Directed homologous recombination for genome engineering in Escherichia coli.

E. coli K-12 is the workhorse of molecular biology and the platform of choice for production of DNA, metabolites and proteins of industrial interest. To construct strains for the multitude of purposes, efficient genome manipulation methods are required. The suicide plasmid-mediated, homologous recombination-based gene replacement method is a convenient genome engineering tool. In consecutive recombination events, genomic integration of the plasmid, carrying the modified allele, is followed by its excision, resulting in either a modified genome or the original wild-type chromosome. Using the lac operon as a chromosomal target, we systematically investigated the effects of several factors influencing the outcome of the procedure. Recombinogenic activity was proportional to the length of the targeting homologous fragments. Presence of a properly oriented Chi site stabilized broken chromosomal ends and stimulated recombination in the downstream genomic region. Introduction of a double-stranded break in the chromosome had a profound stimulatory effect on recombination of the free DNA ends. These results shed light on some details of the complex events of intra- and intermolecular homologous recombination in the E. coli genome. Taking into account these findings at the assembly of the targeting plasmid constructs, serial genomic modifications can be created with enhanced efficiency and speed.

Role of DNA minor groove interactions in substrate recognition by the M.SinI and M.EcoRII DNA (cytosine-5) methyltransferases.

The SinI and EcoRII DNA methyltransferases recognize sequences (GG(A)/(T)CC and CC(A)/(T)GG, respectively), which are characterized by an (A)/(T) ambiguity. Recognition of the A.T and T.A base pair was studied by in vitro methyltransferase assays using oligonucleotide substrates containing a hypoxanthine.C base pair in the central position of the recognition sequence. Both enzymes methylated the substituted oligonucleotide with an efficiency that was comparable to methylation of the canonical substrate. These observations indicate that M.SinI and M.EcoRII discriminate between their canonical recognition site and the site containing a G.C or a C.G base pair in the center of the recognition sequence (GG(G)/(C)CC and CC(G)/(C)GG, respectively) by interaction(s) in the DNA minor groove. M.SinI mutants displaying a decreased capacity to discriminate between the GG(A)/(T)CC and GG(G)/(C)CC sequences were isolated by random mutagenesis and selection for the relaxed specificity phenotype. These mutations led to amino acid substitutions outside the variable region, previously thought to be the sole determinant of sequence specificity. These observations indicate that (A)/(T) versus (G)/(C) discrimination is mediated by interactions between the large domain of the methyltransferase and the minor groove surface of the DNA.

Genome sequence of enterohaemorrhagic Escherichia coli O157:H7.

The bacterium Escherichia coli O157:H7 is a worldwide threat to public health and has been implicated in many outbreaks of haemorrhagic colitis, some of which included fatalities caused by haemolytic uraemic syndrome. Close to 75,000 cases of O157:H7 infection are now estimated to occur annually in the United States. The severity of disease, the lack of effective treatment and the potential for large-scale outbreaks from contaminated food supplies have propelled intensive research on the pathogenesis and detection of E. coli O157:H7 (ref. 4). Here we have sequenced the genome of E. coli O157:H7 to identify candidate genes responsible for pathogenesis, to develop better methods of strain detection and to advance our understanding of the evolution of E. coli, through comparison with the genome of the non-pathogenic laboratory strain E. coli K-12 (ref. 5). We find that lateral gene transfer is far more extensive than previously anticipated. In fact, 1,387 new genes encoded in strain-specific clusters of diverse sizes were found in O157:H7. These include candidate virulence factors, alternative metabolic capacities, several prophages and other new functions--all of which could be targets for surveillance.

Markerless gene replacement in Escherichia coli stimulated by a double-strand break in the chromosome.

A simple and efficient gene replacement method, based on the recombination and repair activities of the cell, was developed. The method permits the targeted construction of markerless deletions, insertions and point mutations in the Escherichia coli chromosome. A suicide plasmid, carrying the mutant allele and the recognition site of meganuclease I- Sce I, is inserted into the genome by homologous recombination between the mutant and the wild-type (wt) alleles. Resolution of this cointegrate by intramolecular recombination of the allele pair results in either a mutant or a wt chromosome which can be distinguished by allele-specific PCR screening. The resolution process is stimulated by introducing a unique double-strand break (DSB) into the chromosome at the I- Sce I site. Cleavage by the nuclease not only enhances the frequency of resolution by two to three orders of magnitude, but also selects for the resolved products. The DSB-stimulated gene replacement method can be used in recombination-proficient E.coli cells, does not require specific growth conditions, and is potentially applicable in other microorganisms. Use of the method was demonstrated by constructing a 17-bp and a 62-kb deletion in the MG1655 chromosome. Cleavage of the chromosome induces the SOS response but does not lead to an increased mutation rate.

Cre/loxP-mediated in vivo excision of large segments from yeast genome and their amplification based on the 2microm plasmid-derived system.

In vivo excision and amplification of pre-determined, large genomic segments, directly from the genome of a natural host, provides an alternative to conventional cloning in foreign vectors. Using this approach, we have devised an in vivo procedure for excising large segments of Saccharomyces cerevisiae genome using Cre/loxP system of bacteriophage P1, followed by amplification of excised circles, as based on the yeast 2microm plasmid-derived ori and Flp/FRT machinery. To provide the excision and replication enzymes, trans-acting genes cre and FLP, which were under a very tight control of GAL1 and GAL10 promoters, respectively, were inserted by homologous recombination into the URA3 gene on chromosome V. Two parallel loxP sequences, which serve as the recognition sites for the Cre recombinase, were also integrated into the genome at pre-determined sites that are 50-100kb apart. Moreover, 2microm ori, REP3 and two inverted FRTs, which serve as a conditional replication system, were also integrated between the loxP sites. The strain carrying all these inserted elements was perfectly stable. Only after the induction by galactose of the Cre excision function, the genomic segment flanked by two loxP sites was excised and circularized. Applying this procedure, the 50-kb LEU2-YCR011c and 100-kb LEU2-YCR035c regions of chromosome III were successfully excised from the S. cerevisiae genome, whereas the 2microm ori, as aided by FRT/Flp, provided the amplification function. Such excised and amplified genomic segments can be used for the sequencing and functional analysis of any yeast genes.

Molecular evolution of a pathogenicity island from enterohemorrhagic Escherichia coli O157:H7.

We report the complete 43,359-bp sequence of the locus of enterocyte effacement (LEE) from EDL933, an enterohemorrhagic Escherichia coli O157:H7 serovar originally isolated from contaminated hamburger implicated in an outbreak of hemorrhagic colitis. The locus was isolated from the EDL933 chromosome with a homologous-recombination-driven targeting vector. Recent completion of the LEE sequence from enteropathogenic E. coli (EPEC) E2348/69 afforded the opportunity for a comparative analysis of the entire pathogenicity island. We have identified a total of 54 open reading frames in the EDL933 LEE. Of these, 13 fall within a putative P4 family prophage designated 933L. The prophage is not present in E2348/69 but is found in a closely related EPEC O55:H7 serovar and other O157:H7 isolates. The remaining 41 genes are shared by the two complete LEEs, and we describe the nature and extent of variation among the two strains for each gene. The rate of divergence is heterogeneous along the locus. Most genes show greater than 95% identity between the two strains, but other genes vary more than expected for clonal divergence among E. coli strains. Several of these highly divergent genes encode proteins that are known to be involved in interactions with the host cell. This pattern suggests recombinational divergence coupled with natural selection and has implications for our understanding of the interaction of both pathogens with their host, for the emergence of O157:H7, and for the evolutionary history of pathogens in general.

Investigation of effect of lesser curvature seromyotomy of the stomach in animal experiments.

The effectiveness of seromyotomy of the lesser curvature of the stomach--the simplified version of proximal selective vagotomy--was investigated in eleven dogs. Decreased acid secretion was proved with congo red test and pH measuring by glass electrode. No significant damage to the motor function of the stomach was found with scintigraphy. Histological examinations revealed neurofibre degeneration peripherally to the seromyotomy line after peripheral vagotomy and vacuolar degeneration in the ganglion cells and amputation neuromas in the seromyotomy line.

Versatile insertion plasmids for targeted genome manipulations in bacteria: isolation, deletion, and rescue of the pathogenicity island LEE of the Escherichia coli O157:H7 genome.

A system of versatile insertion plasmids was constructed that permits efficient delivery of the target sites of an ultra-rare-cutting endonuclease and the recombinase FLP into preselected sites of the bacterial genome. With the help of this system, the pathogenicity island LEE of the Escherichia coli O157:H7 genome was excised and isolated in vitro, deleted in vivo, rescued as a plasmid, and transferred into another strain.

Complications of laparoscopic cholecystectomy in Hungary: a multicentre study of 13,833 patients.

OBJECTIVE: To report our countrywide experience of laparoscopic cholecystectomy with particular reference to complications. DESIGN: National multicentre retrospective study. SETTING: 71 university departments and public hospitals in Hungary. SUBJECTS AND INTERVENTIONS: 13,833 patients operated on between 1 January 1990 and 31 December 1993. Follow up by questionnaire. RESULTS: 732 patients (5.3%) required conversion to open cholecystectomy, urgently because of intraoperative complications in 204 (1.5%), electively for acute or chronic inflammation or obscure anatomy in 441 (3.2%), for unexpected findings in 72 (0.5%) and for technical problems in 15 (0.1%). There were postoperative complications in 598 (4.3%) but reoperation was indicated in only 154 patients (1.1%). There were bile duct injury in 81 (0.6%) and 199 bleeds (1.4%) which required conversion in 102 patients (0.7%) and reoperation in 38 (0.3%). 36 of the 181 postoperative recognized bile leaks required reoperation (20%). 19 patients died (0.1%). CONCLUSIONS: The morbidity and the mortality of laparoscopic cholecystectomy are better than after the open operation. The 2-6 times higher risk of bile duct injury mentioned in early studies was not confirmed.

Transabdominal preperitoneal herniorraphy: technique and results.

As enthusiasm for laparoscopic surgery has grown, laparoscopic approaches to the groin hernia have evolved. The most widely accepted laparoscopic repair employs the placement of a large sheet of mesh in a preperitoneal position to cover potential hernia spaces. Between March 1994 and February 1997 160 inguinal and 3 femoral hernia were operated of an transabdominal preperitoneal (TAPP) polipropylen mesh. 131 patients were operated (128 males and 3 females, ranging in age from 19 to 82 years), 31 (23%) of them had bilateral hernias. Recurrent hernia was the indication in 52 (32%) cases. Average operating time for unilateral repair was 80 minutes and for bilateral repairs was 108 minutes. Postoperative complications included 7 (4.3%) cases of transient neuralgias, 20 (12%) cord/scrotal transient seromas-hematomas and 2 (1.2%) hydrocele. The 5 (3.1%) early recurrences were considered to be caused by technical inexperience and/or too small prosthetic patch. The laparoscopic hernioplasty has definitive advantage: minimal postoperative pain, short hospital stay (average postoperative time of hospitalization 3.1 days) and early restoration of full physical activity (in 1 to 2 weeks). The method should be considered as a potential "best option" in patients with recurrences and bilateral inguinofemoral hernias.

A broad-host-range in vivo pop-out and amplification system for generating large quantities of 50- to 100-kb genomic fragments for direct DNA sequencing.

A prerequisite for sequencing large genomes is to obtain 30- to 150-kb genomic DNA fragments in adequate quantity. Previously, we developed a system which enables one to excise and amplify in vivo such segments directly from the Escherichia coli genome. This system, which employed the yeast Flp/FRT elements for excision and the plasmid R6K-based replication machinery for DNA amplification, permits one to bypass conventional cloning [Pósfai et al. (1994) Nucleic Acids Res. 22, 2392-2398]. To extend the applicability of such a system to many species, we describe here a broad-host-range (bhr) system in which the amplification of the excised DNA fragment depends on the oriV element and the Rep (TrfA) protein from the promiscuous RK2/RP4 plasmid. We have constructed insertion plasmids which carry the FRT and oriV sites. To introduce such plasmids into the appropriate position in the host genome, a short genomic sequence homologous to this position was cloned into the multiple cloning site (MCS) of the FRT/oriV insertion plasmid and then recombined into this position in the genome by RecA-mediated recombination. In such a manner, many strains with single FRT/oriV insertions at various positions could be generated. Subsequent genetic crosses or phage transduction allow two neighboring FRT/oriV sites (less than 150 kb apart) to be brought into a single genome. In the present report, the lacZ and phoB sites, which are 51 kb apart in the E. coli genome, were used for the introduction of the FRT/oriV sites. To deliver the Flp (excision) and Rep (amplification) functions in trans, the yeast FLP and RK2 plasmid trfA genes were placed under the control of the Ptet promoter/operator which is tightly regulated by the TetR repressor. The addition of heated chlortetracycline (cTc) inactivates TetR, turning on the synthesis of Flp and TrfA, which respectively, execute (i) excision of the 51-kb genomic segment between the two FRT sites (in lacZ and in phoB), and (ii) its amplification.

[New possibility in inguino-femoral hernia repair: laparoscopic herniaplasty]. / Uj lehetóség az inguino-femoralis sérvek rekonstrukciójában: a laparoscopos hernioplastica.

The development of minimally invasive surgery brought up the challenge: to repair the frequent inguino-femoral hernias laparoscopically. The authors performed 65 laparoscopic hernioplasties in one year: "transabdominal preperitoneal" technique was used in 61 cases und "intraperitoneal onlay mesh" in 4 cases. Fifty-three patients were operated on, 12 of them had bilateral hernias. Recurrent hernia was the indication in 22 patients (34%). The average operating time was 102 and 144 minutes in the unilateral and the bilateral cases, respectively. There was no wound infection, or general complication. Spontaneously dissolving seroma/hematoma of the spermatic cord was noticed and detected by ultrasound in 5 patients (7.7%). The neuralgia caused by the irritation of the nerves of the region in 4 patients (6.1%) disappeared without sequels after treatment with vitamins B. The 2 early recurrences (3.2%), considered to be caused by technical inexperience, these patients were treated successfully with the "intraperitoneal onlay mesh" technique. In the authors' opinion there are definite advantages of laparoscopic hernioplasty, namely the minimal postoperative pain, early mobilization, shorter hospital stay and early restoration of full physical activity (in 1-2 weeks) as well as the known disadvantages of this technique (narcosis, longer operative time, intraperitoneal procedure, higher costs).

Long-term clinical results of highly selective vagotomy performed between 1980 and 1990.

A retrospective analysis was conducted of 778 patients who underwent highly selective vagotomy between 1980 and 1990. Surgery was performed for duodenal ulcers without any complications in 485 (62.3%) patients; for duodenal ulcers with complications such as stenosis, bleeding, or perforation in 270 (34.7%); for combined duodenal and ventricular ulcers in 12 (1.5%), and for ventricular ulcers alone in 11 (1.4%). Pyloroplasty was additionally performed in the presence of complications only. The incidence of intraoperative complications proved to be as high as 1.4%, occurring in 11 patients, while postoperative complications developed in 247 patients (31.7%). Although the overall mortality was 0.6% (5 patients), the mortality rate of those patients who underwent surgery for uncomplicated ulcer disease was 0.2% only (2 patients). The patients comprised 554 men (71.2%) and 224 women (28.8%) with an average age of 41.4 +/- 0.7 years. The average duration of duodenal ulcer disease was 9.5 years, and 643 (83.2%) of the patients were able to be regularly followed up for between 3 and 13 years. Recurrence developed in 62 patients (9.6%): in the duodenum in 57 patients (91.9%), and in the stomach in 5 (8.1%). The rate of recurrence according to sex was 9.4% in men and 10.3% in women, being 42 and 20 patients, respectively. The average duration until recurrence appeared was 27.06 +/- 3.44 months. A reoperation proved necessary in 28 of these 62 patients (45.1%). The clinical results were evaluated by means of a modified Visick classification, according to which 81.8% of the patients belonged to groups 1 or 2, 7.9% to group 3, and 10.3% to group 4.

A new method to repair inguino-femoral hernias: laparoscopic hernioplasty.

The development of minimally invasive surgery has accepted the challenge by having tried to repair inguino-femoral hernias laparoscopically. The authors performed 65 laparoscopic hernioplasties in one year. "Transabdominal preperitoneal" technique was applied in 61 cases and "intraperitoneal onlay mesh" in 4 cases. Fifty-three patients were operated, 12 of them had bilateral hernias. Recurrent hernia was the indication in 22 patients (34%). The average operating time was 102 and 144 minutes in the unilateral and the bilateral cases, respectively. There was neither wound infection, nor general complication. Spontaneously dissolving seroma/haematoma of the spermatic cord was noticed (detected by ultrasound) in 5 patients (7.7%). The neuralgia caused by the irritation of the nerves of the region in 4 patients (6.1%) disappeared without sequels after treatment with vitamins B. The 2 early recurrences (3.2%) were considered to be caused by technical unexperience; the affected patients were treated successfully with the "intraperitoneal onlay mesh" technique. It is emphasized that laparoscopic hernioplasty has definite advantages, namely minimal postoperative pain, early mobilization, short hospital stay and early restoration of full physical activity (in 1 to 2 weeks). On the other hand, general anaesthesia and intraperitoneal invasion are required and the operation consumes much time and cost.

Surgical relations of Crohn's disease and the frequency of recurrence.

There is no disease that would have as many and variable complications as Crohn's disease. One of the most common complications is bowel obstruction which can be caused by the angulation of the bowel or by inflammation, or by formation of granulation tissue (32.3%). Very common is the formation of fistulae amongst the bowels involved and other abdominal organs, and also entero-cutaneous fistulae occur frequently (11.3-14.4%). The frequency of complications is increasing with the duration of the illness. If they are life-threatening, only surgical treatment can help. Surgical treatment is also indicated when conservative treatment fails. The most commonly used surgical interventions are bowel resection and, recently, the plasty of stenotic areas. The operative mortality (3.7%) is influenced by the length of the disease and by the numbers of complications. Recurrence is very common in Crohn's disease (30.1-34.9%). The mortality rate of the second operation is 17.5%. The prognosis is usually poor because recurrence can occur years after the primary operation. In Hungary, the frequency of surgically treated patients with Crohn's disease is low, they count for 0.06% of all general surgical operations.

Thoracoscopic lung resection by the help of Nd:YAG laser.

The Swedish surgeon Jacobaeus was the first to use a lighted cystoscope for the lysis of pleural adhesions /5/. The same author reported, in 1921, five cases of intrathoracic malignancies diagnosed by thoracoscopy /6/. After the appearance of video-assisted cholecystectomy and abdominal surgery, video-assisted thoracic surgery (VATS) also occupied a major share in thoracic surgery. It was long ago that we introduced a thoracoscope (not the video-assisted type) for thoracic intervention. In 1988 we published 24 cases of thoracic sympathectomies in which we used the thoracoscope /16/.

In vivo excision and amplification of large segments of the Escherichia coli genome.

In vivo excision and amplification of large segments of a genome offer an alternative to heterologous DNA cloning. By obtaining predetermined fragments of the chromosome directly from the original organism, the problems of clone stability and clone identification are alleviated. This approach involves the insertion of two recognition sequences for a site-specific recombinase into the genome at predetermined sites, 50-100 kb apart. The integration of these sequences, together with a conditional replication origin (ori), is targeted by homologous recombination. The strain carrying the insertions is stably maintained until, upon induction of specifically engineered genes, the host cell expresses the site-specific recombinase and an ori-specific replication protein. The recombinase then excises and circularizes the genomic segment flanked by the two insertions. This excised DNA, which contains ori, is amplified with the aid of the replication protein and can be isolated as a large plasmid. The feasibility of such an approach is demonstrated here for E. coli. Using the yeast FLP/FRT site-specific recombination system and the pi/gamma-ori replication initiation of plasmid R6K, we have devised a procedure that should allow the isolation of virtually any segment of the E. coli genome. This was shown by excising, amplifying and isolating the 51-kb lacZ--phoB and the 110-kb dapX--dsdC region of the E. coli MG1655 genome.

[The effect of lesser curve seromyotomy of the stomach in animal experiments]. / A gyomron végzett kisgörbületi seromyotomia hatásának vizsgálata állatkísérletekben.

The authors investigated the efficiency of the seromyotomy of the lesser curvature of the stomach in 11 dogs. Reduction of the acid production was verified by glass electrode pH measuring of the gastric mucosa. Scintigraphic examination showed that the operation did not damage the motor function (motility) of the stomach. The fact of peripheral vagotomy was established by histological examination: degeneration of nerve fibres was detected on the peripheral side of the line of the seromyotomy, vacuolar degeneration was found in the cells of parasympathetic ganglions, and amputation neurinomas were shown in the line of the seromyotomy.

[Laparoscopic cholecystectomy]. / A laparoscopos cholecystectomiáról.

Traditional cholecystectomy has been the standard surgical treatment of the gallstone disease for more than 100 years. The technical development led to a new surgical procedure and its rapid acceptance. This is laparoscopic cholecystectomy. Its application is becoming widespread in therapy too. But most of the surgeons are lack of technical experiences in this field. Currently it restricts the indications those are anyway the same of standard cholecystectomy. Besides its many advantages, laparoscopic cholecystectomy has its own disadvantages and being an invasive procedure, there are possibilities of complications. The latest can be reduced by the adequate choice of patients, the careful learning of the operative technic and by turning to open surgery (conversion) when it is necessary. Its morbidity is nearly equal to complications of standard cholecystectomy, but mortality rate is lower (0.05-0.2%). Our morbidity of performed 300 laparoscopic cholecystectomies was 6.4%. We had no death. The hospitalization became as short as 4 days. Our early clinical results (90%) are the same of traditional cholecystectomy. Laparoscopic cholecystectomy as a new surgical procedure involves the efficiency of the standard cholecystectomy and the noninvasive endoscopic technic. Laparoscopic cholecystectomy performed by well trained surgeons is a safe surgical procedure, its early results are excellent and makes the choice of surgical treatment, used in bile surgery richer.

Complementation by detached parts of GGCC-specific DNA methyltransferases.

Individually inactive N- and C-terminal fragments of the m5C-methyltransferase M.BspRI can complement each other resulting in specific, in vivo methylation of the DNA. This was shown by cloning the coding regions for N- and C-terminal parts of the enzyme in compatible plasmids and co-transforming them into E.coli cells. The enzyme could be detached at several different sites, producing either non-overlapping or partially overlapping fragments capable of complementation. Reconstitution of the active methyltransferase from inactive fragments was demonstrated in vitro, as well. Another GGCC-specific methyltransferase, M.BsuRI, showed a similar complementation phenomenon. Moreover, interspecies complementation was observed between appropriate fragments of the two closely related enzymes M.BspRI and M.BsuRI. Fragments of structurally and functionally more different methyltransferases were unable to complement each other.