Modes of DNA replication primed with the oligomer of palindrome

What is the precise molecular mechanism of semi-conservative DNA replication? After the great efforts of the past 20 years, molecular biology has now established the discontinuous syntheses of daughter DNA on both of the parental strands. In order to explain this type of discontinuous replication, we introduce the concept of a palindromic primer.First we focus our attention on various oligomers (RNA or DNA) which appear usually or occasionally in the process of replication. Then we propose the palindromic nature of these oligomers so as to serve as the primer of DNA synthesis. This postulation gives a theoretical reasoning for the discontinuities of both new strands in the fork region of replication.Subsequently we consider Watson's concatemeric intermediate theory, proposed for the explanation of replicative synthesis of phage T7 DNA. By considering the contribution of some sequence-specific endonuclease(s), we suggest the existence of partial palindromic sequences of bases at the connecting region(s) in which the redundant ends of the respective phage DNA molecules are overlapping. Another theory on the replication of linear chromosomal DNA including the concept of the terminal palindromic sequence of bases is also analyzed from the viewpoint of palindromic primer. Further, some recent experimental approaches, especially on the origin(s) of DNA replication, are shown to favour the concept of a palindromic primer.     

Cloning and characterization of the alkaline phosphatase positive regulator gene (phoB) of Escherichia coli

Summary The positive regulator gene (phoB) for alkaline phosphatase of Escherichia coli was cloned into the EcoRI site of pBR322 from the E. coli chromosome by a shotgun method. phoB was then constructed in vitro by replacing the C fragment of gtC by the phoB chromosomal fragment obtained from the hybrid plasmid. When the phoB mutant was lysogenized by phoB, the lysogen became PhoB⁺. The integration site of the phage was identified by P1 phage transduction to be around phoB site on the chromosome. From these results, we conclude that the cloned gene is phoB and not a gene which suppresses phenotypically phoB mutation when it is in a multi-copy state. The restriction map was constructed. Based on this information, several PhoB deletion plasmids and smaller PhoB⁺ plasmids were constructed in vitro. By examining PhoB phenotype when these plasmids were introduced into phoB mutant, we could define the phoB gene locus in 2 kb on the restriction map of the cloned chromosomal fragment. Cells carrying the multi-copy phoB gene produced alkaline phosphatase qualitatively under normal phosphate regulation. The phoB gene product was identified by the maxicell method as a protein with a molecular weight of approximately 31,000 daltons.     

The amphipathic membrane proteins associated with gangliosides: The Paul-Bunnell antigen is one of the gangliophilic proteins

Two amphipathic protein fractions soluble in organic solvents as well as in water have been isolated from the ganglioside fraction of bovine erythrocyte membranes by successive chromatography in chloroform-methanol mixture on DEAE-Sephadex, silicic acid, and α-hydroxypropylated Sephadex G50 (LH60) columns. These two fractions contained a similar low molecular weight protein but with distinctively different amino acid composition. One of these proteins has been characterized by having a strong Paul-Bunnell antigen activity and had a binding affinity to ganglioside. A similar protein without Paul-Bunnell antigen activity was isolated as the major ganglioside-associated protein.     

Relationship between hormone content and autonomy in various autonomous tobacco cells cultured in suspension

Various autonomous cultured tobacco cells including crown gallwere examined for their contents of growth regulators by meansof Avena curvature test, cell-division induction test, and tobaccopith callus test. The crown gall cells derived from cv. Hicks produced auxin andcytokinin in the high levels of 300–500 µg IAA equivalentsand 40–80 µg kinetin equivalents per kg, respectively.The major auxin was identified as indole-3-acetic acid basedon mass spectrometry and gas chromatography. These cells alsoproduced methyl indole-3-acetate as a minor component. One ofthe cytokinins was identified as ribosyl-trans-zeatin by meansof both gas chromatography-mass spectrometry and high performanceliquid chromatography. Auxin and cytokinin activities were not detected in the followingthree suspension cultured tobacco cells: cells requiring neithercytokinin nor auxin derived from the callus of N. tabacum cv.Bright Yellow and cells requiring auxin but not cytokinin derivedfrom the calluses of cv. Bright Yellow and cv. Hicks. Theirauxin and cytokinin contents per kg were less than 1 µgIAA equivalent and less than 0.1 µg kinetin equivalent,respectively. The results obtained in this study indicate that enhanced hormonalcontent is not the only reason for autonomous growth. (Received August 16, 1979; )     

On the role of recA gene product in genetic recombination: an analysis by in vitro packaging of recombinant DNA molecules formed in the absence of protein synthesis.

Summary The role of the recA gene product of Escherichia coli in genetic recombination was examined in a system where recombination takes place in the absence of protein synthesis. recA200 bacteria were infected with two mutant strains of phage lambda in the presence of chloramphenicol and rifampin, and the resulting recombinant DNA molecules were measured by in vitro packaging. When recA200 bacteria grown at a temperature that is permissive for RecA phenotype were transferred to a temperature that is restrictive for RecA phenotype in the presence of the inhibitors, recombination of the infecting phages was severely blocked. This result shows that the recombination activity of the recA200 cells is inactivated by the change of temperature even in the absence of protein synthesis. The most likely explanation of this result is that the recA protein is directly involved in the recombination detected in the presence of chloramphenicol and rifampin.     

Mass fragmentographic determination of lofepramine and its metabolites in human plasma and urine using deuterated internal standards

A sensitive and specific method for the determination of lofepramine and its metabolites, desipramine and 2-hydroxydesipramine, in human plasma and urine is described. Lofepramine, desipramine and 2-hydroxydesipramine were derivatized to ethyl p-chlorobenzoate, the bis(heptafluorobutyryl) derivative and the N,O-bis(trifluoroacetyl) derivative, respectively, and then analysed by gas chromatography—mass fragmentography. Corresponding deuterated compounds were used as internal standards. Determination was possible at levels as low as 2 ng/ml for lofepramine and desipramine and 20 ng/ml for 2-hydroxydesipramine.     

Mechanism of metabolic regulation in photoassimilation of propionate in Euglena gracilis z

Euglena gracilis showed a typical photoassimilation of propionate when cultured on propionate as a sole carbon source. While the acid is metabolized by the methylmalonyl-coenzyme A (CoA) pathway under illumination, supporting growth of Euglena (K. Hosotani, A. Yokota, Y. Nakano, and S. Kitaoka, 1980, Agr. Biol. Chem.44, 1097–1103), it does not allow the protozoon to grow in the dark although it was actively taken up and metabolized. Kinetics of incorporation of radioactivity of labeled propionate, trapping effect of exogenous lactate in the incorporation of labeled propionate and radiorespirometric pattern revealed that propionate was metabolized by the lactate pathway in Euglena in the dark. Enzymes involved in the lactate pathway were located in mitochondria. The reason why Euglena can not grow on propionate in the dark is explained by the failure of producing C₄ dicarboxylic acids essential for biosynthesis of amino acids and sugars, like the mitochondrial oxidation of fatty acids in higher animals. The Euglena cells cultured in the dark contained enzymes of both methylmalonyl-CoA and lactate pathways, but lack of photosynthetically generated ATP has been suggested to force Euglena to select the less-ATP-requiring but futile pathway.     

Different responses of CR(−) and CR(+) B cells to LPS stimulation

Proliferative response of B cells with or without CR [CR(+) or CR(?) B cells] was compared in their polyclonal response when they were stimulated with lipopolysaccharide (LPS). CR(+) B cells responded better in proliferation and more poorly in polyclonal antibody formation than did CR(?) B cells. The dissociation between proliferation and antibody formation in LPS response was not due to the shift of the time kinetics nor the exhaustion of the culture medium. T cells and macrophages did not take part in the dissociation, since macrophage depletion from nu/nu mouse spleen cells could not modify the dissociation. The polyclonal antibody response of CR(?) B cells was more resistant to irradiation than that of CR(+) B cells. These results suggest that among LPS-responsive B cells there are CR(?) B-cell subset(s) more mature than CR(+) B cells.     

Molecular cloning and nucleotide sequence determination of the regulator region of mecA gene in methicillin-resistant Staphylococcus aureus (MRSA).

Molecular cloning and nucleotide sequence analysis were performed for the identification of the regulator genes of methicillin resistance in the genome of a MRSA strain N315. Two open reading frames (orfs) were identified in the 5'-flanking region of the mecA gene. Predicted amino acid sequences of these orfs showed extensive homology to the co-inducer and the repressor protein of the penicillinase (PCase) production in Staphylococcus aureus as well as in Bacillus licheniformis. These orfs are considered to encode putative co-inducer and repressor proteins specific for the regulation of methicillin resistance in MRSA.