The univector plasmid-fusion system, a method for rapid construction of recombinant DNA without restriction enzymes.

BACKGROUND: . Modern biological research is highly dependent upon recombinant DNA technology. Conventional cloning methods are time-consuming and lack uniformity. Thus, biological research is in great need of new techniques to rapidly, systematically and uniformly manipulate the large sets of genes currently available from genome projects. RESULTS: . We describe a series of new cloning methods that facilitate the rapid and systematic construction of recombinant DNA molecules. The central cloning method is named the univector plasmid-fusion system (UPS). The UPS uses Cre-lox site-specific recombination to catalyze plasmid fusion between the univector - a plasmid containing the gene of interest - and host vectors containing regulatory information. Fusion events are genetically selected and place the gene under the control of new regulatory elements. A second UPS-related method allows for the precise transfer of coding sequences only from the univector into a host vector. The UPS eliminates the need for restriction enzymes, DNA ligases and many in vitro manipulations required for subcloning, and allows for the rapid construction of multiple constructs for expression in multiple organisms. We demonstrate that UPS can also be used to transfer whole libraries into new vectors. Additional adaptations are described, including directional PCR cloning and the generation of 3' end gene fusions using homologous recombination in Escherichia coli. CONCLUSIONS: . Together, these recombination-based cloning methods constitute a new comprehensive approach for the rapid and efficient generation of recombinant DNA that can be used for parallel processing of large gene sets, a feature that will facilitate future genomic analysis.

Activation of Xenopus MyoD transcription by members of the MEF2 protein family.

Members of the MEF2 family of DNA binding proteins interact with a set of AT-rich sequences commonly found in the promoters and enhancers of muscle-specific genes. We have shown that a MEF2 binding site precisely overlaps the TFIID binding site (TATA box) in the Xenopus MyoDa (XMyoDa) promoter and appears to play an important role in muscle-specific activity of this promoter. To further investigate the potential role of MEF2 in the regulation of XMyoDa transcription, we have analyzed the appearance of factors that interact with the XMyoDa TATA/MEF2 site during early amphibian development. Proteins that bind specifically to this site were present at low levels during early development and increased in abundance during gastrulation and neurulation. Two related cDNAs were isolated that encode proteins that recognize the XMyoDa TATA motif. Both proteins are highly homologous to each other, belong to the MADS (MCM1 agamous deficiens SRF) protein family, and are most highly related to the mammalian MEF2A gene products. Xenopus MEF2A (XMEF2A) transcripts accumulated preferentially in forming somites after the appearance of XMyoD transcripts. Ectopic expression of XMEF2A and other members of the MEF2 gene family activated transcription of a reporter gene controlled by the XMyoDa promoter. Transcriptional activation of the XMyoDa promoter required only the conserved DNA binding domain of XMEF2A and was independent of a domain necessary for activity when this factor was bound to multiple upstream sites. These results suggest that the XMyoDa promoter can be activated by binding of MEF2 to the XMyoDa TATA motif and indicate that MEF2-dependent transcriptional activation occurs by different mechanisms depending on the location of the MEF2 binding site. We suggest that XMEF2 expression in myogenic cells contributes to the activation and stabilization of XMyoDa transcription during muscle cell differentiation.

Binding of TFIID and MEF2 to the TATA element activates transcription of the Xenopus MyoDa promoter.

Members of the MyoD family of helix-loop-helix proteins control expression of the muscle phenotype by regulating the activity of subordinate genes. To investigate processes that control the expression of myogenic factors and regulate the establishment and maintenance of the skeletal muscle phenotype, we have analyzed sequences necessary for transcription of the maternally expressed Xenopus MyoD (XMyoD) gene. A 3.5-kb DNA fragment containing the XMyoDa promoter was expressed in a somite-specific manner in injected frog embryos. The XMyoDa promoter was active in oocytes and cultured muscle cells but not in fibroblasts or nonmuscle cell lines. A 58-bp fragment containing the transcription initiation site, a GC-rich region, and overlapping binding sites for the general transcription factor TFIID and the muscle-specific factor MEF2 was sufficient for muscle-specific transcription. Transcription of the minimal XMyoDa promoter in nonmuscle cells was activated by expression of Xenopus MEF2 (XMEF2) and required binding of both MEF2 and TFIID to the TATA motif. These results demonstrate that the XMyoDa TATA motif is a target for a cell-type-specific regulatory factor and suggests that MEF2 stabilizes and amplifies XMyoDa transcription in mesodermal cells committed to the muscle phenotype.

Sequential expression of multiple POU proteins during amphibian early development.

The octamer motif is a common cis-acting regulatory element that functions in the transcriptional control regions of diverse genes and in viral origins of replication. The ability of a consensus octamer motif to stimulate transcription of a histone H2B promoter in frog oocytes suggests that oocytes contain a transcriptionally active octamer-binding protein(s). We show here that frog oocytes and developing embryos contain multiple octamer-binding proteins that are expressed in a sequential manner during early development. Sequences encoding three novel octamer binding-proteins were isolated from Xenopus cDNA libraries by virtue of their homology with the DNA binding (POU) domain of Oct-1. The predicted POU domains of these proteins were most highly related to mammalian Oct-3 (also termed Oct-4), a germ line-specific gene required for mouse early development. Transcripts from these amphibian POU-domain genes were most abundant during early embryogenesis and absent from most adult somatic tissues. One of the genes, termed Oct-60, was primarily expressed as a maternal transcript localized in the animal hemisphere in mature oocytes. The protein encoded by this gene was present in oocytes and early embryos until the gastrula stage of development. Transcripts from a second POU-domain gene, Oct-25, were present at low levels in oocytes and early embryos and were dramatically upregulated during early gastrulation. In contrast to the Oct-60 mRNA, translation of Oct-25 mRNA appeared to be developmentally regulated, since the corresponding protein was detected in embryos during gastrulation but not in oocytes or rapidly cleaving embryos. Transcripts from the third POU protein gene, Oct-91, were induced after the midblastula transition and reached their highest levels of accumulation during late gastrulation. The expression of all three genes decreased during late gastrulation and early neurulation. By analogy with other members of the POU-domain gene family, the products of these genes may play critical roles in the determination of cell fate and the regulation of cell proliferation.