Key terms in gene technology

Bacteriophage: A virus cloning vector used to insert foreign genes into bacteria. Most common ones are lambda- and M13-phages.

Bacterial artificial chromosome BAC: An artificially created bacterial plasmid, which includes all the areas needed for replication (ORI and the needed coding DNA). It copies itself only once or twice inside the host cell, but compared to a yeast artificial chromosome BAC is more stable, easy to transform, easier to clean and grows well in E. coli.

Cap-structure: mRNA is capped. The cap-structure is added to the 5'-end of the pre-mRNA during transcription, and seems to be involved in several aspects of pre-mRNA and mRNA metabolism, in most cases related to identifying the 5'-end of the RNA.

Cloning vector: A sequence to which foreign genetic material has been attached, and which then is transferred into a host cell. Most commonly the vector is a bacterial plasmid or a virus. Forget mathematical vectors or vector graphics, this vector is more of a "vessel". Plasmids can carry inserts up to 10 bp, bacterial artificial chromosomes up to 300 and yeast artificial chromosomes up to 200-2000 bp.

Codon/anticodon: A codon is a base triplet in the mRNA, and anticodon is its counterpart in tRNA. By matching an anticodon to a codon the correct amino acids can be added to the protein chain. Only the two first bases are important, causing a wobble-effect (the third codon can be different, and still the amino acid is recognized correctly). Each protein synthesis starts with a start codon (AUG) and ends in a stop codon (UAG, UGA, UAA).

Cosmid vector: A plasmid to which a cos-area from a lambda-phage (see bacteriophage) has been inserted. The cos area enables packing the cosmid inside a protein coating of the phage. Used commonly when building cDNA-libraries. 

DNA ligase: An enzyme which attaches two single strands of DNA together. It does not create a dual-stranded DNA, but only elongates an existing single strand by adding another piece to it. It is used in DNA transcription to close the gaps between Okazaki fragments, and when attaching DNA strands cut with restriction enzymes back together or to a vector. Needs ATP and Mg2+ to work.

DNA polymerase: A DNA polymerase is a cellular or viral polymerase enzyme that synthesizes DNA molecules from their nucleotide building blocks. DNA polymerases are essential for DNA replication, and usually function in pairs while copying one double-stranded DNA molecule into two. DNA polymerases also play key roles in other processes within cells, including DNA repair, genetic recombination, reverse transcription. (Wikipedia)

Expression vector: A vector built specifically for producing proteins. An expression vector includes the needed DNA-insert for coding the protein, a promoter to start the DNA transcription and start and end sites for the actual protein synthesis. Expression vectors are host cell specific.

Exon: A part of a gene which codes proteins. When a finished DNA-strand is spliced, exons are included in the end product. Some exons may be left out to achieve different variations of the gene.

Intron: A part of a gene which does not code any protein, or include functional DNA. Introns are left out when splicing.

Structure of a nucleotide.
(c) Wikipedia

Nucleotide: Consists of three parts: a base, a 5-ring sugar and 1-3 phosphates. Nucleotides with sugars with a hydroxyl group in their 3' carbon are ribonucleotides, while those with only hydrogen in 3' carbon are deoxyribonucleotides. In DNA and RNA, there are five nucleotides: adenine, uracil, thymine, cytocine and guanine. A and C are purines, G, T and U pyrimides. Purines always pair with pyrimides: A-T, C-G and A-U. Nucleotides are joined to one another with phosphodiester-bonds.

PCR (polymerase chain reaction): A method to replicate DNA automatically in a test tube using DNA polymerase. It has three stages: DNA denaturation, binding of the primers and DNA synthesis. A simple animation of it can be found from the website of Wiley. Many polymerases are sensitive to the Mg2+ concentration, so it has to be optimized for the polymerase used in the PCR.

Peptide bond: A bond formed between two amino acids in protein synthesis, when a carboxyl group and an amino group bind together and one molecule of water (H20) is lost.

Plasmid: A small, circular dual-strand of bacterial DNA, which is often used as a cloning vector. They replicate independently inside the bacterial cell. Plasmids always have a point of origin (ORI), which is the starting point for the replication. Plasmids altered for use in biotechnics have a cloning site, which includes restriction sites for several different restriction enzymes. Recombinant plasmids also have a selection gene, which is used to distinguish recombinants from wild types.

Polysome: A site for protein synthesis. Polyribosomes (or polysomes) are a cluster of ribosomes, bound to a mRNA molecule. Many ribosomes read one mRNA simultaneously, progressing along the mRNA to synthesize the same protein. (Wikipedia)

Primer: An RNA/DNA sequence serving as a start point for DNA replication. It's 18-22 bp long, and can be designed and built artificially. The polymerase starts replication at the 3'-end of the primer, and copies the opposite strand. Primers are used in PCR to give the polymerase a place to bind to. Primers are "independent" strands, which bind to the DNA to be used as a model, while promoters are sequences in the actual DNA.
Promoter: An organism-specific site where the polymerase enzyme binds and where the DNA- or RNA-replication starts. There are three types of promoters: 1. constitutive promoters, which work all the time in every tissue type in the organism, 2. tissue- or time-specific promoters which work only in some tissue types or during a certain time, 3. inducible promoters, which work only when a certain stimuli is present. (PatentLens) Eucaryotic cells have a promoter called TATA box, located 25 bases upstream from the start point of the transcription. Promoters can control the efficinecy of RNA synthesis.

RNA types: rRNA: ribosomal RNA. Exist in ribosomes, where they hold the mRNA steady and assist in protein synthesis. mRNA: messenger RNA is a copy of genetic information (DNA), which is used as a template for protein synthesis. tRNA: transfer RNA partakes in protein synthesis by matching a base triplet (a codon) to a correct amino acid, which it brings to the synthesis site.

Reading frame: Reading frame means how the DNA/RNA is "split" into three bases for reading. A sequence of AACTGTAC could be read for example as AAC|TGT|AC or as A|ACT|GTA|C. If there are over 50 amino acids between the start and stop codons, the protein has an open reading frame.

Enzymes used in recombinant DNA technology.
(c) Lecture material, origin unknown.

Recombinant DNA/RNA: Recombinant DNA/RNA includes foreign DNA/RNA, which has been inserted into the genome artificially or naturally via fagosytosis. A recombinant vector or a cloning vector is a sequence to which foreign genetic material has been attached, and which then is transferred into a host cell. Most commonly the vector is a bacterial plasmid or a virus.



Replication: A process where a dual-strand DNA is opened, and both strands are used as a template to create a new dual-strand DNA. The process is semiconservative, meaning that both new strands have 50 % of the original genetic material.

A replication fork. (c) Wikipedia

Restriction enzyme: An enzyme, which recognizes a specific sequence of DNA and cuts it. The recognized sequence is called a restriction site. Chromosomes and bacterial plasmids have hundreds or thousands of restriction sites for different enzymes. Restriction enzymes are very useful in gene technology, where the genes can be cut from precise points for sequencing or ligating to another chromosome. Restriction endonucleases cut the strand from the middle, while exonucleases cut it from the ends of the strand.

Restriction site: A palindromic site where a restriction enzyme makes the cut. Restriction sites 4 bases long appear approximately once every 300 nucleotides, so enzymes using these cut the DNA into very short strands. Restriction site of 6 bp appear once in 4000 nucleotides, and those with 8 bp very rarely.

RNA structure: One stranded, consists of adenine, uracil, cytocine and guanine. The 3' carbon of the sugar ring has a hydroxyl group (-OH). It may bind on its own, creating loops and hairpins.

Transformation of bacteria: The process of a bacteria accepting pure DNA into its cell. If the inserted DNA includes an ORI site, the DNA will be replicated inside the cell. The plasmid determines how many copies are made. Before transformation the bacteria has to be made competent, i.e. ready to accept DNA. This can be done by CaCl-treatment, which causes the cells to swell. Then pure DNA is added into the competent cells in a water solution.

Transformation efficiency: How many transformants (recombinant plasmids) are created per a microlitre of foreign DNA introduced.

Translation: The process of translating an mRNA into a protein. Watch a clear animation of it by St. Olaf's college.

UTR: Untranslated region. UTR 5' and UTR 3' are found between a gene-coding region and its promoter / poly-A -tail.

EU and the Common Agricultural Policy from 1950 to 2000

The Second World War ravaged Europe from 1939 until 1945. It left behind a burning continent, its cities in rubbles and inhabitants poor, injured and lost. Several contracts and agreements were made in effort to rebuild Europe, and one of those contracts was the Treaty of Rome in 1957. The Treaty was signed by Italy, Belgium, Netherlands, Luxembourg, France and Western Germany. To stabilize food production and to increase competitiveness, the Treaty formulated the basic objectives for a common agricultural policy (CAP):

1.  increase agricultural productivity by promoting techical progress and optimizing factors of production
2. ensure a fair standard of living for the agricultural community
3. stabilize markets
4. assure availability of supplies
5. ensure reasonable prices for the consumers.

The three principles behind common agricultural policy were common internal markets, community preference and common financing through EU budget.

1960

Financing CAP took over 90 % of the yearly EU budget. The budget was divided into support (export subsidies, intervention storage) and guidance (less favourable areas -subsidies, environmental subsidies, investment subsidies).

Pricing system was developed during the 60s. Common target prices were agreed upon, starting with wheat in 1967. The governments of each EU country bought excess products from the producest for a specific intervention price. Producer price, the price gained by the producer by selling their product, varied between the target prices and the intervention price. The world market price was a lot lower than the prices within EU, so import tariffs were set to make it more difficult for other countries to get into the EU markets.

In 1968 the agricultural commissioner Sicco Mansholt demanded the intervention prices are lowered and farms have to grow in size to be competitive. He feared that the current price system would lead to overproduction, since everything produced was also bought. His ideas received heavy resistance from other politicians and farmers.

1970

In 1972 only a fraction of Mansholt's plan was implemented. Mansholt called it "consensus politics overrun economic rationale”. A year later, in 1973, the EU grew for the first time, when Great Britain, Denmark and Ireland joined it. Ireland and Norther England had infertile, rough farm lands, for which a new subsidy was formed: the Less Favourable Areas subsidy (LFA). The number of farmers grew, farms grew in size, and became more productive. As Mansholt had feared, this resulted in overproduction. EU had changed from a heavy importer into a net exporter of agricultural products.

1980

In the 80s the EU expanded when Greece joined in 1981, and Spain and Portugal in 1986.
Limitations to production were set to decrease the overproduction. Milk quota was introduced in 1984and producer co-operation levies were set for sugar and grains. Farmers with over 92 hectares of farmland had to lay 10 % of it fallow. Governments bought some of the overproduce to stores and the rest was exported, but both required vast sums of money as intervention prices and export subsidies.

1990

The EU was producing too much, it's expenses were growing and the negotiations to free agricultural trade were heating up. Agricultural commissioner Ray MacSharry started a reform, which became known as the MacSharry-reform. The price for wheat was decreased 35 % during 1993-1995, but new subsidies compensated the entire economical loss for farmers. Countries with largest yields (largest losses due to the price cut) received the largest subsidies:  In 1999, The Netherlands received over 350 euros / ha and Belgium 340 euros/ha, while Spain got only 140 euros/ha. 20 % of all the farmers ended up receiving 80 % of the subsidies.

EU expanded again in 1995 when Finland, Sweden and Austria joined it. It was agreed that new member nations can use their own natinal budget to support their agriculture - this was not allowed in old member countries. A new reform, Agenda 2000, was being planned for 2000-2006. 

2000

Agenda 2000 was to separate developing the countryside from developing agriculture, and to emphasize environmental issues. Agenda 2000 aimed at improving  competitiveness by decreasing prices, ensuring the quality and safety of food and quaranteeing a fair income for farmers. Prices were decreased 15 % for wheat, 20 % for beef and 15 % for milk. Milk quota and the requirement to set aside farmland remained. The price cuts caused monetary losses to farmers, but this time they were only partially compensated with subsidies.

Agenda 2000 was noticeably more difficult to set up than the previous reforms, because EU had expanded very much. The member countries had many differences, and there was great variance in:

• importance of agriculture
• structure of agriculture (farm sizes, crops grown, products produced)
• importance of sustainability vs intensive production
• importing vs exporting agricultural products
• amount of money paid/received from EU

 Agenda 2000 presented two new models of agriculture: the European Model of Agriculture (EMA) and Multifunctional Agriculture (MA). EMA means that farmers are granted financial assistance in exchange for undertaking rural stewardship activities such as environmentally-friendly farming, instead of subsidizing commodity production. Multifunctionality, or multifunctional agriculture are terms used to indicate generally that agriculture can produce various non-commodity outputs in addition to food (such as landscapes, travelling possibilities etc).



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