Sets of genes improved by directed evolution can be recombined in vitro to produce further improvements in protein function. Recombination is particularly useful when improved sequences are available; costs of generating such sequences, however, must be weighed against the costs of further evolution by sequential random mutagenesis. Four genes encoding para-nitrobenzyl (pNB) esterase variants exhibiting enhanced activity were recombined in two cycles of high-®delity DNA shuf¯ing and screening. Genes encoding enzymes exhibiting further improvements in activity were analyzed in order to elucidate evolutionary processes at the DNA level and begin to provide an experimental basis for choosing in vitro evolution strategies and setting key parameters for recombination. DNA sequencing of improved variants from the two rounds of DNA shuf¯ing con®rmed important features of the recombination process: rapid ®xation and accumulation of bene®cial mutations from multiple parent sequences as well as removal of silent and deleterious mutations. The ®ve to sixfold further enhancement of total activity towards the para-nitrophenyl (pNP) ester of loracarbef was obtained through recombination of mutations from several parent sequences as well as new point mutations. Computer simulations of recombination and screening illustrate the trade-offs between recombining fewer parent sequences (in order to reduce screening requirements) and lowering the potential for further evolution. Search strategies which may substantially reduce screening requirements in certain situations are described.# 1997 Academic Press Limited

The SIS/PDGFB gene, encoding the B polypeptide of platelet-derived growth factor (PDGF-B), is transcriptionally activated (>50 fold) in human K562 erythroleukemia cells when they are induced to differentiate into megakary ocytic cells by treatment with phorbol 12-myristate 13-acetate ("tetradecanoylphorbol acetate," TPA). A 250-bp PDGF-B gene promoter attached to a reporter gene was shown to reproduce this TPA-induced activation. In a series of mutants that we constructed, a 10-bp linker sequence was systematically moved across the 250-bp PDGF-B promoter sequence, and the effect upon luciferase reporter activity was measured to Identify a site through which this TPA-induced nsciptional activation occurred. We identified a site, which we named the SIS proMal element (SPE), at positions -58 to -39 rdative to the PDGF-B mRNA initiation site that was esential for the TPA-induced activation. The SPE site contains two repeated sequences (TCTC and CACC) arranged in an ABBA configuration. The SPE sequence was not found in the exsting list ofconsensus sequences for transcription factor binding sites. Gel mobllity-shft assays using an SPE oligonucleotide and K562 cell nuclear extracts showed three shifted complexes, one of which was formed only following TPA treatment of K562 cells. In a tme-course study, TPA induction of the endogenous PDGF-B mRNA and formation of the TPA-inducible complex occurred over the same time frame, and both events were specifically blocked by the addition ofcycloheximide. The 20-bp SPE sequence was highly conserved (19/20) in both the cat and the mouse PDGF-B promoter, and conserved portions of the SPE sequence were also found at two sites within the human PDGF-A promoter. The role of the SPE in regulating the concurrent expression of the PDGF-B and PDGF-A genes in megakaryocytes, as well as various human tumor cells, is considered.

No abstract

No abstract