Optimized BlaM-transposon shuttle mutagenesis of Helicobacter pylori allows the identification of novel genetic loci involved in bacterial virulence

Helicobacter pylori is an important etiologic agent of gastroduodenal disease in humans. In this report, we describe a general genetic approach for the identification of genes encoding exported proteins in H. pylori. The novel TnMax9 mini-blaM transposon was used for insertion mutagenesis of a H. pylori gene library established in Escherichia coli. A total of 192 E. coli clones expressing active β-lactamase fusion proteins (BlaM⁺) were obtained, indicating that the corresponding target plasmids carry H. pylori genes encoding putative extracytoplasmic proteins. Natural transformation of H. pylori P1 or P12 using the 192 mutant plasmids resulted in 135 distinct H. pylori mutant strains (70%). Screening of the H. pylori collection of mutant strains allowed the identification of mutant strains impaired in motility, in natural transformation competence and in adherence to gastric epithelial cell lines. Motility mutants could be grouped into distinct classes: (i) mutant strains lacking the major flagellin subunit FlaA and intact flagella (class I); (ii) mutant strains with apparently normal flagella, but reduced motility (class II), and (iii) mutant strains with obviously normal flagella, but completely abolished motility (class III). Two independent mutations that exhibited defects in natural competence for genetic transformation mapped to different genetic loci. In addition, two independent mutant strains were isolated by their failure to bind to the human gastric carcinoma cell line Katoill. Both mutant strains carried a transposon in the same gene, 0.8 kb apart, and showed decreased autoagglutination when compared to the wild-type strain.     

Characterization of Two Chromosome 12 Cosmid Libraries and Development of STSs from Cosmids Mapped by FISH

We have constructed and characterized two related human chromosome 12-specific cosmid libraries. DNA from flow-sorted chromosomes from a somatic cell hybrid was cloned into a cosmid vector. Approximately 61% of the cosmids in the nearly 26,200 member arrayed libraries (LLt2NC01 and LLt2NC02) contain human DNA inserts, and 31% of the cosmids derived from human DNA contain CA repeats. One hundred and fifty-two cosmids isolated from the libraries have been mapped by fluorescence in situ hybridization (FISH). Cosmids containing human DNA inserts were localized by FISH exclusively to chromosome 12, confirming the chromosomal specificity of the libraries. The cosmids have been localized to all parts of this chromosome, although some regions are more highly represented than others. Partial sequence information was obtained from 44 mapped cosmids, and oligonucleotide primer pairs were synthesized that define unique sequence tagged sites (STSs). These mapped cosmids, and unique STSs derived from them, provide a set of useful clones and primer pairs for screening YAC libraries and developing contigs centered on regions of interest within chromosome 12. In addition, 120 of the mapped cosmids contain CA repeats, and thus they also provide a useful resource for defining highly polymorphic simple tandem repeat elements that serve as genetic markers for linkage analysis and disease gene localization.     

Physical map of the Streptomyces lividans 66 genome and comparison with that of the related strain Streptomyces coelicolor A3(2).

A physical map of the chromosome of Streptomyces lividans 66 ZX7 was constructed by ordering the macrorestriction fragments generated from the genomic DNA with the restriction enzymes AseI and DraI. AseI and DraI linking cosmids (i.e., recombinant cosmids including either AseI or DraI sites) were isolated from a gene bank and used as hybridization probes against Southern transfers of pulsed-field gel electrophoresis (PFGE) restriction patterns. The DraI sites were precisely mapped by PFGE analyses of AseI-DraI double digests and hybridization with the AseI junctions. The 16 AseI and 7 DraI fragments were aligned as a single chromosome of about 8,000 kb. The data supported the interpretation that the chromosome is a linear structure. The related strain Streptomyces coelicolor A3(2) M145, recently mapped by H. Kieser, T. Kieser, and D. A. Hopwood (J. Bacteriol. 174:5496-5507, 1992), was compared with S. lividans at the level of the genomic structure by hybridizing the linking cosmids to Southern transfers of PFGE patterns. In spite of little apparent similarity in their restriction patterns, the comparison of the physical maps revealed a common structure with an identical ordering of the cosmid sequences. This conservation of the map order was further confirmed by assigning genetic markers (i.e., cloned genes and DNA elements relevant to the unstable region) to the AseI fragments.     

Isolation and organization of genes for nitrogen fixation in Rhodopseudomonas capsulata

Summary A library of Rhodopseudomonas capsulata chromosomal DNA was constructed in the broad host range cosmid vector pLAFR1. The library was used to isolate nitrogen fixation genes by complementation of R. capsulata Nif^- mutants. Four complementing regions were localized on different cloned DNA fragments by Tn5 and mini-Mu mutagenesis. Additional nif genes were identified by recombination of transposons from the nif cosmids into the R. capsulata chromosome resulting in the creation of new Nif^- mutations. Most of the newly cloned DNA fragments containing nif genes were found to be unlinked to any other by Southern hybridization of the cloned DNA to chromosomal DNA blots. One of the new fragments was linked to the nifHDK genes. Another cluster spanning 10–12 kilobase pairs contained a number of nif genes, possibly as many as eight.     

Rapid mapping of cosmid clones on pig chromosomes by fluorescence in situ hybridization

Nineteen cosmids have been mapped to pig chromosomes by fluorescence in situ hybridization. Two kinds of cosmid clones were isolated as potential physical and genetic markers for the pig genome. Anonymous cosmids were obtained by screening a commercial cosmid library and were localized to Chromosomes (Chrs) 1, 2, 6, 7, 8, 10, 11, 12, 13, and 14. Some of these cosmids were found to reveal RFLP type DNA polymorphism. Microsatellite-containing cosmid clones were isolated by screening a pig cosmid library with a (CA)₁₀ probe and were regionally mapped to Chrs 2, 6, 7, 13, and 14. Ten of the 19 chromosomes in the pig were labeled with these probes. Two-color fluorescence in situ hybridization was used to increase the efficiency of the cosmid localizations.     

Physical map of the genome of Rhodobacter capsulatus SB 1003.

A map of the chromosome of Rhodobacter capsulatus was constructed by overlapping the large restriction fragments generated by endonucleases AseI and XbaI. The analyses were done by hybridization of single fragments with the restriction fragments blotted from pulsed-field gels and by grouping cosmids of a genomic library of R. capsulatus into contigs, corresponding to the restriction fragments, and further overlapping of the contigs. A technical difficulty due to a repeated sequence made it necessary to use hybridization with cloned genes and prior knowledge of the genetic map in order to close the physical circle in a unique way. In all, 41 restriction sites were mapped on the 3.6-Mb circular genome and 22 genes were positioned at 26 loci of the map. Cosmid clones were grouped in about 80 subcontigs, forming two groups, one corresponding to the chromosome of R. capsulatus and the other corresponding to a 134-kb plasmid. cos site end labeling and partial digestion of cosmids were used to construct a high-resolution EcoRV map of the 134-kb plasmid. The same method can be extended to the entire chromosome. The cosmid clones derived in this work can be used as a hybridization panel for the physical mapping of new genes as soon as they are cloned.     

Physically mapped,cosmid-derived microsatellite markers as anchor loci on bovine chromosomes

To identify physical and genetic anchor loci on bovine chromosomes, 13 cosmids, obtained after the screening of partial bovine cosmid libraries with the (CA)_n microsatellite motif, were mapped by fluorescence in situ hybridization (FISH). Eleven cosmid probes yielded a specific signal on one of the bovine chromosomes and identified the following loci: D5S2, D5S3, D6S3, D8S1, D11S5, D13S1, D16S5, D17S2, D19S2, D19S3, D21S8. Two cosmids produced centromeric signals on many chromosomes. The microsatellite-containing regions were subcloned and sequenced. The sequence information revealed that the two centromeric cosmids were derived from bovine satellites 1.723 and 1.709, respectively. A cosmid located in the subtelomeric region of Chromosome (Chr) 17 (D17S2) had features of a chromosome-specific satellite. Primers were designed for eight of the nonsatellite cosmids, and seven of these microsatellites were polymorphic with between three and eight alleles on a set of outbred reference families. The polymorphic and chromosomally mapped loci can now be used to physically anchor other bovine polymorphic markers by linkage analysis. The microsatellite primers were also applied to DNA samples of a previously characterized panel of somatic hybrid cell lines, allowing the assignment of seven microsatellite loci to defined syntenic groups. These assignments confirmed earlier mapping results, revealed a probable case of false synteny, and placed two formerly unassigned syntenic groups on specific chromosomes.     

Variability of gene order in different Helicobacter pylori strains contributes to genome diversity

Considerable genomic microdiversity has been reported previously among Helicobacter pylori isolates. We have constructed genome maps of four unrelated H. pylori strains (NCTC11637, NCTC11639, UA802 and UA861) using pulsed-field gel electrophoresis (PFGE) with Notl and Nrul, hybridization with extracted PFGE DNA fragments and probing with 17 gene probes. These strains of H. pylori were compared with a fifth unrelated H. pylori strain NCTC11638 mapped previously. Considerable diversity in gene arrangement was evident among the five H, pylori maps, and no consistent gene clustering was found. The association of only four genes, katA (catalase gene), vacA (vacuo-lating cytotoxin gene), hpaA (a putative adhesin gene), and pfr (bacterial ferritin gene) were generally conserved within approximately the same 25% of the genome; however, the order of these genes also varied. Our study demonstrates that macrodiversity, i.e. variability in gene order, in addition to microdiversity, is a characteristic of the H. pylori genome.     

A plasmid-based vector system for the cloning and expression of Helicobacter pylori genes encoding outer membrane proteins

Helicobacter pylori produces a number of proteins associated with the outer membrane, including adhesins and the vacuolating cytotoxin. We observed that the functional expression of such proteins is deleterious to Escherichia coli, the host bacterium used for gene cloning. Therefore, a general method was developed for the functional expression of such genes on a shuttle vector in H. pylori, which has been termed SOMPES (Shuttle vector-based Outer Membrane Protein Expression System). The intact, active gene is reconstituted by recombination in H. pylori from partial gene sequences cloned on an E. coli-H. pylori shuttle vector. This system was established in an H. pylori strain carrying a precise, unmarked chromosomal deletion of the vacA gene, which was constructed by adapting the streptomycin sensitivity system to H. pylori. It is based on the expression of the H. pylori rpsL gene as a counterselectable marker in the genetic background of an rpsL mutant. The utility of this approach is demonstrated by the expression of a recombinant gene encoding vacuolating cytotoxin (vacA) and a recombinant gene encoding an adherence-associated outer membrane protein (alpA) in H. pylori. Received: 10 May 1999 / Accepted: 7 July 1999     

Molecular analysis of the rat MHC. II. Isolation of genes that map to the RT1.E-grc region

An initial mapping analysis of growth and reproduction complex (grc) and grc+ genomic DNA identified several restriction fragment length polymorphisms specific for the grc region of the MHC. To analyze further the genomic organization and structure of the grc, a cosmid library was constructed from a grc+-bearing strain (R21). One cosmid cluster, encompassing 41.4 kb of DNA, contained four, or possibly five, class I genes that mapped to the RT1.E-grc region Two unique non-class I fragments were isolated from certain cosmids within this cluster. These fragments were hybridized to genomic DNA derived from five rat strains (BIL/2, R18, R21, R22, and BIL/1), and the results showed that grc-bearing rats have a deletion of at least 3.1 kb of DNA in the region immediately adjacent to the MHC. The loss of the genes in this region is probably the cause of the growth and reproductive defects in these animals and probably also of their increased susceptibility to chemical carcinogens.     

Identification,characterisation and clinical applications of cosmids from the telomeric and centromeric regions of the long arm of chromosome 22

Using human telomeric repeats and centromeric alpha repeats, we have identified adjacent single copy cosmid clones from human chromosome 22 cosmid libraries. These single copy cosmids were mapped to chromosome 22 by fluorescence in situ hybridisation (FISH). Based on these cosmids, we established contigs that included part of the telomeric and subtelomeric regions, and part of the centromeric and pericentromeric regions of the long arm of human chromosome 22. Each of the two cosmid contigs consisted of five consecutive steps and spanned approximately 100–150 kb at both extreme ends of 22q. Moreover, highly informative polymorphic markers were identified in the telomeric region. Our results suggest that the telomere specific repeat (TTAGGG) _n encompasses a region that is larger than 40 kb. The cosmid contigs and restriction fragment length polymorphism markers described here are useful tools for physical and genetic mapping of chromosome 22, and constitute the basis of further studies of the structure of the subtelomeric and pericentromeric regions of 22q. We also demonstrate the use of these clones in clinical diagnosis of different chromosome 22 aberrations by FISH.     

Genome-wide DNA methylation profiles in noncancerous gastric mucosae with regard to Helicobacter pylori infection and the presence of gastric cancer

Background and Aims: To determine genome‐wide DNA methylation profiles induced by Helicobacter pylori (H. pylori) infection and to identify methylation markers in H. pylori‐induced gastric carcinogenesis. Methods: Gastric mucosae obtained from controls (n = 20) and patients with gastric cancer (n = 28) were included. A wide panel of CpG sites in cancer‐related genes (1505 CpG sites in 807 genes) was analyzed using Illumina bead array technology. Validation of the results of Illumina bead array technique was performed using methylation‐specific PCR method for four genes (MOS, DCC, CRK, and PTPN6). Results: The Illumina bead array showed that a total of 359 CpG sites (269 genes) were identified as differentially methylated by H. pylori infection (p < .0001). The correlation between methylation‐specific PCR and bead array analysis was significant (p < .0001, Spearman coefficient = 0.5054). Methylation profiles in noncancerous gastric mucosae of the patients with gastric cancer showed quite distinct patterns according to the presence or absence of the current H. pylori infection; however, 10 CpG sites were identified to be hypermethylated and three hypomethylated in association with the presence of gastric cancer regardless of H. pylori infection (p < .01). Conclusions: Genome‐wide methylation profiles showed a number of genes differentially methylated by H. pylori infection. Methylation profiles in noncancerous gastric mucosae from the patients with gastric cancer can be affected by H. pylori‐induced gastritis. Differentially methylated CpG sites in this study needs to be validated in a larger population using quantitative methylation‐specific PCR method.     

Helicobacter pylori hspA-hspB heat-shock gene cluster: nucleotide sequence, expression, putative function and immunogenicity

All Helicobacter pylori isolates synthesize a 54 kDa immunodominant protein that was reported to be associated with the nickel-dependent urease of H. pylori. This protein was recently recognized as a homologue of the heat-shock protein of the GroEL class. The gene encoding the GroEL-like protein of H. pylori (HspB) was cloned (plLL689) and was shown to belong to a bicistronic operon including the hspA and hspB genes. In Escherichia coli. the constitutive expression of the hspA and hspB genes was initiated from a promoter located within an IS5 insertion element that mapped upstream to the two open reading frames (ORFs). IS5 was absent from the H. pylori genome, and was thus acquired during the cosmid cloning process. hspA and hspB encoded polypeptides of 118 and 545 amino acid residues, corresponding to calculated molecular masses of 13.0 and 58.2 kDa, respectively. Amino acid sequence comparison studies revealed that, although H. pylori HspA and HspB proteins were highly similar to their bacterial homologues, the H. pylori HspA featured a striking motif at the C-terminus. This unique motif consists of a series of cysteine and histidine residues resembling a nickel-binding domain, which is not present in any of the other bacterial GroES homologues so far characterized. When the plLL689 recombinant plasmid was introduced together with the H. pylori urease gene cluster (plLL763) into an E. coli host strain, an increase of urease activity was observed. This suggested a close interaction between the HspA and HspB proteins and the urease enzyme, and a possible role for HspA in ihe chelation of nickel ions. The genes encoding each of the HspA and HspB polypeptides were cloned, expressed independently as proteins fused to the maltose-binding protein (WIBP) and purified in large scale. The MBP-HspA and MBP-HspB fusion proteins were shown to retain their antigenic properties. Both HspA and HspB represent antigens that are specifically recognized by the sera from H. pylori-infected patients. Whereas HspB was known to be immunogenic in humans, this is the first demonstration that HspA per se is also immunogenic as proteins fused to the maltose-binding protein (WIBP) and purified in large scale. The MBP-HspA and WlBP-HspB fusion proteins were shown to retain their antigenic properties. Both HspA and HspB represent antigens that are specifically recognized by the sera from H, py/or/-infected patients. Whereas HspB was known to be immunogenic in humans, this is the first demonstration that HspA per se is also immunogenic.     

Virulence of infecting Helicobacter pyloristrains and intensity of mononuclear cell infiltration are associated with levels of DNA hypermethylation in gastric mucosae

DNA methylation changes are known to occur in gastric cancers and in premalignant lesions of the gastric mucosae. In order to examine variables associated with methylation levels, we quantitatively evaluated DNA methylation in tumors, non-tumor gastric mucosae, and in gastric biopsies at promoters of 5 genes with methylation alterations that discriminate gastric cancers from non-tumor epithelia (EN1, PCDH10, RSPO2, ZIC1, and ZNF610). Among Colombian subjects at high and low risk for gastric cancer, biopsies from subjects from the high-risk region had significantly higher levels of methylation at these 5 genes than samples from subjects in the low risk region (p ≤ 0.003). When results were stratified by Helicobacter pylori infection status, infection with a cagA positive, vacA s1m1 strain was significantly associated with highest methylation levels, compared with other strains (p = 0.024 to 0.001). More severe gastric inflammation and more advanced precancerous lesions were also associated with higher levels of DNA methylation (p ≤ 0.001). In a multivariate model, location of residence of the subject and the presence of cagA and vacA s1m1 in the H. pylori strain were independent variables associated with higher methylation in all 5 genes. High levels of mononuclear cell infiltration were significantly related to methylation in PCDH10, RSPO2, and ZIC1 genes. These results indicate that for these genes, levels of methylation in precancerous lesions are related to H. pylori virulence, geographic region and measures of chronic inflammation. These genes seem predisposed to sustain significant quantitative changes in DNA methylation at early stages of the gastric precancerous process.     

Analyses of the cag pathogenicity island of Helicobacter pylori

Most strains of Helicobacter pylori from patients with peptic ulcer disease or intestinal-type gastric cancer carry cagA, a gene that encodes an immunodominant protein of unknown function, whereas many of the strains from asymptomatically infected persons lack this gene. Recent studies showed that the cagA gene lies near the right end of a ≈37 kb DNA segment (a pathogenicity island, or PAI) that is unique to cagA⁺ strains and that the cag PAI was split in half by a transposable element insertion in the reference strain NCTC11638. In complementary experiments reported here, we also found the same cag PAI, and sequenced a 39 kb cosmid clone containing the left ‘cagII’ half of this PAI. Encoded in cagII were four proteins each with homology to four components of multiprotein complexes of Bordetella pertussis (‘Ptl’), Agrobacterium tumefaciens (‘Vir’), and conjugative plasmids (‘Tra’) that help deliver pertussis toxin and T (tumour inducing) and plasmid DNA, respectively, to target eukaryotic or prokaryotic cells, and also homologues of eukaryotic proteins that are involved in cytoskeletal structure. To the left of cagII in this cosmid were genes for homologues of HslU (heat-shock protein) and Era (essential GTPase); to the right of cagII were homologues of genes for a type I restriction endonuclease and ion transport functions. Deletion of the cag PAI had no effect on synthesis of the vacuolating cytotoxin, but this deletion and several cag insertion mutations blocked induction of synthesis of proinflammatory cytokine IL-8 in gastric epithelial cells. Comparisons among H. pylori strains indicated that cag PAI gene content and arrangement are rather well conserved. We also identified two genome rearrangements with end-points in the cag PAI. One, in reference strain NCTC11638, involved IS605, a recently described transposable element (as also found by others). Another rearrangement, in 3 of 10 strains tested (including type strain NCTC11637), separated the normally adjacent cagA and picA genes and did not involve IS605. Our results are discussed in terms of how cag-encoded proteins might help trigger the damaging inflammatory responses in the gastric epithelium and possible contributions of DNA rearrangements to genome evolution.     

Role of γ‐glutamyltranspeptidase in the pathogenesis of Helicobacter pylori infection

γ‐Glutamyltranspeptidase and asparaginase have been shown to play important roles in Helicobacter pylori colonization and cell death induced by H. pylori infection. In this study, the association of γ‐glutamyltranspeptidase and asparaginase was elucidated by comparing activities of both deamidases in H. pylori strains from patients with chronic gastritis, gastric and duodenal ulcers, and gastric cancer. γ‐Glutamyltranspeptidase activities in H. pylori strains from patients with gastric cancer were significantly higher than in those from patients with chronic gastritis or gastric ulcers. There was a wide range of asparaginase activities in H. pylori strains from patients with gastric cancer and these were not significantly than those from patients with other diseases. To identify the contributions of γ‐glutamyltranspeptidase and asparaginase to gastric cell inflammation, human gastric epithelial cells (AGS line) were infected with H. pylori wild‐type and knockout strains and inflammatory responses evaluated by induction of interleukin‐8 (IL‐8). IL‐8 response was significantly decreased by knockout of the γ‐glutamyltranspeptidase‐encoding gene but not by knockout of the asparaginase‐encoding gene. Additionally, IL‐8 induction by infection with the H. pylori wild‐type strain was significantly decreased by adding glutamine during infection. These findings indicate that IL‐8 induction caused by γ‐glutamyltranspeptidase activity in H. pylori is mainly attributable to depletion of glutamine. These data suggest that γ‐glutamyltranspeptidase plays a significant role in the chronic inflammation caused by H. pylori infection.     

Genome mapping in halobacteria

The goal of our research is to produce an ordered set of cosmid clones for each of several species of halobacteria for use in physical and genetic mapping. These maps will answer questions about genome evolution and about gene organization and regulation in this archaebacterial lineage. Progress in cloning and mapping the genome of Halobacterium volcanii DS2 (synonym Haloferax volcanii DS2) is reported. Overlapping cosmids are recognized by a strategy which makes use of the distinctive restriction fragments around relatively rare restriction sites. Each site recognized by the infrequently cutting restriction enzymes is a landmark from which to identify different regions of the genome. The main advantage of this strategy is that only a small overlap (10-20%) between cosmid clones is required, resulting in a correspondingly small number of cosmid clones to be analyzed. The certainty of overlap is high, and computation is simple. The final 5-10% of each genome is cloned, linked, and identified by chromosome walking methods. Hybridization of cloned homologous or heterologous genes and of stable RNAs to the minimal cosmid set localizes these genes on the physical map. Additional genes have been and will be cloned by complementation of auxotrophic mutants, or as determinants of resistance to antibiotics.     

Type I Restriction–Modification Loci Reveal High Allelic Diversity in Clinical Helicobacter pylori Isolates

Background: A remarkable variety of restriction‐modification (R‐M) systems is found in Helicobacter pylori. Since they encompass a large portion of the strain‐specific H. pylori genes and therefore contribute to genetic variability, they are suggested to have an impact on disease outcome. Type I R‐M systems comprise three different subunits and are the most complex of the three types of R‐M systems. Aims: We investigated the genetic diversity and distribution of type I R‐M systems in clinical isolates of H. pylori. Material and methods: Sixty‐one H. pylori isolates from a Swedish hospital based case‐control study and 6 H. pylori isolates of a Swedish population‐based study were analyzed using polymerase chain reaction for the presence of the three R‐M systems' subunits. Representative gene variants were sequenced. Results: Although the hsdM and hsdR genes appeared conserved in our clinical H. pylori isolates, the sequences of the hsdS loci were highly variable. Despite their sequence diversity, the genes per se were present at high frequencies. We identified a number of novel allelic hsdS variants, which are distinct from corresponding hsdS loci in the sequenced H. pylori strains 26695, J99 and HPAG1. In analyses of paired H. pylori isolates, obtained from the same individuals with a 4‐year interval, we observed genetic modifications of hsdS genes in patients with atrophic gastric mucosa. Discussion: We propose that the genetic variability of hsdS genes in a bacterial population will give rise to new specificities of these enzymes, which might lead to adaptation to an ever‐changing gastric environment.     

The linkage mapping of cloned restriction fragment length differences in Caenorabditis elegans

Summary The genomic DNA of two closely related strains of the nematode, Caenorhabditis elegans, Bristol (N2), and Bergerac (Bo), has different restriction endonuclease sites (Emmons et al. 1979). Since these two strains interbreed, it is possible to regard the restriction fragment length differences (RFLDs) as mutant variants. The N2 and Bo pattern can be segregated and mapped using clasical genetic techniques.Utilizing a number of genetic markers existing in the N2 strain, we have constructed hybrid populations homozygous for either Bristol or Bergerac over a given chromosomal region with random Bristol-Bergerac composition for the remainder of the genome. Genomic restriction digests from these hybrid populations were probed with random cloned fragments of Bristol DNA. In this way, fragments were mapped to genetically well characterized regions of the C. elegans genome. 27 probes which hybridize to a total of 310 Kb of DNA were found to exhibit six restriction fragment differences. Four of these differences have been mapped, providing probes for four different genomic regions. We have combined classical genetics and recombinant DNA technology to construct linkage maps of cloned DNA fragments using restriction fragment length differences. We are pursuing this approach in order to advance the knowledge of the genetic organization of C. elegans and to provide a means of cloning genes in an organism which provides an experimental model for the study of many biological systems. It is hoped that this approach will also provide a practical solution to some difficult problems in nematode strain identification. Furthermore, the characterization of the families of transposable elements responsible for generating many of the RFLDs will undoubtedly contribute to the understanding of the biological significance of these elements.