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PubMed2002

Differential requirements for COPI coats in formation of replication complexes among three genera of Picornaviridae.

Picornavirus RNA replication requires the formation of replication complexes (RCs) consisting of virus-induced vesicles associated with viral nonstructural proteins and RNA. Brefeldin A (BFA) has been shown to strongly inhibit RNA replication of poliovirus but not of encephalomyocarditis virus (EMCV). Here, we demonstrate that the replication of parechovirus 1 (ParV1) is partly resistant to BFA, whereas echovirus 11 (EV11) replication is strongly inhibited. Since BFA inhibits COPI-dependent steps in endoplasmic reticulum (ER)-Golgi transport, we tested a hypothesis that different picornaviruses may have differential requirements for COPI in the formation of their RCs. Using immunofluorescence and cryo-immunoelectron microscopy we examined the association of a COPI component, beta-COP, with the RCs of EMCV, ParV1, and EV11. EMCV RCs did not contain beta-COP. In contrast, beta-COP appeared to be specifically distributed to the RCs of EV11. In ParV1-infected cells beta-COP was largely dispersed throughout the cytoplasm, with some being present in the RCs. These results suggest that there are differences in the involvement of COPI in the formation of the RCs of various picornaviruses, corresponding to their differential sensitivity to BFA. EMCV RCs are likely to be formed immediately after vesicle budding from the ER, prior to COPI association with membranes. ParV1 RCs are formed from COPI-containing membranes but COPI is unlikely to be directly involved in their formation, whereas formation of EV11 RCs appears to be dependent on COPI association with membranes.

Animals↗

PubMed2002

Evidence that there are two copies of subunit c" in V0 complexes in the vacuolar H+-ATPase.

The proton-translocating core of eukaryotic vacuolar H(+)-ATPase (V-ATPase), V(0) consists of a hexameric arrangement of transmembrane alpha-helices formed from the related polypeptides, subunit c and subunit c". The former is comprised of four transmembrane alpha-helices, whilst the latter has an extra transmembrane domain at its N-terminus. In addition, the fungal form of V(0) contains a minor subunit c-related polypeptide, subunit c'. All three are required for activity of the proton pump in Saccharomyces cerevisiae. We have introduced cysteine residues in the N-terminal extension of subunit c" in a cysteine-free form. All mutant forms are active in the V-ATPase from S. cerevisiae. Oxidation of vacuolar membranes containing the cysteine-replaced forms gave a cross-linked product of 42000Da. Analysis of this species showed it to be a dimeric form of subunit c", and further studies confirmed there are two copies of subunit c" in the V-ATPases in which it is present. Co-expression of double cysteine-replaced forms of both subunit c and c" gave rise to only homotypic cross-linked forms. Also, subunit c oligomeric complexes are present in vacuolar membranes in the absence of subunit c", consistent with previous observations showing hexameric arrangements of subunit c in gap-junction-like membranes. In vitro studies showed subunit c" can bind to subunit c and itself. The extent of binding can be increased by removal of the N-terminal domain of subunit c". This domain may therefore function to limit the copy number of subunit c" in V(0). A deletion study shows that the domain is essential for the activity of subunit c". The results can be combined into a model of V(0) which contains two subunit c" protomers with the extra transmembrane domain located toward the central pore. Thus the predicted stoichiometry of V(0) in which subunit c" is present is subunit c(3):subunit c'(1):subunit c"(2). On the basis of the mutational and binding studies, it seems likely that two copies of subunit c" are next to each other.

Cell Membrane↗

PubMed1997

Direct and GTP-dependent interaction of ADP ribosylation factor 1 with coatomer subunit beta.

A site-directed photocrosslink approach was used to elucidate components that interact directly with ADP- ribosylation factor (ARF)-GTP during coat assembly. Two ARF mutants were generated that contain a photolabile amino acid at positions distant to each other within the ARF molecule. Here we show that one of the two positions specifically interacts with coatomer subunit beta both on Golgi membranes and in isolated coat protein complex type I (COPI)-coated vesicles. Thus, a direct and GTP-dependent interaction of coatomer via beta-coat protein complex (COP) with ARF is involved in the coating of COPI-coated vesicles. These data implicate a bivalent interaction of the complex with the donor membrane during vesicle formation.

ADP-Ribosylation Factors↗

PubMed2000

Role of COPI in phagosome maturation.

Phagosomes mature by sequentially fusing with endosomes and lysosomes. Vesicle budding is presumed to occur concomitantly, mediating the retrieval of plasmalemmal components and the regulation of phagosomal size. We analyzed whether fission of vesicles from phagosomes requires COPI, a multimeric complex known to be involved in budding from the Golgi and endosomes. The role of COPI was studied using ldlF cells, that harbor a temperature-sensitive mutation in epsilon-COP, a subunit of the coatomer complex. These cells were made phagocytic toward IgG-opsonized particles by heterologous expression of human FcgammaRIIA receptors. Following incubation at the restrictive temperature, epsilon-COP was degraded in these cells and their Golgi complex dispersed. Nevertheless, phagocytosis persisted for hours in cells devoid of epsilon-COP. Retrieval of transferrin receptors from phagosomes became inefficient in the absence of epsilon-COP, while clearance of the FcgammaRIIA receptors was unaffected. This indicates that fission of vesicles from the phagosomal membrane involves at least two mechanisms, one of which requires intact COPI. Traffic of fluid-phase markers and aggregated IgG-receptor complexes along the endocytic pathway was abnormal in epsilon-COP-deficient cells. In contrast, phagosome fusion with endosomes and lysosomes was unimpaired. Moreover, the resulting phagolysosomes were highly acidic. Similar results were obtained in RAW264.7 macrophages treated with brefeldin A, which precludes COPI assembly by interfering with the activation of adenosine ribosylation factor. These data indicate that neither phagosome formation nor maturation are absolutely dependent on COPI. Our findings imply that phagosomal maturation differs from endosomal progression, which appears to be more dependent on COPI-mediated formation of carrier vesicles.

Animals↗

PubMed1988

The active form of the cytochrome d terminal oxidase complex of Escherichia coli is a heterodimer containing one copy of each of the two subunits.

The cytochrome d complex is a component of the aerobic respiratory system of Escherichia coli. The enzyme functions as a terminal oxidase, oxidizing ubiquinol-8 within the cytoplasmic membrane and reducing oxygen to water. The enzyme is of particular interest because it is a coupling site in the electron transfer chain. The electron transfer reaction catalyzed by this enzyme is coupled to the translocations of protons across the membrane (H+/e-approximately equal to 1). The oxidase contains two subunits by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, with molecular weights of 58,000 and 43,000. In this paper, the question of the quaternary structure is addressed. Quantitative N-terminal analysis of the isolated enzyme and relative mass quantitation following sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicate the subunits are present in equimolar amounts. Sedimentation velocity and sedimentation equilibrium studies were used to characterize the hydrodynamic properties of the purified enzyme solubilized in Triton X-100, under conditions where the enzyme is active. It is concluded that the active enzyme in Triton X-100 is a heterodimer, containing one copy of each subunit. This is likely the structure of the enzyme in the E. coli membrane.

Algorithms↗

PubMed2005

SNARE complexes and neuroexocytosis: how many, how close?

Regulated secretion is an essential process in all eukaryotic cells. The release of molecules contained inside exocytic granules and synaptic vesicles is mediated by the assembly of a SNARE complex formed by the coil-coiling of three proteins: SNAP-25, syntaxin and VAMP/synaptobrevin. It seems that SNARE complexes assemble together in rosette-shaped super-complexes but there is controversy on the actual number (N) of copies of SNARE complexes that are necessary to mediate exocytosis. We discuss attempts to determine the value of N and suggest that N varies with the type of exocytic vesicles. In addition, we propose that the N value in neuroexocytosis can be estimated by the comparative use of different types of botulinum neurotoxins.

Animals↗

PubMed1995

Structural analysis of the p62 complex, an assembly of O-linked glycoproteins that localizes near the central gated channel of the nuclear pore complex.

The p62 complex is an oligomeric assembly of O-linked glycoproteins of the nuclear pore complex that interacts with cytosolic transport factors and is part of the machinery for nuclear protein import. In this study we have purified the p62 complex from rat liver nuclear envelopes and analyzed its structure and composition. The p62 complex consists of four distinct polypeptides (p62, p58, p54, and p45) and has a mass of approximately 234 kDa, calculated from its hydrodynamic properties and supported by chemical cross-linking and scanning transmission electron microscopy. These data suggest that the p62 complex contains one copy of each constituent polypeptide. Analysis of preparations of the p62 complex by electron microscopy using rotary metal shadowing and negative staining revealed donut-shaped particles with a diameter of approximately 15 nm. Immunogold electron microscopy of isolated rat liver nuclear envelopes demonstrated that p62 occurs on both the nucleoplasmic and cytoplasmic sides of the pore complex near the central gated channel involved in active transport of proteins and RNAs. The properties and localization of the p62 complex suggest that it may be involved in binding transport ligands near the center of the nuclear pore complex and in subsequently transferring them to the gated transport channel.

Animals↗

PubMed2001

Methylation of the FSHD syndrome-linked subtelomeric repeat in normal and FSHD cell cultures and tissues.

Facioscapulohumeral muscular dystrophy (FSHD) has an unusual molecular etiology. In a putatively heterochromatic subtelomeric region of each chromosome 4 homologue (4q35), unaffected individuals have 11 to about 95 tandem copies of a complex 3.3-kb repeat (D4Z4). Most FSHD patients have less than 10 copies at one allelic 4q35. This has been proposed to lead to the loss of heterochromatinization and, thereby, inappropriate gene expression by position effects, explaining the dominant nature of FSHD and the role of a decreased number of copies of D4Z4 at 4q35 but not at 10q26. Consistent with the proposed heterochromatinization of this repeat, by Southern blot analysis, we found that SmaI, MluI, SacII, and EagI sites in D4Z4 are highly methylated in normal and FSHD cell lines and somatic tissues, including skeletal muscle. Like repeated DNA sequences in the juxtacentromeric heterochromatin of chromosomes 1, 9, and 16, D4Z4 was hypomethylated at numerous CpGs in sperm and in cell lines from patients with an unrelated DNA methyltransferase deficiency syndrome (ICF; immunodeficiency, centromeric region instability, facial anomalies) in contrast to its hypermethylation in non-ICF postnatal somatic tissues. Our data on FSHD samples suggest that the disease-associated 4q35 D4Z4 repeats, which constitute a small percentage of the total D4Z4 repeats, are not generally hypomethylated relative to the other repeats of this sequence. However, in individuals not affected with FSHD, the hypermethylation of tandem, high-copy-number D4Z4 repeats might help stabilize heterochromatinization at allelic 4q35 regions just as hypermethylation elsewhere in the genome has been linked to chromatin compaction.

Bacterial Proteins↗

PubMed2005

A hypothetical pathway from the RNA to the DNA world.

If the DNA world was preceded by a RNA world as widely suggested a rational pathway should be discernable to link the two. This report uses as a starting point a membrane-enclosed ribozyme capable of polymerising itself and its counterpart copy. As molecular complexity increased, it is suggested that a consortia of the initial ribozyme polymerase and chaperone molecules formed a complex specifically for RNA replication. A mutation in one of several copy-genomes coding for these replication machines then led in step-wise fashion to a proto-ribosome that increasingly inserted specific amino acids instead of nucleotides into a growing RNA chain, the driving force being selection for improved or new function. Eventually the nucleotides would be entirely displaced in this proto-ribosome, after which the ribose-phosphate linkage would be replaced by peptide linkage. The final steps would be the formation of DNA from the RNA genomic material via reverse transcriptase, coupled with the evolution of enzymes for DNA polymerisation and transcription. At this point the original RNA-replicator machinery would be redundant and eliminated, the RNA genomic material would become mRNA and the present-day function of the ribosome would be fixed. In the scenario described a mechanism for the selection for L: -amino acids becomes evident.

Amino Acids↗

PubMed2025

Coalescing single-cell genomes and transcriptomes to decode breast cancer progression.

Understanding epithelial lineages of breast cancer and genotype-phenotype relationships requires direct measurements of the genome and transcriptome of the same single cells at scale. To achieve this, we developed wellDR-seq, a high-genomic-resolution, high-throughput method to simultaneously profile the genome and transcriptome of thousands of single cells. We profiled 33,646 single cells from 12 estrogen-receptor-positive breast cancers and identified ancestral subclones in multiple patients that showed a luminal hormone-responsive lineage, indicating a potential cell of origin. In contrast to bulk studies, wellDR-seq enabled the study of subclone-level gene-dosage relationships, which showed near-linear correlations in large chromosomal segments and extensive variation at the single-gene level. We identified dosage-sensitive and dosage-insensitive genes, including many breast cancer genes as well as sporadic copy-number aberrations in non-cancer cells. Overall, these data reveal complex relationships between copy number and gene expression in single cells, improving our understanding of breast cancer progression.

Breast Neoplasms↗

PubMed2006

Distorted relation between mRNA copy number and corresponding major histocompatibility complex ligand density on the cell surface.

The major histocompatibility complex (MHC) presents peptides derived from degraded cellular proteins to T-cells and is thus crucial for triggering specific immune responses against viral infections or cancer. Up to now, there has been no evidence for a correlation between levels of mRNA (the "transcriptome") and the density of MHC-peptide complexes (the "MHC ligandome") on cells. Because such dependences are of intrinsic importance for the detailed understanding of translation efficiency and protein turnover and thus for systems biology in general and for tumor immunotherapy in practical application, we quantitatively analyzed the levels of mRNA and corresponding MHC ligand densities in samples of renal cell carcinomas and their autologous normal kidney tissues. Relative quantification was carried out by gene chip analysis and by stable isotope peptide labeling, respectively. In comparing more than 270 pairs of gene expression and corresponding peptide presentation ratios, we demonstrate that there is no clear correlation (r = 0.32) between mRNA levels and corresponding MHC peptide levels in renal cell carcinoma. A significant number of peptides presented predominantly on tumor or normal tissue showed no or only minor changes in mRNA expression levels. In several cases, peptides could even be identified despite the virtual absence of the respective mRNA. Thus we conclude that a majority of epitopes from tumor-associated antigens will not be found in approaches based mainly on mRNA expression studies as mRNA expression reflects a distorted picture of the situation on the cell surface as visible for T-cells.

Amino Acid Sequence↗

PubMed1997

Genotyping by PCR-ELISA of a complex polymorphic region that contains one to four copies of six highly homologous human VH3 genes.

The Humhv3005 human VH gene is located in an intricate locus that encompasses for each haplotype a combination of one to four copies of six highly homologous VH3 genes. To assess the complexity of this region, we developed a polymerase chain reaction-enzyme-derived immunosorbent assay (PCR-ELISA) method capable of detecting each of the VH3 genes. The method consisted of amplification of selected germline VH3 genes with a biotinylated primer, covalent capture of the amplicons onto streptavidin-coated wells, and quantitative typing of the bound VH3 genes with diagnostic oligonucleotides. Pilot studies of two DNA samples with known presence or absence of hv3005 [according to a characteristic BamH1 restriction fragment-length polymorphism (RFLP)] yielded the expected results. Subsequent analysis of 100 additional DNA samples with the known EcoR1 RFLP of hv3005 showed a complete match between the absence of the 9.4-kb hybridizing band and lack of hv3005-like genes, as determined by PCR-ELISA. Importantly, the PCR-ELISA analyses of these 102 genomic DNA samples revealed two new haplotypes in the complex hv3005 region. Combined, these data demonstrate the usefulness and efficiency of this new technique to ascertain the presence or absence of six highly homologous genes in an unusually heterogeneous duplication-insertion-deletion region. In the future, a similar strategy may be used to dissect other similarly complex VH genetic loci.

Amino Acid Sequence↗

PubMed1998

Random inheritance of the replication complex by one of two daughter lambda plasmid copies after a replication round in Escherichia coli.

There are two pathways for replication of plasmids derived from bacteriophage lambda (so-called lambda plasmids) in Escherichia coli. One pathway is based on the assembly of the new replication complex at ori lambda, and the second requires activity of the replication complex inherited by one of two daughter plasmid copies after each replication round. Although these two replication pathways proceed at the same time in the host cell, we previously found conditions for specific elimination of the pathway based on the assembly of the new replication complex; thus, replication is restricted to that carried out by the heritable replication complex. These conditions are (i) the relaxed response to amino acid starvation and (ii) temperature upshift of the culture of cells harboring the lambda crotsPts1 plasmid. Here we asked whether the replication complex is inherited randomly by one of two daughter plasmid copies or whether the inheritance is preferred by one particular copy, that containing the parental DNA r strand or that bearing the l strand. We performed density shift experiments which allowed us to separate plasmid DNA molecules replicated by the heritable replication complex from those devoid of the replication complex and therefore not able to replicate. Then, [3H]thymidine-labelled plasmid DNA strands were separated and hybridized to membrane-bound ssDNA containing a fragment of either the r or l strand of lambda DNA. We found roughly equal efficiency of hybridization to both r and l strands in all experimental systems used. Therefore, we conclude that the lambda replication complex is randomly inherited by one of two daughter plasmid copies rather than preferentially inherited by either the copy carrying the parental r strand or that containing the l strand.

Bacteriophage lambda↗

PubMed1977

Relationship between nuclear and polysomal RNA populations of Achlya: a simple eucaryotic system.

The relationship between hnRNA and mRNA in the water mold Achlya has been investigated in several ways. Analysis of the nuclear and polysomal poly(A) RNA by sucrose density gradient centrifugation under denaturing and nondenaturing conditions showed that the populations have indistinguishable size distributions. The number average sizes were calculated to be 1150 nucleotides for nuclear and 1140 nucleotides for polysomal poly(A) RNA. Selective inhibition of rRNA synthesis was used to investigate the size distribution of hnRNA without regard to poly(A) content. Very little hnRNA was observed which sedimented more rapidly than polysomal poly(A) RNA. Hybridization experiments in which an excess of nuclear DNA was reacted with 3H-poly(A) hnRNA or 3H-poly(A) mRNA showed that both populations contain repetitive transcripts (9-10%) as well as single-copy transcripts (44%). Analysis of hybrids on hydroxyapatite in the presence of 8 M urea demonstrated that the poly(A) RNA complementary to repetitive DNA sequence components represented a population of molecules distinct from the population complementary to single-copy DNA. The complexity of whole cell, nuclear and polysomal RNA was determined by saturation hypbridization to single-copy 3H-DNA. All three populations were complementary to essentially the same fraction of the DNA. Terminal hybridization values were 3.84, 3.76 and 3.76% for whole cell, nuclear and polysomal RNA, respectively, representing a complexity of 2.1 X 10(6) nucleotides. These data suggest that the composition of the hnRNA and mRNA populations are essentially identical. No evidence for selective turnover of any sequence component or size class within the nucleus was observed.

Base Sequence↗

PubMed1998

COPI in ER/Golgi and intra-Golgi transport: do yeast COPI mutants point the way?

Coat complexes facilitate the formation of transport vesicles which are essential for proper trafficking of protein and lipids through the secretory pathway. Since its initial identification in the mid-1980s, the COPI coat complex has been credited with mediating multiple distinct transport events and intracellular processes in the exocytic pathway. Not surprisingly, the diversity of these functions has led to significant debate concerning the primary function of COPI. Specifically, within the ER/Golgi and intra-Golgi systems, does COPI mediate anterograde protein transport, retrograde protein transport, or both? This review will focus on the in vivo roles of COPI, primarily examining data from studies of yeast COPI mutants but also including evidence from mammalian systems as appropriate. Some of the current controversies surrounding whether COPI acts directly or indirectly in anterograde and retrograde transport will also be addressed. Because recruitment of COPI to membranes requires the small GTP-binding protein ARF, we will also discuss ARF and proteins that regulate ARF function, and how these proteins might modulate both COPI-driven events and overall membrane composition. Finally, we will point out some of the links still missing from our understanding of COPI-driven events and discuss possible future directions for studies of COPI function.

Animals↗

PubMed1983

Regulation of protein synthesis in Tetrahymena. RNA sequence sets of growing and starved cells.

The complexity of messenger RNA in growing or starved Tetrahymena thermophila is similar and unusually high (approximately 4.5 X 10(7) nucleotides). The complexity of nuclear RNA in growing cells (approximately 7.8 X 10(7) nucleotides) is only about 1.7 times that of mRNA. The concentration of complex class (rare) messages (approximately 53 copies/growing cell and approximately 11 copies/starved cell) is low in comparison to the size of the cell. The concentration of complex nuclear transcripts is also very low (approximately 0.7 copies/growing cell nucleus and approximately 2.6 copies/starved cell nucleus) considering that the macronucleus contains 45 to 90 copies of each single copy sequence. The complex sequence sets found on polysomes of growing and starved cells overlap about 80% and about 60% of the complex nuclear transcripts appear to be held in common. About 60% of macronuclear single copy DNA is transcribed in one or both physiological states. Although growing and starved cells have extremely different fractions of their messages loaded onto polysomes, within each cell type the complex messages in polysomal and nonpolysomal cytoplasmic fractions are indistinguishable, suggesting that exchange may occur between loaded and unloaded messages. Although T. thermophila DNA has an unusually low G + C content (23%), sequences coding for complex RNAs have base ratios similar to those of total DNA. Therefore, codon usage in Tetrahymena must be extremely biased towards adenine- and uridine-rich codons.

Animals↗

PubMed2026

Distinct Effects of Complement C4A and C4B Copy Numbers in Systemic Sclerosis Serological and Clinical Subtypes.

OBJECTIVE: Complement component 4 (C4), encoded by C4A and C4B within the major histocompatibility complex (MHC) on chromosome 6, regulates the immune response and clears immune complexes. The variable copy number (CN) of C4 genes and retroviral human endogenous retrovirus K (HERV-K) element influence its function. Given the relationship of C4 CN with systemic sclerosis (SSc) risk, we assessed associations with SSc clinical and serologic subtypes. METHODS: We compared imputed C4 CNs across SSc subgroups (4,049 anticentromere positive [ACA+]; 2,200 anti-topoisomerase I [ATA+]; 577 anti-RNA polymerase [ARA+]; 1,078 triple-negative [TN] patients; 6,295 limited cutaneous SSc [lcSSc]; and 2,946 diffuse cutaneous SSc [dcSSc]) and 17,991 controls. We evaluated associations with SSc subtypes, identifying C4-independent HLA alleles. RESULTS: Lower C4 CN and higher HERV-K CN were associated with increased risk in all SSc subgroups. ATA+ patients showed the strongest association, particularly with C4A (odds ratio = 1.88), and differences in C4A CN association were more pronounced between autoantibody subgroups (ATA+ vs ACA+, P = 4 × 10-11) than between clinical subgroups (dcSSc vs lcSSc, P = 1 × 10-4). In ACA+ patients, only low C4B CN showed a significant association to SSc risk (P = 1.23 × 10-5). We also observed sex-biased associations: dcSSc, ATA+, and ARA+ male patients showed stronger effects for C4A and ACA+ and lcSSc female patients for C4B. Finally, our results suggest that the HLA alleles associated with SSc subgroups are independent of C4 CN. CONCLUSION: This study highlights distinct genetic contributions of C4A and C4B in SSc subtypes susceptibility. Our findings suggest that lower C4 CNs, particularly C4A, increase the risk of the severe dcSSc subtype, potentially through a mechanism involving immune complex clearance.

Humans↗

PubMed2006

Characterization of ExsC and ExsD self-association and heterocomplex formation.

Expression of the Pseudomonas aeruginosa type III secretion system (T3SS) is induced by calcium depletion and is positively regulated by the ExsA transcriptional activator and negatively regulated by the ExsD antiactivator. Under conditions permissive for expression of the T3SS, the negative regulatory activity of ExsD is antagonized by a direct binding interaction with ExsC. In the present study, the ExsC-ExsD binding interaction was characterized. Individually, both ExsC and ExsD form self-associated complexes, as judged by bacterial monohybrid and gel filtration experiments. A mixture of purified ExsC and ExsD readily formed a complex that elutes from gel filtration medium as a single included peak. The calculated molecular weight of the ExsC-ExsD complex is consistent with a complex containing multiple copies of ExsC and ExsD. Isothermic titration calorimetry experiments found formation of the ExsC-ExsD complex to be thermodynamically favorable, with a Kd of approximately 18 nM and a likely binding ratio of 1:1. To identify amino acid residues important for the regulatory activities of ExsC and ExsD, self-association, and complex formation, charged-cluster mutagenesis was performed. Two of the resulting ExsD charged-cluster mutants (DM2 and DM3) demonstrated a hyperrepressive phenotype for expression of the T3SS. By two-hybrid and copurification assays, the DM3 mutant was found to be impaired in its interaction with ExsC. This finding demonstrates that the binding of ExsC to ExsD is required for transcriptional induction of the T3SS under calcium-limiting growth conditions.

Artificial Gene Fusion↗