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The site-specific recombinases FLP and Cre are useful for genomic engineering in many living systems. Manipulation of their enzymatic properties offers a means to improve their applicability in different host organisms. We chose to manipulate the thermolability of FLP recombinase. A lacZ-based recombination assay in Escherichia coli was used for selection in a protein evolution strategy that relied on error-prone PCR and DNA shuffling. Improved FLP recombinases were identified through cycles of increasing stringency imposed by both raising temperature and reducing protein expression, combined with repetitive cycles of screening at the same stringency to enrich for clones with improved fitness. An eighth generation clone (termed FLPe) showed improved properties in E. coli, in vitro, in human 293- and mouse ES-cells.

Gene therapy studies require techniques that allow alteradapted an inducible homologous recombination system ation of human genomic DNA sequences. Bacterial artifor use in E. coli DH10B cells, the host strain for BACs ficial chromosome cloning systems (BACs/PACs) bridge and PACs. Using this system, we have introduced PCR the gap between vectors with small inserts and yeast artifragments carrying a selectable marker and a reporter ficial chromosomes (YACs). We report the use of a second gene downstream of the IVS I-110 splicing mutation into a generation BAC vector, pEBAC, containing eukaryotic selspecific site within the ␤-globin gene sequence. The use ectable markers and combining some of the best features of this inducible system minimises the risk of unwanted of the BAC, PAC and HAEC systems, into which a 185 kb rearrangements by recombination between repetitive sequence containing the human ␤-globin gene cluster was elements and allows the introduction of relevant modifiretrofitted. To permit the introduction of mutations correcations or reporters at any specific sequence within sponding to those causing human pathology, we have BACs/PACs in E. coli DH10B cells.Keywords: homologous recombination; ␤-globin; PAC; BAC; recE; recT PACs 1 and BACs 2 are used increasingly for long-range physical mapping, 3,4 positional cloning of disease genes, 5 whole genome sequencing projects 6 and functional studies. 7,8 However, the lack of convenient techniques for performing genetic manipulations on BACs/PACs in the host E. coli DH10B strain imposes serious limitations for functional analysis. Homologous recombination in an F plasmid-based vector in E. coli was first used in 1989 to join overlapping Drosophila cosmid fragments to form a 125 kb fragment. 9 Homologous recombination has also been used recently to introduce targeted modifications in BACs, 10-12 but the available techniques either require the construction of shuttle plasmids or are not directly applicable in DH10B cells.The bacteriophage Red-Gam and the RecE systems promote homologous recombination between linear DNA fragments and circular plasmid molecules or the host chromosome. 13,14 The reaction catalysed by the RecE pathway, termed ET cloning, 14 could be transferred to other recBC strains by cloning part of the recET operon into the l-arabinose-inducible expression plasmid pBAD24 to give plasmid pBAD24-trecET (YZ and AFS, 14 with the gam gene of bacteriophage being used as a natural inhibitor of recBCD nuclease, to allow ET cloning in recBC+ strains of E. coli. This plasmid enabled targeted modification of a 76 kb Drosophila P1 clone in its recBC+ host strain (NS3145) with PCR fragments having about 50 bp of homology in each arm.A second generation BAC/PAC cloning vector, pEBAC140, (Figure 1a, PAI and J-M Vos), combining features of the first generation PAC 1 and BAC 2 cloning systems and the HAEC system 15 was used in the initial efforts to optimize homologous recombination. EBAC/148␤ (Figure 1b) contains the entire ␤-globin locus (about 73 kb) in a 185 ...

Chromatin architecture plays an important regulatory role in nuclear transcription in eukaryotes. In particular, a preset chromatin organization has been proposed to prime rapidly inducible genes for response to extracellular signals. We analyzed the chromatin architecture of the rapidly inducible human c-fos gene using a combination of nuclease digestion studies. Several regions of nuclease sensitivity in the c-fos gene are observed: (i) a hypersensitive site at position -1900; (ii) a region centered at -350 that corresponds to the locations of the serum response element (SRE) and the sis-inducible element (SIE); (iii) a region around the transcriptional start site that includes the TATA box, the cAMP-responsive element (CRE) and a directly repeated sequence (DRE); and (iv) two sites at positions +250 and +550 that appear to delineate the linker regions of two positioned nucleosomes. In contrast, the region from -280 to -90 is strongly resistant to nuclease digestion. We identify a highly positioned nucleosome in this region of the c-fos promoter that may contribute to a defined higher-order nucleoprotein structure containing the nucleosome and proteins bound to the SIE, SRE, DRE, and CRE elements and TATA box. This structure may lead to a functional juxtaposition of the regulatory elements of the c-fos promoter and suggests that such a chromatin architecture is particularly suited for presetting promoters for rapid responsiveness.

The !'i-casein gene is a member of a small gene family encoding the calcium-sensitive caseins, which are specifically synthesized and secreted by the mammary gland during lactation in response to both peptide and steroid hormones. The caseins are involved in the transport of calcium phosphate in milk, which is important for bone development in the infant mammal. We report here the organization and complete DNA sequence of the 8·5 kb long bovine !'i-casein gene. Comparison with the rat !'i-casein gene reveals that the exons of both genes correspond exactly. The 5' flanking sequences of all Ca-sensitive casein genes are conserved within the proximal 200 bp and contain several elements that probably function as cis-acting regulatory elements, including an octamer-like motif, an SV40-type core enhancer and a sequence that appears to be common to all lactoprotein genes. The latter sequence is flanked on either side by 12 bp direct repeats. These direct repeats are themselves each part of sequences that display two-fold symmetry. The first 30 nucleotides of the 3' flanking regions in the bovine and rat !'i-caseins are well conserved, indicating that they are likely to be involved in the mechanism of 3' end processing of the primary transcript.

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