Intracellular Lambda Research Articles

Structure-Function Relationships of Novel Benzimidazole-Based Inhibitors of the Isoprenoid Biosynthetic Pathway in Myeloma Cells

Malignant plasma cells are characterized by the production of monoclonal protein (MP). Our work has demonstrated that disrupting intracellular MP trafficking by targeting the geranylgeranylation of Rab GTPases results in endoplasmic reticulum stress, induction of the unfolded protein response pathway and apoptosis of myeloma cells. Rab proteins are geranylgeranylated by the enzyme geranylgeranyl transferase II (GGTase II) which uses geranylgeranyl diphosphate as the isoprenoid substrate. We have pursued the development of specific GGTase II and geranylgeranyl diphosphate synthase (GGDPS) inhibitors as potential anti-myeloma agents. A family of compounds incorporating a benzimidazole heterocycle and either a carboxyphosphonate or bisphosphonate was prepared by modification of the appropriate heterocycles. The core benzimidazole phosphonate structure was modified with substituents at either the alpha-carbon position or at the 5 and 6 positions on the benzimidazole (Figure). These compounds were subjected to in vitro enzyme assays for GGTase II and the related prenyltransferases farnesyl transferase (FTase) and GGTase I as well as the synthases farnesyl diphosphate synthase (FDPS) and GGDPS. Cellular disruption of protein prenylation was assessed via immunoblot analysis for Ras (a representative farnesylated protein) and for unmodified Rap1a (a representative geranylgeranylated protein) in human myeloma cells (RPMI-8226, MM.1S). The ability of the novel compounds to disrupt MP trafficking was determined by measuring changes in intracellular lambda light chain levels via ELISA. Confirmation of disruption of Rab geranylgeranylation was achieved by Triton X-114 lysis to obtain detergent (membrane) fractions which were subjected to Rab6 (a representative Rab protein) immunoblot analysis. These studies demonstrated that the nature of the phosphonate head group has a profound impact on enzyme specificity: the carboxyphosphonates have specific activity as GGTase II inhibitors while the bisphosphonate analogues have specific activity as FDPS inhibitors with no cross-reactivity against any of the other tested enzymes. The most potent GGTase II and FDPS inhibitors were the unsubstituted analogues. The carboxyphosphonate 1 was found to have an IC50 of 230 μM against GGTase II and cellular activity starting at 2.5 mM. This represents a two-fold improvement in potency compared with the known GGTase II inhibitor 3-PEHPC. The bisphosphonate 5 has an IC50 of 24 nM against FDPS and cellular activity at ≥ 50 μM. The addition of a methyl group at the alpha-carbon position abrogated the activity of the carboxyphosphonate (2) as a GGTase II inhibitor and the activity of the bisphosphonate (6) as a FDPS inhibitor. The addition of substituents at the 5 and 6 positions (compounds 3, 4, 7 and 8)did not improve potency compared with the parent compounds. Co-incubation with low concentrations of the HMG-CoA reductase inhibitor lovastatin (0.1-1 μM), concentrations which on their own are not sufficient to disrupt protein prenylation, potentiated the cellular activity of the lead GGTase II and FDPS inhibitors. In conclusion, these benzimidazole phosphonates represent novel classes of GGTase II and FDPS inhibitors. These studies highlight the importance of the identity of the phosphonate head-group in dictating enzyme specificity. Further studies are underway to prepare additional benzimidazole analogues in order to improve their potency, advance the understanding of the structure-function relationship, and assess the potential of these compounds as anti-myeloma agents. [Display omitted] DisclosuresWiemer:Terpenoid Therapeutics, Inc: Other: Founder. Holstein:Celgene: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy.

Abstract 4271: Standardized approach for monitoring plasma cell disorders

Abstract Multi-color flow cytometry using consensus surface markers (CD138, CD38, CD19, CD45, CD27, CD56, CD81, and CD117), intracellular kappa and lambda, as well as other auxiliary markers is routinely being used for the identification and characterization of plasma cells. Here, we describe the development of a modular 10-color plasma cell panel for immunophenotyping of the plasma cell compartment. The panel was developed as an 8-color dried backbone, which can be supplemented with 2 liquid (drop in) reagents. Lyophilized or dried reagent cocktails in a unit test format are known to drive workflow efficiency in clinical settings by eliminating the need for manual cocktailing (of reagents), and frequent batch to batch verification procedures. Longer shelf life and room temperature storage are additional appealing features for lyophilized and dried products. We have developed a dried-down 8-color plasma cell panel, which is manufactured in a single test-per-tube, ready to use format, and can be supplemented with liquid (drop in) reagents. The flexibility of being able to add 2 liquid reagents to the dried backbone provides the option of using intracellular kappa and lambda to examine clonality or using CD38-multi-epitope and/or anti-p63 (VS38c) reagent(s). The single test-per-tube format is ideally suited for MRD applications, where sample size is limited and there is a need for acquiring a large number of events for data analysis. Also, the dried reagent panel is comprised of multiple high performing polymer dyes, which are brighter than conventional dyes, and afford high resolution of the relevant cell populations. Furthermore, the dried reagent tubes have a long shelf life (12 months) and significantly enhance the workflow efficiency. Comparison of the liquid and dried versions of the 10-color plasma cell panel using peripheral blood from healthy donors (as well as control cell lines) showed similar staining patterns, comparable population statistics, and fluorochrome brightness. The dried plasma cell tube(s) were also tested on bone marrow samples from healthy and diseased subjects in conjugation with intracellular staining for kappa and lambda. Our results demonstrate that the 10-color plasma cell panel can be used for identification of the plasma cell compartment (based on CD38 & CD138 expression), assessment of aberrant marker expression (CD19, CD27, CD81, CD117, CD45, CD56) for the identification of normal and abnormal plasma cells, and examining clonality in multiple patient samples. Citation Format: Ulrika Johansson, Na Li, Suraj Saksena, Natalie Golts, Brent Gaylord. Standardized approach for monitoring plasma cell disorders [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4271.

Contribution of human smooth muscle cells to amyloid angiopathy in AL (light-chain) amyloidosis

ABSTRACTObjective: Amyloid light-chain (AL) amyloidosis is a disease process that often compromises the peripheral vascular system and leads to systemic end-organ dysfunction. Although amyloid formation in vessel walls is a multifaceted process, the assembly of the native light chains (LCs) into amyloid fibrils is central to its pathogenesis. Recent evidence suggests that endocytosis and endolysosomal processing of immunoglobin LCs by host cells is essential to the formation of amyloid fibrils that are deposited in at least some tissues. The aim of this study was to elucidate the role of vascular smooth muscle in amyloid angiopathy. Methods: Human coronary artery smooth muscle cells (SMCs) were grown on coverslips, four chamber glass slides, and growth factor-reduced Matrigel matrix in the presence of 10 µg/ml of ALs (λ and κ isotypes), nonamyloidogenic LCs, and culture medium (negative control) for 48 and 72 hours. Thereafter, a detailed light microscopic, immunohistochemical, and ultrastructural evaluation was conducted to verify amyloid deposition and characterize the role of SMCs in the formation of amyloid deposits in the various experimental conditions. Results: Amyloid deposits were detected extracellulary as early as 48 hours after exposure of vascular smooth muscle cells (VSMCs) to AL-LCs (amyloidogenic light chains) as confirmed by affinity to Congo red dye, thioflavin T fluorescence, and transmission electron microscopy. No amyloid was present in the cultures of SMCs treated with medium alone or nonamyloidogenic LCs. SMCs associated with amyloid deposits exhibited CD68, lysosome-associated membrane protein 1-1, and intracellular lambda light chain expression and only focal smooth muscle actin and muscle-specific actin positivity. Electron microscopy revealed these cells to have an expanded mature lysosomal compartment closely associated with deposits of newly formed amyloid fibrils. Conclusions: The interaction of amyloidogenic LCs with VSMCs is necessary for the formation of amyloid fibrils that are deposited in peripheral vessels. VSMCs participate in the formation of amyloid by the intracellular processing of AL-LCs, which is possible due to their transformation from a smooth muscle to a macrophage phenotype. The formation of amyloid fibrils occurs in the mature lysosomal compartment of transformed cells. The amyloid that is formed is then extruded into the extracellular matrix.

Abstract 4768: Determination of Rab GTPase-mediated pathways critical for the antimyeloma activity of Rab GGTase inhibitors

Abstract Multiple myeloma (MM) is a plasma cell malignancy characterized by the production of high levels of monoclonal protein (MP). This leads to an increased protein folding burden in the endoplasmic reticulum (ER) in MM cells, resulting in a constitutively upregulated unfolded protein response (UPR) and a lower threshold for ER stress-mediated apoptosis. We have previously shown that inhibitors of Rab GGTase, the enzyme responsible for Rab GTPase geranylgeranylation, disrupt MP trafficking in MM cells, leading to an accumulation of MP in the ER and induction of the UPR and apoptosis. There are over 60 different Rab GTPases in mammalian cells involved in regulating a wide range of membrane trafficking events including exocytic, endocytic, and lysosomal pathways. In this study, we aimed to further define how disruption of RabGTPase function in MM cells leads to apoptosis by knocking down individual Rab proteins. Given the accumulation of MP and induction of UPR, our initial focus was on secretory Rabs. Knockdown of Rab1A/B (>50%) using siRNA in RPMI 8226 cells lead to moderate increases in intracellular lambda light chain in Rab1A/B knockdowns after 72 hours (∼120-140% of control levels by ELISA). However, despite the increase in intracellular MP, no increases in UPR or apoptosis markers were seen by western blot analysis or annexin-V/propidium iodide flow cytometry. Extended time course experiments (up to 120 hours) also did not exhibit any increase in UPR or apoptosis. Knockdown of Rab6A (>60%), another RabGTPase associated with the secretory pathway, did not lead to increases in intracellular lambda light chain or induction of apoptosis. These results suggest that although individual knockdown of Rab1 is sufficient to partially disrupt MP trafficking, either more complete knockdown of the individual Rabs or knockdown of multiple Rabs is necessary for induction of the UPR and apoptosis. Additional optimization of the knockdowns will be performed in order to verify these results. These studies support the further development of Rab GGTase inhibitors that globally effect Rab GTPase geranylgeranylation as a novel anti-myeloma strategy. Citation Format: Kaitlyn M. Dykstra, Cheryl L. Allen, Sarah A. Holstein. Determination of Rab GTPase-mediated pathways critical for the antimyeloma activity of Rab GGTase inhibitors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4768.

Interaction Between Heat Shock Protein Inhibitors and Isoprenoid Biosynthetic Pathway Inhibitors In Multiple Myeloma Cells

AbstractAbstract 2895Multiple myeloma (MM) is characterized by the production of substantial quantities of monoclonal protein (MP) by malignant plasma cells. We have previously demonstrated that select inhibitors of the isoprenoid biosynthetic pathway (IBP) induce apoptosis of MM cells via inhibition of Rab geranylgeranylation, leading to disruption of MP trafficking and induction of the unfolded protein response (UPR) pathway. Inhibitors of the molecular chaperone heat shock protein 90 (HSP90) have also been shown to induce components of the UPR and apoptosis in MM cells and are currently under clinical investigation. Given the sensitivity of MM cells to stressors which activate the UPR, we hypothesized that combining agents that disrupt protein folding (HSP90 inhibitors) with agents that disrupt protein trafficking (IBP inhibitors) would yield enhanced cytotoxicity via their effects on MP levels and the UPR. We therefore evaluated the effects of combining IPB and HSP90 inhibitors in MM, Waldenstrom macroglobulinemia (WM), and IgM-secreting cells. Studies were performed in 2 lambda light chain-secreting human MM cell lines (RPMI-8226, U266), a WM cell line (WM-WSU), and the IgM-secreting RL lymphoblastoid cell line. IBP inhibitors included lovastatin (Lov) (HMG-CoA reductase inhibitor), digeranyl bisphosphonate (DGBP) (geranylgeranyl synthase inhibitor), and 3-PEHPC (geranylgeranyl transferase II inhibitor), while 17-AAG and 17-DMAG were used as HSP90 inhibitors. The combination of Lov and 17-AAG resulted in enhanced cytotoxicity in all tested cell lines at both 24 & 48 h as determined by MTT assays. Isobologram analysis revealed a synergistic interaction for the RPMI-8226, RL, and WM cells and an additive interaction for the U266 cells. Pre-treatment with Lov for 24 h prior to the addition of 17-AAG resulted in a synergistic interaction in all tested cell lines. The enhanced cytotoxicity was prevented by co-incubation with either mevalonate or geranylgeranyl pyrophosphate. Pre-treatment with DGBP yielded a synergistic interaction between DGBP and 17-AAG in the RPMI-8226 cell line. The addition of 3-PEHPC to either 17-AAG or 17-DMAG resulted in a synergistic interaction in the RPMI-8226 cells. Treatment with 17-AAG and 17-DMAG led to a concentration-dependent decrease in secreted lambda light chain (RPMI-8226, U266) or IgM (RL) levels after 24 h as measured with ELISA. As we have previously demonstrated, Lov decreased secreted MP levels. The combination of Lov and the HSP90 inhibitors resulted in a further decrease in secreted MP. Interestingly, 17-AAG and 17-DMAG decreased intracellular lambda light chain levels in the RPMI-8226 & U266 lines while increasing intracellular IgM levels in the RL line. The increase in l light chain induced by Lov in the MM lines was partially prevented by co-incubation with either of the HSP90 inhibitors. The increase in HSP70 levels induced by HSP90 inhibition was not significantly altered by co-incubation with Lov; however, the accumulation of unmodified Rap1a (a marker for inhibition of geranylgeranylation) induced by Lov was diminished in the presence of the HSP90 inhibitors. Pre-treatment with Lov prior to the addition of 17-AAG resulted in enhanced apoptosis, as determined by PARP and calnexin cleavage, in the MM cells while significant changes were not observed following 24 h concurrent treatment. As a measure of induction of the UPR, levels of phosphorylated eIF2a (p-eIF2a) were assessed. While no significant changes were noted following 24 h concurrent treatment, pre-treatment with Lov did yield an increase in p-eIF2a levels compared with 17-AAG alone. In conclusion, these studies are the first to demonstrate synergistic cytotoxic interactions between select IBP inhibitors and HSP90 inhibitors in MM cells and to evaluate the effects of these agents on MP levels. That the effects of these agents on components of the UPR and apoptosis are most evident following pre-incubation with Lov suggests that inhibition of prenylation is required for the interaction. Further studies will be required to determine whether the observed decrease in intracellular and secreted light chain levels induced by the HSP90 inhibitors is a consequence of increased protein degradation or decreased protein synthesis. These studies form the basis for further pre-clinical and clinical investigation into the use of this novel combination in MM and related MP-secreting disorders. Disclosures:No relevant conflicts of interest to declare.

Chronic lymphocytic leukemia cytoplasmic inclusions

American Journal of HematologyVolume 85, Issue 4 p. 275-276 Images in HematologyFree Access Chronic lymphocytic leukemia cytoplasmic inclusions† Namrata Setia, Namrata Setia Department of Pathology, Baystate Medical Center, Tufts University School of Medicine, Springfield, MassachusettsSearch for more papers by this authorTimothy Connolly, Timothy Connolly Department of Pathology, Baystate Medical Center, Tufts University School of Medicine, Springfield, MassachusettsSearch for more papers by this authorWilliam Levine, William Levine Department of Pathology, Baystate Franklin Medical Center, Tufts University School of Medicine, Springfield, MassachusettsSearch for more papers by this authorLiron Pantanowitz, Corresponding Author Liron Pantanowitz [email protected] Department of Pathology, Baystate Medical Center, Tufts University School of Medicine, Springfield, Massachusetts Department of Pathology, Baystate Franklin Medical Center, Tufts University School of Medicine, Springfield, MassachusettsDepartment of Pathology, Baystate Medical Center, 759 Chestnut Street, Springfield, MA 01199Search for more papers by this author Namrata Setia, Namrata Setia Department of Pathology, Baystate Medical Center, Tufts University School of Medicine, Springfield, MassachusettsSearch for more papers by this authorTimothy Connolly, Timothy Connolly Department of Pathology, Baystate Medical Center, Tufts University School of Medicine, Springfield, MassachusettsSearch for more papers by this authorWilliam Levine, William Levine Department of Pathology, Baystate Franklin Medical Center, Tufts University School of Medicine, Springfield, MassachusettsSearch for more papers by this authorLiron Pantanowitz, Corresponding Author Liron Pantanowitz [email protected] Department of Pathology, Baystate Medical Center, Tufts University School of Medicine, Springfield, Massachusetts Department of Pathology, Baystate Franklin Medical Center, Tufts University School of Medicine, Springfield, MassachusettsDepartment of Pathology, Baystate Medical Center, 759 Chestnut Street, Springfield, MA 01199Search for more papers by this author First published: 16 July 2009 https://doi.org/10.1002/ajh.21502Citations: 1 † Conflict of interest: Nothing to report. AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. References 1 McCann SR,Whelan A,Greally J. Intracellular lambda light chain inclusions in chronic lymphocytic leukaemia. Br J Haematol 1978; 38: 367– 371. 2 Ralfkiaer E,Hou-Jensen K,Geisler C, et al. Cytoplasmic inclusions in lymphocytes of chronic lymphocytic leukaemia. A report of 10 cases. Virchows Arch A Pathol Anat Histol 1982; 395: 227– 236. 3 Berrebi A,Talmor M,Vorst E, et al. IgM lambda globular cytoplasmic inclusions in chronic lymphocytic leukaemia resembling immunocytoma. Scand J Haematol 1983; 30: 43– 49. 4 Dorion RP,Shaw JH. Intracytoplasmic filamentous inclusions in the peripheral blood of a patient with chronic lymphocytic leukemia. A bright-field, electron microscopic, immunofluorescent, and flow cytometric study. Arch Pathol Lab Med 2003; 127: 618– 620. 5 Djaldetti M,Eisbruch A,Perek J,Hart J. Cytoplasmic inclusions and X-ray microprobe analysis in a case of prolymphocytic leukemia. J Cancer Res Clin Oncol 1984; 107: 229– 232. Citing Literature Volume85, Issue4April 2010Pages 275-276 ReferencesRelatedInformation

Altered biological properties of cell membranes in Escherichia coli dnaA and seqA mutants.

We present evidence that biological properties of cell membranes are altered in dnaA and seqA mutants of Escherichia coli relative to wild-type bacteria. We found that bacteriophage lambda forms extremely large plaques on the dnaA seqA double mutants. On the single mutants, dnaA and seqA, the plaques are also bigger than those formed on the wild-type host. However, no significant differences in intracellular phage lambda development were observed between wild-type and mutant hosts, indicating that differences in burst size do not account for the observed differences in plaque size. On the other hand, more efficient release of the phage lytic proteins and/or higher sensitivity of the cell membranes to these proteins may result in more efficient cell lysis. We found that the efficiency of adsorption of bacteriophage lambda to the dnaA seqA mutant cells is decreased at 0 degrees C , but not at 30 degrees C, relative to the wild-type strain. A considerable increase in the permeability of membranes of the mutant cells for beta-galactosidase is demonstrated. The dnaA and seqA mutants are more sensitive to ethanol (an organic solvent) than wild-type bacteria, and the seqA strain and the double mutant dnaA seqA are very sensitive to deoxycholate (a detergent). We conclude that lesions in the genes dnaA and seqA result in alterations in cell membranes, such that the permeability and possibly also other properties of the membranes are significantly altered relative to wild-type bacteria.

Role of R loops in recA-independent homologous recombination of bacteriophage lambda.

We have previously shown that the DNA-dependent RNA polymerase of Escherichia coli can promote homologous recombination of bacteriophage lambda independently of the recA function. To detect this recombination, we jointly infected the cells with a pair of lambda phages in the presence of chloramphenicol, extracted intracellular lambda DNA molecules, packaged them in vitro, and measured the number of resulting recombinant phage particles. We showed that the recombination of DNA molecules takes place in vitro after extraction of DNA from the cells. We fractionated recombinogenic forms of intracellular lambda DNA and showed that they carry RNA. These and other results suggest that the R (RNA) loop structures, generated by transcription within the cells, promote homologous recombination in vitro. We discuss possible mechanisms of this recombination and compare those with the other forms of general recombination.

Physical evidence for the temporal transition of transcription in bacteriophage lambda.

A high proportion of intracellular lambda DNA molecules are found to have D-loops, when isolated under four different conditions: (1) lambda Ots after 7 min at 31 degrees C in the presence of chloramphenicol; (2) lambda Ots after 7 min at 31 degrees C without chloramphenicol; (3) lambda Ots after 30 min at 42 degrees C; and (4) lambda cIIcIII after 50 min at 37 degrees C. The great majority of these D-loops contain RNA and are produced by E. coli RNA polymerase. In the presence of chloramphenicol, D-loops are mostly limited to the immediate early regions of the major leftward and rightward operons. At early times, with no chloramphenicol present, D-loops map primarily within the delayed early regions of the two major operons. At late times, D-loops are found mostly within the major late operon of the bacteriophage DNA. This physical evidence corroborates evidence of the temporal transition in lambda transcription obtained by other means. Chloramphenicol is shown to block the transition from immediate early to delayed early transcription.

Shutoff of lambda gene expression by bacteriophage T4: role of the T4 alc gene.

Bacteriophage T4 normally contains 5-hydroxymethylcytosine instead of cytosine in its DNA. Multiple mutants of T4 which synthesize DNA with cytosine do not transcribe their late genes due to the action of the T4 alc gene (Snyder et al., Proc. Natl. Acad. Sci. U.S.A. 73:3098--3102, 1976), which is also responsible for unfolding the host nucleoid after T4 infection (Sirotkin et al., Nature [London] 265:28--32, 1977; Tigges et al., J. Virol. 24:775--785, 1977). It seems reasonable that T4 alc function plays a role in shutting off host transcription, and the observation that some of the RNA made after infection with a T4 alc mutant hybridizes to Escherichia coli DNA (Sirotkin et al., Nature [London] 265:28--32, 1977; Tigges et al., J. Virol. 24:775--785, 1977) supports this hypothesis. Although it is likely that the roles of the alc function in the blocking of some types of transcription and in the unfolding of the host nucleoid are related, it is not known how these effects are achieved or, in fact, whether all types of transcription are affected equally by the alc function. In an attempt to answer these questions, we studied the effect of T4 alc function on bacteriophage lambda transcription and on the structure of intracellular lambda DNA. We found that the alc function is responsible for the shutoff of lambda late transcription but probably not for the shutoff of lambda early transcription. We also found that alc does not block lambda transcription by directly removing the supercoils from circular lambda DNA via either a nicking or topoisomerase activity. Furthermore, we conclude that T4 infection also prevents the translation of non-T4 mRNA because late lambda mRNA's were made after superinfection by a T4 alcs mutant and were of normal length but were not translated into lambda late proteins.

Evidence of RNA in D loops of intracellular lambda DNA.

If lambda DNA replication is blocked by mutation in any one of several genes essential for replication, intracellular lambda DNA often shows short three-stranded regions called D loops. In this report we show that one arm of a D loop is an RNA . DNA hybrid, whereas the remaining arm is made up of single-stranded DNA. The RNA can be partially removed by RNase A and totally removed by RNase H. Also, D loops do not appear if infections are made in cells treated with rifampin, a potent inhibitor of transcription by Escherichia coli RNA polymerase. Several genes associated with recombination, including the host recA gene, are not essential for D-loop formation.

Antagonists of DNA gyrase inhibit repair and recombination of UV-irradiated phage lambda.

Intracellular lambda DNA (from EDTA-sensitive tandem duplication phages) was extracted from infected rec+ bacteria and scored for infectivity and recombination (loss of duplication) by transfection of recA recB spheroplasts and subsequent assay for EDTA resistance. When phage development was blocked by repressor or by antibiotics (chloramphenicol and/or rifampin), the apparent recombination frequency was about 0.1% above the background value for recA infections. Prior irradiation of the phage greatly stimulated recombination; the frequency was 20% when UV fluence was 140 J/m2. Repair (recovery of infectivity) and recombination of irradiated phage DNA proceeded readily in the presence of chloramphenicol and rifampin. Inhibitors of DNA gyrase (coumermycin and oxolinic acid) blocked repair and reduced recombination. UV-stimulated recombination was very low in recA but nearly normal in recB cells: repair was reduced in both mutant strains. The recombination remained high as phage/cell ratios less than unity.

Visualization of a novel junction in bacteriophage lambda DNA.

At early times after infection of a recA derivative of Escherichia coli with lambdab221c126red270a42 phage, a low but significant proportion of intracellular lambda molecules show a novel junction. These junctions are also present, although in reduced numbers, in a lysate obtained at late times after infection of a recA+ host with lambdacIIcIII phage. Fine structure and denaturation mapping analyses showed that these junctions occur at homologous positions and that they are compatible with the occurrence of a cross-strand exchange between lambda DNA duplexes similar to the type proposed in most molecular models for genetic recombination. However, the results are also consistent with the structures expected if a replicating growing point undergoes branch migration.

Electron microscopy of DNA-containing plasms: IV. Glutaraldehyde—uranyl acetate fixation of virus-infected bacteria for thin sectioning

Fixation of bacteria and bacteria containing developing intracellular phage with a mixture of glutaraldehyde (2–5%) and uranyl acetate 0.1% gives sections which are equally as good as the conventional fixation in OsO 4 according to Ryter and Kellenberger. The results further suggest that the fixation is more efficient for structures susceptible to lose easily their DNA content. For instance, intracellular lambda is now shown in numbers compatible with physiological data. The shape of the nuclear area of infected bacteria is, however, not well preserved.

Concatemers in DNA replication: Electron microscopic studies of partially denatured intracellular lambda DNA

We have verified, by identification of individual molecules in the electron microscope, that λ DNA concatemers are long linear molecules containing repeats of the mature phage DNA sequence. The molecules are not made up of whole multiples of the length of mature λ DNA and do not seem to contain specific start or end points. The concatemers, comprising about 20% of the molecular forms extracted late in infection, can be found in the absence of genetic recombination and in the presence or absence of maturation defects. It may be concluded that concatemers are normal intermediates in the late stage of λ replication. A second form of λ DNA, comprising from 40 to 60% of the late intracellular molecules and having the same length as DNA from mature phages, has been observed. This form seems to contain circular permutations of the mature sequence, as though derived by random breakage of circular DNA molecules. We propose that both molecular forms could be generated from a rolling circle (earlier proposed for late replication) which has fallen apart at its growing point. Evidence from sucrose-gradient sedimentation analyses supports the view that some intracellular λ molecules were preferentially broken during purification.

The effect of superinfection by T4r+ coliphage on the deoxyribonucleeases of induced Escherichia coli Y10.

In studying the Escherichia coli Y10 (λ)-T4r+ coliphage system, it was found that T4 excluded intracellular lambda, or lambda excluded T4, depending upon the experimental conditions. Either event was correlated with a significant increase in the specific activity of free intracellular DNase (deoxyribonuclease). During the first 20 minutes after induction of lambda by ultraviolet light, there was no increase in DNase specific activity and the cells responded to T4 as if they were non-induced. Adsorption of T4, or of T4 inactivated by ultraviolet light, led to a rapid increase in free intracellular DNase, the arrest of lambda replication, and infection of the cells by T4. From 20 to 60 minutes after induction there was a progressive increase in free intracellular DNase, accompanied by an increasing ability of the cells to resist T4 infection.The RNase (ribonuclease) treatment of all cellular extracts, whether from normal cells or from cells infected with T4 or lambda, led to a considerable enhancement of DNase activity. This treatment showed that an increase in total DNase activity occurred by 40 to 60 minutes after lambda induction, but failed to reveal any such increase in total DNase activity resulting from successful T4 superinfection.

Purification and properties of intracellular lamba DNA rings

Intracellular lambda DNA has been purified from bacteria infected with lambda phage. The prediction from sedimentation properties that one intracellular form of lambda DNA is a twisted, closed-circular molecule has been confirmed by observation in the electron microscope. Two unique forms of lambda DNA are present in infected cells: twisted, closed-circular molecules (component I) and open-circular molecules (component II). One single-strand scission is sufficient to convert component I to component II. Both components I and II, either native or denatured, are able to infect spheroplasts. The infective entity present after denaturation of component I is a molecule in which strand separation has not occurred. That present after denaturation of component II is a single-strand ring; single-stranded linear molecules are not infective. The ionic strength dependence of the sedimentation rate of components I and II and phage lambda DNA has been studied. For comparative purposes, similar studies have been made with φX174 replicative form. A new consequence of DNA circularity is evident from these studies.

The exclusion of intracellular lambda from Escherichia coli Y10 (lambda) by T4r+ coliphage.

The exclusion of lambda development in induced Y10 (λ) by infection with T4r+ was examined. The slow rate of adsorption of phage to cells was improved by propagation of the bacteria in nutrient broth containing glucose. Under conditions of satisfactory adsorption, it was found that T4 and ultraviolet irradiated T4 nearly completely excluded lambda when added to induced cells as late as 30 minutes after induction. Less than 2% of the induced cells were refractory to T4 infection. By 50 minutes after induction T4 still excluded lambda from 50% of the cells, but by 70 minutes most of the cells were committed to the production of lambda.