SalI Digests Research Articles

Repression of Death Receptor–Mediated Apoptosis of Hepatocytes by Hepatitis B Virus e Antigen

Hepatitis B virus (HBV) e antigen (HBeAg) is associated with viral persistence and pathogenesis. Resistance of HBV-infected hepatocytes to apoptosis is seen as one of the primary promotors for HBV chronicity and malignancy. Fas receptor/ligand (Fas/FasL) and the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) system plays a key role in hepatic death during HBV infection. We found that HBeAg mediates resistance of hepatocytes to FasL or TRAIL-induced apoptosis. Introduction of HBeAg into human hepatocytes rendered resistance to FasL or TRAIL cytotoxicity in a p53-dependent manner. HBeAg further inhibited the expression of p53, total Fas, membrane-bound Fas, TNF receptor superfamily member 10a, and TNF receptor superfamily member 10b at both mRNA and protein levels. Incontrast, HBeAg enhanced the expression of soluble forms of Fas through facilitation of Fas alternative mRNA splicing. In a mouse model, expression of HBeAg in mice injected with recombinant adenovirus-associated virus 8 inhibited agonistic anti-Fas antibody-induced hepatic apoptosis. Xenograft tumorigenicity assay also found that HBeAg-induced carcinogenesis was resistant to the proapoptotic effect of TRAIL and chemotherapeutic drugs. These results indicate that HBeAg may prevent hepatocytes from FasL and TRAIL-induced apoptosis by regulating the expression of the proapoptotic and antiapoptotic forms of death receptors, which may contribute to the survival and persistence of infected hepatocytes during HBV infection.

Analysis of Membrane Topology and Identification of Essential Residues for the Yeast Endoplasmic Reticulum Inositol Acyltransferase Gwt1p

Glycosylphosphatidylinositol (GPI) is a post-translational modification that anchors cell surface proteins to the plasma membrane, and GPI modifications occur in all eukaryotes. Biosynthesis of GPI starts on the cytoplasmic face of the endoplasmic reticulum (ER) membrane, and GPI precursors flip from the cytoplasmic side to the luminal side of the ER, where biosynthesis of GPI precursors is completed. Gwt1p and PIG-W are inositol acyltransferases that transfer fatty acyl chains to the inositol moiety of GPI precursors in yeast and mammalian cells, respectively. To ascertain whether flipping across the ER membrane occurs before or after inositol acylation of GPI precursors, we identified essential residues of PIG-W and Gwt1p and determined the membrane topology of Gwt1p. Guided by algorithm-based predictions of membrane topology, we experimentally identified 13 transmembrane domains in Gwt1p. We found that Gwt1p, PIG-W, and their orthologs shared four conserved regions and that these four regions in Gwt1p faced the luminal side of the ER membrane. Moreover, essential residues of Gwt1p and PIG-W faced the ER lumen or were near the luminal edge of transmembrane domains. The membrane topology of Gwt1p suggested that inositol acylation occurred on the luminal side of the ER membrane. Rather than stimulate flipping of the GPI precursor across the ER membrane, inositol acylation of GPI precursors may anchor the precursors to the luminal side of the ER membrane, preventing flip-flops.

HPV Genotype Detection Using Hybrid Capture Sample Preparation Combined with Whole Genome Amplification and Multiplex Detection with Luminex XMAP

Infection with a high-risk carcinogenic type of human papillomavirus (HPV) is necessary for the development of cervical cancer. The digene HC2 HPV Test (HC2) is an important screening tool but lacks genotyping capability. To address this issue, we developed an assay for the rapid genotyping of HPV in cervical specimens. The three steps of this assay include Hybrid Capture target enrichment, whole-genome amplification, and Luminex XMAP detection. The assay includes the simultaneous detection of two genomic regions from each of 17 high-risk and two low-risk HPV types most associated with disease. The assay performance was tested on HPV plasmids as well as clinical specimens. An analytical limit of detection of 100 copies or less was demonstrated for linear, circular, and integrated HPV DNA. This finding is at least 1 log lower than the HC2 assay limit of detection. There was no cross-reactivity among the HPV types up to 1,000,000 copies. There was also no substantial assay interference from substances in cervical specimens. Although the clinical performance of the assay was not formally tested, the assay had good agreement (Cohen's kappa equal to 0.72) with both a PCR-based HPV genotyping assay (n = 131) and the HC2 assay (n = 502) using representative cervical specimens. This assay may be easy to automate and could be applied for the detection of other targets in future studies.

Genomic Subtraction Recovers Rye-Specific DNA Elements Enriched in the Rye Genome

Repetitive DNA sequence families have been identified in methylated relic DNAs of rye. This study sought to isolate rye genome-specific repetitive elements regardless of the level of methylation, using a genomic subtraction method. The total genomic DNAs of rye-chromosome-addition-wheat lines were cleaved to short fragments with a methylation-insensitive 4-bp cutter, MboI, and then common DNA sequences between rye and wheat were subtracted by annealing with excess wheat genomic DNA. Four classes of rye-specific repetitive elements were successfully isolated from both the methylated and non-methylated regions of the genome. Annealing of the DNA mixture at a ratio of the enzyme-restricted fragments:the sonicated fragments (1:3-1:5) was key to this success. Two classes of repetitive elements identified here belong to representative repetitive families: the tandem 350-family and the dispersed R173 family. Southern blot hybridization patterns of the two repetitive elements showed distinct fragments in methylation-insensitive EcoO109I digests, but continuous smear signals in the methylation-sensitive PstI and SalI digests, indicating that both of the known families are contained in the methylated regions. The subtelomeric tandem 350-family is organized by multimers of a 380-bp-core unit defined by the restriction enzyme EcoO109I. The other two repetitive element classes had new DNA sequences (444, 89 bp) and different core-unit sizes, as defined by methylation-sensitive enzymes. The EcoO109I recognition sites consisting of PyCCNGGPu-multi sequences existed with high frequency in the four types of rye repetitive families and might be a useful tool for studying the genomic organization and differentiation of this species.

Construction and Analysis of A Directional Xenopus laevis Embryonic cDNA Library for Yeast Two-hybrid Using Modified Random Primers

Here we describe a new strategy for cDNA library construction, in which the random primers directing the first strand cDNA synthesis contain additional d (AC) at their 5'-ends, and the linkers which will be added to the double strand cDNA contain d(GTCG) at the 5′-ends. When the linker is added to the 3'-end of the cDNA, a complete SalI site d(GTCGAC) will form at the 3'-end but not at the 5′-end of the cDNA fragment. After SalI digestion, the 3'-cohesive and the pre-existing 5'EcoRI cohesive ends are used to introduce the double stranded cDNA into the linearized plasmid vectors. The ligation products were used to transform E.coli to produce a cDNA library. Using this method, we constructed a directional Xenopus laevis embryonic cDNA library for yeast two-hybrid. We checked the ratio of empty vectors, the size of inserts and existence of several genes, the results showed that cDNA library construction was successful.

Evolutionarily Conserved Mammalian Adenine Nucleotide Translocase 4 Is Essential for Spermatogenesis

The adenine nucleotide translocases (Ant) facilitate the transport of ADP and ATP by an antiport mechanism across the inner mitochondrial membrane, thus playing an essential role in cellular energy metabolism. We recently identified a novel member of the Ant family in mouse, Ant4, of which gene configuration as well as amino acid homology is well conserved among mammals. The conservation of Ant4 in mammals, along with the absence of Ant4 in nonmammalian species, suggests a unique and indispensable role for this ADP/ATP carrier in mammalian development. Of interest, in contrast to its paralog Ant2, which is encoded by the X chromosome and ubiquitously expressed in somatic cells, Ant4 is encoded by an autosome and selectively expressed in testicular germ cells. Immunohistochemical examination as well as RNA expression analysis using separated spermatogenic cell types revealed that Ant4 expression was particularly high in spermatocytes. When we generated Ant4-deficient mice by targeted disruption, a significant reduction in testicular size was observed without any other distinguishable abnormalities in the mice. Histological examination as well as stage-specific gene expression analysis in adult and neonatal testes revealed a severe reduction of spermatocytes accompanied by increased apoptosis. Subsequently, the Ant4-deficient male mice were infertile. Taken together, these data elucidated the indispensable role of Ant4 in murine spermatogenesis. Considering the unique conservation and chromosomal location of the Ant family genes in mammals, the Ant4 gene may have arisen in mammalian ancestors and been conserved in mammals to serve as the sole and essential mitochondrial ADP/ATP carrier during spermatogenesis where the sex chromosome-linked Ant2 gene is inactivated.

The Swi2/Snf2 Bromodomain Is Required for the Displacement of SAGA and the Octamer Transfer of SAGA-acetylated Nucleosomes

The SWI/SNF and SAGA chromatin-modifying complexes contain bromodomains that help anchor these complexes to acetylated promoter nucleosomes. To study the importance of bromodomains in these complexes, we have compared the chromatin-remodeling and octamer-transfer activity of the SWI/SNF complex to a mutant complex that lacks the Swi2/Snf2 bromodomain. Here we show that the SWI/SNF complex can remodel or transfer SAGA-acetylated nucleosomes more efficiently than the Swi2/Snf2 bromodomain-deleted complex. These results demonstrate that the Swi2/Snf2 bromodomain is important for the remodeling as well as for the octamer-transfer activity of the complex on H3-acetylated nucleosomes. Moreover, we show that, although the wild-type SWI/SNF complex displaces SAGA that is bound to acetylated nucleosomes, the bromodomain mutant SWI/SNF complex is less efficient in SAGA displacement. Thus, the Swi2/Snf2 bromodomain is required for the full functional activity of SWI/SNF on acetylated nucleosomes and is important for the displacement of SAGA from acetylated promoter nucleosomes.

Involvement of a Putative [Fe-S]-cluster-binding Protein in the Biogenesis of Quinohemoprotein Amine Dehydrogenase

Quinohemoprotein amine dehydrogenase (QHNDH) of Paracoccus denitrificans contains a peptidyl quinone cofactor, cysteine tryptophylquinone, as well as intrapeptidyl thioether cross-links between Cys and Asp/Glu residues within the smallestgamma-subunit of the alphabetagamma heterotrimeric protein. A putative [Fe-S]-cluster-binding protein (ORF2 protein) encoded between the structural genes for the alpha- and gamma-subunits of QHNDH in the n-butylamine-utilizing operon likely belongs to a Radical SAM (S-Ado-Met) superfamily that includes many proteins involved in vitamin biosynthesis and enzyme activation. In this study the role of ORF2 protein in the biogenesis of QHNDH has been explored. Although the wild-type strain of Paracoccus denitrificans produced an active, mature enzyme upon induction with n-butylamine, a mutant strain in which the ORF2 gene had been mostly deleted, neither grew in the n-butylamine medium nor showed QHNDH activity. When the mutant strain was transformed with an expression plasmid for the ORF2 protein, n-butylamine-dependent bacterial growth and QHNDH activity were restored. Site-specific mutations in the putative [Fe-S]-cluster or SAM binding motifs in the ORF2 protein failed to support bacterial growth. The alpha- and beta-subunits were both detected in the periplasm of the mutant strain, whereas the gamma-subunit polypeptide was accumulated in the cytoplasm and stained negatively for redox-cycling quinone staining. Matrix-assisted laser desorption ionization time-of-flight mass spectrometric analysis revealed that the gamma-subunit isolated from the mutant strain had not undergone posttranslational modification. These results unequivocally show that the putative [Fe-S]-cluster- and SAM-binding ORF2 protein is necessary for the posttranslational processing of gamma-subunit, most likely participating in the formation of the intrapeptidyl thioether cross-links.

PCR Amplification of the IS 6110 Insertion Element of Mycobacterium tuberculosis in Fecal Samples from Patients with Intestinal Tuberculosis

PCR amplification of insertion element IS6110 of Mycobacterium tuberculosis in fecal samples was evaluated in the diagnosis of intestinal tuberculosis (ITB). The numbers of samples that tested positive by PCR with SalI digestion were 16/18 untreated-ITB samples, 0/8 treated-ITB samples, 12/14 smear-positive pulmonary tuberculosis samples, and 0/30 control samples. The sensitivity, specificity, positive predictive value, and negative predictive value of fecal PCR were 88.8%, 100%, 100%, and 93.7%, respectively.

Genetic Divergence of Campylobacter fetus Strains of Mammal and Reptile Origins

Campylobacter fetus is a gram-negative bacterial pathogen of both humans and animals. Two subspecies have been identified, Campylobacter fetus subsp. fetus and Campylobacter fetus subsp. venerealis, and there are two serotypes, A and B. To further investigate the genetic diversity among C. fetus strains of different origins, subspecies, and serotypes, we performed multiple genetic analyses by utilizing random amplification of polymorphic DNA (RAPD), pulsed-field gel electrophoresis (PFGE), and DNA-DNA hybridization. All 10 primers used for the RAPD analyses can distinguish C. fetus strains of reptile and mammal origin, five can differentiate between C. fetus subsp. fetus and C. fetus subsp. venerealis strains, and four showed differences between type A and type B isolates from mammals. PFGE with SmaI and SalI digestion showed varied genome patterns among different C. fetus strains, but for mammalian C. fetus isolates, genome size was well conserved (mean, 1.52 +/- 0.06 Mb for SmaI and 1.52 +/- 0.05 Mb for SalI). DNA-DNA hybridization demonstrated substantial genomic-homology differences between strains of mammal and reptile origin. In total, these data suggest that C. fetus subsp fetus strains of reptile and mammal origin have genetic divergence more extensive than that between the two subspecies and that between the type A and type B strains. Combining these studies with sequence data, we conclude that there has been substantial genetic divergence between Campylobacter fetus of reptile and mammal origin. Diagnostic tools have been developed to differentiate among C. fetus isolates for taxonomic and epidemiologic uses.

Spermine Synthesis Is Required for Normal Viability, Growth, and Fertility in the Mouse

Spermidine is essential for viability in eukaryotes but the importance of the longer polyamine spermine has not been established. Spermine is formed from spermidine by the action of spermine synthase, an aminopropyltransferase, whose gene (SpmS) is located on the X chromosome. Deletion of part of the X chromosome that include SpmS in Gy mice leads to a striking phenotype in affected males that includes altered phosphate metabolism and symptoms of hypophosphatemic rickets, circling behavior, hyperactivity, head shaking, inner ear abnormalities, deafness, sterility, a profound postnatal growth retardation, and a propensity to sudden death. It was not clear to what extent these alterations were due to the loss of spermine synthase activity, since this chromosomal deletion extends well beyond the SpmS gene and includes at least one other gene termed Phex. We have bred the Gy carrier female mice with transgenic mice (CAG/SpmS mice) that express spermine synthase from the ubiquitous CAG promoter. The resulting Gy-CAG/SpmS mice had extremely high levels of spermine synthase and contained spermine in all tissues examined. These mice had a normal life span and fertility and a normal growth rate except for a reduction in body weight due to a loss of bone mass that was consistent with the observation that the derangement in phosphate metabolism is due to the loss of the Phex gene and was not restored. These results show that spermine synthesis is needed for normal growth, viability, and fertility in male mice and that regulation of spermine synthase content is not required.

Phenotypic and molecular characterization of bovine Campylobacter fetus strains isolated in Brazil

The objective of the present study was to characterize the phenotypic and molecular aspects of Campylobacter fetus strains isolated from bovine herds with reproductive problems. Thirty-one Brazilian field isolates, together with one reference strain of each subspecies of C. fetus, were analyzed. The strains were submitted to phenotypic identification followed by subspecies characterization using the polymerase chain reaction (PCR) and numeric evaluation of restriction fragment length polymorphism (RFLP) separated by pulsed-field gel electrophoresis (PFGE). Phenotypically, 4 isolates (12.1%) were classified as C. fetus subsp. fetus, and 29 isolates (87.9%) were classified as C. fetus subsp. venerealis. However, according to molecular analysis, only 1 isolate (3.0%) was classified as C. fetus subsp. fetus (the reference strain), whereas 32 isolates (97.0%) were considered C. fetus subsp. venerealis. SalI digestion of C. fetus genomic DNA, obtained from the 33 strains, yielded 7–10 DNA fragments ranging in size from 40 to 373 kb, with 12 distinct patterns. Furthermore, the numeric analysis by neighbor-joining of the DNA from the 33 strains resulted in a dendrogram in which 2 distinct groups were identified. It was concluded that phenotypic characterization of C. fetus subspecies might lead to erroneous classification of field isolates. Although RFLP-PFGE is a powerful and reliable technique to characterize C. fetus, it has the inconvenience of being time consuming and laborious. Whereas PCR, besides providing rapid results, was found to be reliable and convenient for the characterization of field isolates of C. fetus.

Distribution of Bm1, a SINE type transposable element, in cecropin B genes of the silkworm, Bombyx mori

In order to investigate the distribution of Bm1, a SINE type transposable element, in cecropin B genes of the silkworm, Bombyx mori, a genomic library was first screened with cecropin B cDNA as a probe. Eighteen out of 275 positive clones were selected at random and SalI digestion patterns were compared. Ten clones which showed different patterns were further analyzed by Southern blotting using a cecropin B cDNA fragment encoding exon 1, exon 2 or the entire coding region as probes. The same SalI digested genomic clones were hybridized with a Bm1 probe. Comparison of positive patterns hybridized with the Bm1 probe to those hybridized with the cecropin B probes indicated that all cecropin B-fragments except one fragment had Bm1. The exceptional fragment contained exon 2 only. The results indicate that Bm1 is widely distributed in cecropin B genes.

Physical and genetic map of the genome of Campylobacter upsaliensis

A physical map of Campylobacter upsaliensis ATCC 43954 was constructed from DNA fragments generated by SalI (5' G/TCGAC), NarI (5' GG/CGCC) and BssHII (5' G/CGCGC) restriction digests separated using pulsed-field gel electrophoresis. The size of the C. upsaliensis genome was approximately 2000 kb, providing evidence of the largest Campylobacter genome sized to date. Twenty-one fragments created from these restriction digests were assembled into a physical map using a combination of complementary methods including cross-Southern hybridization, hybridization fingerprint analysis and hybridization with homologous and heterologous (from Campylobacter jejuni) gene probes. The position of ten genetic loci, including that of the iron-uptake regulatory (fur) gene, were localized to the physical map. A genomic library of C. upsaliensis ATCC 43954 was constructed in lambda Gem-11 vector. Fifty thousand recombinants with an average size of 16 kb represent a library about 200 times the size of the genome. Using C. jejuni DNA probes, clones representing C. upsaliensis flaA, fur and ftsZ genes were isolated and localized to the physical map.

A mapping study of 13 genes on human chromosome bands 4q11→q25

A chromosome band 4q21 gene (MLLT2, formerly called AF-4/FEL) involved in a reciprocal translocation with chromosome band 11q23 in t(4;11) acute leukemia has been cloned. To provide better definition of gene order and relationships in this region where MLLT2 resides, we used pulsed field gel electrophoresis (PFGE) to investigate 13 genes (including MLLT2) with physical locations in bands 4q11-->q25. Somatic cell hybrids derived from RS4;11, a leukemic cell line carrying the t(4;11)(q21;q23), were also used to localize genes in relation to MLLT2. Linkage of the interleukin 8 (IL8), albumin (ALB), and platelet factor 4 (PF4) genes was confirmed by NotI, SalI and SacII digests. The maximum distance between PF4 and ALB is 210 kb and between ALB and IL8 is 420 kb. The alcohol dehydrogenase, class I (ADH2, ADH3) gene cluster can be linked to the alcohol dehydrogenase, class III gene (ADH5) by SacII, NruI, and EagI digests. The maximum distance between them is 590 kb. Our study indicated that ALB, alpha-fetoprotein (AFP), PF4, beta-thromboglobulin (PPBP), GRO1 (encoding a cytokine also called melanoma growth-stimulatory activity), and IL8 genes can be physically linked. In this study the gamma-interferon induced protein 10 (INP10), bone morphogenetic protein 3 (BMP3), annexin III (ANX3), KIT, amphiregulin (AREG), immunoglobulin J polypeptide (IGJ), deoxycytidine kinase (DCK) and MLLT2 genes were not linked to one another or to the above two groups of genes. Our analysis using somatic cell hybrids combined with previous reports demonstrated that the ADH gene cluster is telomeric to MLLT2 and KIT, ALB, AFP, PF4, beta TG, GRO1, IL8, ANX3, AREG and DCK are centromeric to MLLT2.

Physical genome map of the unicellular cyanobacterium Synechococcus sp. strain PCC 7002

A physical restriction map of the genome of the cyanobacterium Synechococcus sp. strain PCC 7002 was assembled from AscI, NotI, SalI, and SfiI digests of intact genomic DNA separated on a contour-clamped homogeneous electric field pulsed-field gel electrophoresis system. An average genome size of 2.7 x 10(6) bp was calculated from 21 NotI, 37 SalI, or 27 SfiI fragments obtained by the digestions. The genomic map was assembled by using three different strategies: linking clone analysis, pulsed-field fragment hybridization, and individual clone hybridization to singly and doubly restriction-digested large DNA fragments. The relative positions of 21 genes or operons were determined, and these data suggest that the gene order is not highly conserved between Synechococcus sp. strain PCC 7002 and Anabaena sp. strain PCC 7120.

Ribotype analysis of Pseudomonas pseudomallei isolates.

No epidemiological typing system to differentiate among Pseudomonas pseudomallei isolates has been available. Ribotype analysis was developed and used to examine 74 clinical and 10 environmental isolates of P. pseudomallei from Thailand. Six P. pseudomallei ribotypes were identified from restriction fragment polymorphisms of EcoRI chromosomal digests. The predominant ribotype, A, was found in 59 of the isolates examined. By using patterns from hybridizations with SalI, HindIII, and PstI restriction digests, isolates of ribotype A were subdivided into a further five subtypes, giving a total of 10 differentiable P. pseudomallei types. In 23 of 34 melioidosis patients studied, multiple P. pseudomallei isolates were present. In all but one of these patients, a single ribotype of the organism was present. Isolation of two different ribotypes of P. pseudomallei from one patient, one each in sputum and urine, suggests that superinfection may have occurred. The ribotype was shown to be conserved during the course of antibiotic treatments in seven patients studied, although the antibiotic sensitivity patterns in the isolates from these patients varied. The prevalence of subtype A1 in clinical and environmental specimens suggests that this strain may be predominant in this geographical location. These results demonstrate the usefulness of the ribotyping method for epidemiological studies of P. pseudomallei.

Molecular dissection of a specific nuclear domain: The chromatin region of the ribosomal gene cluster in Xenopus laevis

Molecular dissection of the nuclear domain corresponding to the ribosomal chromatin cluster was investigated. The experimental scheme was based on the ability of restriction enzymes to digest the whole genome without affecting this region (several megabases in length). Such a strategy involved the judicious choice of restriction enzymes, which is possible in Xenopus laevis, where the rDNA sequence is known and the repeated units are organized into one unique cluster. SalI, XhoI, and EcoRV digestion produced frequent cutting of the genome leaving the ribosomal cluster intact. Isolation of the rDNA cluster was confirmed by separation of the digested DNA by pulsed-field eleetrophoresis. When applied to purified nuclei, this approach allowed the isolation of the ribosomal chromatin cluster under very mild conditions: no cleavages (either enzymatic or mechanical) were detectable. Since the purification scheme depends only on the DNA sequence outside of the rDNA cluster, it permits the obtention of this domain in different functional states. Electron microscopic analysis demonstrated that the domain organization is substantially preserved and maintains its looped organization (the size and the full number of loops were preserved). This purification scheme provides a powerful tool for studying the structure-function relationships within the ribosomal nuclear domain.

Conjugal transfer in Lactococcus lactis of a 68-kilobase-pair chromosomal fragment containing the structural gene for the peptide bacteriocin nisin.

Nisin-producing transconjugants were generated by mating nisin-producing strains of Lactococcus lactis subsp. lactis with derivatives of L. lactis subsp. lactis LM0230. The sucrose-utilizing ability and reduced bacteriophage sensitivity were also transferred with the nisin-producing character. Pulsed-field gel electrophoretic analysis of genomic DNA from donor, recipient, and nisin-producing transconjugants indicated that 68 kbp of DNA was transferred from the chromosome of the donor into the chromosome of the recipient in the conjugation process. The location of the transferred nisin structural gene spaN in the transconjugant HID500 was not stable, and cultures of strain HID500 were a mixture of different genotypes in which spaN was located at different positions in the chromosome on different SmaI fragments. ApaI, BglI, BssHII, NciI, SalI, and SmaI digests of genomic DNA were used to map the location of spaN in a donor (DL11) and a nisin-producing transconjugant (HID504).

Use of pulsed-field agarose gel electrophoresis to size genomes of Campylobacter species and to construct a SalI map of Campylobacter jejuni UA580

To determine the physical length of the chromosome of Campylobacter jejuni, the genome was subjected to digestion by a series of restriction endonucleases to produce a small number of large restriction fragments. These fragments were then separated by pulsed-field gel electrophoresis with the contour-clamped homogeneous electric field system. The DNA of C. jejuni, with its low G+C content, was found to have no restriction sites for enzymes NotI and SfiI, which cut a high-G+C regions. Most of the restriction enzymes that were used resulted in DNA fragments that were either too numerous or too small for genome size determination, with the exception of the enzymes SalI (5' ... G decreases TCGAG ... 3'), SmaI (5' .... CCC decreases GGG .... 3'), and KpnI (5' ... GGTAC decreases C .... 3'). With SalI, six restriction fragments with average values of 48.5, 80, 110, 220, 280, and 980 kilobases (kb) were obtained when calibrated with both a lambda DNA ladder and yeast Saccharomyces cerevisiae chromosome markers. The sum of these fragments yielded an average genome size of 1.718 megabases (Mb). With SmaI, nine restriction fragments with average values ranging from 39 to 371 kb, which yielded an average genome size of 1.726 Mb were obtained. With KpnI, 11 restriction fragments with sizes ranging from 35 to 387.5 kb, which yielded an average genome size of 1.717 Mb were obtained. A SalI restriction map was derived by partial digestion of the C. jejuni DNA. The genome sizes of C. laridis, C. coli, and C. fetus were also determined with the contour-clamped homogeneous electric field system by SalI, SmaI, and KpnI digestion. Average genome sizes were found to be 1.714 Mb for C. coli, 1.267 Mb for C. fetus subsp. fetus, and 1.451 Mb for C. laridis.