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Transcript
Fundamental Molecular Biology
BL 424 Ch 4 Molecular Biology
Student Learning Outcomes:
1. Explain essential principles of molecular biology:
expression of genetic information: DNA → RNA → protein.
2. Explain basic tools of recombinant DNA:
gene cloning, DNA sequencing, PCR.
3. Describe tools to detect specific nucleic acids and proteins:
Southern, Northern, Western, hybridization
4. Describe how tools of recombinant DNA permit detailed
analysis of gene function in prokaryotes and eukaryotes,
including construction of transgenic organisms
The structure of DNA
DNA is genetic material: (Figs. 4.5, 4.6)
•
•
•
•
•
double-helical structure (antiparallel chains),
complementary bases A-T, C-G
semi-conservative replication
5’ → 3’ direction of synthesis;
leading, lagging strands
Fig 4.2 Chromosomes at meiosis and fertilization
Eukaryotes: most cells of plants, animals are diploid:
– 2 copies of each chromosome.
Meiosis segregates chromosomes → haploid gametes;
Fertilization restores diploid progeny.
Haploid prokaryotes
duplicate DNA;
• divide by fission
Fig. 4.2
Heredity, Genes, and DNA
Classic Mendelian transmission genetics:
Gene: determines polypeptide or structural RNA
Alleles: alternate versions of genes, encode traits
One copy (allele) specifying each trait is inherited
from each parent.
Genotype: genetic makeup of an individual.
Phenotype resulting physical appearance.
Fig 4.1 Inheritance of dominant and recessive genes
Ex: Parental strains with identical alleles of gene specifying
yellow (Y) or green (y) seeds, are crossed: YY x yy.
Progeny (F1 generation) are hybrids: yellow seeds:
yellow is termed dominant, green recessive.
• Genotype of F1 generation is Yy.
• Phenotype is yellow.
Phenotype of F2 shows
• Recessive and dominant:
• 2 alleles per individual
• 1 allele per gamete
Fig. 4.1
Fig 4.3 Gene segregation and linkage
Dihybrid crosses:
• Genes on different chromosomes segregate independently
• Genes on same chromosome mostly stay together - linked
Fig. 4.3
Fig 4.8 Colinearity of genes and proteins
Colinearity of genes and proteins:
• revealed by positions of mutations
• 5’-end of gene is NH2-end of protein
• 3’ end of gene is COOH- end of protein
Fig. 4.8 mutations of TrpA gene of E. coli
Central dogma
Central dogma of molecular biology:
Genetic information DNA → RNA → Protein
• RNA polymerase synthesizes RNA from
DNA templates (transcription):
•
complementary base pairing T-A, A-U; C-G
• Proteins are synthesized on ribosomes from
mRNA templates (translation)
• Ribosomal RNA (rRNA) sites of
protein synthesis on ribosomes
• Transfer RNAs (tRNAs) adaptor
molecules that align charged
amino acids on mRNA template
Triplet code 3 nucleotides specify
1 amino acid; degenerate code
Fig. 4.9
Fig. 4.10
Fig 4.13 Reverse transcription and retrovirus replication
Retroviruses, group of RNA tumor viruses replicate
via synthesis of a DNA intermediate,
• Forms DNA provirus that integrates in host (Ex. HIV)
• RT carried by virus; critical for forming DNA copy
Reverse transcriptase
(RT) can make DNA copies
of any RNA molecule
(cDNA from mRNA)
• Clone copy of mRNAs of
eukaryotic cells to study
Fig. 4.13
Recombinant DNA
Recombinant DNA technology (gene cloning)
• Permits isolation, sequence, analysis and
manipulation of individual genes from any cell.
• Enables detailed molecular studies of structure and
function of genes and genomes
• Revolutionized understanding of cell biology
• Series of tools:
•
•
•
•
•
Restriction enzymes, ligase
Plasmids, other vectors
Gel electrophoresis
Transformation of bacteria,
Introduction of DNA into other cell types
Fig 4.14 EcoRI digestion and gel electrophoresis of λ DNA
Restriction endonucleases (RE):
• Enzymes cleave DNA at specific sequences
– Ex. EcoRI cleaves 5’-GAATTC-3’
• About 100 different enzymes
for specific recognition:
• Fragments separated by
gel electrophoresis
• Smaller molecules move
more rapidly
• Stain DNA to visualize
Fig. 4.14
Fig 4.16 Generation of a recombinant DNA molecule
Recombinant DNA: gene cloning
DNA fragment inserted
into DNA molecule
(a vector such as a plasmid)
capable of independent replication
in host cell.
Recombinant plasmids introduced
into E. coli (transformation);
Select plasmid (antibiotic resistance)
Plasmid replicates with bacteria:
get millions of copies in culture
Fig. 4.16
Fig 4.17 Joining of DNA molecules
• RE often cleave staggered sites, leaving
overhanging single-stranded regions (5’-PO4: 3’-OH)
• DNA ligase seals ends (5’-PO4: 3’-OH)
Fig. 4.17
Fig 4.18 cDNA cloning
• Cloned inserts can be
genomic DNA or cDNA
•
mRNA is copied using
reverse transcriptase (RT)
• Specific primer is often
poly(dT) for eukaryotes
(binds poly(A) on mRNA)
• Add linker sequences for
easier cloning.
Fig. 4.18
Fig 4.19 Cloning in plasmid vectors
* Review molecular cloning:
Fig. 4.19
Fig 4.21 Expression of cloned genes in bacteria
Bacterial expression vectors
contain regulatable promoters
Inserted genes are expressed
at high levels
Expression in eukaryotic cells
may be needed if
posttranslational modifications
(phosphorylation, sugars)
are required
(also needs eukaryotic promoters).
** Consider cloning
Fig. 4.21
DNA sequencing
DNA sequencing gives order of bases
understand genes, genomes, structure, function
Dideoxy method uses premature termination of DNA synthesis.
DNA synthesis is initiated with synthetic primer.
Dideoxynucleotides included with normal nucleotides;
each ddNTP labeled different fluorescent dye
ddNTPs stop DNA synthesis because
no 3 OH group for addition of next dNTP.
Fig. 4.20
ddNTP
Fig 4.20 DNA sequencing (Part 2)
Dideoxynucleotides stop DNA synthesis because no 3 OH
Get series of fragments, partial copies of target, terminated.
Fragments separated by gel electrophoresis; laser beam
excites fluorescent dyes, and records color at each position.
Detection of Nucleic Acids and Proteins
3.Detection of specific nucleic acids, proteins
• Polymerase chain reaction (PCR) amplifies DNA
• Nucleic acid hybridization detects nucleic acids:
• Southern – DNA on gel
• Northern – RNA on gel
• Microarrays - all the mRNAs
• Antibodies detect proteins
• Western – proteins on gel
• Immunofluorescence
• Immunoprecipitation
Detection of Nucleic Acids and Proteins
Polymerase chain reaction (PCR) amplifies DNA
•
•
•
•
•
Repeated replication of segment of DNA: specific primers
Rounds of denature at 95oC,
anneal to primer (55oC)
synthesis of DNA (68oC)
Heat-stable DNA polymerase
from bacteria of hot springs
(Thermus aquaticus (Taq)
Fig. 4.23 PCR
Fig 4.24 Detection of DNA by nucleic acid hybridization
Nucleic acid hybridization
uses complementary
base pairing to
detect specific
nucleic acid sequences
•
DNA or RNA probes
Fig. 4.24
Fig 4.25 Southern blotting
Southern blotting detects specific genes (DNA).
•
•
•
•
DNA digested with RE,
Fragments separated by gel electrophoresis.
DNA fragments transferred to membrane (blotted).
Filter incubated with labeled nucleic acid probe
Northern blotting
detects RNA:
separate RNA on gel,
transfer, hybridize
with specific probe
• Sizes, amount mRNA
• Different tissues
Fig. 4.25
Fig 4.26 Screening a recombinant library by hybridization
Recombinant DNA libraries: collections of clones
containing all genomic or mRNA sequences of
particular cell type. (vector can be plasmid, virus)
Ex. Clone random fragments in vector, test for specific gene
Fig. 4.26
Fig 4.27 DNA microarrays
Hybridization to DNA microarrays allows 1000s of
genes analyzed simultaneously.
• DNA microarray on glass slide has oligonucleotides or
fragments of cDNAs printed by robotic system in tiny spots
• Compare expression
in two cell types
(cancer vs. normal)
• Isolate mRNA
• Use RT then PCR
with different dyes
Ex. Cancer red,
Normal green
If equal, yellow color
Fig. 4.27
Fig 4.28 Fluorescence in situ hybridization
In situ hybridization detects homologous DNA or
RNA sequences in chromosomes or intact cells.
Hybridization of fluorescent probes to specific cells or
subcellular structures
• seen by microscope
Different probe for
each human
chromosome
Fig. 4.28
Detection of Nucleic Acids and Proteins
Antibodies detect specific proteins
Antibodies - proteins from immune cells (B lymphocytes) react to foreign molecules (antigens).
• Different antibodies recognize unique antigens
Antibodies can detect proteins in intact cells.
Cells stained with antibodies
labeled with fluorescent dyes,
or tags visible by electron microscopy.
Fig. 4.31 Human Cells in
culture: actin (blue), tubulin
(yellow), nuclear stain (red)
Fig 4.29 Western blotting
Immunoblotting (Western blotting).
Proteins separated by size on SDS-polyacrylamide
gel electrophoresis (SDS-PAGE).
SDS detergent binds,
denatures proteins,
gives – charge
Small proteins faster
Transfer to membrane
Antibodies bind to
specific proteins
Fig. 4.29
Fig 4.30 Immunoprecipitation
Immunoprecipitation
Purifies specific proteins.
Cells (radioactive proteins)
incubated with antibodies
Antigen-antibody complexes
are isolated and
electrophoresed.
Co-immunoprecipitation asks
which proteins are bound
together in complexes;
Antibody purifies one, ask
which other proteins
Fig. 4.30
Gene Function in Eukaryotes
Analysis of gene function:
• Revealed by altered phenotypes of mutant organisms.
• Study function of cloned gene by reintroducing it into
eukaryotic cells
• Can use specific mutations in genes, deletions of genes,
or add specific genes (can have conditional (ts) mutants)
• Use embryonic stem cells in culture, then transfer to whole
animals or plants
Transgenic organisms have altered genomic DNA
Genetically modified organisms (GMO)
Fig 4.32 Cloning of yeast genes
Model eukaryote yeast:
Transform yeast with plasmids carrying selectable genes
(prototrophic, LEU+)
Yeast vectors are shuttle vectors that reproduce in E. coli
Yeast:
• grow as haploid or
diploid
• easily grown in
culture, reproduce
rapidly (90 min),
• small genome.
• Mutants available for
every gene
• ts mutants for
essential genes
Fig. 4.32
Fig 4.33 Introduction of DNA into animal cells
Cloned DNA can be introduced into plant and
animal cells (gene transfer, transfection).
In most cells, DNA is
transcribed for
several days:
transient
expression.
In 1% or less of cells,
DNA integrates
into genome and is
stably transferred
to progeny cells
(can select)
Fig. 4.33
Fig 4.34 Retroviral vectors
Animal viruses, especially retroviruses, are vectors
to introduce cloned DNAs into cells.
Fig. 4.34
Fig 4.35 Production of transgenic mice
Transgenic mice model system:
Cloned genes in germ line of multicellular organisms
Microinject cloned DNA into pronucleus of fertilized egg;
Check offspring for gene (fur color, check by Southern blot).
Easier to add a new gene – can be inserted anywhere
Fig. 4.35
Fig 4.36 Introduction of genes into mice via embryonic stem cells
Embryonic stem (ES) cells for transgenic mice:
• Cloned DNA put into ES cells in culture – select drug-R
• Stably transformed cells introduced into mouse embryos
• Check gene is in germline, transfer to progeny
Similar techniques to make other transgenic animals
Fig. 4.36
Transgenic plants
Transgenic plants (genetically modified crops, GMOs)
have specific genes added or deleted.
Add DNA to cells in culture with DNA gun,
or use Ti plasmid with Agrobacterium (root nodule symbiont).
Many plants can
regenerate from
callus tissue
Fig. 4.37
Many GFP transgenic animals and plants now exist
Widespread applications
of GFP
Fig 4.39 Gene inactivation by homologous recombination
Specific mutagenesis - homologous recombination
of synthetic DNA to make particular mutations:
• Powerful tool in studying function of eukaryotic genes
• Mutate one copy of gene to be cancer-causing oncogene
• More difficult to delete both copies (knockout)
• Easier to add a gene
Fig. 4.39 specific mutagenesis
Fig 4.40 Production of mutant mice by homologous recombination in ES cells
Knockout mice
Transgenic mice with both copies of a gene mutated:
•
Powerful tool
•
•
May be lethal
Techniques to have KO
only in some tissues
Fig. 4.40
Fig 4.41 Inhibition of gene expression by antisense RNA or DNA
Antisense nucleic acids
• Use RNA or single-stranded DNA complementary to mRNA
of the gene of interest (antisense).
• Hybridize with mRNA and block translation into protein
RNA interference (RNAi) (discovered in C. elegans):
• injection of double-stranded RNA inhibited expression of
gene with complementary mRNA sequence
• Involves RISC complex binding mRNA, cleaving (Fig. 4.36)
Fig. 4.35
antisense
Chapter 5
BL 424 Chapter 5 Genomes brief:
Student learning outcomes:
• Sequences of many genomes known
• Explain structure of eukaryotic chromosomes
includes telomeres, centromeres
• Describe how eukaryotic DNA is linear, is
compacted on nucleosomes (by histones)
• Explain that eukaryotic genes have introns, exons
– much of DNA is noncoding
– Splicing occurs on the primary transcript
– Alternative splicing provides additional proteins
Fig 5.2 The structure of eukaryotic genes
• Gene coding sequences (exons) are separated by
noncoding sequences (introns).
• Entire gene is transcribed to RNA; introns removed
by splicing; only exons are included in mRNA.
• Average human gene 8 introns (gene 27 kb, coding 2.5 kb)
Fig. 5.2
Alternative splicing
Alternative splicing
• provides diversity of final proteins
• different tissues, different times of development
Fig. 5.3
DNA is organized in nucleosomes in eukaryotes
• Eukaryotic DNA is linear, organized in nucleosomes
• Histones (basic small proteins) bind DNA
Fig. 5.11
Review
Review questions:
4.7. Starting with 2 sperm, how many copies of a specific gene
sequence will be obtained after 10 cycles of PCR? After 30
cycles?
4.12. Nucleic acids have net negative charge and are separated
by electrophoresis on basis of size. Proteins have different
charges, and so how are they separated by size in
electrophoresis?
4.11. What is critical feature of cloning vector that permits
isolation of stably transfected mammalian cells?
5.1. Many eukaryotic organisms have genomic sizes much
larger than their complexity would seem to require; explain the
paradox.