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At least 1,009 records · Page 56Linked to original sources
PubMed2005

Energetics of structural transitions of the addiction antitoxin MazE: is a programmed bacterial cell death dependent on the intrinsically flexible nature of the antitoxins?

The Escherichia coli mazEF addiction module plays a crucial role in the cell death program that is triggered under various stress conditions. It codes for the toxin MazF and the antitoxin MazE, which interferes with the lethal action of the toxin. To better understand the role of various conformations of MazE in bacterial life, its order-disorder transitions were monitored by differential scanning calorimetry, spectropolarimetry, and fluorimetry. The changes in spectral and thermodynamic properties accompanying MazE dimer denaturation can be described in terms of a compensating reversible process of the partial folding of the unstructured C-terminal half (high mean net charge, low mean hydrophobicity) and monomerization coupled with the partial unfolding of the structured N-terminal half (low mean net charge, high mean hydrophobicity). At pH<or=4.5 and T<50 degrees C, the unstructured polypeptide chains of the MazE dimer fold into (pre)molten globule-like conformations that thermally stabilize the dimeric form of the protein. The simulation based on the thermodynamic and structural information on various addiction modules suggests that both the conformational adaptability of the dimeric antitoxin form (binding to the toxins and DNA) and the reversible transformation to the more flexible monomeric form are essential for the regulation of bacterial cell life and death.

Antitoxins↗
PubMed2003

Distinction of sperm-binding site and reactive site for trypsin inhibition on p12 secreted from the accessory sex glands of male mice.

Six variants of P12, a Kazal-type trypsin inhibitor in the secretion of male mouse accessory sexual glands, were made using single-site mutations including R19L, Y21V, D22G, R43G, K44S, and R45T, based on one-letter-code mutation of amino acids. The other two variants, Nd10 and Cd8, were made using the deletion of 10 and 8 residues from the N- and C-terminals, respectively. Their CD profiles revealed maintenance of the P12 conformation in the seven variants, excluding Cd8, which became unfolded. Only R19L entirely lost the ability while the other variants were as strong as P12 in inhibiting the trypsin digestion of N-benzoyl-Phe-Val-Arg 7-amido-4-methylcoumarin. The immunocytochemical results demonstrated that D22G and Cd8 failed to bind to sperm, Y21V very weakly did so, and the other variants retained their sperm-binding abilities. Concomitantly, the immunocytochemical stainability of each ligand was parallel to its inhibitory effect on 125I-P12-sperm binding, and a synthetic oligopeptide corresponding to residues 18-24 of P12 was able to inhibit P12-sperm binding. The data together concluded that R19 was essential for protease inhibition and D22 and/or Y21 mainly being responsible for the binding of P12 to sperm. The steric arrangement of R19, Y21, and D22 on the tertiary structure of P12 is discussed.

Amino Acid Sequence↗
PubMed1985

Structure and expression of the egg-laying hormone gene family in Aplysia.

Transcription of the egg-laying hormone (ELH) gene family was examined by characterizing homologous cDNA clones from abdominal ganglion and atrial gland cDNA libraries. All cDNAs contain an exon that spans the coding region (exon III) and one or two additional exons. The tissue-specific expression of the ELH gene family was confirmed by the observation that exon III encodes the ELH precursor protein in the bag cell transcripts and either the A or B precursor proteins in the atrial gland transcripts. The cDNA clones also contain 5' untranslated exons not present in the previously isolated genomic clones. One type of transcript has a 40-base pair segment, designated exon I, contiguous with exon III. A second type of transcript has an additional 149 base pairs of DNA, designated exon II, located between exons I and III. Several genomic clones containing exons I and II were isolated. DNA sequence analysis reveals that exons I and II are directly linked and that they are separated from exon III by an intervening sequence of at least 5 kilobases (kb). Consensus sequences for a putative promotor region and also for RNA splicing and polyadenylation were identified. From this work we can describe a prototype ELH gene complete with identified sequences necessary for the proper initiation of transcription and the subsequent processing of the transcript.

Aplysia↗
PubMed2005

Identification of membrane-bound and secreted proteins from Echinococcus granulosus by signal sequence trap.

The signal sequence trap technique was applied to identify genes coding for secreted and membrane bound proteins from Echinococcus granulosus, the etiologic agent of cystic hydatid disease. An E. granulosus protoscolex cDNA library was constructed in the AP-PST vector such that randomly primed cDNAs were fused with a placental alkaline phosphatase reporter gene lacking its endogenous signal peptide. E. granulosus cDNAs encoding a functional signal peptide were selected by their ability to rescue secretion of alkaline phosphatase by COS-7 cells that had been transfected with the cDNA library. Eighteen positive clones were identified and sequenced. Their deduced amino acid sequences showed significant similarity with amino acid transporters, Krebs cycle intermediates transporters, presenilins and vacuolar protein sorter proteins. Other cDNAs encoded secreted proteins without homologues. Three sequences were transcribed antisense to E. granulosus expressed sequence tags. All the mRNAs were expressed in protoscoleces and adult worms, but some of them were not found in oncospheres. The putative E. granulosus secreted and membrane bound proteins identified are likely to play important roles in the metabolism, development and survival in the host and represent potential targets for diagnosis, drugs and vaccines against E. granulosus.

Alkaline Phosphatase↗
PubMed1988

Immunologic modeling of a 75-kDa malarial protein with carrier-free synthetic peptides.

A protein of 75 kDa is produced in large quantities by the human malarial parasite Plasmodium falciparum and is present on the surface of the merozoite, whose function is to infect erythrocytes. Based on nucleotide sequence coding for 40% of this protein, two nonoverlapping model peptides 13 and 19 residues long were synthesized, coupled to a keyhole limpet hemocyanin carrier, and used to immunize rabbits. Although both antisera had high titers of anti-peptide antibodies, only that raised against the 13-residue peptide showed good reactivity against the original protein. Although the 19-mer adopted the helical secondary structure predicted for the corresponding protein region, antisera against this peptide reacted with the native protein weakly or not at all. Concluding that the poor anti-protein reactivity was due to modification of lysine-containing epitopes by glutaraldehyde conjugation, we used a carrier-free 28-residue peptide presented as a 56-residue disulfide-bonded dimer to model the same region. This peptide, in contrast to the conjugated 19-mer, stimulated the production of IgG antibodies that reacted at high dilution with the authentic protein in immunoblots, ELISA, and radioimmunoprecipitation assays. These data indicate that large carrier-free peptides may be successfully used as immunogens. In addition, our results show that this strategy may greatly improve the ability of conjugation-sensitive peptides to stimulate antibodies reactive with the original protein and therefore has substantial practical application.

Animals↗
PubMed2002

Tomato LeAGP-1 arabinogalactan-protein purified from transgenic tobacco corroborates the Hyp contiguity hypothesis.

Functional analysis of the hyperglycosylated arabinogalactan-proteins (AGPs) attempts to relate biological roles to the molecular properties that result largely from O-Hyp glycosylation putatively coded by the primary sequence. The Hyp contiguity hypothesis predicts contiguous Hyp residues as attachment sites for arabino-oligosaccharides (arabinosides) and clustered, non-contiguous Hyp residues as arabinogalactan polysaccharide sites. Although earlier tests of naturally occurring hydroxyproline-rich glycoproteins (HRGPs) and HRGPs designed by synthetic genes were consistent with a sequence-driven code, the predictive value of the hypothesis starting from the DNA sequences of known AGPs remained untested due to difficulties in purifying a single AGP for analysis. However, expression in tobacco (Nicotiana tabacum) of the major tomato (Lycopersicon esculentum) AGP, LeAGP-1, as an enhanced green fluorescent protein fusion glycoprotein (EGFP)-LeAGP-1, increased its hydrophobicity sufficiently for chromatographic purification from other closely related endogenous AGPs. We also designed and purified two variants of LeAGP-1 for future functional analysis: one lacking the putative glycosylphosphatidylinositol (GPI)-anchor signal sequence; the other lacking a 12-residue internal lysine-rich region. Fluorescence microscopy of plasmolysed cells confirmed the location of LeAGP-1 at the plasma membrane outer surface and in Hechtian threads. Hyp glycoside profiles of the fusion glycoproteins gave ratios of Hyp-polysaccharides to Hyp-arabinosides plus non-glycosylated Hyp consistent with those predicted from DNA sequences by the Hyp contiguity hypothesis. These results demonstrate a route to the purification of AGPs and the use of the Hyp contiguity hypothesis for predicting the Hyp O-glycosylation profile of an HRGP from its DNA sequence.

Amino Acid Sequence↗
PubMed1979

Cloning the argF gene from Escherichia coli K-12 with simian virus 40.

We have inserted a gene coding for ornithine transcarbamylase (OTCase) from Escherichia coli K-12 into the late gene region of simian virus 40 (SV40) DNA and propagated the hybrid molecules as free episomes or by co-infection with an SV40 tsA helper virus. In the first case, the E. coli argF gene was inserted via the EcoRI and BamHI termini in the late gene region of SV40 and the recombinant molecules were used to transfect monkey kidney cells. The hybrid DNA, which was too large to be encapsidated, was replicated for a short time (14 days) but was eventually lost from the surviving cells. In order to allow the argF gene to be packaged into virions, we purified two SV40 vectors containing large deletions of late gene region sequences. One was a 3325 base pair segment from a HaeII + BamHI digest. The argF gene was joined to both vectors at the BamHI site and these linear molecules were used to transfect monkey cells in the presence of SV40 tsA58 DNA as helper. These hybrid DNAs were replicated and packaged into virions. Late in the lytic infection of monkey cells, polyadenylated, cytoplasmic argF transcripts were detected, but significant translation of these trancripts was not observed.

Cloning, Molecular↗
PubMed1989

Functional relationship among TATA sequences, gene induction and transcription initiation in the beta-galactosidase, LAC4, gene from Kluyveromyces lactis.

In the 5' non-coding region of the beta-galactosidase, LAC4, gene of Kluyveromyces lactis, three TATA-like sequences are present at -230, -170 and -142 from the ATG translation start site. By means of deletion mutations in the TATA region, at least two of these TATA sequences, those at -230 and -142, were shown to be required for normal gene expression. Evidence is presented for a functional hierarchy and cooperation between these TATA sequences. The deletion or a change in the position of the TATA sequences affects both beta-galactosidase induction and the location of RNA initiation sites. The TATA sequence at -230 alone is sufficient for correct gene induction when it is moved to a position 41 bp from the major RNA initiation sites located around -110; the -142 TATA alone contributes only partly to gene induction. We suggest a functional distinction between these two related regulatory sequences. This functional distinction might be established by sequence differences and/or targets of unlike specific DNA binding protein(s). A conformational analysis of the LAC4 promoter showed that under torsional stress the functional elements UAS, TATA boxes RNA initiation sites and ATG can be detected as P1-sensitive sites. Possible functions of DNA structural alterations on gene expression are discussed.

Chromosome Deletion↗
PubMed1998

Molecular cloning and expression of active Ole e 3, a major allergen from olive-tree pollen and member of a novel family of Ca2+-binding proteins (polcalcins) involved in allergy.

A cDNA encoding Ole e 3, a major allergen from olive-tree pollen, has been cloned and sequenced. A strategy based on two-step PCR amplification towards the 5' end and 3' end, with an internal specific primer, has been used. The isolated cDNA contains an open reading frame coding for a polypeptide of 84 amino acids, which is in agreement with the composition and molecular mass of the natural allergen, exhibiting two 12-residue segments homologous to Ca2+-binding sites of EF-hand type. The cDNA was inserted into the pET-11b expression vector and over-expressed in Escherichia coli. The purified recombinant protein shows identical secondary structure to that of the natural allergen and is able to bind both IgE from sera of patients allergic to olive pollen and polyclonal antibodies raised against olive-pollen Ole e 3. The capacity of binding Ca2+ has been demonstrated for both natural and recombinant allergens. RNA transcripts of Ole e 3 were only detected in pollen tissue. Northern-blot and Western-blot analyses of poly(A)+ RNA and protein extracts, respectively, obtained from a variety of olive-tree-related and nonrelated mature pollens demonstrated the presence of Ole e 3 homologous proteins. This indicates a sequence conservation and widespread distribution for this family of Ca2+-binding proteins that can be responsible for allergenic cross-reactivity. We suggest the tentative generic name of polcalcins for the members of this family of Ca2+-binding proteins from pollen.

Allergens↗
PubMed2005

A novel thermostable hemoglobin from the actinobacterium Thermobifida fusca.

The gene coding for a hemoglobin-like protein (Tf-trHb) has been identified in the thermophilic actinobacterium Thermobifida fusca and cloned in Escherichia coli for overexpression. The crystal structure of the ferric, acetate-bound derivative, was obtained at 2.48 A resolution. The three-dimensional structure of Tf-trHb is similar to structures reported for the truncated hemoglobins from Mycobacterium tuberculosis and Bacillus subtilis in its central domain. The complete lack of diffraction patterns relative to the N- and C-terminal segments indicates that these are unstructured polypeptides chains, consistent with their facile cleavage in solution. The absence of internal cavities and the presence of two water molecules between the bound acetate ion and the protein surface suggest that the mode of ligand entry is similar to that of typical hemoglobins. The protein is characterized by higher thermostability than the similar mesophilic truncated hemoglobin from B. subtilis, as demonstrated by far-UV CD melting experiments on the cyano-met derivatives. The ligand-binding properties of Tf-trHb, analyzed in stopped flow experiments, demonstrate that Tf-trHb is capable of efficient O2 binding and release between 55 and 60 degrees C, the optimal growth temperature for Thermobifida fusca.

Actinobacteria↗
PubMed1994

Sequence, assembly and evolution of a primordial ferredoxin from Thermotoga maritima.

A gene coding for the ferredoxin of the primordial, strictly anaerobic and hyperthermophilic bacterium Thermotoga maritima was cloned, sequenced and expressed in Escherichia coli. The ferredoxin gene encodes a polypeptide of 60 amino acids that incorporates a single 4Fe-4S cluster. T. maritima ferredoxin expressed in E. coli is a heat-stable, monomeric protein, the spectroscopic properties of which show that its 4Fe-4S cluster is correctly assembled within the mesophilic host, and that it remains stable during purification under aerobic conditions. Removal of the iron-sulfur cluster results in an apo-ferredoxin that has no detectable secondary structure. This observation indicates that in vivo formation of the ferredoxin structure is coupled to the insertion of the iron-sulfur cluster into the polypeptide chain. Sequence comparison of T. maritima ferredoxin with other 4Fe-4S ferredoxins revealed high sequence identities (75% and 50% respectively) to the ferredoxins from the hyperthermophilic members of the Archaea, Thermococcus litoralis and Pyrococcus furiosus. The high sequence similarity supports a close relationship between these extreme thermophilic organisms from different phylogenetic domains and suggests that ferredoxins with a single 4Fe-4S cluster are the primordial representatives of the whole protein family. This observation suggests a new model for the evolution of ferredoxins.

Amino Acid Sequence↗
PubMed1975

Phage T4 lysozyme. Physical properties and reversible unfolding.

Phage T4 lysozyme has been used extensively in studies of the genetic code. However, little work has been done on the characterization of the purified enzyme. Therefore, we determined the spectral properties of native T4 lysozyme and used these properties to follow the unfolding transition. The ultraviolet absorption spectrum and solvent perturbation difference spectrum indicate that the aromatic amino acids are extensively exposed to solvent. The CD and ORD spectra are characteristic of a high fraction of helix. Guanidine hydrochloride denaturation results show that over a T4 lysozyme concentration range of 0.07-1 g/l the c-m equals 2.7 M guanidine hydrochloride at pH 5 and that the transition is 100% reversible as judged by enzymatic assay and four different spectrophotometric criteria: CD at 295 nm, CD at 223 nm, fluorescence intensity at 350 nm and wavelength of maximum fluorescence. Guanidine hydrochloride denaturation at pH 2.5 was followed using fluorescence emission and has a c-m equals 1.7 M guanidine hydrochloride, indicating a strong pH dependence of chemical unfolding. Reversible thermal denaturation conditions were located at acid pH, 0.2 M NaCl, 10-4 M dithiothreitol and 10-6 M T4 lysozyme. The CD signal at 223 nm was used to measure the unfolding. Thermodynamic analysis of the thermal data showed an increase in T-m, increment H-unf and increment S-unf with increasing pH.

Binding Sites↗
PubMed2003

The patchwork nature of rolling-circle plasmids: comparison of six plasmids from two distinct Bacillus thuringiensis serotypes.

Bacillus thuringiensis, the entomopathogenic bacteria from the Bacillus cereus group, harbors numerous extrachromosomal molecules whose sizes vary from 2 to more than 200kb. Apart from the genes coding for the biopesticide delta-endotoxins located on large plasmids, little information has been obtained on these plasmids and their contribution to the biology of their host. In this paper, we embarked on a detailed comparison of six small rolling-circle replicating (RCR) plasmids originating from two major B. thuringiensis strains. The complete nucleotide sequences of plasmid pGI1, pGI2, pGI3, pTX14-1, pTX14-2, and pTX14-3 have been obtained and compared. Replication functions, comprising, for each plasmid, the gene encoding the Rep-protein, double-strand origin of replication (dso), single-strand origin of replication (sso), have been identified and analyzed. Two new families, or homology groups, of RCR plasmids originated from the studies of these plasmids (Group VI based on pGI3 and Group VII based on pTX14-3). On five of the six plasmids, loci involved in conjugative mobilization (Mob-genes and origin of transfer (oriT)) were identified. Plasmids pTX14-1, pTX14-2, and pTX14-3 each harbor an ORF encoding a polypeptide containing a central domain with repetitive elements similar to eukaryotic collagen (Gly-X-Y triplets). These genes were termed bcol for Bacillus-collagen-like genes.

Amino Acid Sequence↗
PubMed2003

Tracking interactions that stabilize the dimer structure of starch phosphorylase from Corynebacterium callunae. Roles of Arg234 and Arg242 revealed by sequence analysis and site-directed mutagenesis.

Glycogen phosphorylases (GPs) constitute a family of widely spread catabolic alpha1,4-glucosyltransferases that are active as dimers of two identical, pyridoxal 5'-phosphate-containing subunits. In GP from Corynebacterium callunae, physiological concentrations of phosphate are required to inhibit dissociation of protomers and cause a 100-fold increase in kinetic stability of the functional quarternary structure. To examine interactions involved in this large stabilization, we have cloned and sequenced the coding gene and have expressed fully active C. callunae GP in Escherichia coli. By comparing multiple sequence alignment to structure-function assignments for regulated and nonregulated GPs that are stable in the absence of phosphate, we have scrutinized the primary structure of C. callunae enzyme for sequence changes possibly related to phosphate-dependent dimer stability. Location of Arg234, Arg236, and Arg242 within the predicted subunit-to-subunit contact region made these residues primary candidates for site-directed mutagenesis. Individual Arg-->Ala mutants were purified and characterized using time-dependent denaturation assays in urea and at 45 degrees C. R234A and R242A are enzymatically active dimers and in the absence of added phosphate, they display a sixfold and fourfold greater kinetic stability of quarternary interactions than the wild-type, respectively. The stabilization by 10 mm of phosphate was, however, up to 20-fold greater in the wild-type than in the two mutants. The replacement of Arg236 by Ala was functionally silent under all conditions tested. Arg234 and Arg242 thus partially destabilize the C. callunae GP dimer structure, and phosphate binding causes a change of their tertiary or quartenary contacts, likely by an allosteric mechanism, which contributes to a reduced protomer dissociation rate.

Allosteric Site↗
PubMed2004

Altered active site flexibility and a structural metal-binding site in eukaryotic dUTPase: kinetic characterization, folding, and crystallographic studies of the homotrimeric Drosophila enzyme.

dUTPase is responsible for preventive DNA repair via exclusion of uracil. Developmental regulation of the Drosophila enzyme is suggested to be involved in thymine-less apoptosis. Here we show that in addition to conserved dUTPase sequence motifs, the gene of Drosophila enzyme codes for a unique Ala-Pro-rich segment. Kinetic and structural analyses of the recombinant protein and a truncation mutant show that the Ala-Pro segment is flexible and has no regulatory role in vitro. The homotrimer enzyme unfolds reversibly as a trimeric entity with a melting temperature of 54 degrees C, 23 degrees C lower than Escherichia coli dUTPase. In contrast to the bacterial enzyme, Mg(2+) binding modulates conformation of fly dUTPase, as identified by spectroscopy and by increment in melting temperature. A single well folded, but inactive, homotrimeric core domain is generated through three distinct steps of limited trypsinolysis. In fly, but not in bacterial dUTPase, binding of the product dUMP induces protection against proteolysis at the tryptic site reflecting formation of the catalytically competent closed conformer. Crystallographic analysis argues for the presence of a stable monomer of Drosophila dUTPase in crystal phase. The significant differences between prototypes of eukaryotic and prokaryotic dUTPases with respect to conformational flexibility of the active site, substrate specificity, metal ion binding, and oligomerization in the crystal phase are consistent with alteration of the catalytic mechanism and hydropathy of subunit interfaces.

Amino Acid Motifs↗
PubMed1997

'Module'-substituted globins: artificial exon shuffling among myoglobin, hemoglobin alpha- and beta-subunits.

Based on the detailed structural analysis of proteins, Go [M. Go, Nature 291 (1981) 90-92] found that protein structures can be divided into some structural units, 'modules,' which correspond to peptides coded by exons. In the present study, to investigate functional and structural roles of modular structures in proteins, we have engineered eight chimera globins, in which the exons are shuffled among human myoglobin, human hemoglobin alpha- and beta-subunits, in addition to the chimera beta beta alpha-globin described previously [K. Wakasugi, K. Ishimori, K. Imai, Y. Wada, I. Morishima, J. Biol. Chem. 269 (1994) 18750-18756]. Although all of the chimera globins stoichiometrically bound the heme and their alpha-helical contents increased by heme incorporation as found for native globins, the alpha-helical contents of the chimera globins were significantly lower than those of native globins, suggesting that 'module' substitutions seriously affect the protein folding and stability in globins. The comparisons among several chimera globins demonstrated that such structural alterations are mainly attributed to loss of some key intermodular interactions for protein folding. By simultaneous substitution of the modules M1 and M4 from the same globin, the protein structure was stabilized, which indicates that the module packing between modules M1 and M4 would be one of the crucial interaction to stabilize the globin fold. Present results allow us to conclude that module substitutions would be available for designing and producing novel functional proteins if we can reproduce the stable modular packing in the 'module'-substituted proteins.

Chromatography, Gel↗
PubMed1997

Thioredoxin from Bacillus acidocaldarius: characterization, high-level expression in Escherichia coli and molecular modelling.

The thioredoxin (Trx) from Bacillus acidocaldarius (BacTrx) was purified to homogeneity by anion-exchange chromatography and gel-filtration chromatography, based on its ability to catalyse the dithiothreitol-dependent reduction of bovine insulin disulphides. The protein has a molecular mass of 11577 Da, determined by electrospray mass spectrometry, a pI of 4.2, and its primary structure was obtained by automated Edman degradation after cleavage with trypsin and cyanogen bromide. The sequences of known bacterial Trxs were aligned at the active site: BacTrx has an identity ranging from 45 to 53% with all sequences except that of the unusual Anabaena strain 7120 Trx (37% identity). The gene coding for BacTrx was isolated by a strategy based on PCR gene amplification and cloned in a plasmid downstream of a lac-derived promoter sequence; the recombinant clone was used as the expression vector for Escherichia coli. The expression was optimized by varying both the time of cell growth and the time of exposure to the inducer isopropyl beta-d-thiogalactoside; expressed BacTrx represents approx. 5% of the total cytosolic protein. CD spectra and differential scanning calorimetry measurements demonstrated that BacTrx is endowed with a higher conformational heat stability than the Trx from E. coli. Nanogravimetry experiments showed a lower content of bound water in BacTrx than in E. coli Trx, and a transition temperature approx. 10 degrees C higher for BacTrx. The three-dimensional model of the oxidized form of BacTrx was constructed by a comparative molecular modelling technique, using E. coli Trx and Anabaena strain 7120 Trx as reference proteins. Increased networks of ion-pairs and shorter loops emerged as major features of the BacTrx structure compared with those of the template proteins. The findings are discussed in the light of the current knowledge about molecular determinants of protein stability.

Amino Acid Sequence↗
PubMed1980

Genes and sites involved in replication and incompatibility of an R100 plasmid derivative based on nucleotide sequence analysis.

The nucleotide sequence of the entire region required for autonomous replication and incompatibility of an R100 plasmid derivative, pSM1, has been determined. This region includes the replication region and all plasmid encoded information required for replication. Numerous reading frames for possible proteins can be found in this region. The existence of one of these proteins called RepA1 (285 amino acids; 33,000 daltons) which is encoded within the region known by cloning analysis to be required for replication is supported by several lines of evidence. These include an examination of the characteristic sequences on the proximal and distal ends of the coding region, a comparison of the sequence of the replication regions of pSM1 and the highly related R1 plasmid derivative Rsc13 as well as other biochemical and genetic evidence. The existence of two other proteins, RepA3 (64 amino acis; 7000 daltons) and RepA2 (103 amino acids; 11,400 daltons) is also consistent with most of the criteria mentioned above. However, the region encoding RepA3, which by cloning analysis is within the region responsible for both replication and incompatibility, has never been demonstrated to produce a 7,000 dalton polypeptide. Since a large secondary structure can be constructed in this region, it is possible that the region contains structure or other information that is responsible for incompatibility. RepA2, encoded entirely within the region identified by cloning analysis to be responsible for incompatibility but not for replication can be visualized in vivo and in vitro. However, the nucleotide sequence of the region encoding RepA2 is completely different in mutually incompatible plasmid derivatives of R1 and R100. It is therefore unlikely that RepA2 plays a major role in incompatibility. Thus, we predict that RepA2 is required to initiate DNA synthesis at the replication origin and that the region proximal to RepA2 either encodes a gene product or structure information that is responsible for incompatibility.

Base Sequence↗