Control of replication of FII plasmids: comparison of the basic replicons and of the copB systems of plasmids R100 and R1

The copy numbers of the FII plasmids R1 and R100 were determined in four different ways and found to be identical. Deletion of one of the copy number control genes, copB, together with its promoter gives rise to plasmid copy mutants with an increased copy number. The increase was found to be 8- and 3.5-fold for plasmids R1 and R100, respectively. These deletion derivatives were found to be extremely sensitive to the presence of CopB activity from their own parent plasmid but not to that of the other plasmid. Hence, the CopB protein and its target are plasmid-specific and not FII-group-specific. These results are consistent with the high degree of nonhomology between plasmids R1 and R100 in a 250-bp region covering the distal part of the copB gene and the repA promoter region, which contains the target for the CopB protein.     

The integrated and free states of Streptomyces griseus plasmid pSG1

A 16.6-kb plasmid-pSG1-was isolated from Streptomyces griseus following transformation of protoplasts with unrelated plasmids. Southern hybridization experiments with radioactive probes prepared from pSG1 fragments and immobilized S. griseus DNA fragments indicated that the plasmid was present in the progenitor strain, in an integrated state. In the pSG1+ isolates plasmid sequences existed both as integrated sequences and as free plasmids. The integrated state of maintenance persisted in strains which have been cured of the free plasmid. The junction site on the plasmid was located on a 0.5-kb EcoRI-SalI fragment. The chromosomal integration site was demonstrated to be the same in all strains derived from S. griseus NRRL3851. The occurrence of both states of plasmid maintenance in the same clones indicates that an integrated pSG1 sequence does not interfere with free plasmid replication and partition. It suggests that the establishment of the free state may involve a replicative excision of pSG1 from the S. griseus chromosome.     

A physical and genetic map of the IncFI plasmid ColV2-K94

A restriction enzyme map of the IncFI plasmid ColV2-K94 was generated using EcoRI, BamHI, HindIII, and XhoI; the genetic features of this element were then mapped from previous heteroduplex studies.     

Relationships of the Col plasmids E2, E3, E4, E5, E6, and E7: Restriction mapping and colicin gene fusions

Thirteen ColE plasmids representing the E2-E7 types have been compared by restriction mapping. Over 80% of their restriction sites were found to be similarly positioned, indicating that these plasmids share a common structure. Three variants are ColE2-CA42 and ColE7-K317, both of which contain 1.8-kb DNA segments in place of a 2.5-kb segment common to the other plasmids, and ColE6-CT14, which has an additional 5.0-kb DNA segment compared to the other plasmids. The colicin (col), immunity (imm), and colicin release (hic) genes of these plasmids have been localized to regions corresponding to those known for ColE3-CA38 and ColE2-P9, with the imm and hic genes adjacent to the 3' end of the col gene. Active colicin is produced from hybrid col genes containing 5' and 3' ends from different E-type plasmids. The 3'-termini of the fused col genes specify the colicin type.     

α-Mannosidase-1 mutants of Dictyostelium dkoideum: Early aggregation-essential genes regulate enzyme precursor synthesis, modification, and processing

The lysosomal enzyme alpha-mannosidase-1 is one of the earliest developmentally controlled gene products in Dictyostelium discoideum. Although this enzyme is synthesized throughout the first 20 h of development, it is not required for complete morphogenesis, since structural gene (manA) mutants lacking activity develop normally. We isolated six strains deficient in alpha-mannosidase-1 activity which, unlike structural gene mutants, fail to aggregate. Fruiting revertants of these strains accumulate wild-type levels of alpha-mannosidase-1 activity, suggesting that both the enzymatic and morphological defects are caused by single mutations in nonstructural genes essential for early development. Direct genetic evidence for mutations outside of the structural gene was obtained by complementation analysis. We used alpha-mannosidase-1-specific monoclonal antibodies to analyze the biochemical defects in these mad (alpha-mannosidase-1-deficient) mutants. All mad mutants show a significantly reduced relative rate of enzyme precursor biosynthesis. The mad-404 mutation results in a complete lack of precursor biosynthesis, as well as a lack of functional alpha-mannosidase-1 mRNA. In some cases, however, the enzymatic defect results from improper post-translational modification which affects precursor processing. We conclude that a small number of aggregation-essential genes are involved in regulating the synthesis, modification, and processing of alpha-mannosidase-1 during development.     

The regulation of''early'' enzymes during the development and dedifferentiation of Dictyostelium discoideum

The specific activities of the enzymes alpha-mannosidase and N-acetylglucosaminidase increase immediately after the initiation of the development of bacterially grown cell cultures of Dictyostelium discoideum. The regulation of these two enzymes was found to be dissociable in the developmental timer mutant, FM-1, which aggregates 4.5 h earlier than wild-type cells due to the absence of the first rate-limiting component of the preaggregative period. The increase in alpha-mannosidase activity occurs in the absence of the first rate-limiting component, but the increase in N-acetylglucosaminidase activity does not. These results indicate the following: (1) the increase in the specific activity of alpha-mannosidase is not related to the timing of subsequent developmental stages; (2) the increase in the specific activity of N-acetylglucosaminidase is not necessary for the subsequent developmental program; and (3) either the increase in the specific activity of N-acetylglucosaminidase is dependent upon progress through the first rate-limiting component, or the increase in this enzyme activity and the first rate-limiting component are both dependent upon an early event for which FM-1 is defective. In addition to early development, we monitored the two enzyme activities during dedifferentiation. The results demonstrate that there is no difference between dedifferentiating wild-type cells and dedifferentiation-defective mutant HI-4 cells. Changes in enzyme specific activity accompanying dedifferentiation are dependent upon the composition of the dedifferentiation-inducing media and are consistent with the levels of these enzymes observed in cells growing in the different nutrient media.     

Expression of monoclonal antibody-defined cell surface antigens during rat brain development

Using single-cell suspensions of mechanically dissociated, prenatal BDIX-rat brain cells (13th, 15th, and 21st days after fertilization) for immunization, we have established a collection of 37 monoclonal antibodies (Mabs) directed against neural cell surface determinants. The developmental-stage-dependent expression of cell-surface antigens recognized by these Mabs was analyzed both on plasma membranes isolated from whole brains of BDIX rats (prenatal days 13-22 and adults) using an indirect 125I solid-phase radioimmunoassay, and on intact BDIX-rat brain cells (prenatal days 13-22) using a fluorescence-activated cell sorter. Different types of developmental stage-dependent profiles of Mab binding were found, these being indicative of the presence of neural cell surface determinants whose expression increases, decreases, or does not change with brain development. Some of the Mab-binding profiles showed transient changes as a function of developmental stage. These Mabs are currently being used for the characterization, reproducible identification, and isolation of neural cell subpopulations of the developing rat brain, with the aim of investigating the cell type dependence and developmental (differentiation) stage dependence of malignant transformation following pulse exposure to the carcinogen N-ethyl-N-nitrosourea at defined stages of brain development.     

Antigenic variation is associated with DNA rearrangements in a relapsing fever Borrelia

Borrelia hermsii, an agent of relapsing fever, undergoes antigenic variation in its host. Surface-exposed proteins with differing primary structures determine the serotype of each organism. Using amino acid sequence data from two of these variable proteins, we synthesized two mixed-sequence oligonucleotides and then used the oligonucleotides to probe mRNA and DNA of three isogenic serotypes of B. hermsii. In Northern blots the probes were specific for the mRNA of the homologous serotype. Southern blots revealed two classes of hybridizing fragments: those common to the three serotypes and those specific for a particular serotype. A serotype-specific DNA fragment, which had hybridized to both oligonucleotide probes, was cloned. Subsequent use of the cloned fragment as a probe provided further evidence that antigenic variation in B. hermsii is associated with DNA rearrangements and with occurrence of expression-linked copies of all, or part, of an antigen-specifying gene.     

On the computation of the tertiary structure of globular proteins—IV. Use of secondary structure information

The distance geometry approach for computing the tertiary structure of globular proteins emphasized in this series of papers (Goelet al., J. theor. Biol. 99, 705–757, 1982) is developed further. This development includes incorporation of some secondary structure information—the location of alpha helices in the primary sequence—in the algorithm to compute the tertiary structure of alpha helical globular proteins. An algorithm is developed which estimates the interresidue distances between chain-proximate helices. These distances, in conjunction with the global statistical average distances obtainable from a database of real proteins and determined by the primary sequence of the protein under study, are used to determine the tertiary structure. Five proteins, parvalbumin, hemerythrin, human hemoglobin, lamprey hemoglobin, and sperm whale myoglobin, are investigated. The root mean square (RMS) errors between the calculated structures and those determined by X-ray diffraction range from 4.78 to 7.56 Å. These RMSs are 0.21–2.76 Å lower than those estimated without the secondary structure information. Contact maps and three-dimensional backbone representations also show considerable improvements with the introduction of secondary structure information.     

Reversible addition of bisulfite buffer to the cytidine ring system

The rate constants for the reversible addition of protons and sulfite to the 5,6 double bond of cytidine and 3-methylcytidine have been spectrophotometrically measured under conditions (25°C, μ = 1.0 ) where the deamination of 5,6-dihydrocytidine-6-sulfonate is minimal. Both the addition and the elimination of sulfite from the ring system are subject to general catalysis of proton transfer. For the reaction in either direction, plots of the pseudo-firstorder rate constants against increasing buffer concentration are biphasic and indicative of at least a two-step reaction pathway with both steps being subject to general acid-base catalysis. Kinetic hydrogen-deuterium isotope effects were measured for both buffer-catalyzed steps of sulfite elimination from 3-methyl-5,6-dihydrocytidine-6-sulfonate and sulfite addition to 3-methylcytidine. Both H₂O and D₂O were used as solvent. For both the addition and the elimination of SO₃²⁻ values of k₂^H/k₂^D were 6.3–7.1 and 2.3–2.6 at low and high imidazole buffer concentration, respectively. The large isotope effects values in the range of 6–7 can be attributed to rate-determining proton transfer to carbon-5 of the cytidine ring system. The smaller values are more likely caused by proton transfer to a electronegative atom such as the oxygen on carbon-2 of the cytidine ring. The equilibrium constants for bisulfite buffer addition to 3-methylcytidine and cytidine at 25°C, μ = 1.0 , pH 7.2, are 10.2 and 1.3 ⁻¹, respectively.