Biotechnology Research

BIT 220MCCC

Chapter 10

TerminologyMolecular Biology/Genetics: the study of gene structure and function at the molecular level

Molecular Biotechnology: the ability to transfer specific units of genetic information from one organism to another

Recombinant DNA Technology= gene splicing= genetic engineering=gene transplantation= gene cloning

Genetics• Science of Heredity

• Explains similarities and differences between organisms

• Two Branches– Classical (Mendelian)

• cells contain pairs of ‘factors’ which determine physical characteristics

• these factors segregate during meiosis, independently

– Molecular

DNA/RNA Molecular Basis of Life

Central Dogma

DNA RNA proteincontrol

Transcription Translation

DNA - Deoxyribonucleic AcidRNA - Ribonucleic Acid

DNA DNA

Replication (mitosis,meiosis)

DNA Structure

ReplicationGene Expression

Mutation

PROPERTIES OF DNA

1.Replicate Genotypic function

2.Mutate –chemically change and transmit these changes to future

generations - if mutant in germ lineevolutionary function

3.Gene expressionDirect the synthesis of proteinsphenotypic function

1. Miescher DNA 1868 isolated DNA from pus cells (NUCLEIN)

2. Frederic Griffith 10.1 and 10.21928 ‘transforming principle’ in pneumococcus

3. Oswald Avery, Colin MacLeod, Maclyn McCarty 10.3•1944 DNA was the active substance in the heat-killed S strain extracts because DNasesdestroyed the activity while RNases and proteases did no.

4. Chargaffthe molar concentration of A = T and G = Cwhich led to the discovery of base pair complementarity.

5. Wilkins and Franklin 10.10X-ray crystallographs (diffraction patterns) of

DNA -Review DNA structure: Figures 10.6, 10.7, 10.8, 10.11, 10.12, 10.13, Table 10.3

6. Watson and Crickdeduced a two-stranded structure wrapped in a right-handed helix with the bases internal, the phosphates external, and an internal repeating subunit separated by 0.34 nm

NucleotideSmallest unit of nucleic acid

Three components1. sugar2. phosphate3. nitrogenous base

Other Nomenclature

Nucleoside : SUGAR and BASE (NO PHOSPHATE)

BASES NUCLEOSIDES NUCLEOTIDES

Adenine Deoxyadenosine Deoxyadenosine 5’-triphosphateGuanine Deoxyguanosine “” (dATP)Cytosine Deoxycytidine “”Thymine Deoxythymidine “”

Double Helix2 strands wrapped around one anotherSPIRAL STAIRCASE

Allows replicationAllows permanence

Bases on one side of helix arecomplementary to other strand

What draws two strands together?1. Bases hydrogen bond2. Bases are hydrophobic

DNA StructureA. Sugar-Phosphate Backbone

covalent bonds between S and Pphosphodiester linkages

B. StairsSugar covalently bound to baseBases hydrogen bound to each otherA::TC:::G

DNA Antiparallel Strands

One strand runs 3’C (OH of sugar) to 5’ C (phosphate);

The other strand 5’ to 3’

Alternate forms

A 11 bp per turn/ right-handed

B 10.4 base pairs per turn/ right handed FOUND IN VIVO -physiological DNA form

Z 12 bp per turn/left handed

SupercoilingIn vivo negatively supercoiled (underwound)

Introduced when one or both strands nicked and strands rotate around one another

Figures 10.14 and 10.15

Chromosome StructureComposed of DNA (RNA viruses) and proteinssingle piece of nucleic acid

Prokaryotic (bacteria and virus)single chromosome/ 3000 genesE coli condenses DNA into loops 10.16no intronsall single copy DNAmonoploid

Eukaryotic1000 times more DNA than bacterianoncoding regions introns30-85% single copymost diploid

Eukaryotic Chromosomes

Chromatina. DNAb. non-histone proteinsc. histone proteins: 10.23 and 10.24

DNA wraps around dimers of each H2a, H2b, H3, and H4 forming nucleosome

H1 sits outside nucleosome

DNA Compaction10.27

Level 1 Nucleosome

Level 2 supercoiling of nucleosomes

Level 3 Scaffold composed of non-histone proteinspacking during metaphase (most condensation)

Centromeres Figures 10.29 and 10.30interchangeable among chromosomes110-120 bp in length.

Telomeres Figure 10.31contain special repeated DNA sequences that enable the ends of the chromosomes to be replicated, inhibit their degradation by DNA degrading enzymes, and prevent fusion with other chromosomes.

In vertebrates, the TTAGGG repeat is highly conserved and in humans 500-3000 repeats occur in telomeres which gradually shorten with age.

•Eukaryotic DNA contains excess DNA, up to 50% or more, that doesnot code for proteins and comprises families of highly repeated sequence

elements or repetitive DNA (satellite DNA)•three classes of DNA

1. unique or single-copy1-10 copies per haploid genome

genes that encode proteinsregulatory sequences

2. moderately repetitive 10-100,000 copies

transposable elements (jumping genes)rRNAhistone

ribosomal genes3. highly repetitive

>100,000 copiestelomeres

centromeresunknown function

Types of repeats

LINEs - Long Interspersed Nuclear Elements

SINEs - Short Interspersed Nuclear Elements

LTRs - Long terminal repeats

DNA transposons (transposable elements)

Denaturation/Renaturation

D: Two strands of DNA separate (Melt)

R: Two strand hydrogen bond through complementary bases (Annealing)

The kinetics of these processes will indicate the amount of repetitive DNA

Higher the copy number; the faster the renaturation occurs

HybridizationRadioactive-labelled sequences

Nucleic Acid probe contains radioactive isotopethis isotope emits charged particles as it decaysthese charged particles captured on emulsion (film)exposed silver halides produce back spots

Fluorecently-labelled sequencesTech Sidelight