FCH 532 Lecture 5

New Homework assignment

Chapter 5

* In addition, only a fractional percentage of bases were methylated (i.e. not every adenine was methylated, for example) and these occurred at very specific sites in the DNA. * A characteristic feature of the sites of methylation, was that they involved palindromic DNA sequences.

* In addition to possessing a particular methylase, individual bacterial strains also contained accompanying specific endonuclease activities. * The endonucleases cleaved at or near the methylation recognition site.

* These specific nucleases, however, would not cleave at these specific palindromic sequences if the DNA was methylated.

Thus, this combination of a specific methylase and endonuclease functioned as a type of immune system for individual bacterial strains, protecting them from infection by foreign DNA (e.g. viruses).

* In the bacterial strain EcoR1, the sequence GAATTC will be methylated at the internal adenine base (by the EcoR1 methylase). * The EcoR1 endonuclease within the same bacteria will not cleave the methylated DNA. * Foreign viral DNA, which is not methylated at the sequence "GAATTC" will therefore be recognized as "foreign" DNA and will be cleaved by the EcoR1 endonuclease. * Cleavage of the viral DNA renders it non-functional.

Figure 5-37 Restriction sites.

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Types of restriction endonucleases

• Type I and Type II restriction enzymes have both endonuclease and methylase activity on the same polypeptide.

• Type I enzymes cleave the DNA at a location of at least 1000 bp away from the recognition sequence.

• Type III enzymes cleave DNA from 24 to 26 bp away from the recognition site.

• Type II restriction enzymes are separate from their methylases. These enzymes cleave DNAs at specific sites within the recognition sequence (table 5-4).

Restriction endonucleases recognize pallindromic sequences• Palindrome-a word, verse, or sentence that reads the

same forwards or backwards.

• Restrction enzymes cleave 2 DNA strands at positions that are symmetrically staggered about the center of the palindromic sequence.

• Yields restction fragments with complementary single-stranded ends (1-4 nt in length called sticky ends).

• The sticky ends can associate by complementary base pairing with other fragments generated by the same restriction enzyme.

Figure 5-37 Restriction sites.

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Restriction maps• After digest with DNA restriction endonuclease the fragments

can be separated according to size by gel electrophoresis.• DNA can be separated according to size by agarose or

polyacrylamide.• Duplex DNA is detected by staining with intercalating, planar,

aromatic cations such as ethidium, acridine orange, or proflavin, between stacked base pairs. New stains like SYBR are available that are notThese exhibit flurorescence under UV light.

• As little as 50 ng of DNA may be detected in a gel by staining it with ethidum bromide.

• Can also be used to visualize single stranded DNA or RNA.• Can be used to generate a restriction map.

Figure 5-38 Agarose gel electrophoretogram of restriction digests.

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23130 bp

9416 bp6557 bp

4361 bp

2322 bp2027 bp

Digest of Agrobacterium radiobacter plasmid pAgK84 digested with:

A. BamHI, B. PstI, C. BglII, D. HaeIII, E. HincII, F. SacI, G. XbaI, H. HpaI. Lane I contains l phage DNA digested with HindIII as standards.

Figure 5-39 Construction of a restriction map.

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Figure 5-40 Restriction map for the 5243-bp circular DNA of SV40.

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Restriction-fragment length polymorphisms

• Individuality in species derives from genetic polymorphism; homologous human chromosomes differ in sequence ~every 1250 bp.

• Restriction enzyme digests of the corresponding segments from homologous chromosomes contain fragments of different lengths (restriction fragment length polymorphisms or RFLPs) which can be used for identification.

Figure 5-41 Restriction-fragment length polymorphisms.

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Figure 5-42 Inheritance of RFLPs according to the rules of Mendelian genetics.

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Circles females, squares males

Plasmid-based cloning vectors

• Plasmids - circular DNA duplexes of 1 to 200 kb that contain the requisite genetic machinery (replication origin) necessary for autonomous replication in bacteria or yeast.

• Types of plasmids are determined by their copy number.

• Stringent control- one to a few copies per cell.

• Relaxed control- 10 to 700 copies per cell-if protein synthesis is inhibited by an antibiotic (chloramphenicol), the plasmid will continue to replicate up to 2000-3000 copies.

Figure 5-43 The pUC18 cloning vector.

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-galactosidaselac repressor permase transacetylase

colorless

Plasmid-based cloning vectors

• Plasmids used in molecular cloning are– relatively small

– replicate under relaxed control

– carry genes for antibiotic resistance

– number of restriction sites (polylinker) for inserting DNA segments.

• Cannot be used to clone DNAs longer than ~10 kb.

• Blue-white screeing.

Figure 5-46 Construction of a recombinant DNA molecule.

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Gene manipulation

• Restriction fragments with matching restriction sites are annealed together and can be connected by the action of ligase.

• If the foreign DNA and cloning vector have no common restriction sites, they may still be spliced using a terminal deoxynucleaotidyl transferase (terminal transferase).

• Mammalian enzyme that adds nucleotides to the 3’-OH group of a DNA chain. It is the only known DNA polymerase that does not require a template.

Figure 5-47 Splicing DNA using terminal transferase.

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Gene manipulation

• Chemically synthesized pallindromic linkers may also be used “insert” a required restriction site onto a DNA sequence.

Figure 5-48 Construction of a recombinant DNA molecule through the use of synthetic oligonucleotide

adaptors.

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Figure 5-50 Detection of DNAs containing specific base sequences by the Southern transfer technique.

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Figure 5-51 A degenerate oligonucleotide probe.

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Figure 5-52 Colony (in situ) hybridization.

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Figure 5-53 Chromosome walking.

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Figure 5-56Construction of a recombinant DNA molecule by directional

cloning.

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Figure 5-54 The polymerase chain reaction (PCR).

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•Thought up by Kerry Mullis in 1985.•Amplify DNA up to 10 kb.•Heat denatured DNA is incubated with

•DNA polymerase•dNTPs•Two oligonucleotide primers

•Heat stable poymerases used

•Taq•Pfu

PCR

• Amplified DNA can be used for RFLP analysis, Southern blotting, and sequencing.

• Can be used for rapid detection of diseases nad mutations.

• Can be used to identify DNA from hair, sperm, blood by amplification of short tandem repeats (STRs)-segments of repeating DNA sequence (2 -7 bp) such as (CA)n and (ATGC)n

• STRs are genetically variable and can be used as markers for individuality. The number of tandem repeats of STR are unique to an individual.

• STRs are amplified from unique sequence outside the tandem repeats.

• RNA can be amplified by PCR; first reverse transcribing it to DNA (cDNA) through reverse transcriptase.

Figure 5-57 Site-directed mutagenesis.

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Allows for the “customization” of a protein.

Oligonucleotide containing a short gene segment with the desired altered base sequence corresponding to the new amino acid sequence is used as a primer in the reaction.

In this case used DNA polymerase I. Can also use PCR to amplify a gene of interest and insert a mutation in the primer.

Production of proteins• Cloned structural genes can be inserted into an

expression vector to produce recombinant protein.• Relaxed control plasmid with an efficient promoter

can produce up to 30% of the total cellular protein as the inserted structural gene.

• Inclusion bodies-large amounts of insoluble and denatured protein. The protein must be extracted and renatured by dissolving in a chaotrope like urea or guanididium chloride and slowly renaturing the protein.

Figure 5-55 Electron micrograph of an inclusion body of the protein prochymosin in an E. coli cell.

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Production of proteins• Can engineer a signal sequence to target the

protein to the periplasmic space of the bacteria so it folds properly.

• Toxic proteins can be placed under an inducible promoter (lac) promoter in a plasmid that also has the gene for the lac repressor protein. – Binding of the lac repressor will prevent the expression

from the lac promoter.– After cells have grown to high density, an inducer

(isopropylthiogalactoside-IPTG, a synthetic nonmetabolizable analog of allolactose) is added to release the lac repressor protein.

Reporter genes can be used to monitor transcription

• Rate at which a gene is expressed dependent on upstream control sequences.

• Replace the gene you want to monitor with a reporter gene.

• Reporter genes encode proteins that can be easily detected by some assay. lacZ can be assayed with x-gal and the production of blue color.

• Another reporter is the green fluorescent protein (GFP) which produces a bioluminiscent protein when irradiated with UV or 400nm light.

lacZ

geneX

Replace geneX with reporter gene in the correct reading frame

In the presence of X-gal, expression will produce the blue color.

Figure 5-58 Use of green fluorescent protein (GFP) as a reporter gene.

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Transgenic organisms

• Organisms expressing a foreign gene are considered transgenic.

• Foreign gene referred to as transgene.• For the change to be permanent, transgene must be

stably integrated into germ cell.• Established in mice by microinjection of DNA into a

pronucleus of a fertilized ovum. • Can also be accomplished in an embryonic stem cell.

Figure 5-59 Microinjection of DNA into the pronucleus of a fertilized mouse ovum.

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Nucleic acid sequencing

• Development of DNA sequencing techniques has spurred the huge amount of DNA sequence data (>35 billion nucleotides in 2003 and growing!)

• Complete genomes determined for over 110 prokaryotes and over 11 eukaryotes.

Nucleic acid sequencing

• Development of DNA sequencing techniques has spurred the huge amount of DNA sequence data (>35 billion nucleotides in 2003 and growing!)

• Complete genomes determined for over 110 prokaryotes and over 11 eukaryotes.