Library

Abstract: A bacterial cloning system for mapping and analysis of complex genomes has been developed. The BAC system (for bacterial artificial chromosome) is based on Escherichia coli and its single-copy plasmid F factor. It is capable of maintaining human genomic DNA fragments of greater than 300 kilobase pairs. Individual clones of human DNA appear to be maintained with a high degree of structural 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.

Abstract: Sequencing of ribosomal DNA clone libraries amplified from environmental DNA has revolutionized our understanding of microbial eukaryote diversity and ecology. The results of these analyses have shown that protist groups are far more genetically heterogeneous than their morphological diversity suggests. However, the clone library approach is labour-intensive, relatively expensive, and methodologically biased. Therefore, even the most intensive rDNA library analyses have recovered only small samples of much larger assemblages, indicating that global environments harbour a vast array of unexplored biodiversity. High-throughput parallel tag 454 sequencing offers an unprecedented scale of sampling for molecular detection of microbial diversity. Here, we report a 454 protocol for sampling and characterizing assemblages of eukaryote microbes. We use this approach to sequence two SSU rDNA diversity markers-the variable V4 and V9 regions-from 10 L of anoxic Norwegian fjord water. We identified 38 116 V4 and 15 156 V9 unique sequences. Both markers detect a wide range of taxonomic groups but in both cases the diversity detected was dominated by dinoflagellates and close relatives. Long-tailed rank abundance curves suggest that the 454 sequencing approach provides improved access to rare genotypes. Most tags detected represent genotypes not currently in GenBank, although many are similar to database sequences. We suggest that current understanding of the ecological complexity of protist communities, genetic diversity, and global species richness are severely limited by the sequence data hitherto available, and we discuss the biological significance of this high amplicon diversity.

Abstract: Recent progress in molecular microbial ecology has revealed that traditional culturing methods fail to represent the scope of microbial diversity in nature, since only a small proportion of viable microorganisms in a sample are recovered by culturing techniques. To develop methods to investigate the full extent of microbial diversity, we used a bacterial artificial chromosome (BAC) vector to construct libraries of genomic DNA isolated directly from soil (termed metagenomic libraries). To date, we have constructed two such libraries, which contain more than 1 Gbp of DNA. Phylogenetic analysis of 16S rRNA gene sequences recovered from one of the libraries indicates that the BAC libraries contain DNA from a wide diversity of microbial phyla, including sequences from diverse taxa such as the low-G1C, gram-positive Acidobacterium, Cytophagales, and Proteobacteria. Initial screening of the libraries in Escherichia coli identified several clones that express heterologous genes from the inserts, confirming that the BAC vector can be used to maintain, express, and analyze environmental DNA. The phenotypes expressed by these clones include antibacterial, lipase, amylase, nuclease, and hemolytic activities. Metagenomic libraries are a powerful tool for exploring soil microbial diversity, providing access to the genetic information of uncultured soil microorganisms. Such libraries will be the basis of new initiatives to conduct genomic studies that link phylogenetic and functional information about the microbiota of environments dominated by microorganisms that are refractory to cultivation. The biosphere is dominated by microorganisms (32), yet most microbes in nature have not been studied. Traditional methods for culturing microorganisms limit analysis to those that grow under laboratory conditions (14, 25). The recent surge of research in molecular microbial ecology provides compelling evidence for the existence of many novel types of microorganisms in the environment in numbers and varieties that dwarf those of the comparatively few microorganisms amenable to laboratory cultivation (7, 13, 31). Corroboration comes from estimates of DNA complexity and the discovery of many unique 16S rRNA gene sequences from numerous environmental sources (8, 10, 28). Collectively, the genomes of the total microbiota found in nature, which we termed the metagenome (11), contain vastly more genetic information than is contained in the culturable subset. Given the profound utility and importance of microorganisms to all biological systems, methods are needed to access the wealth of information within the metagenome. Cloning large fragments of DNA isolated directly from microbes in natural environments provides a method to access soil metagenomic DNA. Previously, we investigated the use of the bacterial artificial chromosome (BAC) vector to express Bacillus cereus genomic DNA (20). The advantage of BAC vectors is that they maintain very large DNA inserts (greater than 100 kb) stably in Escherichia coli (23), facilitating the cloning of large fragments of DNA. Our results demonstrated that expression of heterologous DNA from B. cereus in an E. coli BAC system was detectable at a reasonable frequency (20), validating the idea that the low-copy BAC vector (one to two per cell) (23) could be used to express foreign DNA from foreign promoters in E. coli. Here we describe the construction and initial screening of two BAC libraries made with DNA isolated directly from soil. We found detectable levels of several biochemical activities from BAC library clones. Sequence analysis of selected BAC plasmids encoding such activities and of 16S rRNA genes in one of the libraries confirms the novelty of the genomic information cloned in our libraries. The results show that DNA extracted directly from soil is a valuable source of new genetic information and is accessible by using BAC libraries. Our results demonstrate that both traditional and functional genomics of uncultured microorganisms can be carried out by this approach and that screening of metagenome libraries for activities or gene sequences can provide a basis for conducting genomic analyses of uncultured microorganisms.

Abstract: This paper describes a plasmid vector for cloning cDNAs in Escherichia coli; the same vector also promotes expression of the cDNA segment in mammalian cells. Simian virus 40 (SV40)-derived DNA segments are arrayed in the pcD vector to permit transcription, splicing, and polyadenylation of the cloned cDNA segment. A DNA fragment containing both the SV40 early region promoter and two introns normally used to splice the virus 16S and 19S late mRNAs is placed upstream of the cDNA cloning site to ensure transcription and splicing of the cDNA transcripts. An SV40 late region polyadenylation sequence occurs downstream of the cDNA cloning site, so that the cDNA transcript acquires a polyadenylated 3' end. By using pcD-alpha-globin cDNA as a model, we confirmed that the alpha-globin transcript produced in transfected cells is initiated correctly, spliced at either of the two introns, and polyadenylated either at the site coded in the cDNA segment or at the distal SV40 polyadenylation signal. A cDNA clone library constructed with mRNA from SV40-transformed human fibroblasts and this vector (about 1.4 X 10(6) clones) yielded full-length cDNA clones that express hypoxanthine-guanine phosphoribosyltransferase (Jolly et al., Proc. Natl. Acad. Sci. U.S.A., in press).