A new bacteriophage P1-derived vector for the propagation of large human DNA fragments.

We have designed a P1 vector (pCYPAC-1) for the introduction of recombinant DNA into E. coli using electroporation procedures. The new cloning system, P1-derived artificial chromosomes (PACs), was used to establish an initial 15,000 clone library with an average insert size of 130-150 kilobase pairs (kb). No chimaerism has been observed in 34 clones, by fluorescence in situ hybridization. Similarly, no insert instability has been observed after extended culturing, for 20 clones. We conclude that the PAC cloning system will be useful in the mapping and detailed analysis of complex genomes.

The complete BRCA2 gene and mutations in chromosome 13q-linked kindreds.

Breast carcinoma is the most common malignancy among women in developed countries. Because family history remains the strongest single predictor of breast cancer risk, attention has focused on the role of highly penetrant, dominantly inherited genes in cancer-prone kindreds (1). BRCA1 was localized to chromosome 17 through analysis of a set of high-risk kindreds (2), and then identified four years later by a positional cloning strategy (3). BRCA2 was mapped to chromosomal 13q at about the same time (4). Just fifteen months later, Wooster et al. (5) reported a partial BRCA2 sequence and six mutations predicted to cause truncation of the BRCA2 protein. While these findings provide strong evidence that the identified gene corresponds to BRCA2, only two thirds of the coding sequence and 8 out of 27 exons were isolated and screened; consequently, several questions remained unanswered regarding the nature of BRCA2 and the frequency of mutations in 13q-linked families. We have now determined the complete coding sequence and exonic structure of BRCA2 (GenBank accession #U43746), and examined its pattern of expression. Here, we provide sequences for a set of PCR primers sufficient to screen the entire coding sequence of BRCA2 using genomic DNA. We also report a mutational analysis of BRCA2 in families selected on the basis of linkage analysis and/or the presence of one or more cases of male breast cancer. Together with the specific mutations described previously, our data provide preliminary insight into the BRCA2 mutation profile.

Genomic characterization of the human heterotrimeric G protein alpha, beta, and gamma subunit genes.

Heterotrimeric guanine nucleotide binding proteins (G proteins) transduce extracellular signals received by transmembrane receptors to effector proteins. Each subunit of the G protein complex is encoded by a member of one of three corresponding gene families. Currently, 16 different members of the alpha subunit family, 5 different members of the beta subunit family, and 11 different members of the gamma subunit family have been described in mammals. Here we have identified and characterized Bacterial Artificial Chromosomes (BACs) containing the human homologs of each of the alpha, beta, and gamma subunit genes as well as a G alpha11 pseudogene and a previously undiscovered G gamma5-like gene. The gene structure and chromosome location of each gene was determined, as were the orientations of paired genes. These results provide greater insight into the evolution and functional diversity of the mammalian G protein subunit genes.

The development and applications of the bacterial artificial chromosome cloning system.

The development of the Bacterial Artificial Chromosome (BAC) system was driven in part by the Human Genome Project as a means to construct genomic DNA libraries and physical maps for genomic sequencing. The BAC system is based on the well-characterized Escherichia coli F-factor, a low copy plasmid that exists in a supercoiled circular form in host cells. The structural features of the F-factor allow stable maintenance of individual human DNA clones as well as easy manipulation of the cloned DNA. BACs are currently used in a wide array of applications from genome sequencing to gene discovery. This paper describes the key elements in the development of the BAC system and its current notable applications.

Conditional-lethal deoxyribonucleic acid polymerase I mutant of Escherichia coli.

A new deoxyribonucleic acid polymerase I mutant of Escherichia coli was isolated among conditional lethal mutants. Deoxyribonucleic acid replication in the mutant ceased in 20 min after the temperature was raised to 43 degrees C, and reinitiated when cells were further incubated at this temperature.

Conditional lethality of deletions which include uvrB in strains of Escherichia coli lacking deoxyribonucleic acid polymerase I.

Deletions of the uvrB gene were not obtained in polA1 strains of Escherichia coli either by selecting for spontaneous deletions or by transduction from strains carrying such deletions. A strain forming a temperature-sensitive deoxyribonucleic acid polymerase I and carrying a deletion of the uvrB gene is inviable at the nonpermissive temperature.

Host requirements for growth of lambda-P22 hybrid in Escherichia coli.

The requirements for growth of bacteriophage lambda containing the deoxyribonucleic acid replication region from Salmonella phage P22 were determined in a burst size experiment. The products of genes dnaE, dnaJ, dnaK, dnaY, dnaZ, and seg were required, but not the products of genes dnaA, dnaB, dnaC, and dnaX. This lambda-P22 hybrid phage was also dependent on polA for growth at 32 degrees C.

Synthesis of bacteriophage lambda DNA in vitro: requirement for O and P gene products.

We have developed a cell-free system to study bacteriophage lambda DNA replication. Maximal DNA synthesis in vitro requires the four deoxynucleoside triphosphates, ATP, and exogenous lambda DNA. DNA synthesis requires the products of the phage O and P genes but is not inhibited by lambda repressor. The kinetics of synthesis is linear for 10-15 min; however, the product of synthesis amounts to only 0.5-1% of the added template DNA. As judged by isopycnic analysis, extensive regions of the template are copied. Sedimentation analysis indicates that all of the product consists of short (11S) DNA chains. Fractions partially purified from lambdaO(+)P(+)-infected cell extracts will complement extracts prepared from lambdaO(-) or lambdaP(-)-infected cells.

DNA polymerase I-dependent mutants of coliphage lambda.

Mutagenized lysates of bacteriophage lambda were screened for mutants unable to plate on DNA polymerase I-deficient (polA(-)) hosts. The mutants obtained were all recombination deficient (red(-)). These mutants, like red(-) and gam(-) mutants previously isolated by others, grow more poorly than wild-type lambda even on polA(+) hosts (burst size 14 to 30% of wild-type lambda.) In a polA(-) host, the burst size of red(-) and gam(-) mutants is reduced an additional five- to tenfold, and lysis is delayed. Wild-type lambda grows normally in polA(-) hosts. Neither lambdaN(-)nin (which doesn't express red or gam) nor lambdabio phages (from which all or part of the red-gam region is deleted) form plaques on polA(-) hosts. Apparent revertants, able to plate on polA(-) hosts, have been selected from both lambdaN(-)nin and lambdabio. Those derived from N(-)nin seem to be N(-)nin cro(-) mutants; whereas those coming from lambda bio have a new bypass mutation (pas) that lies between genes P and Q.

Construction and characterization of the IGF Arabidopsis BAC library.

A bacterial artificial chromosome (BAC) library has been established for Arabidopsis thaliana (ecotype Col-0) covering about seven haploid nuclear genome equivalents. This library, called the Institut für Genbiologische Forschung (IGF) BAC library, consists of 10,752 recombinant clones carrying inserts (generated by partial EcoRI digestion) of an average size of about 100 kb in a modified BAC vector, pBeloBAC-Kan. Hybridization with organellar DNA and nuclear repetitive DNA elements revealed the presence of 1.1% clones with mitochondrial DNA, 0.2% clones with plastid DNA, 3.2% clones with the 180 bp paracentromeric repeat, 1.6% clones with 5S rDNA, and 10.8% clones with the 18S-25S rDNA repeat. With its extensive genome coverage, its rather uniformly sized inserts (80 kb < 85% < 120 kb) and low contamination with organellar DNA, this library provides an excellent resource for A. thaliana genomic mapping, map-based gene cloning, and genome sequencing.

Deoxyribonucleic acid polymerase II activity in an Escherichia coli mutator strain.

The polB gene encoding deoxyribonucleic acid (DNA) polymerase II has been located close to a mutator gene, mutT1, in Escherichia coli. We find the DNA polymerase II prepared from mutT1, strains to be normal in reaction requirements, heat stability, and ability to remove mismatched bases at termini. Recombinants formed from a mutant defective in DNA polymerase II (polB100) and mutT1 are deficient in polymerase II and have the same mutator phenotype as mutT1. Our linkage analysis indicates that mutT1 and polB100 are not isoallelic.

Isolation of a specialized transducing bacteriophage lambda carrying the polC locus of Escherichia coli.

We have isolated a specialized transducing phage carrying the polC locus of E. coli K-12. A strain of E. coli lacking the lambda attachment site was infected with bacteriophage lambda. Lysogens carrying lambda at the tonA locus were isolated by selecting lambda-immune, T5-resistant strains. Transducing phages of dapC, dapD and polC, which map within 0.2 min of tonA, were obtained in lysates prepared from two of the lysogens. The isolated phage, lambdadpolC, is defective but can transduce four different polC temperature-sensitive mutants. After lysogenation with the transducing phage, DNA polymerase III activity is restored to normal levels in extracts of a polC strain lacking polymerase III activity. However, attempts to obtain increased levels of DNA polymerase III in extracts of induced lysogens carrying lambdadpolC have been unsuccessful.

Mapping of a mutation, polB100, affecting deoxyribonucleic acid polymerase II in Escherichia coli K-12.

Direct assay for deoxyribonucleic acid polymerase II in extracts has been used to screen recombinants for the polB allele in Hfr x F(-) crosses, F-ductants in episome transfer, and transductants in generalized transduction by phage P1. The polB gene is located at 2 min on the Escherichia coli linkage map; it is 39 to 64% co-transducible with leu. A mutant, E. coli polA1 polB100 polC (ts), deficient in deoxyribonucleic acid polymerases I and II and having a thermolabile deoxyribonucleic acid polymerase III, has been prepared by the P1-mediated cross: P1 (HMS85 polB100) x E. coli BT1026 polA1 polC (ts) leu(-).

Bacterial plasmids in antarctic natural microbial assemblages.

Samples of psychrophilic and psychrotrophic bacteria were collected from sea ice, seawater, sediments, and benthic or ice-associated animals in McMurdo Sound, Antarctica. A total of 155 strains were isolated and tested for the presence of plasmids by DNA agarose gel electrophoresis. Thirty-one percent of the isolates carried at least one kind of plasmid. Bacterial isolates taken from sediments showed the highest plasmid incidence (42%), and isolates from seawater showed the lowest plasmid incidence (20%). Plasmids were significantly more frequent in the strains which had been first isolated from low-nutrient media (46%) than in the strains which had been isolated from high-nutrient media (25%). Multiple forms of plasmids were observed in two-thirds of the plasmid-carrying strains. A majority of the plasmids detected were estimated to have a mass of 10 megadaltons or less. Among 48 plasmid-carrying strains, 7 showed antibiotic resistance. It is concluded that bacterial plasmids are ubiquitous in natural microbial assemblages of the pristine marine ecosystem of Antarctica.

Heat-labile alkaline phosphatase from Antarctic bacteria: Rapid 5' end-labeling of nucleic acids.

A heat-labile alkaline phosphatase has been purified to near homogeneity from HK47, a bacterial strain isolated from Antarctic seawater. The active form of the enzyme has a molecular weight of 68,000 and is uniquely monomeric. The optimal temperature for the enzymatic activity is 25 degrees C. Complete and irreversible thermal inactivation of the enzyme occurs in 10 min at 55 degrees C. By using this heat-labile enzyme for dephosphorylation followed by a 10-min heat treatment, rapid end-labeling of nucleic acids by T4 polynucleotide kinase has been achieved.

Characterization of uncultivated prokaryotes: isolation and analysis of a 40-kilobase-pair genome fragment from a planktonic marine archaeon.

One potential approach for characterizing uncultivated prokaryotes from natural assemblages involves genomic analysis of DNA fragments retrieved directly from naturally occurring microbial biomass. In this study, we sought to isolate large genomic fragments from a widely distributed and relatively abundant but as yet uncultivated group of prokaryotes, the planktonic marine Archaea. A fosmid DNA library was prepared from a marine picoplankton assemblage collected at a depth of 200 m in the eastern North Pacific. We identified a 38.5-kbp recombinant fosmid clone which contained an archaeal small subunit ribosomal DNA gene. Phylogenetic analyses of the small subunit rRNA sequence demonstrated it close relationship to that of previously described planktonic archaea, which form a coherent group rooted deeply within the Crenarchaeota branch of the domain Archaea. Random shotgun sequencing of subcloned fragments of the archaeal fosmid clone revealed several genes which bore highest similarity to archaeal homologs, including large subunit ribosomal DNA and translation elongation factor 2 (EF2). Analyses of the inferred amino acid sequence of archaeoplankton EF2 supported its affiliation with the Crenarchaeote subdivision of Archaea. Two gene fragments encoding proteins not previously found in Archaea were also identified: RNA helicase, responsible for the ATP-dependent alteration of RNA secondary structure, and glutamate semialdehyde aminotransferase, an enzyme involved in initial steps of heme biosynthesis. In total, our results indicate that genomic analysis of large DNA fragments retrieved from mixed microbial assemblages can provide useful perspective on the physiological potential of abundant but as yet uncultivated prokaryotes.

Bacterial artificial chromosome cloning and mapping of a 630-kb human extrachromosomal structure.

We have cloned and mapped a circular 630-kb human extrachromosomal structure (termed amplisome) using the bacterial artificial chromosome (BAC) cloning system. Twenty-one BACs were isolated from an amplisome-enriched library by colony hybridization. The insert sizes range from 25 to 143 kb, with an average size of 82 kb. The coverage of the amplisome in clones is approximately 2.7-fold. To construct a physical map of the amplisome, we used three different but complementary methods: hybridization, STS content mapping, and fingerprinting. In addition, we compared the advantages and the drawbacks of these techniques in mapping the amplisomal BACs. The 21 BACs were grouped into two contigs and the two small gaps (3.5 and 26.5 kb) were filled by screening of a human genomic BAC library. The organization of the amplisome revealed by the BAC-based physical map is consistent with the long-range restriction map reported previously. Our results demonstrate that a 630-kb region can be rapidly cloned and mapped into contigs by use of the BAC system. Because of the low frequency (<0.1%) of chimerism and rearrangement, these BAC clones are ready for DNA sequencing and functional analysis.

Framework for a physical map of the human 22q13 region using bacterial artificial chromosomes (BACs).

Detailed physical maps of entire chromosomes based on combined genetic, cytogenetic, and structural information are essential components for positional cloning and genomic sequencing. Despite the wealth of genetic information of the known diseases in the chromosome 22q13, the construction of a detailed physical map of the terminal region is difficult due to the sparsity of the genetic markers. We present here a map of bacterial artificial chromosome (BAC) contigs that cover a number of genetic loci in the 22q13 region. One hundred thirty-six BACs with an average insert size of 140 kb are assembled into 35 contigs defined by 64 markers in 22q13-qter. Twenty-three anonymous markers are now linked to the previously mapped genetic anchor points.

Cloning of contiguous genomic fragments from human chromosome 21 harbouring three trefoil peptide genes.

A group of small peptides with a typical cysteine-rich domain (termed trefoil motif or P-domain) is abundantly expressed at mucosal surfaces of specific normal and neoplastic tissues. Their association with the maintenance of surface integrity was suggested. The first known human trefoil peptide (pS2) was isolated from breast cancer cells (MCF7). Its oestrogen-inducible gene, and the human homologue to the porcine spasmolytic peptide gene (hSP/SML1) appear synchronously expressed in healthy stomach mucosa and several carcinomas of the gastrointestinal tract. Both genes were shown to be localised at 21q22.3. A new trefoil peptide from human intestinal mucosa (hITF/hP1.B) and its gene were described recently. By using suitable oligonucleotide primers and PCR and isolating large (110-250 kb) genomic recombinants cloned in the bacterial artificial chromosome (BAC) system, we present a genomic region from chromosome band 21q22.3 cloned in contiguous sequences and encoding all three members of human P-domain/trefoil peptides proving a physical linkage of all three trefoil peptide genes. Such genomic sequences will provide useful experimental material for analysis of gene regulation, for gene modification experiments and for establishing transgenic cells or animals.