Cloning positional

Figure 3.10 Typical screening plate for cellulase and amylase positive clones from enrichment cultures. Clear halos indicate the presence of positive clones and the halos diameters give a first idea about the overall activities of the respective enzymes.

Source Vector Total size of DNA screened Number of positive clones Number of clones screened Reference... 

Alcohol oxidoreductases capable of oxidizing short chain polyols are useful biocatalysts in industrial production of chiral hydroxy esters, hydroxy adds, amino adds, and alcohols [83]. In a metagenomic study without enrichment, a total of 24 positive clones were obtained and tested for their substrate specifidty. To improve the detedion frequency, enrichment was performed using glycerol or 1,2-propanediol and further 24 positive clones were deteded in this study. 

In industrial processes, 1,3-propanediol is used for the production of polyester fibers, polyurethanes and cydic compounds [85]. 1,3-Propanediol can be produced from glucose with the limiting step catalyzed by glycerol dehydratase. A metagenomic survey for glycerol hydratases from the environment resulted in seven positive clones, one of which displayed a level of catalytic efficiency and stability making it ideal for application in the produdion of 1,3-propanediol from glucose. 

Total pectinase, cellulase and lipase activities secreted by colonies were detected on BSM plates containing respectively 1% of citrus pectin, 2% Walseth cellulose and 1% olive oil + rhodamine. After few days at 30°C, pectin plates were covered by 1% CTAB for Ihour, positive colonies became surrounded by a clear halo walseth plates are not stained the halo is visible directly on positive clones lipase activity is revealed under UV on oil-rhodamine plates. 

The primary goal in the treatment of CML is to eradicate the Ph-positive clones. Elimination of the Ph is termed cytogenetic complete remission. If treatment produces RT-PCR-negative disease, this is termed molecular complete remission and indicates a several-log reduction over cytogenetic complete remission. An early goal of therapy is to achieve hematologic complete remission or to normalize peripheral blood. 

To obtain the sequence for CBP, a cDNA library from the silk gland was prepared and screened using a anti-CBP antibody. One positive clone was identified from 200,000 plaques, and rapid amplification of 5 complementary DNA ends (5 -RACE) was performed to obtain the full length of the CBP cDNA (Figure 24.2a). The predicted sequence encodes a 297-residue polypeptide of... 

Populations, gene flow between, 55-56 Population structure, 55-56 Positional cloning, 35, 36 Positive drug response, 262 Positive predictive value (PPV), 169-171 Postmarketing surveillance, 94—95, 324-325... 

Positive clones A phrase usually used to describe those hybridoma clones which bind reasonably to their respective hapten in an enzyme-linked immunosorbent assay, thereby eliminating non-specific antibodies raised to different epitopes of the hapten/carrier conjugate. 

By far, the most widely used application of the yeast two-hybrid system as intimated in Introduction is the identification of protein partners for a test protein of either known or unknown function. Here, the DNA encoding the test protein or the domain of a test protein is cloned in frame into the bait vector. The fish vector contains cDNA and it is constructed so that there is one cDNA molecule per vector. Fish and bait vectors are cotransformed into the appropriate strain of competent yeast, and the resultant transformed yeast cells are screened for growth on SD media and for reporter gene activities. Putative positive clones are then isolated and characterized further. In the next section, each of these stages is discussed in detail. 

Selection of putative positive clones on synthetic defined media... 

Sequencing of positive clones to determine identity and frame... 

Kere, J., and Laitinen, T. (2004) Positionally cloned susceptibility genes in allergy and asthma. Curr. Opin. Immunol. 16, 689-694. 

Allen, M., Heinzmann, A., Noguchi, E., et al. (2003) Positional cloning of a novel gene influencing asthma from chromosome 2ql4. Nat. Genet. 35, 258-263. 

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