We have cloned and sequenced the promoter-proximal region of the Bacillus subtilis operon containing the pbpB gene, encoding essential penicillin-binding protein PBP2B. The first two genes in the operon, designated yllB and yllC, are significantly similar to genes of unknown function similarly positioned upstream of pbpB in Escherichia coli. Both B. subtilis genes are shown to be nonessential. The third B. subtilis gene, yllD, is essential, as is the correspondingly positioned ftsL gene of E. coli. The predicted product of yllD is similar to FtsL in size and distribution of charged residues but is not significantly related in primary amino acid sequence. The major promoter for the cluster lies upstream of the first gene, yllB, but at least one minor promoter lies within the yllC gene. The operon is transcribed throughout growth at a low level.Vegetative cells of Bacillus subtilis, like those of most other rod-shaped bacteria, grow by lengthwise extention and divide by ingrowth of a centrally positioned septum. A number of genes involved in formation of the septum have been identified, mainly by the isolation and characterization of temperature-sensitive mutants that form nonseptate filaments at the nonpermissive temperature (5). In Escherichia coli several of these fts genes are clustered in the 2-min region of the chromosome, along with many other genes involved in division and cell wall synthesis (DCW) (26). Apparent homologs of most genes in the cluster are grouped together on the B. subtilis chromosome. Alignment of the two DCW regions reveals that the linear order of genes, over 25 kbp or so, is generally conserved, although some open reading frames (ORFs) occur in only one of the organisms (8).At the upstream margin of the known sequence in B. subtilis, a likely homolog of the E. coli pbpB (ftsI) gene was found (25). In E. coli, this gene encodes the penicillin-binding protein PBP3, which is essential for synthesis of septal peptidoglycan (19). In B. subtilis the equivalent gene is duplicated. The downstream copy, called spoVD, encodes a protein specialized for synthesis of modified cell wall material that forms the spore cortex (3). The upstream copy was previously shown to encode PBP2B, which seems to be the functional homolog of PBP3 (4, 25). Here we extend the sequence of the B. subtilis DCW cluster upstream of pbpB and describe the remainder of the pbpB operon. The operon contains four genes, which we argue are probably all functionally homologous to equivalently positioned genes in E. coli. The third gene in the operon is shown to be essential for cell viability and therefore is probably functionally equivalent to the E. coli ftsL (mraR) gene (12, 24), even though it shows negligible primary sequence conservation. The first two genes in the operon, which are very similar to the equivalent (but uncharacterized) genes in E. coli, are shown to be dispensable in B. subtilis. MATERIALS AND METHODSBacterial strains. The bacterial strains, phages, and plasmids used in this study are listed in Table 1. The i...
The Saccharomyces cerevisiae high mobility group protein HMO1 has two DNA binding domains, box A and box B, and a lysine-rich C-terminal extension. Among other functions, HMO1 has been implicated as a component of the RNA polymerase I transcription machinery. We report here that HMO1 promotes DNA apposition as evidenced by its stimulation of end-joining in the presence of T4 DNA ligase. Analysis of truncated HMO1 variants shows that enhanced DNA end-joining requires the C-terminal domain but that box A is dispensable. The efficiency of joining DNA ends with different nucleotide content parallels that of DNA ligase, and optimal ligation efficiency is attained when DNA is effectively saturated with protein, implying that HMO1 binds internal sites in preference to DNA ends. Removal of the C-terminal tail does not attenuate the self-association characteristic of HMO1 but alters the stoichiometry of binding and prevents intramolecular DNA cyclization. This suggests that the C-terminal domain mediates an accretion of HMO1 on DNA that causes in-phase DNA bending and that binding of HMO1 lacking the C-terminal domain results in out-of-phase bending. Taken together, our results show that HMO1 shares with mammalian HMGB proteins the ability to promote DNA association. Notably, the C-terminal domain mediates both DNA end-joining and an accretion of multiple HMO1 protomers on duplex DNA that produces in-phase DNA bending. This mode of binding is reminiscent of that proposed for the mammalian RNA polymerase I transcription factor UBF.
The reaction of MCl 2 (M = Pd, Pt) with one mole-equivalent ofconfirmed the complexes to be monomeric with square-planar geometry about the metal centre. Variable-temperature NMR spectroscopy showed that the complexes undergo fluxional processes in solution, which
The activation induced cytosine deaminase (AID) mediates diversification of B cell immunoglobulin genes by the three distinct yet related processes of somatic hypermutation (SHM), class switch recombination (CSR), and gene conversion (GCV). SHM occurs in germinal center B cells, and the transcription factor Bcl6 is a key regulator of the germinal center B cell gene expression program, including expression of AID. To test the hypothesis that Bcl6 function is important for the process of SHM, we compared WT chicken DT40 B cells, which constitutively perform SHM/GCV, to their Bcl6-deficient counterparts. We found that Bcl6-deficient DT40 cells were unable to perform SHM and GCV despite enforced high level expression of AID and substantial levels of AID in the nucleus of the cells. To gain mechanistic insight into the GCV/SHM dependency on Bcl6, transcriptional features of a highly expressed SHM target gene were analyzed in Bcl6-sufficient and -deficient DT40 cells. No defect was observed in the accumulation of single stranded DNA in the target gene as a result of Bcl6 deficiency. In contrast, association of Spt5, an RNA polymerase II (Pol II) and AID binding factor, was strongly reduced at the target gene body relative to the transcription start site in Bcl6-deficient cells as compared to WT cells. However, partial reconstitution of Bcl6 function substantially reconstituted Spt5 association with the target gene body but did not restore detectable SHM. Our observations suggest that in the absence of Bcl6, Spt5 fails to associate efficiently with Pol II at SHM targets, perhaps precluding robust AID action on the SHM target DNA. Our data also suggest, however, that Spt5 binding is not sufficient for SHM of a target gene even in DT40 cells with strong expression of AID.
Allogeneic off-the-shelf cell therapies offer distinct advantages over conventional autologous cell therapies in terms of scaled manufacturing, on-demand availability and optimization of cellular starting material. A unique consideration in the use of allogeneic cell therapies is the potential for immune cell-mediated recognition of the allogeneic cell product by the patient's immune system. CAR T-cell therapies are commonly combined with conditioning chemotherapies that suppress a patient's immune system, creating a suitable window of activity to elicit clinical response. However, protracted lympho-conditioning also affects immune reconstitution and can negatively impact the rate of infection. Alternative approaches to prevent allorejection may therefore help to enhance the efficacy of the therapy while preserving the immune system of the patient. Elimination of cell-surface human leukocyte antigen (HLA) molecule expression by genetic knockout (KO) has long been known to abrogate T-cell reactivity. However, loss of class I HLA elicits NK cell-mediated recognition and clearance, and therefore must be combined with other immune-modulating strategies to limit host NK cell reactivity. Allogeneic models combining class I HLA deletion with NK cell inhibitory molecules, such as HLA-E and CD47, have been shown to abrogate NK cell reactivity in mouse models. However, HLA-E is the canonical activator of NKG2C, a dominant activating receptor found on human NK cells. Likewise, the expression of signal regulatory protein alpha (SIRPα), the major interactor for CD47, is mostly restricted to macrophages and dendritic cells and not human NK cells, and the observed effects of this immune-modulating strategy in the mouse system may only offer partial or incomplete immune evasion in the human system. In this study, we provide details of a bona fide off-the-shelf strategy where iPSC-derived NK (iNK) cell therapy is multiplexed engineered with a novel combination of immune-evasion modalities; beta 2 microgobulin (B2M) KO to prevent CD8 T-cell mediated rejection; class II transactivator (CIITA) KO to prevent CD4 T-cell mediated rejection; and CD38 KO to enable combination with anti-CD38 mAbs, which can be administered to deplete host alloreactive lymphocytes, including both NK and T cells. In vitro mixed lymphocyte reaction (MLR) data demonstrated that upon co-culture with allogeneic PBMCs, B2M KO iNK cells stimulated less T-cell activation than their B2M sufficient counterparts as evidenced by reduced CD38, 41BB, and CD25 levels on T cells. Additionally, B2M KO iNK cells impaired T-cell expansion over the duration of co-culture, resulting in a 50% decrease in expansion at the peak of the control response. However, B2M KO iNK cells were depleted over time, suggesting activation of an NK cell "missing self" response by the peripheral blood NK (pbNK) cells. In contrast, when the assay was performed in the presence of anti-CD38 mAb, depletion of B2M KO iNK cells was blocked, and instead B2M KO iNK cell numbers increased by 3.5-fold, comparable to the iNK cell numbers found in the control arm (cultured without allogeneic PBMCs). Interestingly, pbNK cell numbers decreased, while T-cell activation and expansion remained lower than in B2M-sufficient MLR cultures. Furthermore, when B2M KO iNK cells were cocultured with tumor cells and anti-CD38 mAb in vitro, ADCC was comparable to the B2M sufficient cells, indicating uncompromised effector function. Finally, in vivo studies suggested that co-administration of anti-CD38 mAbs can significantly enhance the persistence of B2M KO iNK cells in the presence of allogeneic pbNK cells as seen in the spleen and bone marrow (Figure 1). Together these data demonstrate that the combination of triple-gene knockout of CD38, B2M and CIITA with a CD38-targeting mAb is an effective strategy to avoid host immune rejection, and highlights the potential advantages of multiplexed engineered iPSCs to facilitate large-scale manufacture of complex engineered, off-the-shelf cellular therapies. Figure 1 Figure 1. Disclosures Williams: Fate Therapeutics: Current Employment. Malmberg: Merck: Research Funding; Vycellix: Consultancy; Fate Therapeutics: Consultancy, Research Funding. Lee: Fate Therapeutics, Inc.: Current Employment. Bjordahl: Fate Therapeutics: Current Employment. Valamehr: Fate Therapeutics, Inc.: Current Employment.
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