Repeated sequences cloning

TES-32 is the most abundant single protein product secreted by the parasite. It is also heavily labelled by surface iodination of live larvae (Maizels et al., 1984, 1987), and is known by monoclonal antibody reactivity to be expressed in the cuticular matrix of the larval parasite (Page et al, 1992a). TES-32 was cloned by matching peptide sequence derived from gel-purified protein to an expressed sequence tag (EST) dataset of randomly selected clones from a larval cDNA library (Loukas et al., 1999). Because of the high level of expression of TES-32 mRNA, clones encoding this protein were repeatedly sequenced and deposited in the dataset (Tetteh et al., 1999). Full sequence determination showed a major domain with similarity to mammalian C-type (calcium-dependent) lectins (C-TLs), together with shorter N-terminal tracts rich in cysteine and threonine residues. Native TES-32 was then shown to bind to immobilized monosaccharides in a calcium-dependent manner (Loukas et al., 1999). 

Studies of overall genome composition based on reassociation kinetics (Simpson et ai, 1982 Cox et ai, 1990 Marx et a/., 2000) and analysis of fully sequenced bacterial artificial chromosome (BAC) clones from the 5. mansoni genome project show that platyhelminth genomes contain abundant highly and moderately repetitive sequence (Fig. 2.1). Much of the repetitive DNA comprises two classes of integrated mobile elements class I elements, which include long terminal repeat (LTR) retrotransposons and retroviruses, non-LTR retro-transposons and short interspersed nuclear elements (SINES) and transpose via an RNA intermediate, and class II elements (trans-posons), which transpose as DNA (Brindley et ai, 2003). Additionally, small dispersed or tandemly repeated sequences are common. A wide variety of these sequences have been isolated and characterized from a variety of taxa (Table 2.4). 

His tag. The ability of the imidazole moiety of the histidine residue to bind divalent metal ions such as nickel, iron, and cobalt can be used to purify histidine-containing proteins on a column which has such divalent metals bound to it. To streamline the purification procedure of any desired protein, such histidines are deliberately added to a protein of choice by cloning a four- to sixfold CAG repeat sequence into the expression construct upstream of the gene of interest so as to express an N-terminal or C-terminal tetra- or hexa-His tag. From experience, especially in E. coli, C-terminal tagging often yields superior results to N-termi-... 

Since the fusion protein partner is substantially larger than the size of the required peptide, overall peptide yield represents only a fraction of the purified fusion protein, even before losses due to subsequent purification. For example, for a fusion protein of 150 amino acids and a peptide of 15 amino acids, the relative levels of products are 91 and 9%, respectively. So, it is useful to increase the proportion of the final fusion protein that comprises peptide sequences. This can be achieved either by using a smaller fusion protein or by increasing the number of peptide sequences cloned in tandem with the fusion partner. As shown in the following examples both approaches have been adopted, but with variable results. Finally, the separation of the tandem repeats of peptides into monomers can be achieved by either chemical cleavage or enzymatic cleavage (Section 1.1.1.3). 

Techniques for the production of individual cDNAs, cloned in bacteria at the rate of one cDNA per bacterium, were multiplexed to produce cDNA libraries , large collections of cDNAs, simply by repeating the cloning procedure with complex cDNA mixtures. These complex cDNA mixtures, now in bacteria, can be surveyed by retrieving and sequencing each individual cDNA, giving a faithful representation of the abundance of each cDNA in the original mixture if sufficient cDNAs are sequenced. 

Second, it is important to consider whether a clone library will be representative of a particular repeated sequence. Besides the genetic factors, above, unusual patterns of restriction sites in some repeated sequences may influence their relative abundance in a library. This would be more likely for a tandemly repeated sequence or a very long repeated sequence than for short, interspersed repeated DNA sequences. Traditional A or plasmid libraries would be sufficient for most studies, but in certain situations it might be necessary to resort to DNA libraries of randomly frag-... 

Figure 2. Repeat sequences encoded by the 14-1 cDNA clone.

Recombinant DNA technology and bacterial fermentation techniques have enabled the design of artificial genes and the production of artificial protein materials. Typically a peptide repeating sequence is designed and translated into a DNA sequence. The DNA monomer is chemically synthesized and enzymatically polymerized and cloned into a bacterial vector or a plasmid, which is later transferred into a bacterial host to express the coded protein polymer. Because the protein products are genetically coded in the DNA sequences. 

Complete genomic sequence of E. coli Complete genomic sequence of S. cerevisiae Complete genomic sequence of D. melanogaster Complete genomic sequences of vertebrate mitochondria Collection of primate Alu repeat sequences Collection of popular cloning vectors... 

Figure 10.2. XGRAIL output using the human BAC clone RG364P16 from 7q31 as the query. The upper window shows the results of the prediction, with the histogram representing the probability that a given stretch of DNA is an exon. The various bars in the center represent features of the DNA (e.g., arrows represent repetitive DNA, and vertical bars represent repeat sequences). Exon and gene models, protein translations, and the results of a genQuest search using the protein translation are shown. (See color plate.)...

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