Construction of a cosmid library of DNA replicated early in the S phase of normal human fibroblasts.

We constructed a subgenomic cosmid library of DNA replicated early in the S phase of normal human diploid fibroblasts. Cells were synchronized by release from confluence arrest and incubation in the presence of aphidicolin. Bromodeoxyuridine (BrdUrd) was added to aphidicolin-containing medium to label DNA replicated as cells entered S phase. Nuclear DNA was partially digested with Sau 3AI, and hybrid density DNA was separated in CsCl gradients. The purified early-replicating DNA was cloned into sCos1 cosmid vector. Clones were transferred individually into the wells of 96 microtiter plates (9,216 potential clones). Vigorous bacterial growth was detected in 8,742 of those wells. High-density colony hybridization filters (1, 536 clones/filter) were prepared from a set of replicas of the original plates. Bacteria remaining in the wells of replica plates were combined, mixed with freezing medium, and stored at -80 degrees C. These pooled stocks were analyzed by polymerase chain reaction to determine the presence of specific sequences in the library. Hybridization of high-density filters was used to identify the clones of interest, which were retrieved from the frozen cultures in the 96-well plates. In testing the library for the presence of 14 known early-replicating genes, we found sequences at or near 5 of them: APRT, beta-actin, beta-tubulin, c-myc, and HPRT. This library is a valuable resource for the isolation and analysis of certain DNA sequences replicated at the beginning of S phase, including potential origins of bidirectional replication.

Low abundance of microsatellite repeats in the genome of the brown-headed cowbird (Molothrus ater).

A cosmid library made from brown-headed cowbird (Molothrus ater) DNA was examined for representation of 17 distinct microsatellite motifs including all possible mono-, di-, and trinucleotide microsatellites, and the tetranucleotide repeat (GATA)n. The overall density of microsatellites within cowbird DNA was found to be one repeat per 89 kb and the frequency of the most abundant motif, (AGC)n, was once every 382 kb. The abundance of microsatellites within the cowbird genome is estimated to be reduced approximately 15-fold compared to humans. The reduced frequency of microsatellites seen in this study is consistent with previous observations indicating reduced numbers of microsatellites and other interspersed repeats in avian DNA. In addition to providing new information concerning the abundance of microsatellites within an avian genome, these results provide useful insights for selecting cloning strategies that might be used in the development of locus-specific microsatellite markers for avian studies.

Human release factor eRF1: structural organisation of the unique functional gene on chromosome 5 and of the three processed pseudogenes.

In lower and higher eukaryotes, a family of tightly related proteins designated eRF1 (for eukaryotic release factor 1) catalyses termination of protein synthesis at all three stop codons. The human genome contains four eRF1 homologous sequences localised on chromosomes 5, 6, 7 and X. We report here the cloning and the structural analysis of the human eRF1 gene family. It appears that the gene located on chromosome 5 alone is potentially functional, whereas the other three sequences resemble processed pseudogenes. This is the first description of the structural organisation of the human eRF1 gene, which has been remarkably conserved during evolution and which is essential in the translation termination process.

New, male-specific microsatellite markers from the human Y chromosome.

Seven novel microsatellite markers have been developed from a new cosmid library constructed from flow-sorted human Y chromosomes. These microsatellites are tetranucleotide GATA repeats and are polymorphic among unrelated individuals. Five of the seven markers are male-specific, with no PCR product being generated from female DNA. One marker produces male-specific, polymorphic PCR products but occasionally produces a much larger, invariant product from female DNA. The remaining marker is polymorphic in both males and females with many shared alleles between the sexes. This report of six new, male-specific markers doubles the number of tetranucleotide markers that are currently available for the human Y chromosome. These new markers will be valuable where nonrecombining, gender-specific DNA markers are desired, including forensic investigations as well as studies of populations and their evolutionary histories.

DNA synapomorphies for a variety of taxonomic levels from a cosmid library from the New World bat Macrotus waterhousii.

An effective method yielding taxon-specific markers from the genome of a single individual would be valuable for many types of scientific investigations, including systematic, forensic, conservation, and evolutionary studies. We explored the use of cosmid libraries, with insert sizes averaging 35 kb, to streamline the process of locating sequences of DNA that can serve as taxonomic markers from the specific to the ordinal levels. By screening approximately 2.6% of the leaf-nosed bat (Macrotus waterhousii) genome, we identified several potential DNA fragments that appear to be synapomorphic for a variety of taxonomic levels. A more thorough analysis of the markers documented that 17 Macrotus-specific clones represent three distinct DNA generic markers, whereas 30 microchiropteran clones represent multiple copies of a single family of repetitive DNA. The Microchiroptera taxon markers hybridize with representatives of most of the Microchiroptera families; however, no hybridization was detected for members of the superfamily Rhinolophoidea. These results demonstrate that cosmid libraries can be a valuable source for isolating taxon-specific markers from mammals even when the insert size is as large as 35 kb.

Large DNA fragment sizing by flow cytometry: application to the characterization of P1 artificial chromosome (PAC) clones.

A flow cytometry-based, ultrasensitive fluorescence detection technique is used to size individual DNA fragments up to 167 kb in length. Application of this technology to the sizing of P1 artificial chromosomes (PACs) in both linear and supercoiled forms is described. It is demonstrated that this method is well suited to characterizing PAC/BAC clones and will be very useful for the analysis of large insert libraries. Fluorescence bursts are recorded as individual, dye stained DNA fragments pass through a low power, focused, continuous laser beam. The magnitudes of the fluorescence bursts are linearly proportional to the lengths of the DNA fragments. The histograms of the burst sizes are generated in <3 min with <1 pg of DNA. Results on linear fragments are consistent with those obtained by pulsed-field gel electrophoresis. In comparison with pulsed-field gel electrophoresis, sizing of large DNA fragments by this approach is more accurate, much faster, requires much less DNA, and is independent of the DNA conformation.

A high-resolution annotated physical map of the human chromosome 13q12-13 region containing the breast cancer susceptibility locus BRCA2.

Various types of physical mapping data were assembled by developing a set of computer programs (Integrated Mapping Package) to derive a detailed, annotated map of a 4-Mb region of human chromosome 13 that includes the BRCA2 locus. The final assembly consists of a yeast artificial chromosome (YAC) contig with 42 members spanning the 13q12-13 region and aligned contigs of 399 cosmids established by cross-hybridization between the cosmids, which were selected from a chromosome 13-specific cosmid library using inter-Alu PCR probes from the YACs. The end sequences of 60 cosmids spaced nearly evenly across the map were used to generate sequence-tagged sites (STSs), which were mapped to the YACs by PCR. A contig framework was generated by STS content mapping, and the map was assembled on this scaffold. Additional annotation was provided by 72 expressed sequences and 10 genetic markers that were positioned on the map by hybridization to cosmids.

Characterization of a flow-sorted human chromosome 10 cosmid library by FISH.

Fluoresence in situ hybridization (FISH) was used to localize cosmids to regions of human chromosome 10. A total of 301 cosmids were selected randomly from a flow-sorted human chromosome 10 cosmid library constructed from human x hamster cell line 762-8A and arrayed in microtiter storage dishes. Over 70% (211/301) of the cosmids mapped to unique regions of chromosome 10. About 7% (22/301) produced multiple hybridization signals indicative of chimeric clones or sequences repeated at low copy number. Three cosmids (3/301, or 1%) hybridized to the centromeric regions of chromosome 10 and one or more other human chromosomes. About 19% (59/301) consisted mostly or entirely of hamster DNA inserts, and about 2% (6/301) appeared to be nonrecombinants.

How bats achieve a small C-value: frequency of repetitive DNA in Macrotus.

Bats possess a genome approximately 50-87% the size of other eutherian mammals. We document that the events that have achieved or maintained a small genome size in the Mexican leaf-nosed bat Macrotus waterhousii have resulted in a lower copy number of interspersed and tandemly repetitive elements. These conclusions are based on examination of 1726 randomly chosen recombinant cosmids, with an average insert size of 35.7 kb and representing 2.6% of the haploid genome of M. waterhousii. Probes representative of microsatellites [(GT)n, (CT)n, (AT)n, (GC)n] and a tandem repeat (rDNA) were used to estimate frequency of repetitive elements in the M. waterhousii genome. Of the four dinucleotides, (GT)n was present in 33.5% of the clones, (CT)n was present in 31.0% of the clones, and (AT)n and (GC)n were not represented in any of the clones examined. The 28S rDNA and a repetitive element from M. californicus were found in three and four clones, respectively. The dinucleotides (GT)n and (CT)n occurred together in the same clone more frequently than expected from chance. Although our data do not allow us to empirically test which mechanisms are maintaining copy number of repetitive DNA in the bat genome, the nonrandom association of these different families of repetitive DNA may provide insight into a mechanism that proportionately reduces diverse families of repetitive DNA that are known to be amplified by very different mechanisms.

Enzymatic elongation of microsatellite oligomers for use in direct-label chemiluminescent hybridizations.

Short, synthetic oligonucleotide sequences representing microsatellites and other short tandem repeats can be elongated (concatamerized) using a simple method in which complementary strands are annealed, phosphorylated, primer extended and ligated. When used in direct-label chemiluminescent hybridizations, the elongated microsatellite sequences provide an approximately 30-fold increase in signal strength compared with microsatellite oligomers that have not been concatamerized. Concatamerization of simple repeat oligomers further enables the use of relatively short oligonucleotide sequences in direct-label chemiluminescent hybridization experiments, thereby reducing the overall need for radioisotopes in certain commonly performed laboratory procedures such as DNA fingerprinting and selection of clones containing microsatellite sequences.

Characterization of a cosmid library from flow-sorted chromosomes 11.

A cosmid library specific for human chromosome 11 has been constructed from flow-sorted chromosomes. The flow-purified chromosomes were prepared from the hamster/human hybrid line J1 which contains chromosome 11 as the only human chromosome. Individual clones were sampled in 187 microtitre plates, resulting in a total of 17,952 colonies. Hybridization analysis revealed that 83.7% of these clones were of human and 10.4% of hamster origin. The average insert size was estimated at 33.6 kb, and only 2.4% of insert fragments appear to be rearranged. This should result in 494,487 kb of cloned human DNA representing 3.5 chromosome 11 equivalents. We have prepared high-density nylon membranes of the arrayed library containing 1,536 single colonies per filter. We have demonstrated the usefulness of the library in the molecular genetic analysis of human chromosome 11 by testing for the presence of possibly polymorphic simple repeat motifs, by identifying cosmids that contain inserts from the telomeric ends of chromosome 11 and by assessing the potential of the library for rapid chromosome walking.

Construction and characterization of partial digest DNA libraries made from flow-sorted human chromosome 16.

In this report, we present the techniques used for the construction of chromosome-specific partial digest libraries from flow-sorted chromosomes and the characterization of two such libraries from human chromosome 16. These libraries were constructed to provide materials for use in the development of a high-resolution physical map of human chromosome 16, and as part of a distributive effort on the National Laboratory Gene Library Project. Libraries with 20-fold coverage were made in Charon-40 (LA16NL03) and in sCos-1 (LA16NC02) after chromosome 16 was sorted from a mouse-human monochromosomal hybrid cell line containing a single homologue of human chromosome 16. Both libraries are approximately 90% enriched for human chromosome 16, have low nonrecombinant backgrounds, and are highly representative for human chromosome-16 sequences. The cosmid library in particular has provided a valuable resource for the isolation of coding sequences, and in the ongoing development of a physical map of human chromosome 16.

Genome organization of repetitive elements in the rodent, Peromyscus leucopus.

To document the frequency and distribution of repetitive elements in Peromyscus leucopus, the white-footed mouse, a cosmid genomic library was examined. Two thousand thirteen randomly chosen recombinants, with an average insert size of 35 kb and representing 2.35% of the haploid genome of P. leucopus, were screened with probes representing microsatellites, tandem repeats, and transposable elements. Of the four dinucleotides, (GT)n was present in 87% of the clones, (CT)n was present in 59% of the clones, and (AT)n and (GC)n each was represented in our sample by a single clone (0.05%). (TCC)n was present in 8% of the clones. Of the tandem repeats, the 28S ribosomal probe and the (TTAGGG)n telomere probe were not represented in the library, whereas a heterochromatic fragment was present in 9% of the clones. A transposable element, mys, was estimated to occur in 4700 copies, whereas a long interspersed element (LINE) was estimated to occur in about 41,000 copies per haploid genome. LINE and mys occurred together in the same clones more frequently than expected on the basis of chance. Hybridizing the library to genomic DNA from P. leucopus, Reithrodontomys fulvescens, Mus musculus, and human produced general agreement between phylogenetic relatedness and intensity of hybridization. However, dinucleotide repeats appeared to account for a disproportionately high number of positive clones in the more distantly related taxa.

Characterization of a human chromosome 8 cosmid library constructed from flow-sorted chromosomes.

A cosmid library for human chromosome 8 has been constructed from flow-sorted chromosomes in the vector sCos-1. This library is 85% human and has been arrayed into 210 microtiter plates representing four genome equivalents. Cosmids have been isolated with 10 of 11 probes representing nine different loci from chromosome 8.

Use of sex-linked minisatellite fragments to investigate genetic differentiation and migration of North American populations of the peregrine falcon (Falco peregrinus).

The M13 repeat detects different levels of genetic variation in falcons. First, this minisatellite probe reveals typically highly variant restriction fragments that show no apparent unequal distribution between the sexes. Secondly, the M13 repeat detects sets of fragments that are only present in DNAs from female falcons. The level of polymorphism displayed by the sex-linked fragments is greatly reduced relative to most autosomal minisatellites. In addition, the size of these fragments (in kilobase pairs) is species-specific among Mauritius kestrels (Falco punctatus) and peregrines (Falco peregrinus). Variation observed at one o of the sex-linked fragments in peregrines has proven to be useful in distinguishing a subset of the tundrius subspecies of this endangered raptor. This correlation has enabled a genetic test to be used to examine the representation of tundrius peregrines during mass migration.

Physical mapping of human chromosomes by repetitive sequence fingerprinting.

We have developed an approach for identifying overlapping cosmid clones by exploiting the high density of repetitive sequences in complex genomes. Individual clones are fingerprinted, using a combination of restriction enzyme digestions followed by hybridization with selected classes of repetitive sequences. This "repeat fingerprinting" technique allows small regions of clone overlap (10-20%) to be unambiguously assigned. We demonstrate the utility of this approach, using the fingerprinting of 3145 cosmid clones (1.25 x coverage), containing one or more (GT)n repeats, from human chromosome 16. A statistical analysis was used to link these clones into 460 contiguous sequences (contigs), averaging 106 kilobases (kb) in length and representing approximately 54% (48.7 Mb) of the euchromatic arms of this chromosome. These values are consistent with theoretical calculations and indicate that 150- to 200-kb contigs can be generated with 1.5 x coverage. This strategy requires the fingerprinting of approximately one-fourth as many cosmids as random strategies requiring 50% minimum overlap for overlap detection. By "nucleating" at specific regions in the human genome, and exploiting the high density of interspersed sequences, this approach allows (i) the rapid generation of large (greater than 100-kb) contigs in the early stages of contig mapping and (ii) the production of a contig map with useful landmarks for rapid integration of the genetic and physical maps.

Isolation and molecular characterization of a highly polymorphic centromeric tandem repeat in the family Falconidae.

An abundant tandem repeat has been cloned from genomic DNA of the merlin (Falco columbarius). The cloned sequence is 174 bp in length, and maps by in situ hybridization to the centromeric regions of several of the large chromosomes within the merlin karyotype. Complementary sequences have been identified within a variety of falcon species; these sequences are either absent or in very low copy number in the family Accipitridae. The cloned merlin repeat reveals highly polymorphic restriction patterns in the peregrine falcon (Falco peregrinus). These polymorphisms, which have been shown to be stably inherited within a family of captive peregrines, can be used to differentiate the Greenland and Argentina populations of this endangered raptor species.