A non-alkalophilic mutant of Bacillus alcalophilus lacks the Na+/H+ antiporter.

A non-alkalophilic mutant strain of $\text{Bacillus}\text{alcalophilus}$ grows on L-malate over a pH range from 5.0 to 9.0. The mutant does not exhibit the energy-dependent efflux of Na⁺ that has been used to assay a $\text{Na}{}^{\mathrm{+}}\text{H}{}^{\mathrm{+}}$ antiporter in the wild type organism. The mutant also fails to transport α-aminoisobutyric acid, at pH 9.0, either in the presence or absence of Na⁺; at pH 5.5, the amino acid analogue is taken up by a Na⁺-independent mechanism. The properties of the mutant constitute strong evidence that the $\text{Na}{}^{\mathrm{+}}\text{H}{}^{\mathrm{+}}$ antiporter is involved in maintaining an acidified cytoplasm in $\text{B}\text{.}\text{alcalophilus}$.     

Distinct nuclear genes for yeast mitochondrial and cytoplasmic methionyl-tRNA synthetases.

Total methionine-tRNA synthetases from wild type $\text{Saccharomyces}\text{cerevisiae}$ can be fractionated on hydroxylapatite into two peaks: Peak I is the mitochondrial, peak II the cytoplasmic isoenzyme. The specificity towards various tRNAs and the antigenic determinants are not identical. A mutant strain, known for its altered cytoplasmic enzyme, contains a mitochondrial species with the same properties as the wild type mitochondrial enzyme, as well as a cytoplasmic isoenzyme with a K_M for methionine about 300 times higher than the corresponding wild type enzyme. Another strain, obtained by back-crossing the mutant with a wild type strain, retains the enzyme pattern found in the mutant. The results are in favor of two distinct nuclear genes for yeast mitochondrial and cytoplasmic methionyl-tRNA synthetases.     

DNA synthesis during the division cycle of three substrains of Escherichia coliBr

The relationship between chromosome replication and the bacterial division cycle has been examined in three substrains of Escherichia coli$\text{B}\text{r}$ obtained from different sources and designated $\text{B}\text{r}$ A, $\text{B}\text{r}$ F and $\text{B}\text{r}$ K. At growth rates greater than 1.0 doubling per hour (μ > 1.0), the time for a round of chromosome replication (C) was 42 minutes in all three substrains, but the time between the end of a round and cell division (D) was 22 minutes in $\text{B}\text{r}$ A, 16 minutes in $\text{B}\text{r}$ F and 14 minutes in $\text{B}\text{r}$ K. At slower growth rates C and D increased, but to significantly different extents in the three substrains. When μ = 0.5, C and D were approximately 80 and 40 minutes in $\text{B}\text{r}$ A, 60 and 20 minutes in $\text{B}\text{r}$ F, and 70 and 20 minutes in $\text{B}\text{r}$ K.As a consequence of the lengths of the C and D periods in the three stocks of E. coli$\text{B}\text{r}$, the patterns of chromosome replication during the division cycle differed. The most obvious difference was that E. coli$\text{B}\text{r}$ F and E. coli$\text{B}\text{r}$ K possessed periods devoid of DNA synthesis at both the beginning and the end of the division cycle during slow growth, whereas E. coli$\text{B}\text{r}$ A contained only one period devoid of DNA synthesis at the end of the cycle.     

A mutant of Escherichia coli with altered inducer specificity for the fad regulon

A novel regulatory mutant of the fatty acid degradation ($\text{fad}$) regulon of $\text{Escherichia coli}$ was isolated. This mutant, D-2, was induced to synthesize the fatty acid β-oxidation enzymes during growth on decanoate and laurate whereas the wild type strain was induced only when fatty acids with a chain length greater than 12 carbon atoms were present in the growth medium. The fatty acid specificity of the acyl CoA synthetase was also changed in strain D-2. The data are consistant with the hypothesis that acyl CoA's themselves are the inducers of the $\text{fad}$ regulon and suggest that strain D-2 may synthesize an altered $\text{fad}$ regulatory protein. The results also suggest that the acyl CoA synthetase may possess regulatory as well as enzymatic activity.     

Repair of deaminated cytosine residues of DNA: biological significance of the absence of uracil from DNA.

Uracil-DNA glycosylase of $\text{Bacillus}\text{subtilis}$ is involved in repair of deaminated cytosine residues of DNA. Survivals of SPO2 phage after treatment with bisulfite and weak alkali are considerably higher in wild type strains than in $\text{urg}$ mutants, which are deficient in the enzyme activity, whereas survivals of bisulfite/alkali-treated PBS1 phage in the two types of cells are essentially the same. The spontaneous mutation frequency of a $\text{urg}$ mutant is three fold higher than is that of a wild type strain.     

Resistance to the peptide-like antibiotic negamycin in Escherichia coli

An $\text{Escherichia coli}$ mutant resistant to the peptide-like antibiotic negamycin carries a mutation, NEG40, which maps at minute 65 on the bacterial genome. Termination of protein synthesis, which is normally blocked by negamycin in wild type cellular extracts, continues with cellular extracts from the mutant in the presence of the drug; indeed, release of complete peptides from the polysomes still proceeds over a wide range of drug concentrations. The data suggest that the NEG40 mutation affects one of the components of the termination complex (ribosome or release factor).     

The src gene product (pp60 src) of avian sarcoma virus rapidly induces DNA synthesis and proliferation of calcium-deprived rat cells

A general characteristic of neoplastic cells, but not their non-neoplastic counterparts, is the ability to proliferate in calcium-deficient medium. NRK cells infected with the transformation-defective, temperature-sensitive, ASV mutant, tsLA23, were unable to proliferate in calcium-deficient medium at the non-permissive 40°C, but they very rapidly initiated DNA synthesis (within 1 hour) and resumed proliferation in this medium after being shifted to 36°C, a temperature permissive for the production of active pp60^src and for neoplastic transformation. These observations suggest that activated pp60^src acts near the $\text{G1}\text{S}$ transition point in the cell cycle to bypass or stimulate a calcium-dependent mechanism required for the initiation of DNA synthesis, which enables the cells to display the neoplastic property of proliferating in calcium-deficient medium.     

Denovo synthesis of prolactin by human decidua

Relatively large amounts of immunoreactive prolactin were measured in homogenates of human decidual tissue obtained immediately after delivery of normal term pregnancies. In order to study the release and possible synthesis of prolactin by this tissue, explants of decidua were incubated for 24 hours at 37°C in oxygenated Gey's buffer containing 20% fetal calf serum. When cycloheximide was added to the medium in concentrations sufficient to prevent $\text{in}$$\text{vitro}$ protein synthesis, 85–90% of the prolactin present in the tissue was released into the medium during the first 3 hours of incubation. No additional prolactin accumulated in either the medium or the tissue during the remainder of the incubation period. In the absence of cycloheximide, the prolactin concentration in the medium increased progressively during incubation, so that after 24 hours the total amount of hormone present in the tissue and medium was significantly greater than that in the tissue and medium prior to incubation (37.6 ± 9.6 ng/ml at 0 time vs 82.2 ± 7.7 ng/ml at 24 hours). When ³H-1-leucine (100 u Ci) was supplied during incubation, radioactive proteins were detected in the medium at 24 hr, 14–20% of which were specifically precipitated by antiserum to human pituitary prolactin. When aliquots of this medium were chromatographed on Sephadex G-100, 80–95% of the ³H-proteins precipitated by antiserum to pituitary prolactin eluted in the same position as did purified, iodinated pituitary prolactin. These data indicate that a species of prolactin which is identical to pituitary prolactin by the criteria of immunoprecipitation and gel chromatography is synthesized by human decidual tissue $\text{in}$$\text{vitro}$.     

Role of proteins and lipids in non-linear Arrhenius plots of Drosophila mitochondrial succinate-cytochrome c reductase studied by rebinding experiments

Abrupt changes in the Arrhenius activation energy of membrane-bound enzymes have often been correlated with changes in the physical state of membrane phospholipids. Similar changes in activation energy have also been found in soluble enzymes. The possibility exists, therefore, that in some of the membrane-bound enzymes the changes might reflect intrinsic changes of the proteins independent of changes in the membrane phospholipids. This hypothesis was investigated using Drosophila mitochondria isolated from wild type and the mutant Ocd^ts-1. In this mutant it has been shown that succinate-cytochrome $\text{c}$ reductase exhibits a change in Arrhenius activation energy at 18°C which is not found in the wild type (Sondergaard, L., Nielsen, N.C. and Smillie, R.M. (1975) FEBS lett. 50, 126–129). A quantitative thin-layer chromatographic analysis of mitochondrial phospholipids showed sphingomyelin to be more abundant in the wild type than in the mutant (5.2% and 4.3% of the total phospholipids, respectively). Since it was shown that the succinate-cytochrome $\text{c}$ reductase had a lipid requirement for full activity, reciprocal rebinding experiments were done. These experiments showed that the reconstituted membranes exhibited the change in activation energy at 18°C only when the protein moiety came from mutant mitochondria, that is, the change was independent of the source of the phospholipids used.     

Binding of the structural protein soc to the head shell of bacteriophage T4

Qβ plus strands with a 70 S ribosome bound to the coat cistron initiation site were used as template for Qβ replicase. Minus strand synthesis proceeded until the replicase reached the ribosome. The ribosome was removed and elongation was continued in a substrate-controlled, stepwise fashion. The nucleotide analog N⁴-hydroxyCMP was introduced into the positions complementary to the third and fourth nucleotides of the coat cistron. The minus strands were elongated to completion, purified and used as template for Qβ replicase. The final plus strand preparation consisted of four species, with the sequences -A-U-G-G- (wild type), -A-U-A-G- (mutant C₃), -A-U-G-A- (mutant C₄) and -A-U-A-A- (mutant $\text{C}{}_3\text{C}{}_4$) at the coat initiation site. The ribosome binding capacity of the mutant RNAs relative to wild type was <0.1 (C₃), 3.2 (C₄) and 0.3 ($\text{C}{}_3\text{C}{}_4$). The finding that mutant C₃ no longer formed an initiation complex suggests that the interaction of the ribosome binding site with fMet-tRNA plays an essential role in the formation of the 70 S initiation complex. The fact that mutant C₄ RNA bound more efficiently than wild type, and that mutant $\text{C}{}_3\text{C}{}_4$ RNA showed substantial ribosome binding capacity whereas the single mutant C₃ did not, can be explained by assuming that an A residue following the A-U-G triplet interacts with a complementary U residue in the anticodon loop sequence. In the case of $\text{C}{}_3\text{C}{}_4$ this additional base-pair may offset the reduced codon-anticodon interaction resulting from the modification of the A-U-G codon.     

Mutational alteration of Bacillus subtilis DNA polymerase 3 to hydroxyphenylazopyrimidine resistance: polymerase 3 is necessary for DNA replication

A spontaneous mutant of $\text{Bacillus}\text{subtilis}$ resistant to killing by two hydroxyphenylazopyrimidines has been isolated. The DNA polymerase III of this mutant is resistant to inhibition by these drugs. The K_i for 6-(p-hydroxyphenylazo)-uracil (HPUra) is 20 μM, about 40 times higher than the K_i of the wild-type enzyme. The mutant and wild-type polymerases behave similarly during purification, are sensitive to N-ethylmaleimide and to 0.1 M KCl, and have the same K_m for dGTP (0.5 μM). The HPUra inhibition of both enzymes is attenuated competitively by dGTP. We conclude that polymerase III is the target for hydroxyphenylazopyrimidines $\text{in}\text{vivo}$, and since the drugs specifically inhibit replicative DNA synthesis, polymerase III is necessary for DNA replication.     

Induction by ouabain of hemoglobin synthesis in cultured friend erythroleukemic cells

Induction of erythroid differentiation in ouabain-resistant murine erythroleukemia cells by ouabain is reported. Ouabain induction results in the appearance of hemoglobin-containing cells 12–24 hr earlier than induction of the same clone by dimethyl sulfoxide. The levels of globin mRNA after ouabain induction are similar in amount to the globin mRNA levels observed after induction by dimethyl sulfoxide. The concentration of ouabain required to induce hemoglobin synthesis depends upon the K⁺ ion levels in the culture medium. Lowering the extracellular K⁺ ion concentration 2–4 fold reduced by 10–40 fold the ouabain concentration necessary for the induction of hemoglobin synthesis. In low K⁺ medium (1.8 mM), ouabain is an effective inducer of hemoglobin synthesis at a concentration of 0.02 mM. This K⁺ effect is specific for ouabain induction, since induction by other inducers, such as dimethyl sulfoxide and dimethyl acetamide, does not exhibit this marked sensitivity to the levels of K⁺ ions in the culture medium. These results suggest that the binding of ouabain to the plasma membrane enzyme, $\text{Na}\text{K}$ ATPase, is required for the induction of erythroid differentiation by ouabain. A small but significant proportion of wild-type, ouabain-sensitive cells also can be induced by ouabain, below ouabain concentrations that are toxic to these cells. The observation that the binding of ouabain to the $\text{Na}\text{K}$ ATPase induces hemoglobin synthesis suggests that changes in the intracellular concentration of K⁺ ions may be involved in the control of erythroid differentiation in Friend erythroleukemic cells.     

DNA synthesis and the cell cycle in cultures of normal and mutant (mdg/mdg) embryonic mouse muscle cells.

The relationship between cell fusion, DNA synthesis and the cell cycle in cultured embryonic normal and dysgenic ($\text{mdg}\text{mdg}$) mouse muscle cells has been determined by autoradiography. The experimental evidence shows that the homozygous mutant myotubes form by a process of cell fusion and that nuclei within the myotubes do not synthesize DNA or undergo mitotic or amitotic division. The duration of the total cell cycle and its component phases was statistically the same in 2-day normal and mutant ($\text{mdg}\text{mdg}$) myogenic cultures with the approximate values: T, 21.5 hr; G₁, 10.5 hr; S, 7.5 hr; and G₂, 2.5 hr. In both kinds of cultures, labeled nuclei appeared in myotubes 15–16 hr after mononucleated cells were exposed to [³H]thymidine, and the rate of incorporation of labeled nuclei into multinucleated muscle cells was comparable in control and dysgenic cultures. Thus, homozygous $\text{mdg}\text{mdg}$ muscle cells in culture are similar to control cells with respect to their mechanism of myotube formation and the coordinate regulation of DNA synthesis and the cell cycle during myogenesis.     

Immunological properties of membrane fractions from wild type and dnaA mutants of Escherichia coli

Membrane vesicles from Escherichia coli wild type and an otherwise isogenic $\text{dna}\text{A}$ mutant were used to immunize rabbits. In addition, a membrane protein fraction, containing the material found deficient in $\text{dna}$A mutants, was purified by preparative polyacrylamide gel electrophoresis in sodium dodecylsulfate, and used for immunization. The antisera produced were analyzed by immunoelectrophoresis and immunofluorescence microscopy. The antisera obtained by immunization with membrane vesicles from either wild type or $\text{dna}\text{A}$ mutant membrane preparations were qualitatively similar in the precipitin bands seen after immunoelectrophoresis. The antisera obtained by immunization with the purified protein fraction contained a subset of the antibodies seen when whole vesicles were used for immunization. In a semiquantitative precipitin assay, the antisera prepared against whole membrane vesicles or the isolated protein fraction both caused the precipitation of more protein from sodium dodecylsulfate-solubilized membranes of wild type than of $\text{dna}\text{A}$ mutants. No difference was seen by immunoelectrophoresis between the protein composition of wild type or $\text{dna}\text{A}$ membrane preparations. Thus, the $\text{dna}\text{A}$ mutant appears to differ from the wild type in the quantitative composition of its membrane proteins, whereas no qualitative differences were detected.Fluorescein-conjugated antiserum preparations were employed to assess the reactivity of intact cells, spheroplasts and membrane vesicles with the antisera studied above. Wild type cells of E. coli have a barrier to reaction with the antisera; this barrier is removed when the cells are converted to spheroplasts or to membrane vesicle. Similarly, a highly permeable mutant of E. coli permits reaction of the antisera with unaltered cells. Antisera to both whole membrane vesicles and to the isolated protein fraction react identically with the cellular and subcellular preparations. Thus, antisera prepared from membrane proteins isolated after sodium dodecylsulfate-polyacrylamide gel electrophoresis can still recognize some antigens present in membrane vesicle preparations.     

An Escherichia coli K12 mutant carrying altered ribosomal protein (S10).

An $\text{Escherichia coli}$ mutant (JE14373) carrying decreased stability of stable RNA species was found to have altered electrophoretic mobility of a 30S ribosomal protein (S10). Recombinants covering $\text{str}$ gene (76 min on $\text{E. coli}$ linkage map by Bachmann, Low and Taylor, 1976 (ref. 1)) obtained from a cross of CSH64 × JE14373, restored normal S10 protein. The size analysis of RNAs labeled for 15 min with [³H]uridine showed 50 to 60 % decrease of 16S RNA in this mutant strain, but almost no decrease of 23S RNA at 10 or 40 min after addition of rifampicin. On the other hand, no change was observed in the stability of both rRNA pieces in its parental PA3092 strain even at 40 min after addition of rifampicin.     

A mutant of Pseudomonas putida with altered regulation of the enzymes for degradation of phenol and cresols

A mutant strain of $\text{Pseudomonas putida}$ NCIB 10015 has been isolated that is unable to utilize $\text{para}$-cresol as a sole carbon source. The mutant strain grows very poorly when $\text{meta}$-cresol is to be utilized but grows rapidly at the expense of phenol or $\text{ortho}$-cresol. Preliminary evidence is presented that this mutant strain has a regulatory element of altered specificity. This regulatory element determines the induction of all known enzymes involved in the metabolism of phenol and $\text{ortho, meta}$ and $\text{para}$-cresol. This is the first report of a mutation affecting the regulation of meta-cleavage enzymes.     

Genetical and biochemical evidence that a novel dinucleoside polyphosphate coordinates salvage and de novo nucleotide biosynthetic pathways in mammalian cells

Studies with ‘wild type’ Chinese hamster ovary cells and mutant derivatives defective in purine salvage and $\text{de novo}$ nucleotide biosynthesis pathways have brought to light the possibility that an unusual dinucleoside polyphosphate, HS-3 (see appendix) is a crucial regulator of these two pathways. Three antitumor drugs, methotrexate, 5-fluorouracil and azaserine as well as L-glutamine, purines and pyrimidines were used to define the loci of HS-3 metabolism. Wild type and salvage pathways mutants accumulated HS-3 in the absence of glutamine. $\text{De novo}$ pathways mutant accumulated HS-3 only when purine was absent. Depletion of HS-3 was induced in wild type and $\text{de novo}$ mutant cell lines by purine compounds. Salvage pathways mutants did not cause depletion of HS-3 when supplied with purines or pyrimidines, except 5-fluorouracil. Data indicate that HS-3 is probably synthesised when an early step in purine nucleotide synthesis is blocked and depleted when the salvage pathways are operative. HS-3 may be an important factor in certain diseases involving nucleotide metabolism.     

The sites of synthesis and the subsequent migration of newly synthesized protein in retina

Radioautographs of rabbit retinas fixed immediately after a 1 or 2 min exposure in vitro to ³H leucine revealed high rates of protein synthesis in receptor cell inner segments, perikarya of ganglion cells, and cells of the inner nuclear layer. If these brieflly labelled retinas were returned to unlabelled medium for periods of up to 6 hr, the radioautographs revealed a progressive dispersion of the labelled proteins from their sites of synthesis. This was largely completed by $\text{1}\text{1}\text{2}$ hr and appeared, in one instance at least, to involve processes other than simple diffusion. Superimposed on the dispersive phenomenon was a process of concentration of the newly formed proteins at two sites quite distant from their synthesis, that was apparent after $\text{1}\text{1}\text{2}$hr. One of these sites was the receptor cell outer segments, as has been previously described, the other was the outer plexiform layer.     

Preferential synthesis of yeast mitochondrial DNA in alpha factor-arrested cells

α factor is a diffusible substance produced by $\text{S. cerevisiae}$ cells of the $\text{α}$ mating type which inhibits cell division (1) and the initiation of nuclear DNA synthesis (2) in cells of the $\text{a}$ mating type. In this report, it is shown that mitochondrial DNA synthesis continues at a normal rate in $\text{a}$ cells for at least 6 hours in the presence of α factor, resulting in a 5-fold increase in the amount of mitochondrial DNA per cell. The continued synthesis of mitochondrial DNA in the absence of nuclear DNA synthesis allows specific labeling of yeast mitochondrial DNA.     

Initiation of chromosome replication in Escherichia coli. I. Requirements for RNA and protein synthesis at different growth rates

The effects of inhibition of protein and RNA synthesis on initiation of chromosome replication in Escherichia coli$\text{B}\text{r}$ were determined by measuring rates of DNA synthesis during the division cycle before and after addition of chloramphenicol and rifampicin. The ability of cells to initiate a round of replication depended upon the pattern of chromosome replication during the division cycle. Initiation in the presence of chloramphenicol (200 μ/ml) and rifampicin (100 gmg/ml) was observed only in slowly growing cells which normally initiated a new round between the end of the previous round and the subsequent division (i.e. in the D period of the division cycle). The cells that initiated were in the D period at the time of addition of the drugs. Rapidly growing cells which normally initiated before the D period and slowly growing cells which normally initiated after the D period did not initiate in the presence of the drugs. The contrasting effects of the drugs in cells possessing different chromosome replication patterns, and the coupling between septum-crosswall formation (the D period) and initiation suggest that the timing of initiation of chromosome replication in E. coli is controlled by the cell envelope.