Display cloning: functional identification of natural product receptors using cDNA-phage display.

BACKGROUND: The identification of cellular targets has traditionally been the starting point for natural product mode of action studies and has led to the understanding of many biological processes. Conventional experimental approaches have depended on cell-based screening and/or affinity chromatography. Although both of these techniques aid in the discovery of protein cellular targets, a method that couples protein identification with gene isolation would be extremely valuable. RESULTS: A procedure for the direct cloning of cellular proteins, based on their affinity for natural products, using cDNA phage display has been developed. The technique is referred to as display cloning because it involves the cloning of proteins displayed on the surface of a bacteriophage particle. The approach has been established by isolating a full-length gene clone of FKBP12 (FK506-binding protein) from a human brain cDNA library using a biotinylated FK506 probe molecule. During the affinity selection, the FKBP12 gene emerged as the dominant library member and was the only sequence identified after the second round of selection. CONCLUSIONS: The development of display cloning greatly facilitates the investigation of ligand-receptor interaction biology and natural product mode of action studies. This procedure utilizes heterologous protein display on infectious phage, which allows the amplification and repeated selection of putative sequences, leading to unambiguous target identification. In addition, the direct connection of a functional protein to its gene sequence eliminates the subsequent cloning step required with tissue homogenate or cell lysate affinity methods, allowing direct isolation of an expressible gene sequence.

Study of accommodative facility testing reliability.

This study was designed to evaluate the reliability of the accommodative facility testing procedure. Sixty-six subjects, ages 8 to 12 years, were studied over 3 consecutive weeks. Monocular and binocular accommodative facility, using plus and minus 2 D lenses, was performed each week. Statistical analysis showed a significant mean increase in cycles per minute (cpm) between initial and subsequent testing periods, both monocular and binocular, for all subjects as a group. The most dramatic increases were observed among subjects who scored below established norms initially. To evaluate the effect of test-retest variability on the subject's pass/fail status the first test period results were used to categorize the subjects as pass or fail. Passing was greater than or equal to 11 cpm monocularly, greater than or equal to 8 cpm binocularly. Failing was less than 1, cpm monocularly, less than 8 cpm binocularly. Subsequent test results were compared to the initial testing results to determine the pass/fail reliability of the testing procedure. No significant differences were found to occur in either the monocular or binocular "pass" category. However, a significant increase in the passing rate from the initial to the subsequent testing periods for both the monocular and binocular facility rates was observed in the "fail" category. Dividing the fail group into "low-fails" (less than 6 cpm monocularly, less than 3 cpm binocularly) and "high-fails" (greater than 6 less than 11 cpm monocularly, greater than 3 less than 8 cpm binocularly) indicated this significant increase was principally in the high-fail group.