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Background Worldwide, an increase in antimicrobial resistance (AMR) of Neisseria gonorrhoeae has been observed. Until now, no protocol for an external quality assessment (EQA) has been available for Germany. The German gonococcal resistance network (GORENET) performed an EQA of primary laboratories in Germany in order to assess quality of antibiotic susceptibility testing, to gain information about laboratory procedures and to assess the impact of these procedures on test results. Methods Laboratories assessed drug susceptibility to cefixime, ceftriaxone, azithromycin, penicillin and ciprofloxacin for five N. gonorrhoeae strains, using their standard laboratory protocols. Minimal inhibitory concentrations (MICs) were compared to World Health Organisation (WHO) consensus results (or, if not available, reference laboratory results), while deviation by +/− one doubling dilution was accepted. Data on laboratory procedures were collected via a standardised questionnaire. Generalized linear models and conditional inference trees (CTREE) were used to assess relationships between laboratory procedures and testing outcomes. Results Twenty-one primary laboratories participated in the EQA in June 2018. 96% of ciprofloxacin MICs were reported within accepted deviations, as well as 88% for cefixime, 85% for ceftriaxone, 79% for penicillin and 70% for azithromycin. The use of interpretation standards and general laboratory procedures like agar base, incubation settings or the use of control strains strongly differed between laboratories. In statistical analysis, incubation time of cultures 2 concentration was associated with correct results regarding azithromycin compared to 3%. CTREE analysis showed that incubation time, humidity and CO2 concentration had the greatest influence on the average deviation from consensus results. Conclusions In conclusion, we report the development of a protocol for N. gonorrhoeae antimicrobial susceptibility testing in Germany. While testing results were in accordance with the expected consensus results in 70–96%, depending on the antibiotic agent, laboratory methodology was heterogeneous and may significantly affect the testing quality. We therefore recommend the development of a standard operating procedure (SOP) for N. gonorrhoeae susceptibility testing in Germany.

Background Emerging antimicrobial resistance (AMR) challenges gonorrhoea treatment and requires surveillance. Aim This observational study describes the genetic diversity of Neisseria gonorrhoeae isolates in Germany from 2014 to 2017 and identifies N. gonorrhoeae multi-antigen sequence typing (NG-MAST) genogroups associated with AMR or some patient demographics. Methods 1,220 gonococcal isolates underwent AMR testing and NG-MAST. Associations between genogroups and AMR or sex/age of patients were statistically assessed. Results Patients’ median age was 32 years (interquartile range: 25–44); 1,078 isolates (88.4%) originated from men. In total, 432 NG-MAST sequence types including 156 novel ones were identified, resulting in 17 major genogroups covering 59.1% (721/1,220) of all isolates. Genogroups G1407 and G10557 (G7072) were significantly associated with decreased susceptibility to cefixime (Kruskal–Wallis chi-squared: 549.3442, df: 16, p

Background German national guidelines recommend ceftriaxone combined with azithromycin for Neisseria gonorrhoeae (NG) treatment since 2014. The Gonococcal-Resistance-Network (GORENET) monitors gonococcal antimicrobial resistance (AMR) in Germany. The aim is to assess whether national guidelines are still effective in Germany and which factors affect higher minimum inhibitory concentrations (MICs). Methods GORENET laboratories sent NG isolates to the conciliar laboratory for centralized retesting of AMR using E-test. We included infection year, sex, age, infection site and clinical service type in the analysis. Geometric means were calculated for MICs for infection year. The effects of infection year, sex, age, infection site and clinical service type on MICs for ceftriaxone, cefixime, and azithromycin were investigated by multiple linear regression. Results Overall, 278 (2014), 303 (2015), 438 (2016) and 409 (2017) isolates were analysed. Of these, 90% of isolates came from men. Median age was 33 years (IQR: 25–44). Cumulative geometric means of MICs 2014–2017 were 0.006 µg/ml for ceftriaxone, 0.022 µg/ml for cefixime, and 0.185 µg/ml for azithromycin. In adjusted analysis, MICs decreased for ceftriaxone, cefixime and azithromycin by 0.74 (CI-95% 0.70–0.79), 0.89 (CI-95% 0.87–0.92) and 0.79 (CI-95% 0.75–0.83) per year, respectively. For ceftriaxone, isolates from urology (1.40; 95%-CI 1.15–1.69) and other service types (1.39; 95%-CI 1.10–1.77) compared to internal medicine, and from women (1.58; 95%-CI 1.14–2.18) were associated with increased MICs. Regarding cefixime isolates collected from urology (1.14; 95%-CI 1.02–1.28) compared to internal medicine, and from women (1.41; 95%-CI 1.18–1.69) were associated with increased MICs. For azithromycin, isolates from urology (0.82; 95%-CI 0.70–0.97) compared to internal medicine, and from women (0.77; 95%-CI 0.59–1.00) were associated with decreased MICs. Conclusion Treatment options as recommended by German national guidelines are still applicable. The lower MICs after 2014 may be due to the change of national treatment guidelines in 2014. Differences in MICs regarding service types and sex need to be further investigated. Disclosure No significant relationships.

Background The widespread antimicrobial resistance (AMR) of Neisseria gonorrhoeae (NG) is a serious problem for the treatment of gonorrhoea. Because NG infections are not reportable in Germany, only limited data on disease epidemiology and antimicrobial susceptibility patterns are available. The Gonococcal Resistance Network (GORENET) monitors trends of NG AMR in Germany and links this to epidemiological data and NG multiantigen sequence typing (NG-MAST) data to guide treatment algorithms and target future prevention strategies. Methods Between April 2014 and December 2018, NG isolates and data on patient-related information were collected from laboratories nationwide and centralized susceptibility testing using E-test was performed. Susceptibility results for cefixime, ceftriaxone, azithromycin, ciprofloxacin and penicillin were defined according to EUCAST 4.0 standards. Results Of 2115 isolates, 91.6% of isolates were from men. The most frequently tested materials among men were urethral (92.4%) and rectal swabs (3.8%), and among women mainly endocervical swabs (80.9%). Resistance to ceftriaxone (MIC ≥0.125 mg/L) occurred only sporadically (0–0.3%) during the entire observation period (2015 and 2018), while 1.0–2.1% of isolates were resistant to cefixime (MIC ≥0.125 mg/L). Proportion of isolates resistant to azithromycin (MIC ≥ 0.5 mg/L) was 11.4% (2014), 11.3% (2015), 4.3% (2016), 3.7% (2017), 9.4% (2018). 53.4–71.7% were resistant to ciprofloxacin, and 14.2–24.3.1% were resistant to penicillin. Conclusion Resistance to ceftriaxone and to cefixime was low, whereas azithromycin resistance showed a discontinuous presentation with partly high levels during the observation period. Rates of ciprofloxacin and penicillin resistance were very high. According to the current national guidelines, ceftriaxone 1-2g IV and azithromycin 1,5g orally are usually used in dual therapy. Shortly revised national guidelines will state that use of azithromycin should be avoided if possible if a test of cure can be guaranteed and a susceptibility test is available. Continued surveillance of NG AMR remains relevant to ensure efficient disease management. Disclosure No significant relationships.

Background Neisseria gonorrhoeae (NG) infections are not reportable in Germany. The Gonococcal Resistance Network (GORENET) is a laboratory network to monitor antimicrobial resistance (AMR) in Germany, linking data from sequence typing to epidemiological data. We described prevalence of gonococcal sequence types in Germany and associations to AMR to improve future treatment and prevention strategies. Methods NG isolates collected between April 2014 and December 2017 were tested by E-test and sequence typed by NG multiantigen sequence typing (NG-MAST). For sequence typing, DNA was extracted and internal fragments of porB and tbpB were amplified by polymerase chain reaction. Fragments were sequenced by Sanger sequencing and evaluated using a global database (www.ng-mast.net). Genogroups were assigned to sequence types which shared one allele and exhibited ≥99% homogeneity in the other allele. Results 1220 isolates were sequence typed (106 in 2014, 96 in 2015, 525 in 2016, and 495 in 2017). In total, we detected 422 different sequence types that grouped into 17 genogroups. Among the most prevalent genogroups were G2400 (6.8%), G1407 (6.8%), G5441 (6.2%), G25 (5.6%), G2992 (5.5%) and G10557 (5.3%). The multi-resistant G1407 and G2400 were most prevalent in 2014 (12.4% and 10.5%, respectively) and declined to 6.1% and 7.3% in 2017. Two new genogroups, G11461 (3.6%) and G17420 (2.1%), emerged showing high prevalence in 2017 and no association to extended-spectrum cephalosporin resistance. Furthermore, a novel genogroup-association with cefixime resistance and reduced cephalosporin susceptibility was identified. Conclusion From 2014 to 2017 prevalence of G1407 declined and two novel extended-spectrum cephalosporin sensitive clones G11461 and G17420 seem to have replaced the multi-drug resistance clone G1407. To verify these results, continuous testing with an increased number of isolates should be performed. Disclosure No significant relationships.

Genome engineering through homologous recombination (HR) is a powerful instrument for studying biological pathways or creating treatment options for genetic disorders. In mammalian cells HR is rare but the creation of targeted DNA double-strand breaks stimulates HR significantly. Here, we present a method to generate, evaluate, and optimize rationally designed endonucleases that promote HR. The DNA-binding domains were synthesized by assembling predefined zinc-finger modules selected by phage display. Attachment of a transcriptional activation domain allowed assessment of DNA binding in reporter assays, while fusion with an endonuclease domain created custom nucleases that were tested for their ability to stimulate HR in episomal and chromosomal gene repair assays. We demonstrate that specificity, expression kinetics, and protein design are crucial parameters for efficient gene repair and that our two-step assay allows one to go quickly from design to testing to successful employment of the custom nucleases in human cells.

Microarray studies revealed that as a first hit the SV40 T/t antigen causes deregulation of 462 genes in mammary gland cells (ME cells) of WAP-SVT/t transgenic animals. The majority of deregulated genes are cell proliferation specific and Rb-E2F dependent, causing ME cell proliferation and gland hyperplasia but not breast cancer formation. In the breast tumor cells a further 207 genes are differentially expressed, most of them belonging to the cell communication category. In tissue culture breast tumor cells frequently switch off WAP-SVT/t transgene expression and regain the morphology and growth characteristics of normal ME cells, although the tumor-revertant cells are aneuploid and only 114 genes regain the expression level of normal ME cells. The profile of retransformants shows that only 38 deregulated genes are tumor-specific, and that none of them is considered to be a typical breast cancer gene.

We recently established transgenic animals (WAP-SV-T/t) carrying the early coding region of Simian Virus 40 (SV40) under the transcriptional control of the whey acidic milk protein promoter (WAP), which restricts the expression of the transgene to mammary gland epithelial cells (ME-cells). SV40 T/t-antigen synthesis causes premature mammary gland involution during late pregnancy by inducing apoptosis and leads to development of mammary tumors after the first lactation period in both p53 positive (WAP-SV-T/t) and p53 negative double transgenic animals (WAP-SV-T/t.p53-/-). The high apoptotic rate persists in all of the T/t-antigen positive breast tumor cells, as well as in established ME-tissue culture cell lines. ME-cells which spontaneously switch off the expression of the WAP-SV-T/t transgene do not undergo apoptosis. However, these cells again exhibit an extensive DNA fragmentation when SV40 T/t-antigen synthesis is reintroduced, which indicates that it is the expression of T/t antigen which is the critical factor for induction of apoptosis. In addition, we isolated several ME-cell lines from different breast tumors which have spontaneously lost the T/t-antigen yet remain maximally transformed. Strikingly, these cells contain a missense mutation of the p53 gene at codon 242 (p53(242)), which substitutes alanine for glycine. This mutation increases p53 stability and it reduces the transactivating function of p53, albeit without affecting the ability of the protein to interact with the DNA. This indicates that p53 missense mutations are selected for in breast tumors initially expressing T/t-antigen. Therefore, the p53(242) mutation is sufficient to maintain the transformed state after the ME-cells have switched off the WAP-SV-T/t transgene. Interestingly, the p53 minus state per se is not sufficient to induce ME-cell transformation since homozygous null mice for the p53 gene (p53-/-) fail to develop breast cancer.

In order to determine whether partial methylation of the herpes simplex virus (HSV) tk gene prevents tk gene expression, the HSV tk gene was cloned as single-stranded DNA. By in vitro second-strand DNA synthesis, specific HSV tk gene segments were methylated, and the hemimethylated DNA molecules were microinjected into thymidine kinase-negative rat2 cells. Conversion of the hemimethylated DNA into symmetrical methylated DNA and integration into the host genome occurred early after gene transfer, before the cells entered into the S phase. HSV tk gene expression was inhibited either by promoter methylation or by methylation of the coding region. Using the HindIII-SphI HSV tk DNA fragment as a primer for in vitro DNA synthesis, all cytosine residues within the coding region, from +499 to +1309, were selectively methylated. This specific methylation pattern caused inactivation of the HSV tk gene, while methylation of the cytosine residues within the nucleotide sequence from +811 to +1309 had no effect on HSV tk gene activity. We also methylated single HpaII sites within the HSV tk gene using a specific methylated primer for in vitro DNA synthesis. We found that of the 16 HSV tk HpaII sites, methylation of 6 single sites caused HSV tk inactivation. All six of these "methylation-sensitive" sites are within the coding region, including the HpaII-6 site, which is 571 bp downstream from the transcription start site. The sites HpaII-7 to HpaII-16 were all methylation insensitive. We further inserted separately the methylation-sensitive HSV tk HpaII-6 site and the methylation-insensitive HpaII-13 site as DNA segments (32-mer) into the intron region of the simian virus 40 T antigen (TaqI site). Methylation of these HpaII sites caused inhibition of simian virus 40 T-antigen synthesis.

Gene knockout in murine embryonic stem cells (ESCs) has been an invaluable tool to study gene function in vitro or to generate animal models with altered phenotypes. Gene targeting using standard techniques, however, is rather inefficient and typically does not exceed frequencies of 10(-6). In consequence, the usage of complex positive/negative selection strategies to isolate targeted clones has been necessary. Here, we present a rapid single-step approach to generate a gene knockout in mouse ESCs using engineered zinc-finger nucleases (ZFNs). Upon transient expression of ZFNs, the target gene is cleaved by the designer nucleases and then repaired by non-homologous end-joining, an error-prone DNA repair process that introduces insertions/deletions at the break site and therefore leads to functional null mutations. To explore and quantify the potential of ZFNs to generate a gene knockout in pluripotent stem cells, we generated a mouse ESC line containing an X-chromosomally integrated EGFP marker gene. Applying optimized conditions, the EGFP locus was disrupted in up to 8% of ESCs after transfection of the ZFN expression vectors, thus obviating the need of selection markers to identify targeted cells, which may impede or complicate downstream applications. Both activity and ZFN-associated cytotoxicity was dependent on vector dose and the architecture of the nuclease domain. Importantly, teratoma formation assays of selected ESC clones confirmed that ZFN-treated ESCs maintained pluripotency. In conclusion, the described ZFN-based approach represents a fast strategy for generating gene knockouts in ESCs in a selection-independent fashion that should be easily transferrable to other pluripotent stem cells.

The engineering of proteins to manipulate cellular genomes has developed into a promising technology for biomedical research, including gene therapy. In particular, zinc-finger nucleases (ZFNs), which consist of a nonspecific endonuclease domain tethered to a tailored zinc-finger (ZF) DNA-binding domain, have proven invaluable for stimulating homology-directed gene repair in a variety of cell types. However, previous studies demonstrated that ZFNs could be associated with significant cytotoxicity due to cleavage at off-target sites. Here, we compared the in vitro affinities and specificities of nine ZF DNA-binding domains with their performance as ZFNs in human cells. The results of our cell-based assays reveal that the DNA-binding specificity--in addition to the affinity--is a major determinant of ZFN activity and is inversely correlated with ZFN-associated toxicity. In addition, our data provide the first evidence that engineering strategies, which account for context-dependent DNA-binding effects, yield ZFs that function as highly efficient ZFNs in human cells.

Introduction Neisseria gonorrhoeae (NG)-infections are not reportable in Germany and only limited data on NG-epidemiology and antimicrobial resistance (AMR) are available. With GORENET we monitor the NG-AMR and patterns of resistance testing in Germany in order to guide treatment algorithms and targeted prevention strategies. Methods We recruited laboratories based on geographic distribution and number of NG-isolates. From April 2014 prospective data on all performed NG-AMR-tests together with patient-related information were collected. Laboratories send a part of the isolates to the national reference laboratory (NRL) for culturing and AMR-testing towards ceftriaxone, cefixime, azithromycin, ciprofloxacin, and penicillin by using E-Test, as well as beta-lactamase. Results are interpreted according to European Committee on Antimicrobial Susceptibility Testing 4.0. We described the reported samples by sex and age and characterised isolates tested in NRL by resistance patterns. We calculated proportions and medians, together with interquartile range (IQR), where appropriate. Results Between April and December 2014 we received information on 651 isolates tested in 19 laboratories. Altogether, 90.3% isolates were from men and 8.5% from women. Median age of tested men was 36 (IQR 29–49) and women 28 (IQR 22–41) years. NRL received 502 isolates, 342 were vital and 253 were tested for AMR. From those 0 were resistant towards ceftriaxone, 1.6% towards cefixime, 11.1% towards azithromycin, 73.1% towards ciprofloxacin, and 30.4% towards penicillin. Further 37.9% and 50.6% isolates were intermediate susceptible to Azithromycin and to Penicillin. From 205 isolates tested for beta-lactamase, 25.9% were positive. Conclusion In Germany isolates tested for NG-AMR were mostly from men. We assume that a substantial proportion of these isolates could be from men having sex with men. NG-AMR to ceftriaxone and cefixime remains low, while resistance and intermediate susceptibility to azithromycin, ciprofloxacin and penicillin is substantial. Monitoring of NG-AMR should be highly prioritised. Disclosure of interest statement The GORENET Project is funded by German Federal Ministry of Health. No conflicts of interest.

Heterologous trans-splicing is a messenger RNA (mRNA) processing mechanism, that joins RNA segments from separate transcripts to generate functional mRNA molecules. We present here for the first time experimental evidence that the proximal segment of the HIV-nef RNA segment can be trans-spliced to both viral (e.g. SV40 T-antigen) and cellular transcripts. Following either microinjection of in vitro synthesized HIV-nef and SV40 T-antigen pre-mRNA or transfection of the HIV-nef DNA into T-antigen positive cells (CV1-B3; Cos7), it was found that recipient cells synthesized HIV-nef/T-antigen hybrid mRNA and protein molecules. To generate the hybrid mRNA, the cells utilized the 5′ cryptic splice sites of the HIV-nef (5′cry 66 and 5′cry 74) and the SV40 T/t-antigen 3′ splice site. To demonstrate that heterologous trans-splicing also occurs between the HIV-nef RNA and cellular transcripts, a cDNA library was established from HIV-nef positive CV1-B3 cells (CV1-B3/13 cells) and screened for hybrid mRNA molecules. Reverse transcription-PCR and Northern blot analysis revealed that a significant portion of the HIV-nef transcript is involved in heterologous trans-splicing. To date, eight independent HIV-nef/cellular hybrid mRNA molecules have been identified. Five of these isolates contain segments from known cellular genes (KIAA1454, PTPκ, Alu and transposon gene families), while three hybrid segments contain sequences of not yet known cellular genes (genes 1–3).

The mammalian genome encodes a DNA cytosine-5-methyltransferase (MTase) of about 170 kDa that is apparently responsible for both de novo and maintenance methylation at CpG sites. Both methylation activities have to be regulated accurately to ensure correct developmental and cell type-specific gene activity. Distorted DNA methylation patterns have been associated with cell aging and diseases such as cancer and fragile X syndrome. Structural and functional in vitro studies of the mouse MTase have indicated that the enzyme has both a regulatory and a catalytic region located in the N-terminal and C-terminal parts of the protein, respectively. The regulatory region includes the nuclear localization signal (NLS), the sequence for DNA targeting and the Zn-binding domain. The catalytic domain carries the ten consensus sequence motifs specific for all known pro- and eukaryotic DNA cytosine-5-methyltransferases. In an attempt to separate regulatory and catalytic functions of the enzyme in vivo, we have tested various deletion mutations by means of transient and stable cell transfection experiments. Expression of the transgenes, all of which retained the C-terminal catalytic domain, was monitored by immunofluorescence staining, Northern blot analysis and SDS gel electrophoresis. Despite high levels of transgene expression, the truncated MTase molecules exhibited neither de novo nor maintenance methylation activity. These findings might indicate that in vivo, an efficient control mechanism prevents the ectopic activity of the DNA MTase that is structurally compromised in its N-terminal regulatory region.

Genome engineering is a powerful instrument to study basic biological questions or to create treatment options for genetic diseases. A safe way to correct genetic mutations is through homologous recombination (HR), during which a mutant sequence is replaced with the corresponding wild-type sequence. While HR is predominant in yeast, it is a rare event in mammalian cells where non-homologous recombination is much more frequent. The creation of a targeted DNA double-strand break (DSB) has been shown to locally stimulate HR several hundred-fold by activating the cellular machinery for double-strand break repair, but it presupposes the availability of a site-specific endonuclease to create the DSB. Some initial experiments with artificial nucleases in human cells revealed that cytotoxicity of such enzymes is a major issue, most likely through cleavage at non-specific sites. Here, we present a rapid two-step method to evaluate and optimize novel, rationally designed endonucleases. In a first step, the DNA-binding domain was synthesized by assembling predefined C2H2-zinc-finger modules and fused to a transcriptional activation domain. Each zinc-finger module is designed to recognize 3 bp of DNA and the modules can be assembled in any order necessary to recognize any given sequence in a target locus. DNA-binding specificity of these artificial transcription factors was assessed in a reporter assay. In a second step, the DNA-binding domain was attached to the catalytic domain of the FokI restriction endonuclease to generate the custom nucleases. Upon co-transfection with a repair matrix, they were assessed for their ability to stimulate HR in an episomal gene repair assay. We have engineered several kinds of custom nucleases and demonstrate that high specificity, fine-tuned expression kinetics and protein design are absolutely critical to promote HR. Importantly, the same parameters are also crucial for a low level of cytotoxicity. After optimization, expression of the custom nucleases stimulated both episomal and chromosomal gene repair and HR was achieved in 35% or 2.5% of transfected cells, respectively. Thus, custom nucleases will be a valuable tool to stimulate gene repair for therapeutic purposes. Moreover, they can be easily combined with viral vector systems for efficient delivery.

In addition to its well-known synaptic function, acetylcholinesterase was recently shown to stimulate neurite outgrowth from cultured chick neurons in a manner unrelated to its catalytic activity. It remained unclear, however, whether each of the variant acetylcholinesterase enzyme forms can promote such process extension and whether this effect of acetylcholinesterase was limited to neurite outgrowth. Using DNA microinjections and stable transfections of cultured glioma cells, we explored the possibility that specific acetylcholinesterase isoforms affect cellular development and morphology of CNS astrocytes. Cells microinjected with human ACHEDNA constructs that differ in their exon-intron composition displayed rapid yet stable induction of cell body enlargement and process extensions. Cells transfected with ACHEDNA carrying the neuronal-characteristic 3'-E6 domain also displayed stable process extensions. However, stable transfections with ACHEDNAs including the 3'-alternative 14/E5 region induced the appearance of small, round cells in a dominant manner. This was associated with expression of 14/E5-ACHEmRNA transcripts and the production of soluble acetylcholinesterase monomers that were catalytically indistinguishable from the 3'-E6 enzyme but displayed higher electrophoretic mobility than that of the 3'-E6 form. Thus, variable expression levels and alternative splicing modes of the ACHE gene correlated in these experiments with glial development in a manner that was apparently unrelated to catalysis.

T-antigen-positive transformation revertant cell lines were isolated from fully simian virus 40 (SV40)-transformed Fisher rat embryo fibroblast cells (REF 52 cells) by methionine starvation. Reversion of the transformed cells (SV-52 cells) was caused by a mutation within the cellular genome. To demonstrate this, we isolated SV40 DNA from the host genome, inserted it into plasmid pSPT18 DNA, cloned it in Escherichia coli, and microinjected it into the nuclei of the REF 52 cells. Fully transformed cells were obtained with the same efficiency (20 to 25%) as after microinjection of wild-type SV40 DNA I. Furthermore, the revertant cells were resistant to retransformation by SV40. Following microinjection of wild-type SV40 DNA I, 42 independent cell lines were isolated. Cells of all analyzed lines acquired additional SV40 DNA copies, but changes in the cell morphology or growth characteristic were not demonstrable. However, the revertants were retransformable with a high efficiency after polyomavirus and adenovirus type 2 infections or microinjection. Also, fusion of the revertant cells with the grandparental REF 52 cells led to restoration of the transformed state.

In this investigation we analysed the maintenance methylation activity of the mammalian cell DNA methyltransferase by microinjection of hemimethylated HSV-tk DNA into thymidine kinase-negative rat 2 cells. We found that the hemimethylated DNA was efficiently converted into symmetrical methylated molecules before DNA replication. Furthermore, integration of the trans-DNA into the host genome is an early event after gene transfer.

Microinjection of early simian virus 40 (SV40) DNA fragments has shown that maximal transformation of rat cells (Ref 52) is a property of the second SV40 T-antigen exon. Expression of this particular T-antigen region was obtained by coinjection of the Taq/Bam DNA fragment with the early promoter/enhancer HpaII/BglI fragment. Microinjection of the DNA fragment mixture induced two categories of transformants; namely, maximally and minimally transformed cells. The maximally transformed cells synthesize two Taq/Bam-specific polypeptides, and the minimally transformed cells only the lower molecular weight form. Both types of transformants contain the cellular p52 protein at high concentrations. Furthermore, maximal transformation of Ref 52 cells requires the carboxy terminus of the T-antigen. Cells transformed by microinjection of the SV40 Pst A-fragment display different parameters of maximally transformed cells but not anchorage-independent growth.

The time-course of intranuclear Simian virus 40 (SV40) tumor (T) antigen synthesis and accumulation in permissive CV1 monkey cells and nonpermissive 3T3 mouse cells has been studied by immunofluorescence and cytofluorometry. CV1 cells accumulate T antigen continuously over a period of 48 h after infection, whereas in 3T3 cells the T-antigen content remains about constant and at a comparatively low level. Only those CV1 cells which have attained a threshold concentration of intranuclear T antigen synthesize viral capsid proteins (V antigen). In nonpermissive 3T3 cells, the T-antigen threshold value for the onset of V-antigen synthesis is higher than in CV1 cells and is never reached by infected cells. However, 3T3 cells microinjected with sufficient amounts of SV40 DNA easily surpass this value and behave permissively.