Genetics & medicine notes

These notes contain the second unit of notes from BIO 1010 at RPI.

DNA

DNA (deoxyribonucleic acid) contains all the instructions used to build a living organism. It is a molecular blueprint for a living organisms, organized in a double helix shape, and first discovered in 1953.

DNA is built of nucleotides - molecules that are like letters in the DNA alphabet. There are four nucleotides, A (adenine), C (cytosine), G (guanine) and T (thymine). Each nucleotide is composed of a phosphate group, sugar (deoxyribose) and nitrogenous base. Each "ladder" in the DNA helix contains two base pairs - A is complementary with T, and C is complementary with G.

Nucleotides can be (confusingly classified) into two categories:

• Purines: adenine and guanine (A, G)
• Pyrimidines: thymine and cytosine (C, T)

The most important functions of DNA include:

• The storage of genetic information
• The ability to replicate to facilitate cell division
• The ability to be translated into amino acids to create proteins
• Generate genetic variation by adding minor alterations to gene information over time

Polarity of DNA

We can describe the direction of DNA by referring to its 5' and 3' ends. The 5' end is the direction of the phosphate attached to the 5th carbon of the sugar, whereas the 3' end is the direction of the free hydroxyl group attached to the 3rd carbon of the sugar.

DNA replication

DNA replication always takes place in the 5' to 3' direction, and takes several steps:

• Proteins bind to locations on the DNA called origins of replication to form the pre-replication complex
• The two strands of DNA are separated
  • Helicase proteins untwist and unzip DNA along both directions of the origin using ATP as their energy source
  • Single-stranded binding proteins prevents DNA from degradation during replication
• Within the replication bubble (the unzipped region of DNA) DNA replication begins:
  • Single-stranded binding protein (SSBP) keeps replication bubble open
  • Primase starts a new stand of RNA primer (a nucleotide sequence that is complimentary to the DNA strand) in the 5' -> 3' direction
  • DNA polymerase III synthesizes DNA from the RNA primers by moving 5' -> 3'
  • Disconnected fragments of strands synthesized by DNA polymerase III are called Okazaki fragments and their RNA is replaced by DNA using DNA polymerase I
  • Ligase creates phosphodiester bonds between Okazaki fragments to link strands together into a continuous strand
  • DNA polymerase III uses proofreading to check for mismatched base pairs

Packaging DNA

The DNA of a single cell completely unwound would take up to 2 meters in length. Thus, DNA is tightly wound around proteins called histones that link together to form nucleosomes. These are then bound together, then looped and condensed to finally form chromosomes.

Central Dogma of Biology

The central dogma of biology is that DNA transcribes to form RNA, RNA translates to form proteins.

The genetic code

The genetic code is the set of rules that converts RNA base sequences to amino acid sequences that are used to make proteins. There are 20 amino acids, a fact first suggested by George Gamow. The code is based on sets of 3 nucleotides, called codons, and the code is redundant, meaning more than one codon can code for the same amino acid.

Genes and genetic variation

A gene is a group of base sequences that code for specific amino acids that form proteins. A gene determines the phenotype (observable characteristics) of an organism and is heritable.

Mutations

A mutation is a change in the nucleotide sequence of DNA caused by random chance. In some cases, it can be base substitutions - one nucleotide could be replaced with another. In other cases, it can be deletions or insertions, which can lead to significant changes in the amino acids sequence and correspondingly the proteins produced. For instance, a single base substitution causes sickle cell anemia.

Transcription & Translation

• RNA polymerase copies DNA to mRNA (messenger RNA)
• mRNA heads outside the nucleus to ribosomes
• Using the mRNA transcript as template, tRNA molecules attach to it and link to amino acids, which are joined into a polypeptide
• The polypeptide is folded into a unique 3D shape to create a protein

RNA vs. DNA

• RNA uses uracil instead of thymine
• RNA's single-stranded nature and 3D shape allows it to serve many different roles in cell functioning unlike DNA which has a single function

Transcription

Each gene has a promoter sequence (which indicates where to start transcription), a terminator sequence (which indicates where to end transcription), and genetic instructions. Transcription begins near the promoter sequence. The RNA polymerase attaches to the promoter sequence, separates the strands of DNA, and then synthesizes the RNA by adding nucleotides that are complementary to the template strand of the DNA. Then, the RNA polymerase continues to move along the DNA until it reaches the terminator sequence.

After transcription, what happens depends on the type of cell. In prokaryote cells, no further processing of the RNA is necessary; in eukaryote cells, a 5' cap, 3' tail is added, and introns are removed by the spliceosome, making the RNA mRNA, and the mRNA floats out of the nucleus and enters the cytoplasm.

Sometimes, genes undergo alternative splicing, which is when certain non-intron sequences are spliced out as well as introns, which allows changing the protein created during translation.

Translation

Ribosomes attach to the ribosome binding sites on mRNA, and are used to synthesize polypeptides that make up proteins. To do this, they take 3-base sequences in RNA, called codons. Molecules called tRNA help translate codons to amino acids. To do this, they have two ends:

• One end has a complementary sequence of 3-base sequences, called anticodons, which attach to the mRNA sequence
• The other end carries an amino acid corresponding to the codon

The first codon translated is always an AUG codon, known as the start codon. As the ribosome moves along the mRNA, it adds more amino acids together by creating peptide bonds to form a polypeptide. It stops once it reaches a stop codon. While being synthesized, polypeptides fold into a unique 3D shape, becoming a protein. The unique shape of a protein allows it to perform its function.

The table of which codons match with which amino acids is known as a codon table, and is shown below:

(Source: ResearchGate)

DNA repair

Every day, errors in DNA accumulate to up to a quintillion a day. DNA repairs are conducted by enzymes. For example, RNA polymerase can sometimes add mismatched bases, but other proteins conduct mismatch repair. Exposure to environmental toxins, UV radiation, etc. can cause chemical errors in DNA, which are fixed in a process called nucleotide excision repair.

Human karyotype

Human has 23 pairs of chromosomes, 22 pairs of autosomal chromosomes and 1 pair of sex chromosomes.

Mitosis vs Meiosis

Both mitosis and meiosis are types of cell replication. Mitosis produces genetically-identical diploid cells via chromosomal duplication, is a 4-stage process, and is used by all somatic (body) cells. Meiosis produces genetically-distinct haploid cells, is a 8-stage process, and is used by gametes (sperm and egg cells)

Gene regulation

Every cell in the body has a copy of the same genome. But each cell has a different function. How do we explain that cells can do different things, if they all use the same genome? The answer is with gene regulation, which determines which genes are turned on and off and thus determines which proteins to be made, changing the function of a cell.

In bacteria, related genes can be placed together to form an operon, which share a promoter and terminator.

Sugars are often used by cells as their source of energy. Lactose must be broken before being metabolized, whereas glucose can be metabolized directly. One common area of gene regulation for bacteria is in producing beta-galactosidase and beta-galactoside permease, two enzymes crucial in being able to transport break down lactose within bacteria. These enzymes are coded for in the lac operon.

One form of gene regulation occurs in transcription. Transcription starts at the promoter, a region of DNA that RNA polymerase attaches to. Next to the promoter is the operator, a section of DNA to which a repressor protein can bind. When lactose is not present, the repressor binds to the operator and prevents the RNA polymerase from transcribing the operon. This represses the gene. When lactose is present, however, lactose binds to the repressor, and changes the shape of the repressor. This makes the repressor unable to bind to the operator, allowing the operon to be transcribed. Mutations in both the repressor and operator can interfere with this process.

Another form of gene regulation also occurs in transcription. Bacteria typically have a preference for glucose over lactose, but when glucose levels are low, levels of cyclic AMP (cAMP) molecule, an indicator of "hunger", increase. The promoter for the lac operon is a weak promoter, which means RNA polymerase binds relatively weakly to the promoter. To counter this, there is an activation site before the promoter, which an activator protein can bind to, in order to make the weak promoter behave like a strong promoter. When cAMP levels are high, cAMP binds to the activator protein, allowing the activation protein to bind. Meanwhile, when cAMP levels are low, cAMP does not bind to the activator protein, preventing the activation protein from binding.

Eukaryotic gene regulation

First, DNA is packed very tightly around histones and bundled in tight coils around chromosome. By keeping DNA tightly packaged, it cannot be transcribed, which suppresses genes.

Second, upstream (before) the promoter are DNA sequences called enhancers. Transcription factors are proteins that can bind to the enhancer to help RNA polymerase bind to the promoter. They can be considered analogous to activator proteins in prokaryotes.

Homeobox genes

A fly's body has 3 sections - head, thorax, and abdomen. HOX (homeobox) genes are transcription factors that regulate the body plan of an embryo to grow along the head-tail direction. The expression of the HOX genes are crucial for many animals, not just flies - humans have it as well - to develop a proper body plan.

Many organisms share much of the same DNA. The reasons for this are:

• Descent from common ancestor
• Common cellular mechanisms across all organisms
• Key genes are conserved because mutations would reduce fitness

Epigenetics

Epigenetics is the study of heritable changes in gene expression that do not involve changes to the underlying DNA sequence. That is, an organism's DNA (genome) can remain unchanged, but gene expression can change. The epigenome is how your genome is expressed. Epigenome is like "software" if genome is "hardware". Many factors can affect the epigenome:

• Methyl groups can attach to DNA and activate or repress gene expressions
• Acetyl groups can attach to histone packaging of DNA and make genes accessible
• Small RNA molecules can attach to genes and turn genes on and off

RNA interference is when intereference RNA (iRNA) molecules destroy mRNA become they reach ribosomes and causes translation to not occur. This is often used to prevent viral mRNA to prevent viruses from injection their DNA into a cell's DNA, thus protecting a cell from a virus. iRNA can be further divided into micro-interference RNA (miRNA), which can silence many genes, and short-interference RNA (siRNA), which silence specific genes.

Genomics

DNA sequencing is the process of reading the sequence of nucleotides of DNA.

The Sanger sequencing method was first invented in 1975. After denaturing DNA using heat, multiple copies are made of a segment using PCR (polymerase chain reaction). Primers are attached, and 4 polymerase solutions are added. This allows the DNA to grow complementary chains until reaching special nucleotides that are missing the hydroxyl on the 3' carbon, at which the DNA chain stops growing. Using electrophoresis followed by autoradiography allows the sequence to be read. Computers helped tremendously with speeding up this process.

Shotgun sequencing was an improvement over Sanger sequencing that allowed for a DNA sequence to be split up into many small fragments that were then Sanger sequenced. The fragments were then assembled back together.

Next-generation sequencing (NGS) was a further improvement on shotgun sequencing that use fluorescently-labeled bases to identify each nucleotide by shining a different wavelength of light.

Sequencing has grown tremendously cheaper over the years since the human genome project in 1988, and costs under $100 a day. Project Encode took the results from the human genome project and assigned a biochemical function to all the functional elements of the human genome. The roadmap of epigenomes did the same for the human epigenome. Finally, the genome 10K project extended sequencing to 10,000 vertebrates, and the Earth biogenome project aims to extend sequencing to all eukaryotic life.

In addition, DNA's ability as a biology storage medium may one day allow for alternative forms of long-term storage of digital information.

Genetic origins of disease

Many diseases are caused by altered or misshapen chromosomes. Nondisjunction refers to the failure of chromosomes or chromatids to separate during meiosis, and can cause zygotes with altered numbers of chromosomes, such as trisomy (3 chromosomes) or monosomy (1 chromosome). Trisomy 21 (trisomy in the 21st chromosome) causes Down syndrome, which is characterized by susceptibility to disease, a shortened life span, and cognitive impairment.

Personalized medicine

The idea of personalized medicine is to customize a treatment for the right patient at the right time. The goal is to improve effectiveness and efficiency of healthcare delivery and improving health outcomes and quality of life.

Single-nucleotide polymorphisms (SNPS) are variants of a single base pair in the DNA, and are the foundation of personalized medicine. A personal SNP profile can be used to know risks of developing diseases. Research areas that contribute to personalized medicine include:

• Discovering biomarkers - chemical signatures of things occuring in the body
• Profiling the microbiome - analyzing an individual's microrganisms to adjust treatments
• Pharmacogenomics - using a patient's genetic profile to predict a drug's efficacy, guide dosage, and improve patient safety
• Clinomics - using computer science to accurately interpret genetic data and guide healthcare decisions
• Epigenomics - examines which factors act on individual genes, and how changes in the epigenome affect our health

Cancer

All cancers derive from uncontrolled cell division of a single cells. DNA in these cells are mutated such that they cause the cells to continually divide. Thus, all cancers are genetic diseases.

Cancer typically developers from an accumulation of mutations rather than a singular mutation. Many of these mutations are in genes coding epidermal growth factor receptors (oncogenes) which binds to cells to cause cells to divide. Additionally, mutations often occur in tumor suppressor genes (which are transcription factors), which are often defective in cancer cells. The Cancer Genome Atlas is one project created to learn more about the specific mutations cause cancer by genotyping tumors. The project caused the realization of that the type of tissue does not define the underlying genetic profile, and drugs must target a specific genetic profile for a given individual.

There are a variety of cancer treatments. Targeted cancer treatments include:

• Hormone therapy - slow or stop the growth of hormone-sensitive tumors
• Signal transduction inhibitors e.g. TKIs (tyrosine kinase inhibitors) - blocks signals passed from one molecule to another inside a cell, leading to cancer cell death
• Angiogenesis inhibitors - blocks the growth of new blood vessels to tumors
• Immunotherapy - trigger the immune system to destroy cancer cells
• Monoclonal antibodies - deliver molecules that bind to cancer cells to mark the cancer cells for destruction

CRISPR

CRISPR - clustered regularly interspaced short palindromic repeats - is a part of the prokaryotic adaptive immune system. It is composed of short repeats of DNA that have spacer DNA in between. The spacer DNA match bacteriophage DNA (viruses that target bacteria), allowing the bacteria to remember past invaders. The bacteria uses it to create proteins that use special sequences to identify foreign DNA and cut the DNA. By injecting custom DNA, genes can be inserted or inactivated.

Stem Cells

Stem cells are undifferentiated cells that can be induced to develop into multiple types of cells. Stem cells can be used to replace damaged bodily tissues and organs, and is called regenerative medicine. Tissue-specific stem cells can be found in most tissues, but typically become one specific type of tissue. Embryonic stem cells, by contrast, can turn into any cells. Induced pluripotent stem cells are regular tissue-specific stem cells that can behave as embryonic stem cells.

Synthetic biology

Synthetic biology is the application of engineering processes to biology to create systems that do not already exist in the natural world. It is a combination of genetic engineering and engineering.

Emerging infectious diseases

An epidemic is a disease that affects a large number in one area. A pandemic is an epidemic that has spread to multiple areas. Zoonotic diseases are infectious diseases that can be transmitted from other vertebrates to humans, and form the majority of diseases. Zoonotic diseases go through 5 stages, where stage 1-2 are animal to human transmission, and 3-4 are human to human transmission. Emerging infectious diseases can be:

• Virus-based: RNA viruses have higher mutation rates than DNA viruses. Viruses spread by attaching and entering into human cells using spike proteins, replicating using the cell's transcription and translation machinery, and releasing many copies of themselves
• Bacterial - antibacterial resistance is a growing issue