A bacterial coning system for mapping and analysis of complex genomes has been developed. The BAC system (for bacterial artificial chromosome) is based on Escherichia colt and its single-copy plasmid F factor. It is capable of maintaining human genomic DNA fragments of >300 kilobase pai-. Individual clones of human DNA appear to be maintained with a high degree ofstructural stability in the host, even after 100 generations of serial growth. Because of high cloning efficiency, easy manipulation of the cloned DNA, and stable maintenance of inserted DNA, the BAC system may facilitate construction of DNA libraries of complex genomes with fuller representation and subsequent rapid analysis of complex genomic structure.There is currently underway an intense effort to construct a high-resolution physical map of each of the human chromosomes. Eventually, these maps will be composed of overlapping fragments of human DNA and will allow the direct acquisition of DNA fragments that correspond to specific genes. Completion of the physical map requires the availability of comprehensive libraries of DNA clones in appropriate vectors. Furthermore, the accuracy and efficiency of physical mapping increase progressively with the size of the clone firgments in these libraries. Thus, the construction of libraries using yeast artificial chromosomes (YACs), which permit cloning of fragments of ;500 kilobase pairs (kb), represents a fundamental advance in our ability to generate physical maps that order DNA over multi-megabase distances (1). However, some difficulties have been encountered with the manipulation of YAC libraries (2-4). Thus, for example, in various libraries a fraction of the clones result from co-cloning events; i.e., they include in a single clone noncontiguous DNA fiagments. We describe here a bacterial cloning system that we refer to as BACs, bacterial artificial chromosomes. This system may provide a supplement and alternative to the YAC system for some applications requiring cloning of large fragments. The BAC system is based on the well-studied Escherichia coli F factor. Replication of the F factor in E. coli is strictly controlled (5). The F plasmid is maintained in low copy number (one or two copies per cell), thus reducing the potential for recombination between DNA fragments carried by the plasmid. Furthermore, F factors carrying inserted bacterial DNA are capable of maintaining fragments as large as 1 megabase pair, suggesting that the F factor is suitable for cloning of large DNA fragments (6). Other bacterial systems for cloning large DNA have been developed. For example, the system based on bacteriophage P1 is in use (7). However, the P1 vector has a maximum cloning capacity of 100 kb. A bacterial system based on F factors has been reported (8). However, in this system, human DNA inserts >120 kb have not been cloned and characterized. The BAC system allows us to clone large DNA from a variety of complex genomic sources into bacteria, where the DNA is stable, easy to manipulate, and represents a si...

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Silk fibers have outstanding mechanical properties. These fibers are insoluble in organic solvents and water, are biocompatible, and exhibit slow biodegradation in vitro and in vivo due to the hydrophobic nature of the protein and the presence of a high content of β‐sheet structure. Regenerated silk fibroin can be processed into a variety of materials normally stabilized by the induction of β‐sheet formation through the use of solvents or by physical stretching. To extend the biomaterial utility of silk proteins, options to form water‐stable silk‐based materials with reduced β‐sheet formation would be desirable. To address this need for more rapidly degradable silk biomaterials, we report the preparation of water‐stable films from regenerated silk fibroin solutions, with reduced β‐sheet content. The keys to this process are the preparation of concentrated (8 % by weight) aqueous solutions of fibroin and a subsequent water‐based annealing procedure. These new materials degrade more rapidly due to the reduced β‐sheet content, as determined in vitro via enzymatic hydrolysis, yet support human adult stem‐cell expansion in vitro in a similar or improved fashion to the crystallized proteins in film form. These new silk‐based materials extend the range of biomaterial properties that can be generated from this unique family of proteins.

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