TSP-1 and TSP-2 are homotrimers, whereas TSP-3, TSP-4 and TSP-5 (usually referred to as cartilage oligomeric matrix protein, COMP) are homopentamers (for reviews, see Lawler, 2000 Adams, 2001 Armstrong and Bornstein, 2002). There are five TSPs in vertebrates, each the product of a different gene. The thrombospondins (TSPs) are large, secreted, multimeric glycoproteins that modulate extracellular matrix (ECM) structure and cell behaviour. Mutations in the T3 repeats of TSP-5/COMP, which cause two human skeletal disorders, are predicted to disrupt the tertiary structure of the T3–CTD assembly. The central architectural role of calcium explains how it is critical for the functions of the TSP C-terminal region. The availability for cell attachment of an RGD motif in T3 repeat 7 is modulated by calcium loading. Disruption of the hairpin structure of T3 repeats 6 and 7 decreases protein secretion and stability. The CTD forms a lectin-like β-sandwich and contains four strictly conserved calcium-binding sites. The T3 repeats lack secondary structure and are organised around a core of calcium ions two DxDxDGxxDxxD motifs per repeat each encapsulate two calcium ions in a novel arrangement. The crystal structure of a cell-binding TSP-1 fragment, spanning three T3 repeats and the CTD, reveals a compact assembly. The most highly conserved region of all TSPs are the calcium-binding type 3 (T3) repeats and the C-terminal globular domain (CTD). In almost all cases you would be using one of these well characterized strains and so would not need to worry about whether there were unknown plasmids.Thrombospondins (TSPs) are extracellular regulators of cell–matrix interactions and cell phenotype. coli) that have been studied for decades. In practice microbiologists have domesticated strains of bacteria (a favorite is Escherichia coli - often abbreviated to E. This is easy to test - we just try growing the bacteria in the presence of ampicillin, if they don't then we can use our plasmid. All we need to know is that the bacteria were are transforming are not already resistant to ampicillin. However this doesn't matter as much as you might think.įor example, assume we are using a plasmid that contains a marker (selectable gene) encoding resistance to ampicillin. We could sequence all the DNA inside the bacteria, but that is still a lot of work. It could be difficult to know if you were just using a random bacteria isolated from nature - especially since there are likely to be many thousands of different plasmids (1730 were present in a sequence database as of 2009). §Note: Polymerase chain reaction - you can learn more about this technique here: ‡Note: For some applications this can be very important, for example if you are using an expression vector you need the insert to transcribed in the correct direction! †Note: There are hundreds of commercially available restriction enzymes recognizing many different sequences (many of which are palindromes, but not all).Īmong these the most commonly used are six-cutters (with 6 bp recognition sites - if you make a bunch of simplifying assumptions you can calculate that these enzymes on average will cut once every 4096 bp. There are many more tricks that have been developed, but adding sites at the ends of primers almost always works, so that is a very good one to know! This amplifies the insert you want and creates a copy of the insert DNA with whatever restriction sites you want added at the ends. If the regions flanking the sequence you want to clone don't contain any useful restriction sites you can instead use primers with restriction sites added to their 5' ends and then amplify the sequence using PCR§. This again greatly increases the number of possible restriction enzyme sites. the different ends mean the insert can only be put into the plasmid in one orientation‡. In fact, it is quite common to use two different enzymes and this allows us to do "directional cloning" - i.e. Third, we don't need to use the same enzyme for both ends. Second, we often don't care if we clone a small amount of extra DNA, this means that we can search over a larger area than you might expect to find appropriate restriction enzymes. First, most vectors will have a region known as the "Multiple Cloning Site" (MCS) that can be cut with many different restriction enzymes† - this gives you more choices of enzyme and makes it more likely that you can find one that cuts near the ends of the region you wish to clone.
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