Molecular, Cellular, and Tissue Level Life: DNA, RNA, Protein Synthesis, and Genetic Aberrations

DNA – The Code of Life

• Nucleic acids are vital for storing information that controls cellular activity and organism development.
• They control protein synthesis, which is crucial for body structure and enzyme production, thereby regulating all living organisms.
• Two types of nucleic acids:
  • Deoxyribonucleic acid (DNA)
  • Ribonucleic acid (RNA)

Deoxyribonucleic Acid (DNA)

• Discovery of DNA structure is a significant biological achievement.

Discovery of DNA's Structure

• Early 1950s, King’s College, London: Maurice Wilkins and Rosalind Franklin used X-ray crystallography to determine DNA structure.
• Cavendish Institute, Cambridge University: Francis Crick and James Watson analyzed Franklin's data.
• They built a model showing DNA as a twisted ladder (double helix).
  • Runners: phosphates and sugars
  • Rungs: pairs of organic bases

Genetic Replication

• Watson and Crick's second Nature article (May 30, 1953) explained genetic replication.
• Base pairing (A=T, C=G) implies that one strand determines the other.
• When strands separate, each acts as a template for a new, complementary chain.
• Crick and Sydney Brenner (1961) provided proof of a triplet code for reading genetic material in DNA.
• Information transfers from the nucleus to the cytoplasm via RNA, where proteins are made.
• DNA’s double-stranded molecule can replicate and carry genetic instructions via the sequence of bases.

Nobel Prize

• Rosalind Franklin had chemistry degrees but died before expressing her views, and before the Nobel Prize was awarded.
• In 1962, Watson, Crick, and Wilkins won the Nobel Prize in physiology/medicine.
• The Nobel Prize is not awarded posthumously and can only be shared among three winners.

Location of DNA

• Mainly in the nucleus as part of chromosomes, forming the chromatin network.
• Chromatin: chromosomal material made of DNA, RNA, and histone proteins in non-dividing cells.
• DNA is coiled to fit within the nucleus; about two meters in each human cell.
• Extranuclear DNA: small amounts found in mitochondria (animals and plants) and chloroplasts (plants).

DNA Structure

• Shape: long, twisted ladder (double helix).
• Polymer of nucleotide monomers.
• Each nucleotide contains:
  • Sugar molecule: deoxyribose (S)
  • Phosphate molecule (P)
  • Nitrogenous base:
    • Adenine (A)
    • Thymine (T)
    • Guanine (G)
    • Cytosine (C)
• These bases instruct cells on synthesizing enzymes and proteins.
• Four bases result in four different nucleotides.

Double Helix Composition

• Outer strands: alternating sugar/phosphate links with strong bonds.
• Rungs: base pairs linked by weak hydrogen bonds.
• Base pairs attach to sugar molecules.

Base Pairing Rules

• Adenine bonds with thymine (or uracil in RNA) via two hydrogen bonds $(A=T; A=U)$.
• Cytosine bonds with guanine via three hydrogen bonds $(C≡G)$.

Classification of Nitrogenous Bases

• Purines: two fused rings of nitrogen, carbon, and hydrogen (e.g., guanine and adenine).
• Pyrimidines: one ring of similar atoms, smaller than purines (e.g., thymine, cytosine, and uracil).
• Base pairs consist of one purine and one pyrimidine.

Uniformity and Variation in Bases

• The four nucleotides are the same across different organisms.
• Differences arise from the sequence in which nucleotides are strung together.
• Nucleotide sequence in certain DNA sections varies among individuals (except identical twins).
• Nucleotide sequence determines an organism’s genetic code.

Role of DNA

• Carry hereditary information as genes in each cell.
• Provide a blueprint for growth and development by coding for protein synthesis.
• Replicate genetic information to daughter cells during cell division, ensuring generational continuity.

Non-Coding DNA

• Less than 2% of human DNA codes for proteins.
• Exons: protein-coding regions.
• Introns: non-coding regions interrupting exons.
• Complex organisms have more non-coding DNA.
• Non-coding regions form functional RNA molecules with regulatory functions.

Extraction of DNA from Organic Matter

• Various methods exist, including commercial DNA extraction kits.
• Technique requirements:
  • Maximize DNA release.
  • Minimize DNA degradation.
  • Be cost-effective, time-saving, and efficient.
• Salt and ethanol extraction:
  • Simple and time-saving.
  • High yielding.
  • However, manual methods yield less pure DNA than commercial kits.

Replication

• Process of creating a new DNA molecule identical to the original from an existing DNA molecule.
• Occurs in the nucleus during interphase.
• Necessary to ensure genetic code is passed on to new daughter cells during cell division.

Replication Mechanism

• Catalyzed by DNA polymerase.
• Double helix unwinds.
• Weak hydrogen bonds break, separating the two strands.
• Each chain of bases is exposed.
• Free nucleotides attach to matching bases.
• A only bonds with T, and C only bonds with G, ensuring identical daughter DNA.
• One DNA double helix becomes two identical double helices.
• Daughter DNA molecules twist into a double helix and wind around histones, forming a chromosome.
• The entire process takes only a couple of seconds.

Ribonucleic Acid (RNA)

• Made in the nucleus by DNA and involved in protein synthesis.

RNA Structure

• Polymer made of nucleotides, but differs from DNA:
  • Single-stranded.
  • Shorter than DNA.
  • Sugar is ribose, not deoxyribose.
  • Uracil replaces thymine.

RNA Function

• Carries instructions from DNA in the nucleus to ribosomes in the cytoplasm.
• Controls the synthesis of proteins from amino acids.

Similarities between DNA and RNA

1. Both are made up of:
   • Polymers.
   • Nucleotides (sugar, phosphate, nitrogen base).
   • Four nitrogenous bases.
2. Both are responsible for protein synthesis.

Mitochondrial DNA (mtDNA)

• Present in chromosomes in the nucleus of nearly every cell.
• Small amounts also in mitochondria in the cytoplasm.

Characteristics of mtDNA

• Double-stranded, ring-shaped molecule in all mitochondria.
• Inherited entirely from the mother (egg cell).
• Has its own genome of about 16,500 base pairs, coding for proteins (enzymes), tRNA, and rRNA. Hence, much shorter than chromosomal DNA.
• Genes are essential for normal mitochondrial function, coding for enzymes that control cellular respiration.

Origin of Mitochondrial DNA

• Mitochondria were originally separate prokaryotes.
• They entered a symbiotic relationship with eukaryotic cells through endosmosis.
• Prokaryotes: organisms without a nucleus or membrane-bound organelles (mostly unicellular bacteria).
• Eukaryotes: organisms with a nucleus and membrane-bound organelles (e.g., animals, plants, fungi, and protists).

Tracing Genetic Links

• During fertilization, only sperm chromosomes enter the egg cell; organelles do not.
• Resultant zygote organelles (e.g., mitochondria) come from the egg cell (mother).
• mtDNA passes from generation to generation, establishing a direct maternal genetic line.
• mtDNA genes of offspring will be the same as the mother’s, grandmother’s, great-grandmother’s, etc.
• mtDNA occasionally mutates (e.g., substitution, forming markers).
• mtDNA profile of two sets of markers can be made.
  • Similar mtDNA shows close relationship.
  • Different mtDNA profiles indicate divergence along different evolutionary pathways.
• Markers can be mapped through generations to trace female lineages (matrilineage).
• Used to track ancestry of human groups/individuals back hundreds of generations.

mtDNA Testing

• Tissue scrape from inside the cheek is sent for mtDNA testing.
• Genetic code is studied at specific locations and compared to reference samples.
• Cambridge Reference Sequence (CRS): common source of information of human mtDNA, particularly in people of European descent.
• Similar genetic sequences indicate a common female ancestor.

Uses of mtDNA Profiles

• Reconstruct family maternal-linked relationships.
• Investigate forensic cases where chromosomal DNA is degraded.
• Determine if siblings have the same mother.

Protein Synthesis

• Proteins, including enzymes, are essential for living systems; cells may have millions active at any moment.

Types of RNA Involved in Protein Synthesis

• Different types of RNA made in the nucleus have specific functions in protein synthesis:
  • Messenger RNA (mRNA)
  • Transfer RNA (tRNA)
  • Ribosomal RNA (rRNA)

Process in the Nucleus: Transcription

• mRNA forms in the nucleus similarly to DNA replication.
• Transcription: Coded message in DNA is carried (transcribed) into mRNA, which carries it to the ribosome.

Transcription of DNA

• Transcription: DNA makes and codes mRNA.
• Process starts when a small DNA piece (gene) unwinds.
• Catalyzed by RNA polymerase, which separates DNA strands by breaking hydrogen bonds between complementary nucleotides.
• Polymerase attaches and moves along one DNA strand (template), pairing new nucleotides with complementary nucleotides.
• Sugar-phosphate backbone is added, and a new mRNA strand forms.
• Nucleotide sequence is determined by the template DNA nucleotides.
• DNA transcribes its genetic code to the mRNA.
• Uracil (not thymine) pairs with adenine.
• mRNA strand breaks away from DNA, which then re-zips.
• mRNA moves through nuclear membrane pores to ribosomes, the sites of protein synthesis.

Determining Protein Synthesis

• Protein is a long chain (polymer) of amino acids (monomers).
• Twenty different amino acids are involved in protein synthesis.
• Combine in various numbers and sequences to form thousands of different proteins; the shortest has 50 amino acids.
• Amino acid order determines the protein made.

Role of mRNA

• Amino acid sequence is determined by the genetic code from DNA passed to mRNA.
• Genetic code is carried as ‘codewords’ (codons) transcribed to mRNA. Each codon contains three bases.
• There are 64 different codons; 61 code for amino acids, and 3 are stop codons (UGA, UAA, UAG).
• The triplet code is the basis of the genetic code; a gene comprises codons that code for a protein.
• The order of codons in mRNA determines the amino acid sequence, which dictates the protein synthesized.

Process at the Ribosomes

• mRNA binds to the ribosome at the start codon.
• mRNA codons act as a template to determine the order in which amino acids are linked.

Role of tRNA

• At least 64 different tRNA molecules are made from nucleotides in the nucleoplasm.
• Each tRNA has an anticodon (three bases at one end) that picks up a specific amino acid from the cytoplasm and transfers it to the ribosome.
• tRNA binds to an amino acid at one end and to mRNA at the other, depositing the amino acid in the correct position to form a specific protein.

Translation of RNA into Proteins

• The ‘start signal’ codon begins protein synthesis from amino acids.
• Three codons act as ‘stop signals,’ indicating the message is over, and the protein chain is complete.
• All other codons code for specific amino acids.
• Anticodon bases link to complementary mRNA codon bases (translation).
  • For example, if mRNA codon is GGA, tRNA anticodon is CCU.
• tRNA releases to carry more of its specific amino acid to the ribosome.
• Enzymes catalyze amino acids linking together via peptide bonds to form a polypeptide chain.
• Polypeptide chains link together to form the final functional protein.
• Translation: process by which a specific protein is formed from a chain of amino acids, due to the sequence of codons in mRNA, coded by DNA.

Role of rRNA

• rRNA is the most common form of RNA, making up ribosomes with proteins.
• rRNA moves from codon to codon along mRNA, reading the code.
• rRNA controls the process of protein synthesis.

How Antibiotics Interfere with Protein Synthesis

• Antibiotics counteract bacterial infections by interacting with bacterial ribosomes and inhibiting protein synthesis.
• Ribosomes of bacteria (prokaryotes) and eukaryotes differ, allowing antibiotics to target bacteria specifically.
• Proteins are essential for new cell production and growth; inhibiting protein synthesis prevents bacteria from making new cells to spread infection.
• Different antibiotics inhibit protein synthesis by interacting with bacterial ribosomes differently:
  • Tetracyclines prevent tRNA attachment.
  • Chloramphenicol prevents peptide bond formation.
• By targeting different mRNA translation stages, antibiotics can be changed if resistance develops.
• Note: A chain of fewer than 50 amino acids is a polypeptide; more than 50 is a protein.
• Protein synthesis involves:
  • Transcription of the DNA code onto mRNA in the nucleus.
  • Translation of the mRNA message into polypeptides and proteins in the ribosomes.

Genetic Aberrations

• Caused by mutations: any alteration in the genetic makeup (genetic code) of an organism.

Factors Leading to Genetic Changes

• Changes in the nucleotide sequence during a lifetime may be caused by:
  1. Random nucleotide damage or loss:
     • Crossing over of paternal and maternal chromosomes in meiosis.
     • Replication of DNA.
     • Transcription of DNA to RNA.
  2. Breakdown of DNA by mutagens:
     • Environmental factors like sunlight, radiation, and smoking.
     • Mutagenic chemicals (e.g., formaldehyde, benzene, carbon tetrachloride).
     • Viruses and microorganisms.
• Mutagen: physical or chemical agents that induce and speed up mutations on DNA.

Gene Mutations

• Small, localized changes in DNA strands.
• Changes involving a single nucleotide are called point mutations.
• May occur by:
  • Substitution: one nucleotide is exchanged for another.
  • Insertions: one or more extra nucleotides are added to the DNA molecule.
  • Deletions: one or more nucleotides are removed from the DNA molecule.
• Altering the nucleotide sequence affects individual codons and the mRNA transcribed.
• This causes the absence of or incorrect form of the protein for which that gene codes.

Application of DNA Technology

• DNA profiling or fingerprinting is one application.

DNA Profiling/Fingerprinting

• Each person has unique DNA (except identical twins), despite 99.9% of human DNA being identical.
• Differences occur in highly variable, non-coding parts of DNA.
• DNA profiling involves extracting and identifying highly variable regions containing short tandem repeats (STRs).
  • STRs: repeating sequences of base pairs (e.g., CAGACAGACAGA is a repeat of CAGA three times).
• The number of repeated sequences varies considerably among individuals at the same DNA point, distinguishing one DNA profile from another.
• 13 to 20 different DNA sites are investigated to confirm the individual’s unique profile.
• Scientists generate DNA profiles using samples from blood, bone, hair, and other body tissues.
• DNA profile: an individual’s unique DNA fragments separated by electrophoresis.

How a DNA Profile Is Made

• Cells are treated with chemicals to extract DNA.
• Restriction enzymes cut at the beginning and end of each repeated sequence, resulting in variable-length fragments.
• Polymerase Chain Reaction (PCR) makes large numbers of these fragments.
  • PCR: a lab technique to make multiple copies of a DNA segment.
• DNA fragments are separated and detected using techniques like electrophoresis.
• Gel electrophoresis: a method to separate large molecules mainly by size and electrical charge.
• A pattern reflecting different numbers of base pair repeats in different individuals is obtained.
• DNA fragment length depends on the number of repeats.
• Separated DNA fragments appear as dark bands on film, creating a DNA fingerprint.
• Each cell carries an identical set of this unique DNA, differing from that of other individuals (except identical twins).
• Identical banding patterns in two genetic profiles indicate they come from the same person.
• Related people may have similar parts, but no one else will have the same sequences in every DNA part.
• DNA is chemically inert, allowing it to be recovered from long-dried blood or semen and even extracted from ancient Neanderthal bones.

Uses of DNA Profiling

• Forensics: using scientific technologies to investigate crimes.

Forensics

• Identifying DNA differences is useful in forensic investigations.
• Unique genetic sequences can accurately identify individuals in the courtroom.
• DNA traces at crime scenes provide crucial evidence (e.g., blood, skin, or semen stains).
• Matching DNA fingerprints can identify criminals and has reversed convictions, freeing innocent individuals.

Diagnosing Inherited Disorders

• DNA profiling helps medical professionals determine hereditary diseases.
• Enables parents to make decisions about affected pregnancies and prepare for treatment.

Identifying Casualties

• DNA fingerprints can identify unrecognizable casualties, such as soldiers.

Paternity Testing

• Establishes paternity in custody and child support disputes.
• Determines parents if babies are switched in maternity homes.

Fight Illegal Trading

• The origin of timber and other products can be identified to fight illegal trading.

Disadvantages of DNA Profiling

Violation of Privacy

• Storing identifiable information is seen as a privacy violation.
• Genetic traits could lead to health insurers denying coverage.

Issues on Accuracy

• Accuracy depends on equipment, personnel competency, and experience.
• Laboratory errors can lead to incorrect information.

Manipulation

• Tampering and irresponsible handling can lead to false information.

Chromosomes and Meiosis

Chromosomes

• Long, thread-like structures that form part of the chromatin network in cell nuclei.
• Made of DNA strands wound around proteins called histones.
• Discovered by chance in 1888; named for their ability to absorb dye.
• Visible under a microscope only as individual threads when a cell is dividing.
• In somatic (body) cells of diploid organisms:
  • The number of chromosomes is the same in each cell.
  • Made up of two sets: one maternal, one paternal (diploid cells or 2n).
  • Each paternal chromosome has a matching maternal chromosome (homologous pair).
  • Chromosomes in a pair are the same size and shape, with the same genes but possibly different alleles.
  • DNA of each chromosome replicates to form two identical threads or chromatids joined by a centromere; happens in interphase.
  • Replication ensures each daughter cell receives a full complement of genetic material during cell division.

Chromosome Number

• Each species has a specific number of chromosomes in its somatic cells.
• Organisms can have identical chromosome numbers without being related.
• Similarities in DNA show relationships, not numbers.

Meiosis

What is Meiosis?

• Cell division in reproductive organs of plants and animals to produce gametes (sex cells) in animals and spores in plants.
• The number of chromosomes is reduced in the daughter cells formed, i.e. the number of chromosomes is halved
• Gametes/spores are haploid cells with one set of chromosomes (n).
• In sexual reproduction, a male haploid gamete fuses with a female haploid gamete during fertilization to form a diploid zygote.

Where Does Meiosis Take Place?

• In animals, meiosis occurs in the reproductive organs, testis, and ovaries.
  • Spermatogenesis: sperm cell formation in the testis.
  • Oogenesis: egg cell formation in the ovaries.
• In plants, meiosis forms spores in sporangia.
  • Microsporangia: pollen sacs in male anthers.
  • Megasporangia: ovules in female ovaries.

The Process of Meiosis

• Parent cell DNA replicates in interphase prior to meiosis.
• Followed by two divisions:
  • Meiosis 1: reduction division, two haploid cells (n).
  • Meiosis 2: copying division, two haploid cells divide by mitosis to form 4 haploid cells.

Meiosis 1 – A Reduction Division

• Early prophase 1:
  • Chromosomes become short and fat and visible as two chromatids joined by a centromere.
• Late prophase 1:
  • Homologous chromosome pairs lie alongside one another, forming a bivalent.
  • Centrioles move to opposite poles.
  • A spindle develops across the cell from centrioles.
  • Crossing over takes place.
• Metaphase 1:
  • Bivalents move to the middle of the cell and line up on the equator.
  • Centromeres attach to the spindle threads.
• Anaphase 1:
  • Centromeres do not split.
  • Bivalents separate, and chromosomes are pulled by contracting spindle threads to opposite poles.
• Telophase 1:
  • Cytoplasm divides (cytokinesis) to form two haploid cells, each with one of each homologous pair of chromosomes.

Meiosis 2 – A Copying Division

• The two chromatids that make up each chromosome need to separate.
• Each of the haploid cells divide again by mitosis.

Note

• 2 chromatids make up a chromosome.
• 2 chromosomes make up a homologous pair.
• A pair of homologous chromosomes in close contact with each other make up a bivalent.

Crossing Over

• Mutual exchange of pieces of chromosome that the whole groups of genes are swapped between maternal and paternal chromosomes.
• Takes place in late prophase of meiosis 1.
• Replicated homologous chromosome pairs come together in synapsis to form bivalents.
• They swap pieces of inner chromatids by breaking and reforming their DNA while paired up.
• Points of crossing over (where chromatids break) are called chiasmata.
• Genes exchange between maternal and paternal chromatids, forming recombinant chromatids.
• The outer, unchanged chromatids are called parentals.

Importance of Crossing Over

• Exchange of genetic material produces chromatids with a unique combination of genes.
• This variation among daughter cells creates new combinations of genetic material.
• Offspring do not look the same (except for identical twins) or like their parents.
• Mistakes during the exchange process may lead to mutations, which can be harmful or beneficial.
• New genes may be introduced into the genetic makeup of a species, influencing evolution.

Importance of Meiosis

1. Gametes are formed by meiosis.
2. Halves the number of chromosomes, keeping the number constant from generation to generation.
3. Results in genetic variation through crossing over and random arrangement of chromosomes at the equator of the cell during metaphase.

Similarities between Mitosis and Meiosis

• Both are types of cell division.
• Parent cell DNA is replicated in interphase before cell division starts.
• In early prophase, chromosomes become short and fat and are visible as two chromatids joined by a centromere.

Differences between Mitosis and Meiosis

Abnormal Meiosis

• Results in chromosome abnormalities.
• Chromosome non-disjunction: chromosomes fail to separate correctly during meiosis.
• If this occurs in sex chromosomes (X and Y), fertilization involving abnormal gametes will result in a zygote with an extra or missing chromosome (aneuploidy).
• Affected individuals have physical and mental characteristics called syndromes.
• Klinefelter’s syndrome and Down syndrome are examples of aneuploidy.
• Chromosome non-disjunction causes the effects.
• Diagnosis: identifying the nature of an event (e.g., an illness).
• Prognosis: predicting how the event (e.g., an illness) will develop.

Klinefelter’s Syndrome

• Example of aneuploidy, occurs in males (XXY).
• Extra copy of the X chromosome in males is cause by the sex chromosomes splitting unevenly in the egg (or rarely sperm).
• Symptoms:
  • Sparse body hair
  • Enlarged breasts
  • Small testicles and penis
  • Not very deep voices
• Usually cannot father children.

Down Syndrome

• Example of aneuploidy, children born with an extra (third) copy of chromosome number 21 (trisomy).
• During oogenesis, number 21 chromosomes do not separate properly during anaphase 1; both go into one daughter cell instead of one into each of the two daughter cells formed.
• An egg with two number 21 chromosomes is fertilized to form a zygote with three number 21 chromosomes (one from the father and two from the mother), giving a total of 47 chromosomes.
• As the new embryo develops by mitosis, all the cells will have 47 chromosomes

Down Syndrome Characteristics

• Varying degrees of intellectual and developmental delay. The severity of symptoms differs widely from person to person
• Flatten facial features with slightly slanting eyes due to folds of skin at the corner of the eyes
• Other physical features include short stubby fingers and toes with the big toes widely spaced from the second tow, a largish head and ears that may develop differently
• Often accompanied by heart defects and other health problems
• Happy, loving nature
• Health problems such as heart defects may be treated through surgery and medication, but there is no cure

How Common Is Down Syndrome?

• Relatively common birth defect, affecting about 1 in 900 births.
• Chances increase with the mother’s age (maternal age effect).
  • A 35-year-old woman has a 1 in 350 chance.
  • A 45-year-old woman has a 1 in 30 chance.

Managing Genetic Abnormalities

• Children with Down and other syndromes tend to grow and develop more slowly than other children.
• In many countries, Down syndrome children attend mainstream schools, needing special classes in some areas.
• Despite changes in attitude, they still need support.
• Parents should work with teachers to help each child learn and live a normal, integrated life.

Aborting the Foetus

• New test combines a blood test with an ultrasound examination early in pregnancy.
• Foetuses with Down syndrome can be pinpointed 11 weeks after gestation.
• Allows women more time to decide whether to undergo follow-up testing or abortion.
• Foetal cells can be extracted from amniotic fluid (amniocentesis) to determine the karyotype.
• Amniocentesis: removing a small fluid sample from the amniotic cavity.

What Is a Karyotype?

• A set of chromosomes in a cell, showing the number, size, and shape of chromosomes during metaphase of mitosis.
• Non-sex chromosomes (autosomes) of similar size are grouped from A to G by length.
• Sex chromosomes (gonosomes) (X – female; Y – male) are placed separately.
• Karyotypes can show:
  • Whether a cell comes from a male or female.
  • Abnormal chromosomes.