CDNA library construction

Several groups have reported the isolation of cDNA clones for myeloperoxidase from cDNA libraries constructed from mRNA purified from HL-60 cells (Morishita et al. 1987 Weil et al. 1987). If such libraries are induced to express the proteins for which the cDNA molecules encode, then they can be screened for particular genes using antibodies. Indeed, this has been the approach used to isolate cDNA clones for myeloperoxidase from HL-60 cells. Full-length clones have been described and sequenced, and their identity confirmed by a variety of means ... 

Fig. 8.1. Generalized selection cycle for in vitro evolution of an RNA catalyst. Random libraries are PCR-amplified, transcribed, modified with a tethered reactant, reacted with a second substrate in solution, and reverse-transcribed. Active RNA/cDNA library constructs are separated from inactive ones so that they can enter the next cycle of selection.

The final SAR gene family, SAR8.2, comprises several highly related cDNAs which were isolated by differential screening of a tobacco cDNA library constructed from RNA of induced resistant leaves (D. Alexander et al., unpublished data). The cDNA encodes a basically-charged protein of about 50 residues, which has not been isolated and whose function is unknown. 

Jiang, G., Harrison, D.J., mRNA isolation for cDNA library construction on a chip. Micro Total Analysis Systems, Proceedings of the 4th TTAS Symposium, Enschede, Netherlands, May 14-18, 2000, 537-540. 

We have cloned the 59 kD trichome PPO from a cDNA library constructed from epidermal mRNA (H. Yu and J. Steffens, unpublished data) and are currently using this clone as a probe to isolate the genomic DNA encoding the S. berthaultii trichome PPO (S. Newman and J. Steffens, unpublished data). These studies may allow us to increase the insect entrapping abilities of cultivated potato, which has retained low Type A trichome densities and no longer possesses the biochemical machinery... 

Efficient extraction of high quality RNA from a variety of plant tissues is an important first step in many procedures, such as analysis of gene expression, cDNA library construction, and in vitro translation. This procedure, which is essentially as described in Draper et al. (1), involves grinding and phenol extraction of plant material followed by differential precipitation of RNA with sodium acetate. The protocol has been successful with leaf material and cultured cells from a large number of species, however, slight adjustments may be necessary to optimize extraction from other tissues. 

Zhu YY, Machleder EM, Chenchik A, li R, Siebert PD (2001) Reverse transcriptase template switching a SMART approach fiar full-length cDNA library construction. Biotechniques 30(4) 892-897... 

Ohara R, Kikuno RF, Kitamura H, Ohara O (2005) CDNA library construction from a small amount of RNA adaptor-ligation approach for two-round cRNA amplification using T7 and SP6 RNA polymerases. Biotechniques 38 451-458... 

We have obtained a cDNA clone of the wound-induced enzyme by screening a cDNA library constructed in an expression vector, pTTQlS, with the antibody. Size of the insert DNA in the clone (pCMW33) was 1.8 kbp, and the transformant bacteria harboring this plasmid produced under the inductive conditions protein of about 58 kDa exhibiting ACC synthase activity. Northern blot analysis of RNA obtained from incubated mesocarp slices with the insert DNA as a probe showed that mRNA for ACC synthase was of 1.9 kb. This mRNA was not detected in fresh tissue, but increased after wounding (unpublished results). 

A mixture ofj degenerate oligonucleotide probes from a 10-ammo acid sequence of a peptide (CYCPSTEVAK m Figure) was used to screen a cucumber Xgtl 1 cDNA library (constructed from polv A+ RNA of 5-day-old seedlings illuminated for 6... 

The two-hybrid system is a yeast-based genetic screen that is used to identify and characterize protein-protein interactions (/). In this system, two fusion proteins are expressed in yeast and their interaction is monitored in vivo using appropriate reporter-gene systems. One hybrid protein, termed the bait, contains the DNA-binding domain (DBD) of a transactivator fused to protein X. The second fusion protein, termed the prey or target, contains an activation domain (ACT) of the transactivator fused to protein Y. If DBD-X and ACT-Y are co-expressed in yeast, and if X and Y interact with one another, then the transactivator is reconstituted in a functionally relevant manner and expression of the reporter gene is enhanced. The power of the system resides in the ability to screen cDNA libraries constructed m the ACT vector to conduct global searches for novel proteins that interact with a protein of interest. 

Expression cloning has had widespread sueeess in discovery of prokaryotic enzymes [32], but relatively few examples of expression cloning for elucidation of plant metabolism have been reported. In one example, a cDNA library constructed from genetic material of pumpkin seedlings Cucurbita maxima L.) was functionally... 

---