Cleavage Of Genomic DNA Research Articles

Programmed cell death of the prothoracic glands of Manduca sexta during pupal-adult metamorphosis

The degeneration of the prothoracic glands of Manduca sexta during pupal-adult metamorphosis was analyzed by both light and transmission electron microscopy, and correlated with a functional analysis of the ability of the prothoracic glands to synthesize ecdysteroids in vitro. Apoptosis of degenerating glandular cells was also examined by in situ indirect immunofluorescein detection of digoxigenin-labeled genomic DNA cleavage (the TUNEL method). The ability of the prothoracic gland to synthesize ecdysteroids reached a peak 4 days after pupation, decreased drastically on day 6 and then decreased further to the basal level 2 days later. Prothoracic gland degeneration was initiated on day 6 and was characterized by nuclear condensation, cytoplasmic budding, giant autophagic vacuoles, the disappearance of the smooth endoplasmic reticulum and intercellular channels, and the fragmentation of the cytoplasm into membrane-bound bodies. The cell debris of these degenerating cells was then engulfed by numerous phagocytic hemocytes. The results of the analysis of apoptosis by immunofluorescence detection are in complete accord with the histological, ultrastructural and radioimmunoassay data, suggesting that apoptosis is the basic mechanism for programmed cell death of the cells comprising these vital glands.

Direct excision of 50 kb pair DNA fragments from megabase-sized fragments produced during apoptotic cleavage of genomic DNA

The DNA of U937 cells exposed to two different apoptotic stimuli, namely the cocktail H 2O 2/3-aminobenzamide (3AB) and etoposide, was analyzed using field inversion gel electrophoresis (FIGE) as well as programmable, autonomously controlled electrode electrophoresis (PACE). The results obtained indicate that FIGE is not appropriate for sizing apoptotic DNA fragments. PACE appears to be more accurate and reliable and the results obtained with this technique strongly suggest that the 50 kb DNA fragments are directly excised from Mb-sized DNA fragments without the intermediate cleavage of 200–300 kb products.

A simple and efficient method for making site-directed mutants, deletions, and fusions of large DNA such as P1 and BAC clones.

This study addresses two important technical problems: how to perform targeted alterations such as site-directed mutagenesis and deletions in large fragments of DNA and how to construct full-length genes from two partly overlapping bacterial artificial chromosome (BAC) plasmids. Given the size and the lack of convenient unique restriction sites in these large-insert bacterial clones, these are nontrivial tasks. Here we describe a simple and efficient protocol based on RecA-assisted restriction endonuclease (RARE) cleavage, a method that enables sequence-specific cleavage of genomic DNA. The same protocol has been used with minor modifications to introduce site-specific mutations into an apolipoprotein-B 90-kb P1 clone, to generate deletions in a 160-kb BAC, and to generate a 160-kb BAC containing the complete 92-kb gene for low-density lipoprotein-related protein-1 (LRP-1) from two smaller overlapping BACs ("BAC marriage").

Gonadotropin suppression of apoptosis in follicle somatic cells.

In each oestrous cycle only a limited number of follicles are selected for ovulation whereas the remaining majority undergo atresia. The earliest and most prominent feature of atresia is the death of granulosa cells. Recent biochemical evidence has demonstrated that granulosa cell death during follicular atresia in swine (Tillyet al., 1992), bovine (Jollyet al., 1994) and rodent (Tillyet al., 1991) ovaries occurs by apoptosis, a process whereby cells die in a controlled manner. A biochemical event considered to be characteristic of apoptotic cell death is the intranucleosomal cleavage of genomic DNA into fragments 180–200 bp in size, which separate into a distinctive ladder-like pattern on agarose gel electrophoresis. Detection of this pattern of oligonucleosomes in DNA provides a marker of apoptotic cell death.

A new approach for the electrophoretic detection of apoptosis.

Apoptotic cell death is often characterized by internucleosomal cleavage of genomic DNA, which exhibits a distinctive ladder upon electrophoresis. However, techniques used for the isolation and detection of DNA to demonstrate laddering may not be sufficiently sensitive, particularly when cleaved DNA is present at modest levels. We propose a new approach for isolating total cellular DNA using a silica-based resin that improves the resolution of DNA laddering. In addition, we introduce a rapid DNA labeling method that can increase the sensitivity of detecting DNA laddering. Each of these methods can be used for DNA from cell cultures or tissues.

DNA fragmentation during thymic apoptosis is catalyzed by DNase γ

The physiological and pathological importance of cell death by apoptosis has recently been recognized. One of the hallmarks of apoptosis is the enzymatic cleavage of genomic DNA into nucleosomal oligomers. The identification of an endonuclease responsible for apoptosis might help to explain how this cell suicide is regulated and why DNA is cleaved. Here, we found that γ type of DNase was retained in apoptotic rat thymocyte nuclei. Homogeneously purified DNase γ (Mr = 33 kDa) from the apoptotic nuclei was revealed to be a Ca2+/Mg2+-dependent endonuclease and inhibited by Zn2+. This enzyme cleaved chromosomal DNA with 3′-hydroxyl (OH) and 5′-phosphoryl (P) ends. The cleavage ends and its divalent cation dependencies match those observed in apoptotic thymocytes induced by X-irradiation or glucocorticoid treatment, indicating that this endonuclease is a central component of the thymic apoptosis machinery.

Involvement of Apoptosis in 4-Vinylcyclohexene Diepoxide- Induced Ovotoxicity in Rats

Previous studies have determined that 4-vinylcyclohexene diepoxide (VCD) causes specific destruction of oocytes contained in small pre-antral (primordial and primary) ovarian follicles of Fischer 344 rats following 30 days of daily dosing with VCD. The purposes of this study were to identify the type of VCD-induced cell death occurring in small pre-antral follicles and to determine the earliest time following the onset of dosing when evidence of follicular destruction could first be detected. A significant decrease in the number of oocytes contained in small pre-antral follicles in ovaries of rats after 15 days of daily dosing (ip) with VCD (80 mg/kg) had been observed in preliminary experiments. Therefore, a study was conducted to determine the time of the onset of this follicular destruction by examination of follicular DNA integrity. Female Fischer 344 rats were dosed daily (80 mg/kg, ip) for 6, 8, 10, 12, or 14 days, and ovaries were removed 1, 4, or 24 hr after the final dose. Small pre-antral follicles (25–100 μm) were isolated by gentle dissociation of ovaries with collagenase, and follicles were sorted with micropipets. Genomic DNA was isolated from follicles and radiolabeled with [32P]dideoxy ATP, and the degree of fragmentation quantified by agarose gel electrophoresis and autoradiography. Degradation of DNA was evaluated by32P content in low-molecular-weight fragments (<4 kilobase pairs). Degradation of DNA was not observed in follicles collected 24 hr after the final dose on any day. However, a random pattern of DNA degradation was observed, and was significantly greater (p< 0.05) compared with controls, when follicles were collected 4 hr following VCD administration on Days 10 and 12, but not on Days 6 or 8, of dosing. Although not significant, there was also evidence of DNA degradation in dosed animals on Day 14. Histological evaluation of small pre-antral follicles in ovarian sections during the early stages of VCD-induced DNA degradation (Day 10; 4 hr) demonstrated margination of chromatin along the nuclear membrane in oocytes and disruptions in focal contact between granulosa cells and oocytes, both features indicative of apoptosis. Furthermore, there was no sign of ruptured membranes in granulosa cells or oocytes or of an inflammatory response, characteristics of necrosis (pathological cell death). Whereas biochemical and morphological evidence of follicular destruction was seen 4 hr after dosing on Day 10, numbers of oocyte-containing primordial and primary follicles in VCD-treated animals were not different from controls at that time. These results demonstrate that the initial evidence of impending destruction of small pre-antral follicles is first consistently visualized following 10 days of daily dosing with VCD, although a measurable reduction in oocyte numbers has not yet occurred. Despite the fact that internucleosomal cleavage of genomic DNA was not observed, morphological evaluations support that granulosa cells and oocytes in primordial and primary follicles are destroyed via the induction of apoptosis.

Apoptosis as a mechanism of cell death induced by different chemotherapeutic drugs in human leukemic T-lymphocytes

The involvement of apoptosis in the mechanism of cell death induced by six clinically relevant anticancer drugs [methotrexate (MTX), doxorubicin (ADR), daunorubicin (DNR), vincristine (VCR), 6-mercaptopurine (6MP), and prednisolone (PRD)] in human leukemic T-lymphocytes (CCRF-CEM and Jurkat) was investigated by analysing changes in cell size and morphology, changes in membrane integrity, alterations in [Ca 2+] i and induction of DNA fragmentation. MTX, ADR, and DNR showed pronounced dose- and time-dependent cytotoxic effects on both cell lines, whereas cell viability was not considerably reduced by 6MP or PRD. On the other hand, the cytotoxic activity of VCR was much higher on Jurkat cells than on CEM cells. With the exception of 6MP and PRD, all the other compounds induced extensive chromatin condensation, nuclear fragmentation, plasma membrane blebbing, and formation of apoptotic bodies and fragmentation of DNA in both cell lines. Occurrence of DNA fragmentation always preceded loss of membrane integrity. These observations are consistent with cell death being mediated by apoptosis. Significant increases in [Ca 2+] i were only observed in CEM cells preincubated with MTX or DNR (10 μM). In contrast, MTX as well as VCR induced a reduction in the basal intracellular Ca 2+concentration in Jurkat T-cells. Although the ability to induce changes in [Ca 2+] i correlated with higher cytotoxic potency of the anticancer drugs, a causal relationship between increased [Ca 2+] iand induction of apoptosis could not be clearly established. These results, therefore, suggest no determinant role for Ca 2+ in triggering the process of endonucleolytic cleavage of genomic DNA in these leukemic T-lymphocytes.

Dolichyl Phosphate, a Potent Inducer of Apoptosis in Rat Glioma C6 Cells

Exposure of rat glioma C6 cells to dolichyl phosphate resulted in cell shrinkage followed by nuclear fragmentation and internucleosomal cleavage of genomic DNA, yielding ladder patterns of oligonucleosomal fragments, all characteristics of apoptosis, This phenomenon occurred in a dose and time dependent manner. Dolichol and prenol failed to induce apoptosis. The inhibitors of N-glycosylation, tunicamycin and swainsonine had no apparent effect on dolichyl phosphate-induced apoptosis. Apoptotic changes were also observed in HL-60 cells, SIRC cells and HeLa cells. Thus, dolichyl phosphate functions as a potential apoptosis inducer as well as an essential carrier lipid in the biosynthesis of N-linked glycoprotein.

Regulation of apoptosis by bcl-2 family proteins and its role in cancer and chemoresistance

In essentially all tissues that have self-renewal capacity, there exists a delicate balance between cell production by mitogenesis and cell loss due to programmed cell death (PCD), which maintains total cell numbers within physiologically appropriate ranges. Genetic alterations that either dysregulate the cell division process, resulting in faster cell proliferation, or that affect physiological cell death mechanisms that cause slower rates of cell loss, occur frequently in human tumor cells and contribute to the clonal expansion of cancer cells in vivo. PCD is an active form of cell suicide that sometimes requires new gene expression for its initiation and that in many, but not all, cases culminates in a characteristic set of biochemical and morphological events. These include genomic DNA cleavage by endonucleases, chromatin condensation (pyknosis), nuclear fragmentation, proteolysis of cytoskeletal and other proteins, plasma membrane blebbing, and cell shrinkage. In many ways, these biochemical events can be viewed as a form of cellular autodigestion in which the macromolecular components of cells are degraded so that their constituent subunits can be recycled in the body. When present in their classical form, the morphological events accompanying this type of cell death are broadly termed apoptosis. Though well known for its role in the normal physiological cell death mechanisms that maintain tissue homeostasis, a wide variety of pathological conditions and external factors can trigger the PCD pathway. Included among these apoptotic stimuli are essentially all chemotherapeutic drugs and radiation, a finding of considerable relevance to our understanding of how currently available treatments of cancer work and for devising strategies for improving them. In this review, the regulation of PCD by members of the bcl-2 family of proteins is discussed, primarily within the context of human cancers where abnormalities in the expression of BCL2 family genes frequently occur and contribute both to the origins of cancer and our difficulty in treating it.

Structure of the terminal 300 kb of DNA from human chromosome 21q

The most distal 300 kb of human chromosome 21q was cloned and mapped using telomeric yeast artificial chromosomes (YACs). The region contains low-copy subtelomeric repeats at the telomeric end, chromosome 21-specific sequences more centromerically, and the S100B gene at a distance of 100–140 kb from the chromosome terminus. RecA-assisted restriction endonuclease cleavage of genomic DNA showed that the cloned fragments correspond to telomere-terminal genomic DNA, and restriction enzyme mapping of the YACs shows that the smaller clone (175 kb) corresponds exactly to the telomeric end of the larger one (300 kb). PCR assays for 21q-specific markers were used to show that COL6A1, COL6A2, and LA161 were all outside of the subtelomeric region spanned by the YACs and thus at least 300 kb from the 21q terminus. The molecular probes provide telomeric closure for existing 21q maps.

Long-range mapping of gaps and telomeres with RecA-assisted restriction endonuclease (RARE) cleavage.

RecA-assisted restriction endonuclease (RARE) cleavage is a method to perform sequence-specific cleavage of genomic DNA, and is useful in physical mapping studies. After making two modifications, we have applied this method to mapping large regions of DNA in several cell types, including a notorious gap near the Huntington disease (HD) locus on chromosome 4. RARE cleavage fragments were analysed by pulsed field gel electrophoresis and Southern blotting and the distances between cleavage sites determined with accuracy. Using RARE cleavage, the gap measured was less than 60 kilobases in length. RARE cleavage is also a straightforward technique to map the distance from a marker to a telomere. The terminal 1.7 megabases of several HD and control cell lines were mapped with no large differences between cell lines in this region.

Induction of apoptotic DNA damage and cell death by activation of the sphingomyelin pathway.

The potential involvement of ceramide-related signaling processes in the induction of apoptosis by tumor necrosis factor alpha was assessed by multiple biochemical strategies in the human leukemic cell lines HL-60 and U937 and the murine fibrosarcoma cell lines L929/LM and WEHI 164/13. Exposure of these cells to tumor necrosis factor alpha resulted in internucleosomal cleavage of genomic DNA, yielding laddered patterns of oligonucleosomal fragments characteristic of apoptosis when resolved by agarose gel electrophoresis; similar responses were observed after exposure to exogenous sphingomyelinase or synthetic ceramides. Quantitative spectrofluorophotometry demonstrated that these treatments promoted time- and concentration-dependent degradation of DNA, resulting in the formation of and eventual release of small DNA fragments (< or = 3.0 kb). Corresponding damage to bulk DNA was demonstrated by enhanced-fluorescence alkaline unwinding analysis. DNA fragmentation was not induced by phospholipase C or synthetic diglyceride; in fact, the effects of sphingomyelinase and ceramide were substantially reduced by coexposure to these agents, suggesting opposing roles for diglyceride- and ceramide-mediated signals in the regulation of apoptosis. Phospholipase A2 and arachidonic acid failed to promote DNA fragmentation, as did phospholipase D. Characterization of DNA strand breaks by alkaline and neutral elution analyses confirmed that ceramide action was restricted to breakage of mature, double-stranded DNA but not of nascent DNA. The induction of DNA damage was associated with appearance of apoptotic morphology and decreased clonogenicity. These results demonstrate that the ceramide-dependent signaling system selectively induces apoptosis and raise the possibility that ceramide-activated enzymes represent important components in a signaling cascade involved in the regulation of programmed cell death.

Ribosomal DNA, peroxidase and extensin variation in genomic DNA of Medicago laciniata (L.) Miller from Australia and Africa

Fifty-four Medicago laciniata (L.) Miller accessions from diverse locations in eastern Australia, Africa and Israel were characterized for genotypic differences by restriction endonuclease cleavage of genomic DNA and probing with radio-labelled DNA sequences to detect restriction fragment length polymorphisms (RFLP). Digestion of the genomic DNA with the restriction endonuclease, Bam HI, and probing with the complete ribosomal DNA (rDNA) repeat unit of soybean (pGmrl) and its subclones revealed that the variable region in M. laciniata spans the Bam H1 sites of the intergenic spacer region between the 18s and 26s DNA. The length variation in the rDNA repeat unit clearly distinguished South African from Australian accessions. The rDNA variants from SA1847 (Morocco) and SA7750 (Tunisia) were the same as the Australian accessions and slightly larger than the rDNA spacer variant unique to SA3428 (Israel). When Eco R1 digested M. laciniata genomic DNA was probed with peroxidase and extensin cDNA clones, all Australian accessions were again characterized by the same RFLP genotype with further differentiation between African and Israeli accessions. An accession of unknown origin, SA3412, possessed the same RFLP genotype as the Australian accessions for all endonuclease and probe combinations. For the African accessions, differences in RFLP genotype were more frequent than differences in phenotype (leaflet laciniation and colour). However, of three South African accessions with the same RFLP genotype, SA26844, SA26847, and SA26848, SA26847 had more pronounced leaflet laciniation.

Solution structure of a pyrimidine.purine.pyrimidine DNA triplex containing T.AT, C+.GC and G.TA triples.

Under certain conditions, homopyrimidine oligonucleotides can bind to complementary homopurine sequences in homopurine-homopyrimidine segments of duplex DNA to form triple helical structures. Besides having biological implications in vivo, this property has been exploited in molecular biology applications. This approach is limited by a lack of knowledge about the recognition by the third strand of pyrimidine residues in Watson-Crick base pairs. We have therefore determined the solution structure of a pyrimidine.purine.pyrimidine (Y.RY) DNA triple helix containing a guanine residue in the third strand which was postulated to specifically recognize a thymine residue in a Watson-Crick TA base pair. The structure was solved by combining NMR-derived restraints with molecular dynamics simulations conducted in the presence of explicit solvent and counter ions. The guanine of the G-TA triple is tilted out of the plane of its target TA base pair towards the 3'-direction, to avoid a steric clash with the thymine methyl group. This allows the guanine amino protons to participate in hydrogen bonds with separate carbonyls, forming one strong bond within the G-TA triple and a weak bond to an adjacent T.AT triple. Dramatic variations in helical twist around the guanine residue lead to a novel stacking interaction. At the global level, the Y.RY DNA triplex shares several structural features with the recently solved solution structure of the R.RY DNA triplex. The formation of a G.TA triple within an otherwise pyrimidine.purine.pyrimidine DNA triplex causes conformational realignments in and around the G.TA triple. These highlight new aspects of molecular recognition that could be useful in triplex-based approaches to inhibition of gene expression and site-specific cleavage of genomic DNA.

Induction of apoptosis in cerebellar granule neurons by low potassium: inhibition of death by insulin-like growth factor I and cAMP.

High levels of extracellular K+ ensure proper development and prolong survival of cerebellar granule neurons in culture. We find that when switched from a culture medium containing high K+ (25 mM) to one containing a low but more physiological K+ concentration (5 mM), differentiated granule neurons degenerate and die. Death induced by low K+ is due to apoptosis (programmed cell death), a form of cell death observed extensively in the developing nervous system and believed to be necessary for proper neurogenesis. The death process is accompanied by cleavage of genomic DNA into internucleosome-sized fragments, a hallmark of apoptosis. Inhibitors of transcription and translation suppress apoptosis induced by low K+, suggesting the necessity for newly synthesized gene products for activation of the process. Death can be prevented by insulin-like growth factor I but not by several other growth/neurotrophic factors. cAMP but not the protein kinase C activator phorbol 12-myristate 13-acetate can also support survival in low K+. In view of the large numbers of granule neurons that can be homogeneously cultured, our results offer the prospect of an excellent model system to study the mechanisms underlying apoptosis in the central nervous system and the suppression of this process by survival factors such as insulin-like growth factor I.

Complement-stabilized D-loop: RecA-catalyzed Stable Pairing of Linear DNA Molecules at Internal Sites

The RecA protein (RecA) of Escherichia coli has the ability to pair a single-stranded DNA to a homologous sequence in a duplex DNA without requiring denaturation of the duplex. This ability has stimulated interest in the use of RecA for targeting to genomic DNA. However, because pairing generally requires that the double-stranded DNA either have a homologous end or be negatively supercoiled, the application of RecA to targeting has been very limited. Here, we show that if the sequence complementary to the probe is also included in the reaction, RecA can pair the two single strands to sites distant from any ends on linear DNA. The resulting structure, termed a complement-stabilized D-loop (csD-loop), is cleavable by restriction endonucleases and, upon removal of RecA, remains stable to temperatures up to the t m of the double-stranded probe. These results indicate that the csD-loop probably consists of two side-by-side Watson-Crick duplexes, much like a replication bubble. This novel reaction of RecA may be useful in gene mapping and isolation, as well in sequence-specific cleavage of genomic DNA, and might have functions in vivo.

Biochemical Identification of Apoptosis (Programmed Cell Death) in Granulosa Cells: Evidence for a Potential Mechanism Underlying Follicular Atresia*

In the present study, we examined the possibility that granulosa cell death during ovarian follicular atresia occurs by apoptosis (programmed cell death). To investigate this possibility, atresia was induced in immature female rats by injecting 15 IU PMSG. Controls received either vehicle or no treatment. PMSG-treated animals were killed on days 1-5 post-injection while controls were killed on days 1 or 5. The onset of atresia was assessed histologically by light microscopic inspection of 5 microns tissue sections and functionally by quantification of serum progesterone and estrogen levels. Apoptosis is characterized by the cleavage of genomic DNA into oligonucleosomal length fragments by a Ca2+/Mg(2+)-dependent endogenous endonuclease. Such fragments form a distinctive ladder pattern when separated electrophoretically. Accordingly, the occurrence of apoptosis in granulosa cells was assessed by examining the pattern of fragmented DNA in cell lysates after agarose gel electrophoresis. Gels were stained with ethidium bromide and DNA visualized by UV transillumination. The earliest morphological signs of atresia were detected 4 days after PMSG injection as evidenced by degeneration and detachment of granulosa cells from the basal lamina. Serum estrogen increased from basal to levels 7-fold over controls by day 3 after PMSG treatment, falling to control values by day 4 and thereafter. In contrast, progesterone remained basal for the first 3 days, rising to levels 3-fold and 8-fold above controls 4 and 5 days after PMSG treatment, respectively. Such shifts in the ratio of estrogen to progesterone production are known to be characteristic of follicular atresia. Finally, electrophoretic analysis of low mol wt DNA in granulosa cell lysates revealed a definitive ladder pattern of oligonucleosomal length DNA fragments (characteristic of apoptosis) on days 4 and 5 after PMSG injection. This pattern was not detectable on days 1 and 2 after treatment. Lysates obtained 3 days after PMSG treatment showed a faint apoptotic-like pattern of DNA fragments; a result consistent with other systems in which DNA cleavage begins before any morphological signs of death. Interestingly, a ladder pattern of DNA fragments was present in control lysates suggesting that granulosa cell death under normal (vs. induced) conditions of atresia in immature rats occurs by apoptosis. These data demonstrate an intimate association between apoptotic-like events and dying granulosa cells and thus support the possibility that apoptosis is involved in the induction of follicular atresia.

An integrative vector exploiting the transposition properties of Tn 1545 for insertional mutagenesis and cloning of genes from Gram-positive bacteria

We have constructed and used an integrative vector, pAT112, that takes advantage of the transposition properties (integration and excision) of transposon Tn 1545. This 4.9-kb plasmid is composed of: ( i) the replication origin of pACYC 184; ( ii) the attachment site ( att) of Tn 1545; ( iii) erythromycin- and kanamycin-resistance-encoding genes for selection in Gram − and Gram + bacteria; and ( iv) the transfer origin of IncP plasmid RK2, which allows mobilization of the vector from Escherichia coli to various Gram + recipients. Integration of pAT112 requires the presence of the transposon-encoded integrase, Int-Tn, in the new host. This vector retains the insertion specificity of the parental element Tn 1545 and utilises it to carry out insertional mutagenesis, as evaluated in Enterococcusfaecalis. Since pAT 112 contains the pACYC184 replicon and lacks most of the restriction sites that are commonly used for molecular cloning, a gene from a Gram + bacterium disrupted with this vector can be recovered in E. coli by cleavage of genomic DNA, intramolecular ligation and transformation. Regeneration of the gene, by excision of pAT 112, can be obtained in an E. coli strain expressing the excisionase and integrase of Tn 1545. The functionality of this system was illustrated by characterization of an IS 30-like structure in the chromosome of En. faecalis. Derivatives pAT113 and pAT114 contain ten unique cloning sites that allow screening of recombinants having DNA inserts by α-complementation in E. coli carrying the ΔM15 deletion of lacZα. These vectors are useful to clone and introduce foreign genes into the genomes of Gram + bacteria.

Pulsed field electrophoresis of genomic restriction fragments for the detection of nosocomial Legionella pneumophila in hospital water supplies

Ten Legionella pneumophila strains isolated from different sources were analyzed according to their restriction fragment patterns obtained by cleavage of genomic DNA with NotI and SfiI and separation by pulsed field electrophoresis. Three L. pneumophila isolates from a nosocomial outbreak in Lübeck (Germany) and three other L. pneumophila strains independently isolated from a water tap located in the care unit where the patients were hospitalized exhibited identical restriction fragment profiles. Therefore, we concluded that these environmental specimens were the source of the Legionnaires disease. Another two isolates from patients and two strains from the environment, all unrelated to the outbreak described, showed different cleavage patterns.