can eventually interfere with the ability to reproduce.

What are the phases of mitosis and their associated events?

Mitosis occurs after DNA replication and ensures that the two sister chromatids of every replicated chromosome are distributed equally to the daughter cells. In prophase, chromosomes become condensed and organized for mitosis. Prometaphase follows as chromosomes become organized for interaction with the spindle. During metaphase, chromosomes line up along the metaphase plate, which is important for accurate segregation. During anaphase, sister chromatids separate and move toward opposite daughter nuclei. During telophase, the separated chromosomes are associated with the two daughter-nucleus regions, followed by cytokinesis, which divides the cell. Mitosis separates sister chromatids rather than homologous chromosomes, and the cells remain diploid, or 2N, throughout the process.

How do cohesins regulate sister chromatid pairing, how are cohesin distributions and dynamics regulated across cell cycle phases, and what are the consequences of dysfunction in cohesin stabilization?

Cohesin complexes maintain contact between sister chromatids so that replicated chromosomes remain paired and can later be separated properly. Cohesin is loaded during G1, before DNA replication. During S phase, ESCO1 or ESCO2 acetylates cohesin, stabilizing the complex and establishing cohesion between the sister chromatids after replication. During prophase, cohesin is partially removed from chromosome arms but remains at the centromeres. At anaphase, Separase removes the remaining cohesin, allowing sister chromatids to separate. If cohesin stabilization is defective, sister chromatids may not remain properly paired, causing abnormal chromosome segregation and chromosome loss.

Robert's syndrome (resulting from mutations in ESCO II) is just one example of many different congenital malformations that affect human populations. How does age-specific mortality from congenital malformations compare to other causes of death? What is meant by "collectively common but individually rare" and what are the implications for studying such disorders?

Congenital malformations are the leading cause of death during the first year of life, exceeding other causes such as low birth weight. The phrase “collectively common but individually rare” means that congenital disorders are common when considered together, but each individual disorder is rare because a specific disorder may result from a particular gene or allele and produce a specific set of traits. This makes these disorders difficult to study because researchers may have very few patients with any one disorder, making it harder to identify causes and organize research.

Though not described explicitly in class, consider what factors might lead to different societal and individual impacts of congenital malformations between locations with substantial medical (and scientific) infrastructure to locations without such infrastructure? To what extent can modern medicine or other factors cure, ameliorate, or avoid these disorders?

Congenital malformations can affect people differently depending on where they live. In places with good medical care, doctors can often find problems before or soon after birth and treat or improve some of them with medicine or surgery. Some can also be prevented, such as certain birth defects by getting enough folic acid during pregnancy. However, not all congenital malformations can be cured, especially when they are caused by changes in development or genetics. In these cases, doctors may only be able to manage the condition and improve the person's quality of life. In places with less medical care, people may not have access to these tests or treatments, so the same condition can have more serious effects.