Chapter 18: Molecular Genetics

Frederick Griffith

Conducted an experiment which demonstrated that bacteria can be reprogrammed through a process called transformation. This suggested that something inside the cell must be responsible for the cell's programming and that this molecule could be transferred from cell to cell. Griffith called this phenomenon the transforming principle, because something from the heat-killed pathogenic bacteria must have

transformed the living non-pathogenic bacteria to make them disease-causing.

Hershey and Chase

In 1952, they conducted a series of experiments to prove that DNA, not protein, was the hereditary material. This involved radio-labelling the proteins and DNA associated with a particular virus and allowing it to infect a bacterium to see which factor would be "injected" to transform the bacterium. They used the T2 bacteriophage.

Friedrich Miesher

A young Swiss physician who coined the term "nucleic acid" to describe a weak acidic acid, a phosphorus-containing substance that he had isolated from the nuclei of white blood cells.

DNA

Deoxyribonucleic acid, the nucleic acid molecule that governs the processes of heredity in plant and animal cells. Same in all cells in an individual except for gametes.

RNA

Ribonucleic Acid plays a role in gene expression and protein synthesis and shares a similar structure with DNA.

- The sugar component of RNA is ribose rather than deoxyribose.

- Instead of Thymine, it has Uracil.

- It remains single-stranded, but this strand can sometimes fold back on itself, producing regions of complementary base pairs.

- It can assume different structures which serve particular functions.

Phoebus Levene

A Russian-born American biochemist who isolated two types of nucleic acid (RNA and DNA). He showed that chromosomes are made of a combination of DNA and proteins. But he did not know which played a role in heredity.

Nucleotide

Individual units that make up the chains of RNA and DNA. Made of a sugar molecule and a single nitrogenous base.

- Nitrogenous bases in DNA = adenine, guanine, cytosine, and thymine.

- Nitrogenous bases in RNA = same as DNA, except instead of thymine is replaced with uracil.

Nucleotides are more complex in order than originally thought.

Chargaff's Rule

When Ukrainian-American biochemist Chargaff found that nucleotides are not present in equal amounts. Instead, they are found in varying but characteristic proportions. The amount of adenine is proportional to thymine, and the amount of cytosine is proportional to guanine.

Purines

Adenine and guanine have two carbon rings, and two hydrogen bonds form between adenine and thymine.

Pyrimidines

Thymine, cytosine, and uracil have one carbon-nitrogen ring, and three hydrogen bonds form between cytosine and guanine. Two hydrogen bonds form between adenine and uracil.

Rosalind Franklin

Used X-ray photography to analyze the structure of DNA. She concluded that DNA has a helical structure with two regularly repeating patterns. She also concluded (from how DNA reacted with water) that nitrogenous bases were located in the DNA, and the sugar phosphate backbone is on the outside.

Watson and Crick

First to publish a structural model for DNA of the double helix.

Complementary Base Pairs

A-T and C-G. Held together by hydrogen bonds.

Antiparallel

In DNA, the phosphate bridges run in opposite directions in the two strands. Each end of a double-stranded DNA molecule contains the 5' end and the 3' end. These ends are important for DNA replication and protein synthesis. The 5' end terminates in a phosphate group while the 3' end terminates in a sugar hydroxyl group.

Gene

A functional sub-unit of DNA that directs the production of one or more polypeptides (originally described as an inheritable trait).

Genome

The sum of all DNA that is carried in each cell of the organism. It includes genes and regions of non-coding DNA.

Replication

The process of creating an exact copy of a molecule of DNA. This occurs during the S phase with only one replication per life. Takes place simultaneously.

Semi-Conservative

What DNA replication is. Each new molecule of DNA contains one strand of the original complementary DNA molecule and one new parent strand. Each new DNA molecule conserves 1/2 of the original molecule.

Meselson Stahl Experiment

Used bacteria and a heavy isotope of nitrogen to track DNA replication. Proved the semiconservative nature of DNA and maintains that the original double helix serves as a template for, but does not contribute to, a new double helix.

Replication Origin

Starts replication, a nucleotide sequence. A group of enzymes called helicases bind to DNA and cleave and unravel a segment of the double helix by breaking down hydrogen bonds between complementary base pairs. A prokaryote has one replication origin, whereas a eukaryote has thousands.

Replication Fork

Each unwound area is created by the unravelling of DNA. Consists of 2 unwound DNA strands that branch into unpaired but complementary single strands. These strands are the template for new DNA. One template strand runs in the 3' to 5' direction, the other runs in the 5' to 3' direction.

DNA Helicases

A group of enzymes that bind to a specific sequence of DNA is referred to as the replication origin. The helicases unravel the double helix by breaking the hydrogen bonds between complementary base pairs.

DNA Polymerase (III)

Synthesizes new DNA strands through complementary base pairing. Adds free nucleotides to the growing strand one at a time in only one direction, always in the 5' to 3' direction. Also acts as a proofreader.

Elongation

The process of joining nucleotides to extend a new strand of DNA. The heart of replication. Relies on the action of DNA polymerase, which attaches new nucleotides to the free 3' hydroxyl end of a pre-existing chain of nucleotides. There are 2 conditions: elongation only takes place in a 5' to 3' direction, and second, a short strand of RNA, known as a primer, must serve as a starting point for the attachment of new nucleotides.

Primase

An enzyme that synthesizes an RNA primer sequence and signals DNA polymerase III where to begin.

Leading Strand

The strand that is synthesized continuously in the 5' to 3' direction toward the replication fork.

Lagging Strand

The strand that is synthesized in short fragments, called Okazaki fragments, in the 5' to 3' direction.

Okazaki Fragments

Short segments that synthesize the lagging strand. Spliced together by an enzyme called DNA ligase.

DNA Ligase

Splices together Okazaki fragments.

DNA Polymerase I

Extends fragments, removes RNA, and also has a proofreading function (can recognize whether hydrogen bonding is taking place between the new base and its complement on the original strand). The absence of hydrogen bonding indicates a mismatch between bases. When this occurs, DNA polymerase excises the incorrect base and adds the correct one.

Replication Machine

The complex of polypeptides and DNA that interacts at the replication fork. Includes polymerase, primase, ligase, helicase, and several other proteins and enzymes required to accomplish DNA replication. Includes enzymes that relieve torsion in the unwinding of, DNA helix, and proteins that bind to exposed segments of single-stranded DNA to keep the molecule from denaturing.

Termination

The completion of new DNA strands and the dismantling of the replication machine. Occurs after newly formed strands are complete and when they rewind automatically into their chemically stable helix structure. Now, replication proceeds until the new strands are complete and the two new DNA strands separate.

DNA Sequencing

The process of identifying the precise nucleotide sequence of a DNA fragment. Virus θX174 was the first to be sequenced. Now, we can sequence genomes of entire organisms.

Human Genome Project

In 2003, a project to sequence the entire human genome.

Frederick Sanger

Established that proteins consist of a sequence of molecules called amino acids. The specific sequence of amino acids determines the chemical properties of each protein. In turn, the specific proteins that are produced by a cell determine the structure, function, and development of the cell.

Genetic Code

The order of base pairs in a DNA molecule. Determines how amino acids are strung together and how proteins are made. It has 3 characteristics:

1) It is redundant as more than one codon can code for the same amino acid. Only 3 do not code for any amino acids (stop codons).

2) It is continuous and has no spaces and overlaps. Knowing where to start and stop translation is essential. A shift of one or two nucleotides in either direction can alter the codon groupings and result in an incorrect amino acid sequence.

3) It is universal; all organisms use the same building blocks for genetic code.

Gene Expression

The process by which the information encoded in a gene is turned into a function.

Central Dogma of Molecular Biology

In order for a segment of DNA to be expressed by a cell, it must first be transcribed into mRNA and then translated into a sequence of amino acids (a protein). This allows different cells to express different genes.

Transcription

During gene expression, DNA is copied into an RNA molecule. In a eukaryotic cell, transcription takes place in the nucleus and involves mRNA. Starts when a section of DNA unzips at a particular location (gene) and mRNA nucleotides attach to the DNA, forming an mRNA strand. This strand carries the information from DNA in the nucleus to the protein synthesis machinery in the cytoplasm of the cell. For each gene, only one of the double-stranded DNA is transcribed. The overall process includes initiation, elongation, and termination.

mRNA

Messenger RNA. Contains codons, a triplet of three nitrogenous bases complementary to the DNA. Carries genetic information from the nucleus to the ribosome in the cytoplasm. Directs the synthesis of a polypeptide with the aid of another RNA molecule, tRNA.

tRNA

Transfer RNA. Contains the anticodon (a triplet of three nitrogenous bases complementary to the mRNA). Helps direct the synthesis of a polypeptide.

rRNA

Ribosomal RNA, a part of the ribosome, is essential for protein synthesis.

Codon

Sets of three gases in a gene. Used to interpret mRNA.

Sense Strand

The one strand of the double-stranded DNA that is not transcribed, the other that is transcribed, is called the antisense strand.

RNA Polymerases

The main enzymes that catalyze the synthesis of RNA. Told by the promoter region where to bind.

Promoter Region

A sequence of nucleotides on the DNA molecule tells the RNA polymerase complex where to bind.

Initiation in Transcription

RNA polymerase binds to the promoter region of DNA, marking the start of a gene. It opens the double helix and begins synthesizing mRNA. The mRNA strand is built in the 5' to 3' direction, adding nucleotides to the 3'-OH group of the previous one.

Elongation in Transcription

RNA polymerase moves along the template (anti-sense) strand, synthesizing a complementary mRNA strand. Uracil (U) is used in RNA instead of Thymine (T). Only one DNA strand is transcribed, so Okazaki fragments are not needed.

Termination in Transcription

RNA polymerase reaches a specific termination signal (stop codon) in the DNA. It detaches from the DNA, releasing the mRNA strand, and the DNA double helix forms.

Translation

The process where codons on an mRNA strand are read and converted into an amino acid sequence to form a protein. After transcription, the mRNA travels to the cytoplasm. There, two key components involved:

- tRNA, acts as a chemical translator by matching mRNA codons with the correct amino acids.

- Ribosomes, the protein synthesis machinery, read the mRNA and join amino acids together in the correct order.

Together, they assemble proteins based on the mRNA's codon sequence.

Anticodon

A stretch of three nucleotides that are complementary to the mRNA codon.

Ribosome

Ribosomes are the site of protein synthesis, assembling amino acids using mRNA, tRNA, and enzymes. Free-floating ribosomes make proteins for use in the cell; bound ribosomes (on the rough ER) make proteins for export. Each ribosome has a large and small subunit that sandwich the mRNA during translation. Codons move through three sites:

- A site: new tRNA arrives

- P site: amino acid is added to the chain

- E site: empty tRNA exits

Initiation in Translation

Begins when mRNA binds to an active ribosome at the start codon (AUG). The exposed start codon pairs with tRNA carrying methionine, which has the anticodon UAC. Ribosomes expose two adjacent codons to begin the reading frame.

Elongation in Translation

A second tRNA with the matching anticodon binds to the next codon on the mRNA. Enzymes form a peptide bond between the first and second amino acids. The first tRNA detaches and recharges with another amino acid. The ribosome shifts one codon forward, exposing the next codon. This cycle repeats, growing the polypeptide chain.

Termination in Translation

The ribosome reaches a stop codon (UAA, UAG, UGA). No tRNA matches the stop codon so translation halts. The polypeptide chain is released and the ribosome mRNA complex disassembles.

Genomics

The study of entire genomes, including the interactions among multiple genes. Closely associated with proteomics.

Proteomics

The study of all the proteins that are produced by a given genome.

5' end

The phosphate group; the end of the DNA ladder that DNA polymerase is UNABLE to build.

3' end

The end of the DNA ladder that DNA polymerase can add nucleotides to during DNA replication. Made of hydroxyl (OH).

Mutation

Mistakes in the coding of genetic information often take place during DNA replication, transcription, and/or translation, resulting in the production of a different sequence than intended. All mutations are inheritable, but not all are passed to future generations. It can be caused by spontaneous errors during replication or gene expression. They can also be caused by mutagenic agents like X-rays, gamma rays, UV radiation, and chemicals that alter DNA (benzene).

Somatic Cell Mutations

Mutations that occur in body cells, a key cause of cancer. They are not passed on to offspring.

Germ-Line Mutation

Mutations that occur in reproductive cells and are passed from one generation to the next. E.g.. Trisomy.

Point Mutation

A chemical change that affects just one or a few nucleotides. It may involve the substitution of one nucleotide. May involve the substitution of one nucleotide for another, or the insertion or deletion of one or more nucleotides. They may or may not change the sequence of amino acids.

Silent Mutation

A mutation that does not affect the function of the cell. There are no amino acid changes due to redundancy in the code.

Missense Mutation

A mutation that leads to a slightly altered but functional polypeptide. Changes the amino acid sequence and alters the resulting protein. They can be harmful, such as the change in a single amino acid, if the polypeptide that makes up hemoglobin can cause sickle cell disease. But missense mutations can play an important role in generating an enormous variety of antibodies for fighting infections.

Non-sense Mutation

Mutations that render the gene unable to code for a functional polypeptide. This often changes codons to "stop". If this results in deleting a start signal or a premature stop signal, the gene is unable to create a protein. If a nucleotide substitution affects a regulatory sequence, the cell may not respond to metabolic signals.

Frameshift Mutation

Causes the entire reading frame of a gene to be altered usually resulting in a nonsense mutation. Occurs when there is an insertion or deletion of base pairs (often more than one).

Chromosomal Mutation

Involves the rearrangement of genetic material, which can impact multiple genes, even those located on different chromosomes, through processes like crossing over or loss/duplication of chromosome portions.

Spontaneous Mutation

Arise from inherent cellular processes, such as errors in DNA replication, where DNA Polymerase incorrectly pairs bases, leading to changes in the DNA sequence.

Induced Mutation

Result from exposure to external factors such as mutagens, which can be physical (like radiation) or chemical substances that increase the rate of DNA alterations.

Mutagen

Any substance or event originating outside the cell that significantly elevates the frequency at which mutations occur within an organism's genetic material.

X-Rays

A form of high-energy radiation. Tears through DNA molecules causing random changes ranging from mutations to the loss of large portions of chromosomes. Hermann Muller shot x-rays at fruit flies and produced several hundred mutants in a day.

Physical Mutagen

Mutagens that cause physical changes in the structure of DNA. This is the most damaging form of mutagen. E.g. X-rays, gamma rays, UV radiation.

Ultraviolet Radiation

Present in sunlight, lower range of energy levels than x-rays, but still is a powerful mutagen. Causes a chemical reaction between C and T, distorting DNA and interfering with replication. Symptoms include melanoma, which is a form of skin cancer.

Chemical Mutagen

A molecule that can enter the nucleus of a cell and induce mutations by reacting chemically with DNA. Can act by inserting into DNA causing nucleotide substitution or frameshift mutation. E.g. some chemical mutagens have a structure similar to nucleotides but with different base pairing properties, examples include nitrides, gasoline fumes, and cigarette smoke.

Carcinogenic

Associated with one or more forms of cancer, what most chemical mutagens are. Results in somatic cell mutations disrupting expression of genes regulating the cell cycle.

Mutation Accumulation Effect

Single mutations often have minimal impact, but the gradual accumulation of both spontaneous and induced mutations over time can lead to significant cellular damage, contributing to diseases like cancer and driving genetic variation in populations.

Non-Universal Genetic Code

Mitochondria and chloroplasts possess their own DNA and utilize a genetic code that differs slightly from the near universal code found in the nuclear DNA of most organisms.

Organelle Independent Genetics

The DNA within mitochondria and chloroplasts is replicated, transcribed, and translated independent of nuclear DNA within the same eukaryotic cell.

Endosymbiont Theory

The unique genetic code of mitochondria and chloroplasts, along with their structural and functional similarities to prokaryotes, supports the theory that they originated as independent prokaryotic cells engulfed by early eukaryotes.

Maternal mtDNA Inheritance

Mitochondrial DNA is primarily inherited from the mother, as the sperm contribute negligible cytoplasm to the zygote making mtDNA useful for tracking maternal lineage.

Non-Coding Stretches

Analysis of these DNA regions, which exhibit higher mutation rates than coding genes, serves as a crucial tool for studying genetic variations.

Genetic Engineering

When scientists manipulate genetic material to alter genes.

Recombinant DNA

A molecule of DNA including genetic material from different sources. Used to produce organisms with desirable traits without the use of selective breeding.

Restriction Enzymes

Catalyze the cleavage of DNA at specific nucleotide sequences. Often used by prokaryotic organisms to defend themselves against infection by foreign DNA.

Restriction Endonucleases

A type of restriction enzyme that cuts within the interior of a DNA molecule, rather than at the ends. They recognize a short sequence of nucleotides (target sequence) within a strand of DNA and cut the strand at a particular point within the sequence known as the restriction site. For any give endonuclease, its target sequence will occur by chance in one or more locations in almost any fragment of DNA.

Restriction Fragments

A DNA segment that results from the cutting of DNA by a restriction enzyme.

Sticky Ends

The uneven ends of a double-stranded DNA molecule that has been cut with a restriction enzyme.

Gel Electrophoresis

A tool used to sort and analyze DNA samples. Used to separate molecules according to mass and charge. Can be used to separate fragments of DNA. To begin, a solution containing fragments is applied at one end of a gel. An electric current passes through causing one end of the gel to have a positive charge and the other negative. DNA is negative so it moves to the positive end. Smaller fragments move faster.

DNA Fingerprint

A pattern of bands made by DNA fragments in gel electrophoresis.

Biotechnology

The use of natural biological systems to create new technologies and products.

DNA Microarray

A chip (usually a glass microscope slide or a polymer membrane) containing a grid of thousands of microscopic cells. Each cell contains a nucleic acid sequence that can bind with one of the mRNA molecules transcribed during gene expression.

1. mRNA is extracted from cell(s) to be studied.

2. mRNA from each cell sample is used to synthesize an artificial form of DNA called copy DNA (cDNA). Each cDNA is marked with fluorescent tags for identification.

3. Labelled cDNA incubated in the microarray bind to microarray locations corresponding to individual genes.

4. Microarray is scanned/analyzed for patterns.

Can compare genes expressed by same cell in different environments or comparing healthy and cancerous cells.

Insulin

Synthesized by transgenic bacterin in 1982. The first example of a genetically engineered pharmaceutical product.

Polychlorinated Biphenyls

(PCBs) Bacteria degrade this toxic substance. Genetic engineering can be used to enhance this function, making this an example of bioremediation.

Bioremediation

The use of living cells for environmental remediation.

Transgenic Plants

Contain recombinant DNA increasing resistance to herbicides, insects, pests, and viruses.

Somatic Cell Nuclear Transfer

Cloning process, exemplified by Dolly, involving the transfer of nucleus from a somatic (body) cell into an egg that has had its own nucleus removed. Cloned animals exhibit high mortality, diseases, premature aging, and metabolic disorders.

Transgenic Animals

Animals that have been modified to carry genes from other species, often to produce useful traits or substances. Such as goats that produce pharmaceutical products in their milk, goats that produce silk protein in their milk, and pigs with modified antigens to act as human organ donors.

Amniocentesis

Used by pregnant women to screen their fetus for diseases. A needle is used to withdraw a small sample of amniotic fluid which is then placed in a nutrient rich medium where cells are multiplied. Then, they can make a karyotype. Cannot be done before 14 weeks.

Chorionic Villi Sampling

Removing cells from the chorion and preparing a karyotype.