UNIT 1: GENETICS: NUCLEIC ACID

DNA

(Campbell & Reece: Chapters, 5, 16)

LOCATION OF DNA IN A CELL

• Chromatin is a complex of DNA and protein, and is found in the nucleus of eukaryotic cells.

• Histones are proteins that are responsible for the first level of DNA packing in chromatin

• The chromatin network in the nucleus of a cell will coil up tightly during cell division and form individual chromosomes.

• Chromosomes are always duplicated during this process (2 sets of identical genetic information to ensure each cell receives identical genetic info to the parent cell during cell division).

• A duplicated chromosome consists of 2 chromatids attached to each other by a centromere.

• Each chromatid consists of several genes.• Genes consists of a long DNA strand.• A string of DNA coiled around a few

histones is called a nucleosome.

LOCATION OF DNA IN A CELL

Locus: Position of gene on chromosome

CHROMOSOME DUPLICATION AND DISTRIBUTION

2. DNA STRUCTURE• DNA molecules are polymers called

polynucleotides.• Each polynucleotide is made of

monomers called nucleotides.• Each nucleotide consists of :

• a nitrogenous base (Adenine, Thymine, Cytosine or Guanine)

• a pentose sugar (DNA = Deoxyribose sugar),

• and a phosphate group.

• Nucleotide monomers are linked together to build a polynucleotide.

• Adjacent nucleotides are joined by covalent bonds that form between the –OH group on the 3’ carbon of one nucleotide and the phosphate on the 5’ carbon on the next nucleotide.

• These links create a backbone of sugar-phosphate units with nitrogenous bases as appendages.

• The sequence of bases along a DNA polymer is unique for each gene.

A polynucleotide and a single nucleotide

The different nitrogenous bases in DNASingle ring structure

Double ring structure

• A DNA molecule has two polynucleo-tides spiralling around an imaginary axis, forming a double helix.

• In the DNA double helix, the two backbones run in opposite 5’ → 3’ directions from each other, an arrangement referred to as antiparallel.

• One DNA molecule includes many genes.• The nitrogenous bases in DNA pair up

and form hydrogen bonds: adenine (A) always with thymine (T), and guanine (G) always with cytosine (C).

A DNA double helix structure

3. DISCOVERY OF THE DNA STRUCTURE

• Early in the 20th century, the identifi-cation of the molecules of inheritance loomed as a major challenge to biologists.

The Search for the Genetic Material: Scientific Inquiry

• T. H. Morgan’s group showed that genes are located on chromosomes, the 2 components of chromosomes are DNA & protein which became the candidates for the genetic material.

• Key factor in determining the genetic material was choosing appropriate experimental organisms - bacteria & the viruses that infect them were chosen.

• Discovery of the genetic role of DNA began with research by Frederick Griffith in 1928.

• Griffith worked with 2 strains of a bacterium, 1 pathogenic (S cells) & 1 harmless (R cells)

• Heat-killed pathogenic strain were mixed with living cells of harmless strain and the result = some living cells became pathogenic.

• This phenomenon was called transformation, now defined as a change in genotype & phenotype due to assimilation of foreign DNA.

• More evidence for DNA as the genetic material came from studies of viruses that infect bacteria.

• Such viruses, called bacteriophages (or phages), are widely used in molecular genetics research.

Bacterial cell

Phage head

Tail sheath

Tail fiberDNA

• 1952: A. Hershey & M. Chase experiments showing that DNA is the genetic material of T2 phage.

• To determine the source of genetic material in the phage, they designed an experiment showing that only 1 / 2 components of T2 (DNA or protein) enters an E. coli cell during infection

• They concluded that the injected DNA of the phage provides the genetic information.

• After most biologists became convinced that DNA was the genetic material, the challenge was to determine how its structure accounts for its role…

• M Wilkins & R Franklin used X-ray crystallography to study molecular structure.

• Franklin produced a picture of the DNA molecule using this technique.

A picture of the DNA molecule using crystallography by Franklin.

• Franklin’s X-ray crystallographic images of DNA enabled Watson to deduce: • that DNA was helical • the width of the helix • the spacing of the nitrogenous

bases

• Width suggested that the DNA molecule was made up of 2 strands, forming a double helix

Representations of DNA molecule

• Watson and Crick: built models of a double helix to conform to the X-rays & chemistry of DNA.

• Franklin concluded there were 2 antiparallel sugar-P backbones, with the N bases paired in the molecule’s interior.

• But: How did bases pair? A-A?/A-T?/ A-C?/A-G?......

• But: How did bases pair? • They worked it out by using the

following image:

Purine + purine: too wide

Pyrimidine + pyrimidine: too narrow

Purine + pyrimidine: width consistent with X-ray data

• W & C noted that the pairing of the Nitrogen bases was specific, dictated by the base structures.

• Adenine (A) paired only with thymine (T), & guanine (G) paired only with cytosine (C)

• The W-C model explains Chargaff’s rules which states that; in any organism the amount of A = T, & the amount of G = C

4. THE ROLE OF DNA

• DNA is vital for all living beings – even plants.

• It is important for:• inheritance, • coding for proteins and • the genetic instruction guide for

life and its processes. DNA holds the instructions for an

organism's or each cell’s development and reproduction and ultimately death.

DNA can replicate itself.

NON-CODING DNA Multicellular eukaryotes have many

introns(non-coding DNA) within genes and noncoding DNA between genes.

The bulk of most eukaryotic genomes consists of noncoding DNA sequences, often described in the past as “junk DNA”

Much evidence indicates that noncoding DNA plays important roles in the cell.

Sequencing of the human genome reveals that 98.5% does not code for proteins, rRNAs, or tRNAs.

About 24% of the human genome codes for introns and gene-related regulatory sequences.

Intergenic DNA is noncoding DNA found between genes: Pseudogenes are former genes that

have accumulated mutations and are nonfunctional

Repetitive DNA is present in multiple copies in the genome

5. DNA REPLICATION• Replication begins at special sites

called origins of replication, where the 2 DNA strands separate, opening up a replication “bubble” (eukaryotic chromosome may have many origins of replication.

• The enzyme helicase unwinds the parental double helix.

• Single stranded binding protein stabilizes the unwound template strands.

• A replication fork forms.• The enzyme: Topoisomerase breaks,

swivels and re-joins the parental DNA ahead of the replication for, to prevent over winding.

• The unwind complimentary strands now act as individual template for 2 new strands.

• RNA nucleotides are added to each DNA template by the enzyme RNA primase to form RNA primers on both templates

• The enzyme DNA polymerase III add free DNA nucleotides to the RNA primer 3’ carbon.

• The free nucleotides bond with H-bonds to their complimentary bases on the DNA templates.

• Along one template strand of DNA, the DNA polymerase synthesizes a leading strand continuously, moving toward the replication fork.

• To elongate the other new strand, called the lagging strand, DNA polymerase must work in the direction away from the replication fork.

• The lagging strand is synthesized as a series of segments called Okazaki fragments.

• Each fragment has an RNA primer and added DNA strand.

• All the fragments are then joined with the help of the enzyme DNA ligase.

• DNA polymerase I then removes RNA primers and replaces it with DNA nucleotides.

6. PROOFREADING AND REPAIRING DNA

• DNA polymerases proofread newly made DNA, replacing any incorrect nucleotides

• In mismatch repair of DNA, repair enzymes correct errors in base pairing.

• DNA damaged by chemicals, radioactive emissions, X-rays, UV light, & certain molecules (in cigarette smoke for example)

• In nucleotide excision repair, a nuclease cuts out & replaces damaged stretches of DNA.

7.BIOTECHNOLOGY AND GENOMICS

DNA CLONINGBIOTECH PRODUCTS

DNA CLONING–Cloning is the reproduction of

genetically identical copies of DNA, cells or organisms through some asexual means.

–DNA cloning can be done to produce many identical copies of the same gene – for the purpose of gene cloning.

–When cloned genes are used to modify a human, the process is called gene therapy.

–Otherwise, the organisms are called transgenic organisms – these organisms today are used to produce products desired by humans.

–A. Recombinant DNA (rDNA) and B. polymerase chain reaction (PCR) are two procedures that scientists can use to clone DNA

CLONING OF AN ORGANISM

A. CLONING A HUMAN GENE HOW IS INSULIN MADE BY DNA CLONING?

(A. RECOMBINANT DNA)

• A large quantity of insulin are being produced by recombinant DNA technology.

• This process is as follows:1. DNA that codes for the production of insulin is removed from the chromosome of a human pancreatic cell.2. Restriction enzymes cut the gene from the chromosome (isolating the gene for insulin) I

Insulin gene – cut out with restriction enzyme

Isolated insulin gene

3. A plasmid (acting as a vector/carrier of new gene) is removed from the bacterium and cut open with a restriction enzyme to form sticky ends

Plasmid removed from bacterium

Cut by restriction enzyme

plasmidNucleus

BACTERIUM CELL

Sticky ends

• 4. Ligase (enzyme) is added to join the insulin gene to the plasmid of the bacterium cell - forming recombinant DNA.

• 5. The recombinant DNA can then be reinserted into the bacterium, the bacterium will then produce more insulin, therefore cloning the gene.

Insulin gene placed in plasmid by enzyme Ligase ( attached to sticky ends)

• 6. When the bacterium reproduces it makes the insulin inserted into the plasmid.

• 7. The bacteria are kept in huge tenks with optimum pH, temperature and nutrient values, where they multiply rapidly, producing enormous amounts of insulin, this is then purified and sold.

Recombinant DNA placed into bacterium cell

B. POLYMERASE CHAIN REACTION

• PCR – Used in genetic profiling.• To solve crimes – criminals usually leave DNA

evidence at the scene of the crime in the form of saliva, blood, skin, semen and hair. These all contain DNA. If only a little bit of DNA is found or the DNA is old, we can make copies of the available DNA by means of PCR.

• From the DNA produced through PCR, DNA fingerprint can be generated.

PCR method• 1. Sample containing DNA is heated in a test

tube to separate DNA into single strands.• 2. Free nucleotides are added to the test tube

with DNA polymerase (enzyme), to allow DNA replication.

• 3. DNA is cooled to allow free nucleotides to form a complementary strand along side each single strand.

• 4. In this way the DNA is doubled giving sufficient amount of DNA to work with.

DNA SCREENING AND FINGERPRINT TECHNIQUE

• 1. Sample of DNA is cut into fragments by means of restriction enzymes.

• 2. Negative charged electrode at one end of a rectangular flat piece of gel and a positive electrode is placed at the other end.

• 3. The DNA is placed at the negative end of the gel and starts to move to the positive end. Smaller fragments move faster than the larger ones. Separation occurs on the basis of size. This process is called gel electrophoresis.

• 4. DNA is then pressed flat against the gel and transferred to filter paper.

• 5. Radioactive probes bind to special DNA fragments.

• 6. X-rays are taken of the filter paper. The DNA probes show up as dark bands on the film. The pattern of these bands is the DNA fingerprint.

DNA fingerprinting

GEL ELECTROPHORESIS

DNA FINGERPRINT

BIOTECHNOLOGY PRODUCTS

• Today transgenic bacteria, plants and animals are called genetically modified organisms (GMO’s).

• The products that GMO’s produce are called biotechnology products.

GENETICALLY MODIFIED BACTERIA

• Recombinant DNA is used to make transgenic bacteria.

• They are used to make insulin, clotting factor VIII, human growth hormone and hepatitis B vaccine.

• Transgenic bacteria is used to protect the roots of plants from insect attack, by producing insect toxins.

GENETICALLY MODIFIED PLANTS

• Example = pomato• Genetically modified to produce potato's below

the ground and tomato's above the ground. • Foreign genes transferred to cotton, corn, and

potato strains have made these plants resistant to pests because their cells now produce an insect toxin.

• Read p. 253 for more examples

GENETICALLY MODIFIED ANIMALS