Linearization of donor DNA during plasmid transformation in Neisseria gonorrhoeae.

We examined the fate of plasmid DNA after uptake during transformation in Neisseria gonorrhoeae. An 11.5-kilobase plasmid, pFA10, was processed to linear double-stranded DNA during uptake by competent cells, but cleavage of pFA10 was not site specific. A minority of pFA10 entered as open circles. A 42-kilobase plasmid, pFA14, was degraded into small fragments during uptake; no intracellular circular forms of pFA14 were evident. Since pFA10 DNA linearized by a restriction enzyme was not further cut during uptake, the endonucleolytic activity associated with entry of plasmid DNA appeared to act preferentially on circular DNA. Although linear plasmid DNA was taken up into a DNase-resistant state as efficiently as circular DNA, linear plasmid DNA transformed much less efficiently than circular plasmid DNA. These data suggest that during entry transforming plasmid DNA often is processed to double-stranded linear molecules; transformants may arise when some molecules are repaired to form circles. Occasional molecules which enter as intact circles may also lead to transformants.     

Relation between the Yersinia phage and bacteriophages isolated from the environment

The bacteriophage designated RD2 has been isolated from the sewage in Rostov-on-Don city and studied. The morphology of bacteriophage particles and the biological properties of the bacteriophage make it related to the plague bacteriophage isolated by D'Errel. The molecular masses of the compared bacteriophages are almost identical being 26.4 +/- 0.4 Md for RD2 and 24.7 +/- 0.2 Md for D'Errel bacteriophage. The DNAs of the bacteriophages share 80% of homology and possess 15 nonhomologous regions scattered along the genomes. The phages are serologically related. The DNAs of both bacteriophages give the similar pattern of hydrolysis by restriction endonuclease EcoRV, but have the different sensitivity to many other restriction endonucleases. The protein specter of bacteriophage RD2 contains 18 polypeptides (11 minor ones), while the one of D'Errel bacteriophage contains 7 polypeptides similar in molecular mass with the polypeptides of RD2. The bacteriophage RD2 cannot be considered one of the plague causative agents of bacteriophages since the region where it has been isolated has a long epidemiological and epizootical record of absence of plague.     

Relation between the number of closed cross-bridges and the load during muscle contraction

Analytic expressions for the relationships between the number of attached cross-bridges and the load and velocity of muscle contraction are obtained from Deshcherevsky's kinetic theory of muscle contractions. The comparison of these expressions with the experimental relationships known from literature is carried out their good agreement is shown.     

Relationship between the DNA content and mesosome number in cells of Bacillus.

In cells of Bacillus there is evidence that deoxyribonucleic acid forms an association with some membranous structure within the cell, possibly mesosomes. Cells of varieties of Bacillus cereus and Bacillus subtilis were examined to see if any quantitative relationship existed between the numbers of mesosomes and DNA content. No direct relationship could be domonstrated. However, cells of Bacillus cereus var. alesti A(-) maintained a characteristic and constant DNA content and number of mesosomes regardless of growth conditions. During sporulation, a variant of A(-), termed A(-)3, SEQUESTERS ITS DNA at both ends of the cell, leaving a small amount of DNA but no mesosomes in the center compartment. Since this center compartment is capableof growth and division upon replacement in fresh medium (rejuventation) it was examinedfor mesosome content as DNA synthesis and division were initiated. In most cells, acentral mesosome was formed at the site of cell septum formation; however, the presenceof a mesosome was not an absolute prerequisite for cell division. We propose that atthe onset of cell growth, mesosomes primarily function in the process of cell septum formation. As growth and division proceed, mesosomes are produced in characteristicnumbers and may act as the site of DNA synthesis and (or) segregation.     

Reaction between isolated lambda phage DNA and membrane structures of Escherichia coli cells

The saccharose density gradient (30--55%) centrifugation technique applied to E. coli membrane preparations was used to show that treatment of the bacteria with Ca2+ in the cold results in the redistribution of the absorbed phage DNA from the cell wall to the cytoplasmic membrane while freezing-thawing of the bacteria leads to equal distribution of the infectious DNA among all membrane fractions. Quantitative estimation of such a redistribution is reported.     

Criss-crossed interactions between the enhancer and the att sites of phage Mu during DNA transposition.

A bipartite enhancer sequence (composed of the O1 and O2 operator sites) is essential for assembly of the functional tetramer of phage Mu transposase (MuA) on supercoiled DNA substrates. A three-site interaction (LER) between the left (L) and right (R) ends of Mu (att sites) and the enhancer (E) precedes tetramer assembly. We have dissected the role of the enhancer in tetramer assembly by using two transposase proteins that have a common att site specificity, but are distinct in their enhancer specificity. The activity of these proteins on substrates containing hybrid enhancers reveals a 'criss-crossed' pattern of interaction between att and enhancer sites. The left operator, O1, of the enhancer interacts specifically with the transposase subunit at the R1 site (within the right att sequence) that is responsible for cleaving the left end of Mu. The right operator, O2, shows a preferential interaction with the transposase subunit at the L1 site (within the left att sequence) that is responsible for cleaving the right end of Mu.     

Relation between the supercoiling of DNA from loach sperm and the environmental temperature

The effect of environmental temperature changes in the physiological range on DNA supercoiling in the sperm of Misgurnus fossilis L. was studied. Living fishes from the Oka and the Danube and isolated gonads were exposed to temperature changes. In the living fishes, both temperature increase from 4 to 14 degrees C and decrease from 19-21 to 14 degrees resulted in a reversible relaxation of DNA superhelices. Upon decreasing the environmental temperature from 19-21 to 4 degrees C the reversibility of changes in DNA supercoiling was not observed during the next 15 days. In isolated gonads the temperature increase from 4 to 14 degrees C had no effect on the sperm DNA supercoiling. The temperature-dependent changes in the sperm DNA supercoiling were not dependent on the loach population. It is assumed that the effect of changes in environmental temperature on the supercoiling of sperm DNA in vivo plays an important role in the activation of the genome after fertilization.     

Relation between the insulin receptor number in cells, autophosphorylation and insulin-stimulated Ras.GTP formation.

We showed previously that upon insulin stimulation of an insulin receptor overexpressing cell line, most of the p21ras was rapidly converted into the GTP bound state (Burgering, B. M. T., Medema, R. H., Maassen, J. A., Van de Wetering, M. L., Van der Eb, A. J., McCormick, F., and Bos, J. L. (1991) EMBO J. 10, 1103-1109). To determine whether this process also occurs in cells expressing physiologically relevant numbers of insulin receptors, insulin stimulated Ras.GTP formation was quantitated in Chinese hamster ovary (CHO)-derived cell lines expressing varying numbers of insulin receptors. In the parental CHO9 cells, expressing only 5.10(3) insulin receptors, insulin stimulation for 3 min increased Ras.GTP levels with 10%. Upon increasing the number of insulin receptors in these cells, Ras.GTP levels increased almost proportionally until a plateau value of 60% is reached at high receptor numbers. These data show that receptor overexpression is not a prerequisite for insulin-stimulated Ras.GTP formation. The yield of Ras.GTP generated is 0.2-1.0 mol/mol autophosphorylated insulin receptor in CHO9- and NIH3T3-derived cell lines, respectively. These values argue against signal-amplifying processes between the insulin receptor and p21ras. To determine whether receptor autophosphorylation is required for Ras.GTP formation, NIH3T3 cells overexpressing insulin receptors were stimulated with a monoclonal antibody which activates the receptor and subsequent glucose transport without inducing detectable autophosphorylation. Also, CHO cells expressing the mutant Ser1200 receptor, which has markedly impaired tyrosyl autophosphorylation but is capable of mediating insulin-stimulated metabolic effects in CHO cells, were used. In both cases, no Ras.GTP formation was observed. Furthermore, Rat-1-derived cell lines expressing mutant p21ras, which is permanently in the active GTP-bound form, still responded to insulin by increasing the glucose uptake. These results support our hypothesis that Ras.GTP formation is activated by the tyrosyl-phosphorylated insulin receptor and suggest that an active Ras.GTP complex does not mediate metabolic signaling.     

Interactions between inner membrane proteins in donor and recipient cells limit conjugal DNA transfer

Conjugation enables horizontal transmission of DNA among bacteria, thereby facilitating the rapid spread of genes such as those conferring resistance to antibiotics. Cell-cell contact is required for conjugative DNA transfer but does not ensure its success. The presence of certain plasmids in potential recipient cells inhibits redundant transfer of these plasmids from competent donors despite contact between donor and recipient cells. Here, we used two closely related integrating conjugative elements (ICEs), SXT and R391, to identify genes that inhibit redundant conjugative transfer. Cells containing SXT exclude transfer of a second copy of SXT but not R391 and vice versa. The specific exclusion of SXT and R391 is dependent upon variants of TraG and Eex, ICE-encoded inner membrane proteins in donor and recipient cells, respectively. We identified short sequences within each variant that determine the exquisite specificity of self-recognition; these data suggest that direct interactions between TraG and Eex mediate exclusion.