In Vitro HIV-1 LTR Integration into T-Cell Activation Gene CD27 Segment and the Decoy Effect of Modified-Sequence DNA

Integration into the host genome is an essential step in the HIV-1 life cycle. However, the host genome sequence that is favored by HIV-1 during integration has never been documented. Here, we report that CD27, a T cell activation gene, includes a sequence that is a target for in vitro HIV-1 cDNA integration. This sequence has a high affinity for integrase, and the target nucleotides responsible for this higher affinity were identified using a crystal microbalance assay. In experiments involving a segment of the CD27 gene, integration converged in the target nucleotides and flanking sequence DNA, indicating that integration is probably dependent upon the secondary structure of the substrate DNA. Notably, decoy modified CD27 sequence DNAs in which the target nucleotides were replaced suppressed integration when accompanying the original CD27 sequence DNA. Our identified CD27 sequence DNA is useful for investigating the biochemistry of integrase and for in vitro assessment of integrase-binding inhibitors.     

The developmental fate of Dictyostelium discoideum cells depends greatly on the cell-cycle position at the onset of starvation

The relationship between the development of Dictyostelium discoideum Ax-2 and the cell cycle at the onset of starvation was analysed with special reference to sorting behaviors during the formation of polarized cell masses (slugs), using a method for inducing good synchrony. Cells starved at different cell-cycle positions showed different developmental features during further culture. For example, cells just before mitosis and dividing cells were sorted out into the anterior prestalk zone of migrating slugs, while cells starved during most of the G2-phase, into the posterior prespore zone. Time courses of cell aggregation and tip formation were also found to vary greatly in a cell-cycle-related manner, and cells starved during the late G2-phase showed the most rapid development. Differential chemotaxis and cohesiveness are generally considered to be important for cell sorting in Dictyostelium development. In fact, remarkable differences in the chemotactic ability to a chemoattractant, cAMP, were detected among cells starved at any particular phase of the cell cycle. EDTA-resistant cohesiveness was also acquired differently depending on the cell cycle, and it was stronger in the cells showing more rapid aggregation. These findings indicate a close relation of the cell cycle to the cell sorting and pattern formation. The possible significance of the cell-cycle-related events presented here is discussed, with special emphasis on the process of cell aggregation.     

Requirement of the Escherichia coli dnaA gene function for ori-2-dependent mini-F plasmid replication.

The mini-F plasmids pSC138, pKP1013, and pKV513 were unable to transform Escherichia coli cells with a dnaA-defective mutation under nonpermissive conditions. The dnaA defect was suppressed for host chromosome replication either by the simultaneous presence of the rnh-199 (amber) mutation or by prophage P2 sig5 integrated at the attP2II locus on the chromosome, both providing new origins for replication independent of dnaA function. The dnaA mutations tested were dnaA17, dnaA5, and dnaA46. dnaA5 and dnaA46 are missense mutations. dnaA17 is an amber mutation whose activity is controlled by the temperature-sensitive amber suppressor supF6. Under permissive conditions in which active DnaA protein was available, the mini-F plasmids efficiently transformed the cells. However, the transformants lost the plasmid as the cells multiplied under conditions in which DnaA protein was inactivated or its synthesis was arrested. As controls, plasmids pSC101 and pBR322 were examined along with mini-F; pSC101 behaved in the same manner as mini-F, showing complete dependence on dnaA for stable maintenance, whereas pBR322 was indifferent to the dnaA defect. Thus, ori-2-dependent mini-F plasmid replication seems to require active dnaA gene function. This notion was strengthened by the results of deletion analysis which revealed that integrity of at least one of the two DnaA boxes present as a tandem repeat in ori-2 was required for the origin activity of mini-F replication.     

cAMP in Anabaena cylindrica: Rapid Changes in Cellular Levels in Response to Changes in Extracellular Environments

The cellular level of cyclic 3',5'-AMP (cAMP) in the cyanobacteriumAnabaena cylindrica changed transiently in response to changesin extracellular environments. When the cells were transferedfrom dark to light, or anaerobic to aerobic conditions in thedark, the cAMP level rapidly decreased within one min and thengradually recovered. Addition of carbonyl cyanide m-chlorophenylhydrazone(CCCP) which inhibits ATP synthesis caused an increase in cAMPlevel in the light but not in the dark. The level of cAMP increasedseveral fold by lowering the pH from 8 to 6. On the contrary,a rise of pH from 6 to 8 caused a decrease in the cAMP level.It is suggested that the change in membrane electrochemicalpotential is involved in the regulation of cellular cAMP concentration. (Received January 27, 1989; Accepted June 19, 1989)     

Difference between amino acid residues in the metal-ligand environments of iron- and manganese-superoxide dismutases

Alignment of the amino acid sequences of the Pseudomonas ovalis and Photobacterium leiognathi iron-superoxide dismutases (Fe-SODs) with the known sequences of the manganese-superoxide dismutases (Mn-SODs) shows that both types of SOD are highly homologous (33-53% identity) and share residues for the metal coordination. The amino acid residues that form the environment of the metal ions appear to be also conserved between the Fe- and Mn-SODs, except that the Phe-84 and Gln-154 in the Mn-SODs are replaced by Tyr and Ala, respectively, in the Fe-enzymes. Since this latter residue contributes to formation of the hydrophobic metal-ligand environment through hydrogen bonding with Trp-133 and Tyr-34 in the Mn-SODs, its substitution by Ala should cause different micro environments between the metal centers of the Fe- and Mn-SODs. This difference may account for the metal specificity of both types of SODs demonstrated by previous reconstitution experiments.     

Pharmacological and biochemical characteristics of partially purified GABAB receptor

Pharmacological and biochemical characteristics of the partially purified -aminobutyric acid (GABA)_B receptor using baclofen affinity column chromatography have been examined. The Scatchard analysis of [³H]GABA binding to the purified GABA_B receptor showed a linear relationship and the K_D and B_max values were 60 nM and 118 pmol/mg of protein, respectively. Although GTP and Mg²⁺ did not affect on the GABA_B receptor binding, Ca²⁺ significantly increased [³H]GABA binding to the purified GABA_B receptor in a dose-dependent manner and showed its maximum effect at 2 mM. The enhancement of the binding by Ca²⁺ was found to be due to the increase of Bmax by the Scatchard analysis. The treatments with pronase and trypsin significantly decreased the binding of [³H]GABA, but phospholipase A₂ had no significant effect on the binding. In addition, treatment with glycosidases such as glycopeptidase A and -galactosidase significantly decreased the binding of [³H]GABA to the purified GABA_B receptor. These results suggest that purification of the solubilized GABA_B receptor by the affinity column chromatography may result in the functional uncoupling of GABA_B receptor with GTP-binding protein. Furthermore, the present results suggest that cerebral GABA_B receptor may be a glycoprotein and membrane phospholipids susceptible to phospholipase A₂ treatment may not be involved in the exhibition of the binding activity.Special issue dedicated to Dr. Eugene Roberts.     

The effects of phosphorylation and dephosphorylation of brain myosin on its actin-activated Mg2+-ATPase and contractile activities

Purified bovine brain myosin contained approximately 1 and 3 mol of protein-bound phosphate/mol myosin in the light chains and heavy chains, respectively. Large portions of this light chain- and heavy chain-bound phosphate (about 0.8 and 2.4 mol, respectively) were removed by incubation with a brain phosphoprotein phosphatase and potato acid phosphatase, respectively. Upon phosphorylation of the dephosphorylated brain myosin with myosin light chain kinase and casein kinase II, about 1.6 and 3.0 mol of phosphate was incorporated into the light chains and heavy chains, respectively, while much lower levels of phosphate were incorporated into the non-dephosphorylated brain myosin under the same conditions. The actin-activated Mg2+-ATPase activity of brain myosin rephosphorylated with myosin light chain kinase was about twice as high as that of dephosphorylated brain myosin (about 30 and 15 nmol phosphate/mg/min, respectively). On the other hand, whereas the rephosphorylated brain myosin superprecipitated rapidly with F-actin, the rate of superprecipitation of the dephosphorylated brain myosin was extremely low. Under appropriate conditions, a loose network of tiny superprecipitates, which formed initially throughout the solution, contracted to form eventually a large and dense particle. These results indicate that phosphorylation of the light chains of brain myosin is a prerequisite for the contraction of brain actomyosin. The role of phosphorylation of the heavy chains by casein kinase II remains to be elucidated.     

Nucleotide sequence and gene organization of ColE1 DNA

The primary structure of the plasmid ColE1 DNA has been determined. The plasmid DNA consists of 6646 base pairs (molecular mass of 4.43 MDa) and is 48.46% in GC content. The phi 80 trp insert of the composite plasmid of ColE1, pVH51, has also been determined. The determination of the nucleotide sequence of ColE1 DNA provides the basis for examining the relationships between the DNA sequence and the gene organization of the plasmid. The focus of this paper is to use this sequence data coupled with a review of the literature and our own work to examine the nine known functional regions of ColE1: imm (colicin E1 immunity), rep (replication function), inc (plasmid incompatibility and copy number control), bom (basis of mobility), rom (modulator of inhibition of primer formation by RNA I), mob (plasmid mobilization), cer (determinant for conversion of plasmid multimers to monomers), exc (plasmid entry exclusion), cea (structural gene for colicin E1), and kil (structural gene for the Kil protein).     

Structure and antitumor activity of an alkali-soluble polysaccharide from Cordyceps ophioglossoides

A water-insoluble extracellular glucan (CO-1) was isolated from the precipitate formed on incubation of the culture filtrate of Cordyceps ophioglossoides at 37 degrees for 19 h. CO-1 was homogeneous as judged by h.p.l.c., electrophoresis, and ultracentrifugation, and the average molecular weight was determined by h.p.l.c. to be 632,000. The 1H- and 13C-n.m.r. and the i.r. spectra indicated that the glucosidic linkages in CO-1 were beta. From the results of methylation analysis, Smith degradation, n.m.r. studies, and enzymic hydrolysis, it was concluded that CO-1 is composed of a backbone of (1----3)-linked beta-D-glucopyranosyl residues with a beta-D-glucopyranosyl group attached to O-6 of every second D-glucopyranosyl residue of the backbone. CO-1 strongly inhibited the growth of Sarcoma 180 solid-type tumor. CO-1 polyalcohol, which was prepared by Smith degradation of CO-1, exhibited more-potent antitumor activity than CO-1. The absorption maximum of Congo Red shifted significantly in the presence of CO-1.