Complementary DNA library

Fig. 4. Steps in making a cDNA library. Cellular mRNA is used as a template to make a complementary DNA. This cDNA is then ligated to a plasmid,...

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... 

The isolation of Poly (A+) mRNA has also led to the synthesis of complementary DNA for the preparation of BBB-cDNA libraries and the construction of BBB-re-... 

The G-protein that has been termed Gp, and that is linked to phospholipase C activation, may in fact be Gaj 2 or Gc. 3. Ga is designated as the G-protein responsible for activation of phospholipase A2, which results in arachidonic acid release. Some experimental evidence indicates that, at least in HL-60 cells, different agonists can preferentially activate different phospholipases, and some of these are responsible for the activation of secretion. In neutrophils, the two pertussis-toxin-sensitive Ga-proteins (Gaj-2 and G j 3) have been identified by peptide mapping of proteolytic digests of the proteins, by peptide sequencing and by immunoblotting. Complementary-DNA clones for the mRNA of these two molecules have also been isolated from an HL-60 cDNA library. Gai-2 is five to ten times more abundant than Gai.3, the former component comprising 3% of the total plasma membrane proteins. It is possible that these two different Ga-subunits are coupled to different phospholipases (e.g. phospholipases C and D). Pertussis toxin inhibits the secretion of O2 after stimulation of neutrophils by fMet-Leu-Phe, but pertussis-toxin-insensitive G-proteins are also present in neutrophils. These may be members of the Gq family and may be involved in the activation of phospholipase Cp (see 6.3.1). 

Complementary DNAs (cDNA) are DNA copies of mRNAs. Reverse transcriptase is the RNA-directed DNA polymerase that synthesizes DNA strand, using purified mRNA as the template. DNA polymerase is then used to copy the DNA strand forming a double-stranded cDNA, which is cloned into a suitable vector. Once a cDNA derived from a particular gene has been identified, the cDNA becomes an effective probe for screening genomic libraries (Cowell and Austin, 1997). Annotated human cDNA sequences can be accessed from HUNT at http // www.hri.co.jp/HUNT (Yudate, 2001). 

Recently, Sedqi et al. [24] were able to clone a delta opioid receptor complementary DNA by expression of cDNA library from activated thymocytes in Cos 7 cells, whose amino acid sequence was similar to the neural counterpart. Interestingly, they also observed that transcripts for kappa and mu opioid receptors were not detected in thymocytes. Furthermore, Gave-riaux et al. [25] demonstrated transcripts for the delta opioid receptor in T-lymphocyte, B-lymphocyte, and monocyte cell lines, as well as in murine splenocytes. However, they observed that the kappa opioid receptor transcript was only found in B-cell lines. These studies may suggest a selective expression of the delta opioid receptor in specific cells and tissues of the immune system and suggests specialized functions in different anatomical regions. 

Fig. 9.7. Homology-independent fragment shuffling (SCRATCHY) is a combination of ITCHY and DNA shuffling, a) The approach starts by creating two complementary ITCHY libraries, b) To maximize the functional competence of the SCRATCHY library, hybrid sequences of approximately parental size that are in the correct reading...

Step 2 Gene isolation (three alternative methods) Nucleic acid triplet sequencing for amino acid sequence mRNA isolation, with reverse transcriptase and complementary DNA DNA probes from genetic library... 

A second subtype was cloned two years later from a human kidney complementary DNA (cDNA) library using the gene for the human a -adrenergic receptor as a probe (56). The gene for this subtype was shown to be located on human chromosome 4 and thus became known as a2-C4 it was initially identified as the a2, -pharmacological subtype, although this was later shown to be in error. 

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