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At least 19 recordsLinked to original sources
PubMed1978

SPP1 DNA replicative forms: growth of phage SPP1 in Bacillus subtilis mutants temperature-sensitive in DNA synthesis.

The development of bacteriophages SPP1 and phi 29 has been studied in several B. sutilis mutants defective in host DNA replication, under non permissive conditions. Several gene products, involved in the synthesis of host DNA, are required for phi 29 replication, while SPP1 seems to require only the host DNA polymerase III. In addition both phages are unable to grow in a dna A mutant (ribonucleotide reductase). Taking advantage of the fact that SPP1 DNA is actively replicated in several dna mutants at non-permissive temperature, we have studied the structure of the replicative intermediates of this phage in the absence of interfering host DNA synthesis. Fast sedimenting forms of SPP1 DNA can be isolated from phage infected cells and evidence of covalently joined concatemers has been obtained, suggesting the presence of terminally repeated sequences.

Bacillus subtilis↗
PubMed1979

Bacteriophage SPP1 polypeptides synthesized in infected minicells and in vitro.

Minicells produced by B. subtilis CU403divIVB1 and infected by SPP1 synthesize at least 46 polypeptides which can be separated by polyacrylamide gel electrophoresis. These polypeptides represent the expression of 86% of the SPP1 genome's coding capacity. Infection of minicells by sus mutants and deletion mutants of SPP1 has permitted a correlation of genetic location with gene product and has shown that SPP1 normally synthesizes at least 8 non-essential polypeptides. Restriction fragments of SPP1 produced by EcoRI digestion of SPP1 DNA have been purified and used as template DNA in a coupled transcription/translation system derived from E. coli to determine the polypeptides encoded by the individual fragments. SPP1 expression in minicells differs from SPP1 expression in nucleated cells (Esche, 1975) in that late syntheses are not dependent on phage DNA replication in infected minicells.

Bacillus subtilis↗
PubMed1975

Gene expression of bacteriophage SPP1. II. Regulatory aspects.

The expression of late SPP1 genes depends on preceding SPP1 DNA replication. This is shown in nonpermissive infection with a mutant defective in DNA replication and after inhibition of DNA synthesis by HPUra. The potential for host gene expression is not significantly influenced by SPP1 infection, as evidenced by the continuation of host protein synthesis and the inducibility of glycerolphosphate dehydrogenase after infection. The involvement of a positive control element in the regulation of SPP1 gene expression is deduced from the observation that chloramphenicol prevents the synthesis of the only class of mRNA which is transcribed from the L-strand.

Bacillus subtilis↗
PubMed1979

The genome of Bacillus subtilis phage SPP1: the arrangement of restriction endonuclease generated fragments.

SPP1 DNA was cleaved by the restriction endonucleases, BglI, BglII, EcoRI, KpnI, SmaI, and SalI. The molecular weights of the DNA fragments obtained by single enzyme digestion or by consecutive digestion with two enzymes were determined by electron microscopic measurements of contour length and by gel electrophoresis. The major fragments from the six digests could be ordered to give a consistent restriction map of SPP1. The electropherograms of several digests indicated that certain fragments occurred in less than stoichiometric amounts or were heterogeneous in size. Such bands carried a major part of radioactivity, when SPP1 DNA was terminally labelled with P32 prior to degradation by restriction enzymes. These results, and studies of the effect of exonuclease III treatment on restriction enzyme patterns define the terminal restriction fragments. All data obtained support the conclusion drawn in the preceding paper (Morelli et al., 1978 b) that the SPP1 genome is terminally redundant and partially circularly permuted.

Bacillus subtilis↗
PubMed2026

Contact hypersensitivity promotes hair regeneration through SPP1-secreting macrophages.

Allergic contact dermatitis, or contact hypersensitivity (CHS), is a pathological adaptive immune response that paradoxically induces hair regeneration, yet its underlying mechanisms remain unclear. We integrated high-resolution spatial transcriptomics and single-cell RNA sequencing to map the intricate interactions between immune cells, stroma, and hair follicles during CHS-induced hair growth in mice. Among all immunocytes, macrophages underwent the most prominent compositional and functional remodeling. We resolved five transcriptionally distinct macrophage subsets, with contact hypersensitivity driving a shift from homeostatic antigen-presenting cells toward a pro-inflammatory CD14+SPP1+ population. Trajectory analysis revealed divergent differentiation paths under homeostatic versus allergic conditions, highlighting the plasticity of skin macrophages. Mechanistically, CD14+SPP1+ macrophages secreted SPP1 (osteopontin), which engaged CD44 on hair follicle stem cells to activate PI3K-AKT signaling and trigger their proliferation. Notably, canonical pro-inflammatory cytokine signaling through TNF-α and IL-1 was dispensable for this process, underscoring the specificity of the SPP1-CD44 axis in immune-mediated hair regeneration. These findings reveal a macrophage-dependent mechanism of immune-mediated hair regeneration, offering therapeutic insights into immune-stem cell crosstalk.

Journal Article↗
PubMed1978

Studies on transduction process by SPP1 phage.

The conditions for optimal transduction efficiency of the Bacillus subtilis phage SPP1 have been investigated. By irradiating transducing lysates with u.v. light we have been able to obtain a fivefold increase in the number of transductants and to reduce strongly the interference caused by infective particles. Any dependence of SPP1 transduction on PBSX induction has been ruled out by the use of xin mutants, which are unable to induce the defective phage. SPP1 mediated transduction is susceptible to the restriction and modification system of B. subtilis. The rec functions involved in the recombination of the SPP1 transduced DNA fragment are probably identical to those required in DNA transformation and heterologous PBS1 transduction.

Bacillus subtilis↗
PubMed1978

Restriction fragment analysis of bacteriophage SPP1 in vitro transcription by host RNA polymerase.

In vitro transcription of SPP1 DNA occurred on only one of the two strands, the same which is predominantly transcribed in SPP1-infected cells. Transcripts were distributed in several size classes. Analysis of elongation kinetics and of size distribution, coupled with hybridization to DNA restriction fragments, showed that some regions of the template have more initiation sites than others; some have none. Some regions were transcribed directly, some were transcribed from initiation sites located in other regions, and one was never transcribed. Several transcription initiation sites on SPP1 DNA are located on EcoRI fragment 1; four to five others are distributed among other fragments. Cutting the DNA with EcoRI did not introduce artifactual initiation sites. In vitro transcription units can be localized and oriented with respect to the EcoRI restriction map of SPP1 DNA.

Bacillus subtilis↗
PubMed1979

The genome of B. subtilis phage SPP1: physical arrangement in phage genes.

41 genes of SPP1 have been delineated by using complementation analyses of 75 conditionally lethal (ts and sus) mutations. The physical locations of these genes on the SPP1 chromosome have been determined by transfection/marker rescue experiments in which restriction endonuclease generated fragments of SPP1 DNA were used as donor DNA. The physical order of these fragments has been previously established (Ratcliff et al., 1979).

Bacillus subtilis↗
PubMed1978

DNA of Bacillus subtilis bacteriophage SPP1: physical mapping and localization of the origin of replication.

The genome of Bacillus subtilis bacteriophage SPP1, a linear, 28.5-megadalton DNA duplex, was mapped by analysis with the restriction endonucleases endo R.Sal I, Sma I, Xba I, Bgl I, Bgl II, and EcoRI. The SPP1 genome, like that of the Salmonella typhimurium phage, P22, was found to be a terminally repetitious, circularly permuted molecule. 6-(p-Hydroxyphenylazo)uracil, a selective, reversible inhibitor of SPP1 DNA synthesis, was exploited to synchronize the initiation of genome replication and to selectively label the site of its initiation with radioactive thymidine. Restriction endonuclease analysis of the distribution of the label located the origin of replicative synthesis at an area approximately 0.2 genome length from one molecular terminus.

Bacillus subtilis↗
PubMed1978

Specific labelling of replicating SPP1 DNA: analysis of viral DNA synthesis and identification of phage DNA-genes.

Specific labelling of replicating bacteriophage SPP1 DNA can be achieved by infection at nonpermissive temperature of a B. subtilis strain carrying the initation mutation dnaB ts134. Under these conditions host DNA synthesis is reduced by 90 to 95%. This technique was used to identify cistrons of SPP1 involved in phage DNA synthesis and to define intermediates in SPP1 replication.

Bacillus subtilis↗
PubMed1975

Transfection with heteroduplex SPP1 DNA: a pyrimidine dimer induced influence on the conversion pattern.

Bacillus subtilis competent cells were transfected with SPP1 heteroduplices having pyrimidine dimers in one of the strands. The data obtained reveal that excision repair of the pyrimidine dimers influences the ratio of wild type versus mutant progeny observed in "normal" heteroduplex transfection. With increased exposure of one strand to UV dose the percentage of infective centers having the unirradiated strand genotype shows an increase. A comparison of the transfection data in her+ and her- host excludes asymmetric replication as the cause of the observed changes in the conversion pattern. The data can be explained on the basis of a dimer induced co-excision of the mismatched region. In addition transfection data from wild type/deletion mutant heteroduplices where the strand of mutant origin was irradiated exclude the possibility of the wild type loop being excised during uptake.

Bacillus subtilis↗
PubMed2025

Multiomics Analysis Reveals Therapeutic Targets for Chronic Kidney Disease With Sarcopenia.

BACKGROUND: The presence of sarcopenia in patients with chronic kidney disease (CKD) is associated with poor prognosis. The mechanism underlying CKD-induced muscle wasting has not yet been fully explored. This study investigates the influence of renal secretions on muscles using multiomics sequencing. METHODS: The kidney transcriptome analysis by RNA-seq and protein profiling by tandem mass tag (TMT), serum TMT and muscle TMT were performed in CKD established using 0.2% adenine and control mice. Spp1 recombinant protein was used to study its effect on myotube atrophy in&#xa0;vitro. In animal experiments on CKD, pharmacological inhibition of Spp1 was used to explore the role of Spp1 in skeletal muscle wasting. Transcriptome analysis was performed to identify differentially expressed genes (DEGs) in the gastrocnemius muscle following Spp1 pharmacological inhibition. RESULTS: In the renal transcriptome and TMT, 503 and 377 proteins/genes respectively were co-upregulated and co-downregulated. In the serum TMT of CKD and normal control (NC) mice, 22 upregulated and 7 downregulated differentially expressed proteins (DEPs) showed the same expression patterns as those in the kidney transcriptome and TMT analysis. Based on bioinformatics analysis and reported studies, we selected Spp1 for further validation. Spp1 recombinant protein was added to C2C12 myotubes in&#xa0;vitro, and the results indicated that Spp1 significantly increased the protein levels of the muscle atrophy marker (Murf-1) and promoted the smaller myotubes (all p&#x2009;<&#x2009;0.05). Compared with NC mice, Spp1 mRNA and protein levels were significantly upregulated in the kidneys of CKD mice, and the serum concentration of Spp1 was also markedly increased (all p&#x2009;<&#x2009;0.05). In animal experiments, pharmacological inhibition of Spp1 increased the weights of gastrocnemius and tibialis anterior muscles (p&#x2009;<&#x2009;0.05) and improved muscle atrophy phenotype. Transcriptome analysis showed that DEGs in the gastrocnemius muscle following Spp1 pharmacological inhibition were enriched in protein digestion and absorption, glucagon signalling pathway, apelin signalling pathway and ECM-receptor interaction pathway. CONCLUSIONS: Our study is the first to establish a regulatory network of kidney-muscle crosstalk to explore the potential mechanism of CKD-related sarcopenia. Employing multiomics analysis, cellular assessment and animal experiments, we have identified that Spp1 could potentialy serve as a promising therapeutic target for CKD patients with sarcopenia.

Sarcopenia↗
PubMed2026

Generation and validation of a Myh11Dre-Spp1Cre intersectional mouse model for lineage tracing of disease-associated smooth muscle cell states.

BACKGROUND: Phenotypic modulation of vascular smooth muscle cells (VSMCs) is a hallmark of vascular remodeling and cardiovascular disease. Recent lineage-tracing and single-cell transcriptomic studies have identified secreted phosphoprotein 1 (SPP1) as a prominent marker associated with disease-associated VSMC states, particularly those linked to fibrotic remodeling and vascular calcification. However, the cellular origins and fate of SPP1-associated VSMC populations remain incompletely understood. METHODS AND RESULTS: We generated a novel Spp1-rSTOPr-Cre (Spp1Cre) knock-in mouse line in which Cre recombinase is expressed from the endogenous Spp1 locus following Dre-mediated excision of a rox-flanked transcriptional STOP cassette. Correct targeting of the knock-in allele was validated by internal, 5' junction, 3' junction, and long-range PCR analyses, as well as Sanger sequencing. To establish an intersectional lineage-tracing strategy, Spp1Cre mice were crossed with Myh11DreERT2 and Rosa26-RSR-LSL-tdTomato-LSL-eGFP reporter mice, enabling permanent labeling of VSMC-derived populations following activation of the endogenous Spp1 locus. Under physiological conditions, eGFP-positive cells were detected at low frequency within the vascular wall and were predominantly negative for the contractile markers ACTA2 and MYH11. As a proof-of-principle application, eGFP-positive cells markedly expanded within atherosclerotic lesions induced by AAV-PCSK9D377Y and high-fat diet feeding. These lineage-traced cells remained largely ACTA2- and MYH11-negative, consistent with a modulated phenotype. Notably, only a minority of eGFP-positive cells expressed SPP1 or fibronectin at the time of analysis, demonstrating the utility of permanent lineage tracing for tracking cells with a history of endogenous Spp1 activation during vascular remodeling. CONCLUSION: We report the generation and validation of a novel Myh11Dre-Spp1Cre intersectional mouse model for lineage tracing of VSMC-derived populations that have activated the endogenous Spp1 locus. This genetic resource provides a valuable platform for investigating the origin, fate, and phenotypic evolution of Spp1-associated VSMC populations during vascular remodeling and cardiovascular disease.

Animals↗
PubMed1975

Gene expression of bacteriophage SPPI. I. Phage directed protein synthesis.

A total of 23 phage specific proteins (including four head and six tail proteins) could be identified after SDS polyacrylamide gel electrophoresis of extracts from phage SPP1 infected Bacillus subtilis cells. The total molecular weight of the proteins amounts to approximately 1.9 X 10(6) daltons, equivalent to the majority of the coding capacity of SPP1 DNA. It can thus be assumed that almost all SPP1 coded proteins have been identified. Protein assignments to phage cistrons were made by analysis of extracts from nonpermissive cells infected with sus-mutants. The SPP1 specified proteins can be subdivided into three groups on the basis of the time of their synthesis during the latent period. Host protein synthesis is not significantly affected by SPP1 infection. Normal expression of host genes appears to be essential for SPP1 growth.

Bacillus subtilis↗