Conditional lethal mutants of the kilB determinant of broad host range plasmid RK2

We have examined the relationship of kilB to the other known determinants which map in the 14'-22' region of RK2. These are trfA, which encodes a diffusible replication function, and tra3, which specifies a function required for plasmid transmissibility. We found that, in addition to kilB, both tra3 and trfA functions are expressed by the cloned 14'-22' region of RK2. Four temperature-sensitive mutants of kilB were isolated by in vitro mutagenesis of the cloned segment. At 42 degrees C these mutant plasmids can be maintained in Escherichia coli cells which lack a korB+ helper plasmid. At 30 degrees C the helper plasmid is required. Our analysis of these mutants revealed that kilB function is distinct from those of trfA and tra3. One mutant plasmid was temperature-sensitive for maintenance of an RK2 ori plasmid, but this phenotype was shown to be independent of the KilB(ts) phenotype. Thus, kilB appears to be a separate new locus in this portion of the RK2 genome. In addition, these mutants allowed us to test for the existence of an essential replication determinant (trfB) in the 50.4'-56.4' region of RK2. Our results demonstrate that this region is non-essential for replication from the RK2 ori in E. coli. We propose an alternative hypothesis to explain the role of the RK2 trfB region for plasmid maintenance in E. coli.     

Experimental studies on a mutant gene (e) preventing the differentiation of eye and normal hypothalamus primordia in the axolotl

The phenotype of axolotls (Ambystoma mexicanum) homozygous for the mutant gene e (“eyeless”) is different from normal in that (1) no optic vesicles develop in $\text{e}\text{e}$ embryos, (2) $\text{e}\text{e}$ larvae from posthatching onward are darker than normal white larvae, and (3) fully grown $\text{e}\text{e}$ animals are sterile.Experiments reported here show that eyelessness in $\text{e}\text{e}$ embryos results from a direct effect of the gene on presumptive forebrain ectoderm; not on the mesoderm that induces the ectoderm to form eyes. Homotopic grafts of normal presumptive ectoderm on $\text{e}\text{e}$ blastula hosts differentiated complete eyes, but reciprocally grafted embryos were always eyeless. Similarly, grafts of either $\text{e}\text{e}$ or normal presumptive prechordal mesoderm into normal hosts gave normal eyes, but in the mutant hosts no eyes developed. Thus the e gene affects only the ectodermal component of the inductive system for eye formation.Genetically eyeless (pigmented) cells, when interspersed prior to gastrulation among genetically eyed (albino) cells in the eye preprimordium, are induced to form clones of pigmented retinal epithelium in the albino host eye.The sterility of $\text{e}\text{e}$ larvae appears also to be due to a direct effect of the e gene on the ectodermal (neural plate) primordium of the hypothalamus. Grafts of normal cells which included the hypothalamic, but not the optic or anterior pituitary primordia, always restored fertility to $\text{e}\text{e}$ recipients.The mutant pigmentation phenotype was demonstrated to be a consequence of eyelessness and, therefore, an indirect effect of the gene. The pigment pattern of normal embryos from which both optic vesicles were removed resembles that of the mutants. In addition, implantation of a single full-sized, functional eye was able to restore the normal pigmentation, but not fertility, to $\text{e}\text{e}$ recipients.     

Application of nitrous acid deamination of hexosamines to the simultaneous GLC determination of neutral and amino sugars in glycoproteins

A method is described for simultaneous gas chromatographic analysis of neutral sugars and hexosamines in glycoproteins. Sugars are hydrolyzed with the aid of Dowex 50-X2 (H⁺) resin and the resin bound glucosamine and galactosamine are deaminated with NaNO₂ to the coresponding neutral 2,5-anhydrohexoses. Hexoses and 2,5-anhydrohexoses are then quantitated as the corresponding alditol acetates. Application of the procedure to several different glycoproteins is presented.     

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.     

Polarity of microtubules of the mitotic spindle

Microtubules are polar structures that grow preferentially at one end. Measurement of their rate of directional growth can be used as a polarity indicator to determine their orientation with respect to a nucleation site. The results are interpreted to signify that the microtubules originating from the centrosomes and chromosomes of the mitotic spindle are antiparallel to each other.     

Interaction of Mg+2 with beef heart mitochondrial ATPase (F1).

The soluble mitochondrial ATPase, F₁, can be slowly inactivated by incubation with Mg⁺² in a manner consistent with the observations of Moyle and Mitchell $\text{(}\text{FEBS}\text{Lett}\text{.}\text{56}$, 55 (1975)). This inhibition results in a low initial rate of ATP hydrolysis upon addition to an ATPase assay medium of F₁ which has been incubated with Mg⁺². This inhibition, however, is completely reversible by Mg·ATP in a time dependent process and results in the rate of ATP hydrolysis increasing during the ATPase assay to reach control levels after 30 sec. The length of the lag is independent of the F₁ concentration in the ATPase assay and the lag is also completely reversed by subsequent incubation with excess EDTA before assay.F₁ is unstable if incubated with EDTA in the absence of free nucleotides or Mg⁺². The rate of inactivation increases with decreasing protein concentration until a limiting rate is reached at high dilution. Mg⁺² in excess of the EDTA or 50 μM ADP stabilize the F₁ against the inactivation but cannot reverse prior denaturation.     

Complementation analysis in Pseudomonas aeruginosa of the transfer genes of the wide host range R plasmid R18

A method of transductional complementation was developed in Pseudomonas aeruginosa to identify the cistrons involved in the conjugal transfer of the wide host range R plasmid R18. This used the P. aeruginosa bacteriophage E79tv-2 and has led to the identification of eight tra cistrons encoded by this plasmid. Plasmids mutant in six cistrons, traA, traB, traC, traD, traE, and traG were resistant to donor-specific phage (Dps^?) while traF and traH mutant plasmids retained phage sensitivity. Some traB mutants were unable to inhibit the replication of phage G101 (Phi(G101)^?) while some were also deficient in entry exclusion (Eex^?). Two traB mutants which were also Eex^? were suppressible by an amber suppressor. Three tra mutants selected directly as being Phi(G101)^? were found to be also Dps^?Eex^? and mutant in traB. These data suggest a relationship between traB, Eex, and Phi(G101). In order to facilitate future genetic comparison of the tra genes of R18 and other wide host range plasmids and the role of the host in conjugation, R18 DNA was compared with that of RP4, by restriction enzyme fragment patterns and found to be identical.     

Quantification of l-glutamine using Escherichia coli glutamate synthase: A sensitive fluorometric assay

A simple, sensitive fluorometric assay for l-glutamine is described. l-Glutamine is quantified by measuring the amount of NADPH oxidized in the presence of a glutamine-containing sample (2–20 nmol), 5 mm α-ketoglutarate, 1 mm EDTA, 63 μM NADPH, and purified Escherichia coli glutamate synthase (EC 2.6.1.53). High concentrations of l-glutamate and ammonia do not interfere. The method has been applied to quantification of l-glutamine in serum.     

Immune responses to hapten--lipopolysaccharide conjugates. 1. Modulation of in vivo antibody responses to the polysaccharide

The immune responses of mice to various lipopolysaccharides (LPS) and hapten-LPS conjugates were compared. We found that some strains of mice ($\text{A}\text{J}$ and BALB/c) produced equivalent amounts of anti-LPS antibody after the injection of either LPS or hapten-LPS conjugates. In contrast, however, other strains of mice (C57BL/6J, C3H/St, DBA/1J, DBA/2J, and Swiss) produced fewer anti-LPS-antibody-secreting cells after stimulation with hapten-LPS conjugates than did mice injected with unsubstituted LPS. The covalent coupling of hapten to LPS changed neither the mitogenic capacity nor the antigenicity of the LPS. The differences in the magnitude of antibody responses to hapten-LPS and LPS in these latter strains of mice occurred in the absence of mature T lymphocytes and was restricted to the primary immune response. Furthermore, these differential responder mice (C57BL/6J) did produce anti-LPS antibody when primed with LPS before challenge with the hapten-LPS conjugate. These data are discussed with respect to both the modulatory capacity of the hapten-LPS in the regulation of the primary immune response to LPS and the biochemical and structural requirements of the hapten-LPS conjugate for immunogenicity.     

Location of a function on RP1 that fertility inhibits Inc W plasmids

Two fertility inhibition (Fi⁺) functions which reduce R388(Inc W) transfer were detected on RP1(Inc P). Neither function affected R388-mediated surface exclusion but they could be distinguished by their effect on pilus production. One of the functions was located in the 6.5-kb Pst1-C region of RP1, part or all of which also occurs on six Fi⁺ but not two Fi^? Inc P plasmids studied.     

Glutamine synthetases from rice: purification and preliminary characterization of two forms in leaves and one form in roots

A relatively rapid five-step procedure was used in purifying to apparent homogeneity the glutamine synthetase from roots and one form of the enzyme (GS_I) from leaves of rice. The steps were: preparation of crude extracts, ammonium sulfate precipitation, filtration on Sepharose 4B, fractionation on DEAE-Sephadex A25, and affinity chromatography on ADP-Sepharose 4B. The purified protein appeared as a single band on polyacrylamide gel electrophoresis. Leaf GS_I and the second type of leaf glutamine synthetase (GS_II) formed distinct peaks when eluted from DEAE-Sephadex (step 4). The root enzyme and leaf GS_I were similar in all the properties which were examined. Both enzymes bound to ADP-Sepharose, had similar biosynthetic (18 μmol P/_img protein/min) and transferase (1324 and 1156 μmol γ-glutamyl hydroxamate/mg protein/min) activities, and the same or nearly the same K_m values for glutamate (2.17 mm), Mg²⁺ (4.5 and 5.0 mm), ATP (286 μm), NH₄⁺ (210 and 135 μm), and ADP (3.8 and 5.3 μm). In contrast, leaf GS_II did not bind to ADP-Sepharose and had much higher K_m values for glutamate (8.3 mm), Mg²⁺ (15 mm), NH₄⁺ (684 μm), and ADP (33 μm).     

The electroretinogram of adult rats following a period of postnatal undernutrition and prolonged nutritional rehabilitation

The electroretinogram (ERG) was used as a tool to estimate the recovery of physiological properties of the adult rat retina resulting from a period of postnatal undernutrition followed by prolonged nutritional rehabilitation. We obtained a characteristic ERG including negative (A) and positive (B) waves. Significant reductions in the response amplitude of the A and B wave were observed. The ratio of the first and second responses to paired photic stimuli (neuronal recovery) was essentially the same in the control and experimental animals. These results indicate that the processes controlling the ERG peak amplitude were permanently affected by a period of postnatal undernourishment, while the functional elements responsible for 1) ERG peak latency and 2) the neuronal recovery were either unaffected by postnatal nutritional deprivation or recovered during subsequent rehabilitation.     

Prostaglandin A1 and E1 inhibit the plating efficiency and proliferation of murine melanoma cells (Cloudman S-91) in soft agar

PGA₁ and PGE₁ reduced the plating efficiency and inhibited proliferation of Cloudman S-91 murine melanoma cells in a dose dependent manner, as assessed by their effects on colony formation in soft agar. PGF_2α did not reduce plating efficiency but was as effective as PGA₁ in raising cAMP and cGMP levels. This data suggests that the inhibition of Cloudman S-91 murine melanoma cell growth occurs via a non-cyclic nucleotide mechanism.     

A near-infrared method for studying hydration changes in aqueous solution: Illustration with protease reactions and protein denaturation

Proteins undergoing protease reactions, heat denaturation, or interactions with sodium dodecyl sulfate (SDS) were used to demonstrate the effectiveness of a near-infrared method for the quantitative study of changes in hydration or water binding during such processes. The spectra of different proteins showed that the liberation of $\text{COO}{}^{\mathrm{?}}\text{and NH}{}_3{}^{\mathrm{+}}$ groups during a protease reaction is associated with a large increase in hydration and excluded volume. On the basis of experiments with model compounds, other spectral changes, including development of continuum absorbance between 1.55 and 1.85 μm and a band with a peak near 2.1 μm, were also attributed to the liberation of these groups. After heat denaturation or in the presence of SDS, the rate of proteolytic hydrolysis was markedly increased, consistent with the view that some preliminary denaturation is necessary for protease activity. The validity of the hydration changes calculated for protease reactions was supported by model studies with l-lysine, and with poly-l-lysine before and after hydrolysis. The near-infrared spectrum of the protein substrate with no added protease was largely unaffected by heat treatment alone, indicating that the hydration as such was not changed to a large extent by the structural modifications of denaturation. In contrast to the protease reaction, the interactions between SDS and the proteins resulted in a decrease in hydration. Results of this paper are compared with those obtained from other methods. Some unique advantages of the near-infrared method for the study of hydration changes during reactions in aqueous solution are described.     

The mitochondrial genome of the fission yeast Schizosaccharomyces pombe. The cytochrome b gene has an intron closely related to the first two introns in the Saccharomyces cerevisiae cox1 gene

The DNA sequence of the cob region of the Schizosaccharomyces pombe mitochondrial DNA has been determined. The cytochrome b structural gene is interrupted by an intron of 2526 base-pairs, which has an open reading frame of 2421 base-pairs in phase with the upstream exon. The position of the intron differs from those found in the cob genes of Saccharomyces cerevisiae, Aspergillus nidulans or Neurospora crassa. The Sch. pombe cob intron has the potential of assuming an RNA secondary structure almost identical to that proposed for the first two cox1 introns (group II) in S. cerevisiae and the p1-cox1 intron in Podospora anserina. It has most of the consensus nucleotides in the central core structure described for this group of introns and its comparison with other group II introns allows the identification of an additional conserved nucleotide stretch. A comparison of the predicted protein sequences of group II intronic coding regions reveals three highly conserved blocks showing pairwise amino acid identities of 34 to 53%. These regions comprise over 50% of the coding length of the intron but do not include the 5' region, which has strong secondary structural features. In addition to the potential intron folding, long helical structures involving repetitive sequences can be formed in the flanking cob exon regions. A comparison of the Sch. pombe cytochrome b sequence with those available from other organisms indicates that Sch. pombe is evolutionarily distant from both budding yeasts and filamentous fungi. As was seen for the Sch. pombe cox1 gene (Lang, 1984), the cob exons are translated using the universal genetic code and this distinguishes Sch. pombe mitochondria from all other fungal and animal mitochondrial systems.     

In vivo regulation of glutamine synthetase by ammonium in the cyanobacterium Anabaena L-31

In cell-free preparations of NH₄⁺-grown cultures of the cyanobacterium Anabaena L-31 the glutamine synthetase activity is only half as much as in N₂-grown cultures. Using a procedure which enables quantitative purification of the enzyme to homogeneity it has been shown that the decrease in the enzyme activity is caused by NH₄⁺-mediated repression. Glutamine synthetase activity in both N₂-grown and NH₄⁺-grown Anabaena remains stable for more than 24 h in the presence of chloramphenicol suggesting low enzyme turnover and an enzyme half-life greater than the generation time (16–18 h) of the cyanobacterium. In N₂-grown cultures, a drastic decrease in the enzyme activity by exogenous NH₄⁺ can be discerned when fresh protein synthesis is prevented by chloramphenicol. The enzyme purified from such cultures has K_m values for NH₄⁺, glutamate Mg²⁺, and ATP similar to those observed for the enzyme from N₂- and NH₄⁺-grown Anabaena, but shows depression in V for all the substrates, leading to drastic decrease in specific activity. The modified enzyme also shows a sharper thermal denaturation profile. These results indicate that NH₄⁺-mediated modification to a less active form may be a means of regulation of glutamine synthetase in N₂-fixing cultures of Anabaena.