Resources Basic concepts for molecular techniques  Note many of these links contain bit maps: they can be big, so don't load them over your phone! Permanently under construction - these resources are intended primarily to provide background material for students taking the courses.	Page overview:    Techniques and concepts    Probes    PCR Techniques    Other molecular biology    Affinity chromatography    Models and definitions    More molecular genetics    Reading list     For detailed overview see index See also the Danish ordliste and English glossary
NB - I have deleted a lot of old lecture notes from the server. Whereas I have taken care to try to trace the relevant links, mistakes may occur. Please inform me of broken links: dbc@kvl.dk. "Basic Plant Pathology" "Disease Resistance" ordliste på dansk A Glossary  in English.

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Index

Molecular biology

• Cloning - Gene banks or libraries - Screening libraries - Probes - Why clone? - PCR - Differential display - In vitro translation (IVT) - cDNA libraries: preparation & Shotgun screening - Southern Blotting - Northern Blotting - Western Blotting - Expression of a cDNA in E.coli - In situ hybridisation - RACE

Molecular Genetics

• Cloning of bacterial avr genes - Map-based cloning including RFLP and AFLP - Transposon mutagenesis - Yeast Two Hybrid System - mutagenesis

Biochemical techniques

• Affinity Chromatography
• Western blotting

Models and definitions in Plant Pathology

• chief features of plant-microbe interactions - Gene-for-gene - Pto - elicitors - phenylpropanoid biosynthetic pathway - Hypersensitive response

Techniques and concepts in Molecular Biology

1. Cloning
   In a molecular genetic sense, cloning is the art of working with individual genes (either as bits of chromosomal (genomic) DNA or as copies of transcripts (cDNA - copy or complementary DNA). The art is to use another organism - e.g. yeast or more usually the gram-negative gut bacteria Escherichia coli - as a factory for replicating your sample DNA. This requires the use of an artificial episome, the vector (i.e. an autonomously replicating bit of DNA). For E.coli, vectors are usually plasmids: self-replicating circular DNA molecules, or viruses, of which the bacteriophage lambda is the most developed. Restriction enzymes (see here) cut DNA molecules at defined sites. This enables them to be joined together again: ligation.
   
2. Gene banks or libraries
   These are collections of individual genes, maintained separately in a host microbe. Their chief use is as the source material in which genes are found.
   1. A cDNA library represents copies of the transcripts expressed in a particular tissue at a particular time. A cDNA clone thus represents the coding sequence of a gene.
   2. Preparation and screening of cDNA libraries.
      The central technique for studying transcripts.
      • cDNA synthesis
      • Shotgun screening of cDNA libraries.
      1. Primary characterisation Characterisation of chalcone synthase CHS 2 from barley, induced in leaves by UV light and a pathogen - the powdery mildew fungus. Illustrates Northern blots and Genomic Southern blots
      2. Complete characterisation Accumulation of different defence-related transcripts in response to Blumeria graminis. Hybridisation of 15 different ³²P-labelled cDNA clones to RNA extracted from inoculated barley leaves (Gregersen et al 1997).
   3. A genomic library represents the entire genome: genes and intergenic regions. The genes contain both the coding sequences and the adjacent regulatory sequences (i.e. the promoter which are responsible for determining both when (in relation to development or in relation to external stimuli) and where (tissue or organ) the gene is expressed.
      Genomic libraries can be made in various types of vectors, depending on the purpose.
      1. Thus for designing hybridisation probes for diagnostic or gene mapping (RFLP) probes, a plasmid vector is most appropriate since the fragments cloned are small (a few hundred to a few thousand bases).
      2. For cloning entire genes, a lambda replacement vector or a cosmid is more appropriate since an entire gene can be carried (fragments from 10 to 35 kB).
      3. For constructing physical maps of chromosome regions, a cosmid, or better a YAC or BAC (yeast or bacterial artificial chromosome vector) allows the cloning of huge fragments of chromosome: 50 to 300 (400) MB of DNA.
   4. Screening libraries
      A library has to be screened in order to find a clone. Where amino acid sequence of a specific protein is known, an oligonucleotide (short piece of DNA) can be designed and either used directly as a hybridization probe or for PCR (see below). If no probe is available, a shotgun screening approach has to be taken, of which differential or subtractive hybridization are frequently used (see Old and Primrose for details).
      
3. Why clone?

   The clone is the basic material for the molecular geneticist and used in many studies with different purposes:

   1. sequenced  -  to work out what it is or might be.
   2. expressed:
      1. to produce its product for in vitro study e.g. preparing antisera or performing enzyme reaction studies.
      2. in another host to test to for in vivo function. How does expression of the gene alter the phenotype of target organism? e.g. tests for functional dominance (cloning avr or resistance genes - see below) or to complement a recessive mutation.

Probes

1. Probes
   Clones can be used as hybridization probes to study gene expression (northern blotting when and where a gene is expressed -see cDNA library above), to genomic blotting (Southern) in order to determine number of copies and to genetically map (RFLP - restriction fragment length polymorphism) and as diagnostic tools. This is possible since DNA is a double-stranded molecule - the two strands are held together by hydrogen bonding between the complementary bases - A to T and C to G (and vice verse!). So you can melt the strands at high temperature (over 68ºC) but they will re-pair at lower temperatures. This means that you use the probe to find the same (complementary!) sequence among a complex mixture. Antibodies can also be used as probes on a western blot. Gel overlay assays are a form of western blot used to confirm protein-protein interactions.

PCR techniques

1. PCR
   The polymerase chain reaction has superseded traditional cloning strategies for many applications. Look it up in either Old and Primrose or Brown. PCR has a major place in human diagnostics, and is gradually emerging as a tool in plant diagnostics, and is now a routine tool in epidemiological and ecological studies, and for genetic mapping - RAPD (random amplification polymorphic DNA) and AFLP.
2. Differential display
   PCR based technique for identifying changes in populations of transcripts. Principal and example
3. RACE
   A PCR-based technique for obtaining an entire cDNA clone from a partial sequence, typically also a PCR product - e.g. from differential display.

Other Molecular Biology

1. In vitro translation (IVT)
   Principal illustrated with a fluorogram of 2D-gel of IVT products from Brassica campestris inoculated with Xanthomonas campestris.
2. In situ hybridisation
   In situ hybridisation to adjacent transverse sections of first barley leaf illustrating the accumulation of an oxalate oxidase-like transcript in the epidermis following inoculation with Blumeria graminis (Wei et al 1998).
3. Translational fusion.
   Expression of a cDNA in E.coli so that a single RNA is produced which encodes for a chimerical protein:
   • that encoded for by the cDNA and
   • a tag which can be purified by affinity chromatography

   in order to:

   • prepare an antiserum for use in e.g. Western blotting.
   • produce proteins which can be studied biochemically
4. Sequencing
   This is a huge topic.
   • ORF open reading frame. Sequence analysis can reveal stretches of DNA sequence which can, hypothetically, be transcribed and translated to give a protein. An ORF is such a stretch of DNA. You do not know whether the protein exists, until it has been identified.
5. Reverse Genetics
   This concerns the process of going from a protein to the gene.
   Illustrated with pea chitinase

Relevant Biochemical techniques

Affinity chromatography

Can use a tag such as:

• lacZ (which binds to a column made with glutathione)
• Glutathione S-transferase (which binds to a column made with glutathione)
• a stretch of histidines (which binds a zinc-containing column)
• a magnetic bead s (use a magnet!!)

Gel overlay assays are a form of western blot used to confirm protein-protein interactions.

Immunoprecipitation is a technique in which an antibody is used to remove the antigen (e.g. a protein) from a complex mixture.

Models and definitions in Plant Pathology

Model summarising chief features of plant-microbe interactions.

1. In monochome and colour. See also The Drastic site at SCRI, especially the response flash animation
2. Molecular models for the "Gene-for-gene" hypothesis
   • Receptor model for race specificity. It is conventional to assume a membrane bound receptor. Evidence for this is lacking.
   • Cytoplasmic or membrane bound receptors? Cytoplasmic or membrane bound receptors? Alternative models for R genes with (1) cytoplasmic or (2) membrane bound receptors. NB. The arrows represent "black boxes" - an unknown number of intermediate steps.
3. Elicitor  
   See here
4. Avirulence genes:
   • Cloning of bacterial avr genes in colour and monochrome. Functional dominance of avr genes - transfer of a cloned avr gene from an avirulent race (or even a non-host pathogen) to a virulent race converts a compatible interaction to incompatibility.
   • A syringolide The AvrD gene product (produced in Pseudomonas syringae pv. tomato and recognised as a specific elicitor in the non-host plant soybean (Glycine max) encodes an enzyme which makes glycosyl lipids: syringolides.
   • AvrBs3 gene product.
5. Physical cloning strategies:
   • Map-based cloning: B&W - colour
   • RFLP
   • RAPD
   • AFLP
6. Resistance genes
   • Structure of R-gene classes including NBS-LRR.
   • An attempt to collate the Pto story into a model.
   • The Cf resistance gene clusters from Lycospersicon spp.
7. Central phenylpropanoid biosynthetic pathway in colour and monochrome - lignin
8. Active oxygen species - the DAB method and here
9. Tissue distribution of barley defence transcripts: Northern blot data, dot blot data and summary of findings.

More Molecular Genetics

1. Yeast two hybrid system
2. Transposon mutagenesis.
   See here and other forms of mutagenesis
3. Protein kinases (and phosphatases). See here.

Reading list

1. Collinge, D.B., and Slusarenko, A.J. (1987). Review: Plant gene expression in response to pathogens. Plant Molec. Biol. 9: 389-410. (copies available from the author)
2. Kaufmann, U., and Bergenholtz, H. (1992). Genteknologisk ordbog, GAD ISBN 87-12-02199-7
   1. Kaufmann, U. and Bergenholtz, H. (1998). Gene Technology Vol I English with spanish equivalents. Lugos Libros, Ontario, Canada, ISBN 1-896266-51-7
   2. Kaufmann, U. and Bergenholtz, H. (1998). Ingenieria Genetica Vol II Español con los euqivalentes en Inglés. Lugos Libros, Ontario, Canada, ISBN 1-896266-51-8
3. Lucas, J.A. (1998). Plant Pathology and Plant Pathogens, Third Edition, Paperback: 0632030461, Price £22.50 in UK, 333 DKK (299,70 Kr. for students) in DSR see: Publisher's information.
4. Neergaard, E. de and Kovács, G. (1995). Plantepatologisk terminologi, DSR Forlag, Copenhagen.
5. Old, R.W. and Primrose S.B. (1994). Principles of gene manipulation, 5th edⁿ Blackwell Scientific, Oxford. ISBN 0-632-03712-1. see: Publisher's information.