Molecular Biology: Genes and DNA

Molecular Biology Definition

• Molecular biology is the study of the composition, structure, and interactions of cellular molecules like nucleic acids and proteins
• These molecules execute the biological processes that are essential for a cell's function and maintenance

Historical Perspective of Molecular Biology

• Although DNA is well-known today, it came to scientists' attention later in biology's history
• Gregor Mendel discovered heredity principles about a century before DNA's discovery
• Scientists rediscovered Mendel's work around the turn of the century and realized life was encoded in genes
• The discovery of DNA structure started a new era, leading to cracking the genetic code and realizing DNA directs protein synthesis within two decades
• Technical advances included DNA sequencing, genetic engineering, and gene cloning
• The complete sequences of many organisms, including the human genome in 2001, have been solved
• The next 50 years of DNA study will focus on the practical benefits of Crick and Watson's discovery for humanity in industry, medicine, food, and agriculture

Timeline of Key Discoveries in Molecular Biology

• 1866: Gregor Mendel published his work on inheritance of traits in peas and is known as the "Father of Modern Genetics"
• 1869: Friedrich Miescher isolated a phosphorus-rich, acidic substance called "nuclein" from white blood cell nuclei
• 1928: Frederick Griffith demonstrated a heritable "transforming principle" that allows bacteria to cause pneumonia in mice
• 1929: Phoebus Aaron Levene characterized and named ribonucleic acid and deoxyribonucleic acid, and a "tetra nucleotide" structure of DNA
• 1952: Alfred Hershey and Martha Chase proved that DNA is genetic material
• 1953: Watson and Crick deduced DNA's double helix conformation
• 1970: Paul Berg developed Recombinant DNA Technology
• 1977: Frederick Sanger developed DNA sequencing
• 1983: Kary Mullis invented In Vitro Amplification of DNA (PCR)
• 2003: The Human Genome Project (HGP) was completed under the leadership of Francis Collins, with support from the U.S. Department of Energy and the National Institutes of Health (NIH)

Classic Experiments Identifying DNA as the Carrier of Genetic Information

• These experiments include Mendel's pea plant experiments, Griffith's bacterial transformation experiments, and the Hershey-Chase experiments

Mendel's Pea Plants

• Mendel cross-bred true-breeding violet flower plants with true-breeding white flower plants (P generation)
• In the F1 generation, all resulting hybrids had violet flowers
• In the F2 generation, around three-quarters of plants had violet flowers, and one-quarter had white flowers
• Individuals possess two alleles for each trait, one from each parent, as stated in the Law of Segregation
• Parents pass only one allele to their offspring
• One allele comes from the female, and the other comes from the male
• Alleles may or may not contain the same information
• Homozygous individuals possess identical alleles for a trait, while heterozygous individuals have different alleles
• An individual's genotype is made up of their many alleles
• An individual’s physical appearance (phenotype) is determined by alleles and the environment

Frederick Griffith's Bacterial Transformation

• In 1928, Frederick Griffith experimented with Streptococcus pneumoniae bacteria in mice, aiming to develop a pneumonia vaccine
• Griffith used two bacterial strains: R and S
• Mice injected with live S strain bacteria invariably died of pneumonia due to the polysaccharide capsule protecting it from the host's immune system
• Mice injected with live R strain bacteria remained healthy, lacking the protective capsule
• Mice injected with heat-killed S-bacteria also remained healthy
• Mice injected with heat-killed S-bacteria combined with live R-bacteria ended up dying
• Griffith concluded that a "transforming principle" allowed nonpathogenic R bacteria to make a polysaccharide coat, evading the mouse immune response

The Hershey-Chase Experiments

• Radioisotopes' availability helped characterize DNA after World War II
• Alfred Hershey and Martha Chase showed in 1952 that the genetic material of bacteriophage T2 is DNA
• Experiment depended on the chemical structure differences between protein and DNA
• DNA contains C, H, O, N, and P, while protein contains C, H, O, N, and S
• Hershey and Chase labeled phage DNA with radioactive Phosphorus-32, which is present in DNA but not in any of the 20 amino acids
• The phages were allowed to infect E. coli, and the transfer of 32P-labeled phage DNA into the bacterium's cytoplasm was observed
• In a second experiment, phages were labeled with radioactive Sulfur-35, which is present in the amino acids cysteine and methionine, but not in DNA
• After infecting E. coli, viral protein shells were sheared off using a high-speed blender and separated via centrifuge
• After separation, the radioactive 35S tracer was found in the protein shells, not in the infected bacteria, suggesting DNA is the infectious genetic material
• After synthesis of phage components and assembly, lysis occurred, and the progeny phage particles contained 32P
• All information needed to make new phage came from injected DNA

Experimental Modeling in Molecular Biology

• Bacteria, bacteriophage, yeast, and animal/plant cells

Bacteria

• Bacteria are prokaryotic, unicellular, free-living cells with a single chromosome not enclosed inside the nucleus but free in the cytoplasm (nucleoid)
• Escherichia coli (E. coli) is a good model because it is easily cultured, relatively simple, has a short generation time (20 minutes), and grows best at 37°C
• The bacteria completes DNA replication, RNA transcription, and protein synthesis quickly

Bacteriophage

• Bacteriophages are the simplest life form, infecting bacteria, as well as animals, plants, and humans
• Bacteriophages are not free-living and are inert until entering a host cell, where they begin replication and are now used as cloning vectors

Yeast

• Yeast is another eukaryotic model with chromosomes inside a nucleus
• Early biochemical research used fermentation
• Yeast mutants help discover genes

Animal and plant cells

• Can be used as models in genetic experiments

Human Genome Project

• The Human Genome Project (HGP) was an international scientific research project completed in 2003 & sequenced the entire three-point-three billion base pair human genome
• The HGP led to bioinformatics' growth
• Sequencing the human genome enables solving the mystery of human disorders and coping with them

Goals of the Human Genome Project

• Optimization of data analysis
• Sequencing the entire genome
• Identification of the complete human genome
• Creating genome sequence databases
• Taking care of legal, ethical, and social issues

Methods of the Human Genome Project

• Expressed sequence tags differentiate genes into those forming part of the genome and those expressing RNAs
• Sequence Annotation involves sequencing the entire genome first, then assigning functional tags

Features of the Human Genome Project

• Contains 3164.7 million base pairs
• Genes average 3000 nucleotides
• The function of over 50% of genes is yet to be discovered
• Proteins are coded by less than 2% of the genome
• Most of the genome has repetitive, non-coding sequences, helping us understand genetic development

Genetics Definition

• Genetics is the biology branch studying genes, genetic variation, and heredity

Inheritance or Heredity Definition

• Inheritance/heredity transmits characters across generations through gametes (sexual) or asexual reproductive bodies, causing similarities

Genome Definition

• The genome includes all DNA in a cell: chromosomes and non-chromosomal sites.
• Bacteria and some fungi have plasmids, and eukaryote mitochondria have functional DNA

Gene Definition

• The gene is the heredity unit: a DNA segment with protein or RNA information encoding genetic characteristics as alleles
• A single DNA molecule contains thousands of genes

Alleles Definition

• Alleles are paired genes at a specific chromosome location controlling a trait or a gene variant controlling a trait in a chromosome region (locus)

Genotype Definition

• Genotype is an individual's genetic makeup determining their phenotype

Phenotype Definition

• Phenotype includes observable traits like height, eye color, and blood type
• A person’s phenotype is determined by genetic make up and environmental factors