Agricultural Molecular Biology

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Agricultural Molecular BiologyTeaching Team

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Agricultural Molecular BiologyCourse Structure

• Credit 3-0 : 45-hour program• Class schedules : Monday 9.00-10.30

Wednesday 9.00-10.30

CAB Lecture room 3rd floor• Evaluation :

– Exam I, II, III 75%, TBA– Presentation 10%– Paper 10%– Class participation 5%

• Grading system : Score-dependent

Agricultural Molecular BiologyCourse Textbooks

• Genes VII, 2000. By Benjamin Lewin.– 5 Reserved books, KPS library

• Molecular Biology, International Edition. 1999. By Weaver RE.

• Genomes, 1999. By TA Brown

What is molecular biology?

• A study of gene structure and function at the molecular level

• Molecular biology grew out of the disciplines of genetics and biochemistry

A brief history

• Transmission of genetics– Mendel’s law of inheritance– Chromosome theory of inheritance– Genetic recombination and mapping– Physical evidence for recombination

• Molecular genetics– Discovery of DNA– Composition of genes– Relationship between genes and proteins– Activities of genes

Transmission of genetics 1856 Gregor Mendel: inheritance of 7 traits in garden pea

Mendel’s laws of inheritance >> principle of segregation

• Genes exist in different forms called alleles.• One allele can be dominant over the other.• Mendel’s work: yellow seed > green seed

parents yellow x green

F1

yellow selfed

F2

3 yellow : 1 green

Mendel’s laws of inheritance >> principle of independent assortment

• 7 genes operated independently• Combinations of 2 different genes 9:3:3:1

Chromosome theory of inheritance

• 1900 rediscovery Mendel’s findings• First chromosome theory:

– Thomas Hunt Morgan: fruit fly (Drosophila melanogaster)

- -red eyed x white eyed

- most red eyed F1

red-eyed males x red-eyed females

1/4 white-eyed males + no white-eyed females

• Eye color is sex-linked, X chromosome– male has 1 copy, female has 2 copies

• Genes are located on chromosome : locus• Diploid organisms have 2 copies of chromosomes

– homozygous and heterozygous– genotype– phenotype : wild-type vs mutant

Genetic recombination and mapping

• Genes on separate chromosome behave independently, same chromosome behave as if they are linked

• Genes on same chromosome not perfect linked• Eye color and miniature wing on X chromosome are 65.5

% linked, with new combination ‘recombinants’• Answer is recombination process, a crossing over betwee

n homologous chromosomes during meiosis• Sturtevant: mathematical relationship between the dista

nce of genes and the recombination frequency• Barbara McClintock : physical evidence for recombinatio

n in maize chromosomes

Molecular genetics

• DNA discovery : 1869 Friedrich Miescher - nuclein– deoxyribonucleic acid (DNA)– ribonucleic acid (RNA) and protein

• Composition of genes– DNA or RNA or protein– Avery proved it was DNA– Bacteria transformation

• virulent dead cells + avirulent living cells• Relationship : genes and protein

Molecular genetics

• Relationship : genes and protein– How do genes work?– Human disease alcaptonuria -- black pigment in urine– Defective enzyme– A gene responsible for enzyme (protein) production

• Activities of genes– How do genes work?– Genes replicate faithfully– Genes direct production of RNAs and proteins– Genes accumulate mutations and allow evolution

Replication

• 1953 Watson and Crick• DNA structure is double helix• 4 bases composition : A, G, T and C

– A - T, G- C• semiconservative replication

Production of polypeptides

• Gene product is either RNA or polypeptides– gene expression

• Transcription : a single copy of DNA strand (RNA)• Translation : protein production

– mRNA carries genetic code to ribosomes– genetic code = codon consisting of 3 bases

• 61 amino acids, 3 stop signals• Accumulate mutations

– one base change -- sickle cell disease– deletions or insertions– transposon

Gene cloning

• Isolate genes and place them in new organisms• Benefits :

– raw materials for studies in molecular biology– protein product ie human insulin– Bt cotton

Genome structure and organisation>>what is genome?

• Genome is the entire DNA content of a cell, including all of the genes and all the intergenic regions

• Most genome are made of DNA• 2 types of living organisms

– prokaryotes: cells lack extensive internal compartmetns

– eukaryotes: cells contain membrane-bound compartments ie. Nucleus, mitochondria, chloroplast

• animals, plants, fungi and protozoa are eukaryotes

Why are genome projects important

Why are genome projects important?

• Genome sequences are the key to the continued development --opens the way to a comprehensive description of the molecular activities of living cells and the ways in which these activities are controlled

• Gene catalog -- isolation and untilisation of important genes

• Additional benefits -- study role of noncoding DNA• Challenge of unknown

Genomes of eukaryotes

• Eukaryotic genome is split into 3 components– nuclear genome– mitochondrial genome– chloroplast genome

• made up of 2 components: nuclear and mitochondria

Human genome

Human genome

• nuclear genome: – 3000 Mb DNA – linear genome divided into 24 (22 autosomes + 2

sex chromosome, X and Y)– shortest 55 Mb and longest 250 Mb

• mitochondrial genome:– circular DNA 16,569 bp

Human genome

• immensity of human genome– analog with normal font size --60 nt = 10 cm– genome sequence stretch for 5,000 km

Nuclear genome

• Genome size ranges <10 Mb to >100,000 Mb

• size broadly coincides with organism complexity

• larger genome --high repeats

Nuclear genome

• Split into a set of linear DNA molecules -chromosomes• chromosome number not related to organism features

Where are genes in a genome?>>Human EST map --expressed sequence tags

Families of genes

• Multigene family --groups of genes of identical or similar sequence– rRNA genes : 5S, 28S, 5.8S and 18S– globin gene families

• alpha-globin family, on chr#16• beta-globin family, on chr#11

• Supergene family– globin supergene family --alpha and beta evolve

from a single ancestral globin gene

Pseudogenes

• Genes which for one reason or another have become nonfunctional– conventional

pseudogenes --inactivation by mutation ie. Create termination codon within coding region

– processed pseudogenes -- abnormality during gene expression

Organelle genomes

• Mitochondria and chloroplasts• Extra chromosomal genes --unusual inheritance patterns• Most mitochondrial and chloroplast genomes are circular

Genetic content of organelle genomes

• Mitochondria display greater variability– number of genes : 12-92– genes for mitochondrial rRNAs, protein components

for respiratory chain• Chloroplasts are less variable

– gene number ~ 200– coding for rRNAs, tRNAs and ribosomal proteins and

proteins involved in photosynthesis

Prokaryotic genome

• Physical structure: size < 5 Mb• Genome is contained in a single circular DNA molecule

in nucleoid• Circular E. coli chromosome circumference is 1.6 mm,

while cell is 1x2 um

Supercoiling

Prokaryotic genome

• Plasmid: small piece DNA, often circular, coexist with main chromosome in a bacteria cell

• plasmids carry genes not present in main chromosome

Prokaryotic genome

• Controversy: should plasmid be part of genome?– E. coli : main chromosme 4.6 Mb+ few kb of plasmid

DNA which are not essential– Borrelia burgdorferi: 910 kb main chromosome

carrying 853 genes + 17 linear and circular plasmids of 533 kb with 430 genes, some of which are essential

Prokaryotic genome

• Genetic organisation: bacterial genomes have compact genetic organisations with little space between genes

Genetic organisation• Operons: an operon is a group of genes located

adjacent to one another in the genome• All genes in an operon are expressed as a single unit

Operon controversy

• Most cases, genes in an operon are functionally related, coding for a set of proteins involved in a single biochemical activity

• Some species, genes in an operon rarely have any biochemical relationship

Repetitive DNA

• Repetitive DNA found in all organisms, in some including humans shows substantial composition

Repetitive DNA

• There are various types of repetitive DNA– tandemly repeated

DNA– interspersed genome-

wide repeats• Tandemly repeats:

– common features of eukaryotes

– also called satellite DNA

Satellite DNA

• Satellite bands in density gradient made up of long series of tandem repeats, hundreds kb

• A genome can contain different types of satellites• Alphoid DNA: one type of satellites in centromere• Minisatellites: form clusters up to 20 kb with repeat

units up to 25 bp – telomeric DNA --important function in DNA

replication

Satellite DNA

• Microsatellites: shorter, usually < 150 bp with repeat unit 4 bp or less– although short, there are many --useful as markers

for mapping– in human, CA repeats make up 0.5% og genome or

15 Mb; A repeats 0.3%– function is mysterious, but useful to geneticists as

they are variable• number of repeats different in different members

of a species due to slippage during DNA replication --insertion or deletion

• No 2 humans have exactly the same combination of microsatellite alleles

Interspersed genome-wide repeats

• Interspersed repeats arise from transposition• Most repeats have inherent transpositional activity