Isolation and mapping of ribosomal RNA genes of Caulobacter crescentus

Ribosomal DNA fragments of 1.0, 3.4, 3.7 and 6.1 kb² produced by EcoRI digestion of the Caulobacter crescentus genome were identified by hybridization to a labeled ribosomal RNA probe. These genomic sequences were further characterized by the isolation of 13 hybrid λ Charon 4 phages with rDNA inserts, and two of the recombinant phages, Ch4Cc773 and Ch4Cc1880, were examined extensively. The Cc773 insert contains EcoRI fragments of 1.0 kb, 3.4 kb and 3.7 kb and the Cc1880 insert contains EcoRI fragments of 1.0 kb, 3.4 kb and 6.1 kb that hybridized to ³²P-labeled rRNA. Thus, the two clones contain different DNA inserts which together account for all of the rDNA fragments detected in digests of the C. crescentus genome. Hybridization with isolated transfer RNA and individual rRNA species indicated that the arrangement of genes in both units is 16 S-spacer tRNA(s)-23 S-5 S, tRNA(s). Homology between the DNA inserts is largely restricted to the rRNA coding regions, which suggests that the two rDNA units are located in different regions of the chromosome. Results of quantitative hybridization experiments are most consistent with a single Cc1880 and Cc773 unit per genome equivalent of 2.7 × 10⁹ daltons. The relatively simple organization of rDNA sequences in the C. crescentus chromosome compared to Escherichia coli is discussed.     

Radioimmunoassay of myelin basic protein in cerebrospinal fluid and its clinical application to patients with neurological diseases

A double antibody radioimmunoassay for myelin basic protein (BP) was developed that detects BP concentration greater than 0.5 ng/ml in cerebrospinal fluid ( CSF ). By this method, the amount of BP in CSF of 62 patients with various neurological disorders including 9 cases of multiple sclerosis was measured. The amount of BP in CSF obtained from 2 cases of multiple sclerosis in the exacerbation had significantly higher values than that in remission or during the gradual recovering stage. Also two of the 6 patients with myelopathy ( etiology unknown ) and one of the 11 patients with cerebrovascular disease, having acute attack, had significantly high BP values in CSF. The amount of BP in CSF from the other neurological patients showed normal level, compared with that from control patients. The presence of cross-reacting materials with bovine BP in CSF appears to be characteristic of acute myelin sheath destruction on not only patients with multiple sclerosis but also those with myelopathy and cerebrovascular disease.     

Formation of protochlorophyll(ide) in wild type and mutant C-2A' cells of Scenedesmus obliquus

Protochlorophyll(ide) was isolated from dark-grown wild typeand mutant C-2A' cells of Scenedesmus obliquus after dark incubationwith 5-aminolevulinate. Proto-chlorophyll(ide) was detectedin mutant cells grown heterotrophically at 29°C or at 21°C.At the latter temperature chlorophyll synthesis was significant.Regulation of chlorophyll synthesis in algae is discussed. ¹Present address: Laboratory of Chemistry, Faculty of Medicine,Teikyo University, Otsuka, Hachioji, Tokyo 192-03, Japan. (Received July 14, 1980; )     

Soluble and bound forms of intracellular acid carboxypeptidase inAspergillus saitoi

The enzymological, physical, and immunological properties of soluble and bound forms of intracellular acid carboxypeptidase isolated from fresh mycelia ofAspergillus saitoi are reported. In the broken mycelia, about 60% of the total activity was found in the 2,000×g precipitate, with most of the remainder in the 100,000×g supernantant. The highly purified enzymes, I_a and I_b, from the 100,000×g supernatant were found to be homogeneous by such criteria as disc gel electrophoresis at pH 9.4 The bound enzyme, II, was solubilized from the 2,000×g precipitate by self-digestion at pH 6.4 and was highly purified by chromotography. The two forms of intracellular enzymes, the soluble enzymes (I_a and I_b) from the 100,00×g supernatant and the solubilized enzyme (II) from the 2,000×g precipitate, were closely related to, but not completely identical with, the extracellular acid carboxypeptidase.     

Induction of hyaluronic acid synthetase by estrogen in the mouse skin

Hyaluronic acid synthetase activity was measured in male mouse skin following the topical application of estradiol in vivo. The enzyme activity increased in parallel with the hyaluronic acid content of the skin, and showed a similar response in the skin of ovariectomized female mice. The increase in enzyme activity was reduced by the anti-estrogen agents, tamoxifene citrate and clomiphene citrate, which block competitively the binding of estrogen to the estrogen receptor. The increase in hyaluronic acid synthetase activity was also reduced by topical application of cycloheximide or by subcutaneous injection of actinomycin D. The results suggest that the stimulation of hyaluronic acid synthesis in mouse skin in response to estrogen treatment is mediated through estrogen receptors and involves the induction of the enzyme hyaluronic acid synthetase.     

Production of prostacyclin-like substance in stroke-prone and stroke-resistant spontaneously hypertensive rats

Activities of aortae to produce prostaglandin (PG) I₂-like substance in stroke-prone spontaneously hypertensive rats (SHRSP), stroke-resistant SHR (SHRSR) and normotensive control rats from the Wistar-Kyoto (WK) colony were compared. PGI₂-like substance was produced by the incubation of the aortic ring in pH 9.0 borate-buffered saline and the amount produced was estimated by comparison of its anti-aggregatory activity with that produced by known amounts of the sodium salt of synthetic PGI₂. Before the development of stroke, amounts of this substance generated in SHRSP and SHRSR were significantly higher than those in WK rats (p<0.01 and p<0.02, respectively). Remarkably reduced capacity to generate PGI₂-like substance was observed in some SHRSP after the development of stroke.     

Characterization of unstable poly (A)-RNA in Caulobacter crescentus

Antabuse (disulfiram) is widely used in the treatment of chronic alcoholism. We have examined the effect of this drug on malignant transformation by Rous sarcoma virus, on eukaryotic cell synthesis, and on nucleic acid binding. It was found that: (1) Disulfiram inhibits the activity of the RNA dependent DNA polymerase of Rous sarcoma virus and inactivates the ability of the virus to malignantly transform chick embryo cells. The monomer of disulfiram, diethyldithiocarbamate does not affect the virus. (2) Disulfiram induced the synthesis of four proteins in normal chick embryo and human foreskin cells. The monomer diethyldithiocarbamate, induced these proteins also. Cellular DNA synthesis is more sensitive to disulfiram than are RNA and protein synthesis. (3) Disulfiram binds to neither DNA or RNA in the presence or absence of copper. However, diethyldithiocarbamate in the presence of, but not in the absence of, copper binds to HeLa cell DNA and to Rous sarcoma virus 70 S genome RNA. These results indicate that this compound, which causes no symptoms in people who do not consume alcohol, may have significant effects on a cellular level.     

Studies on polypeptide-chain-elongation factors from an extreme thermophile, Thermus thermophilus HB8. 3. Molecular properties.

Molecular properties of the polypeptide chain elongation factors from Thermus thermophilus HB8 have been investigated and compared with those from Escherichia coli. 1. As expected, the factors purified from T. thermophilus were exceedingly heat-stable. Even free EF-Tu not complexed with GDP was stable after heating for 5 min at 60 degrees C. 2. GDP binding activity of T. thermophilus EF-Tu was also stable in various protein denaturants, such as 5.5 M urea, 1.5 M guanidine-HCl, and 4 M LiCl. 3. Amino acid compositions of EF-Tu and EF-G from T. thermophilus were similar to those from E. coli. On the other hand, amino acid composition of T. thermophilus EF-Ts was considerably different from that of E. coli EF-Ts. 4. In contrast to E. coli EF-Tu, T. thermophilus EF-Tu contained no free sulfhydryl group, but one disulfide bond. The disulfide bond was cleaved by sodium borohydride or sodium sulfite under native conditions. The heat stability of the reduced EF-Tu . GDP, as measured by GDP binding activity, did not differ from that of the untreated EF-Tu . GDP. 5. T. thermophilus EF-Ts contained, in addition to one disulfide bond, a sulfhydryl group which could be titrated only after complete denaturation of the protein. 6. Under native conditions one sulfhydryl group of T. thermophilus EF-G was titrated with p-chloromercuribenzoate, while the rate of reaction was very sluggish. The sulfhydryl group appears to be essential for interaction with ribosomes, whereas the ability to form a binary GDP . EF-G complex was not affected by its modification. The protein contained also one disulfide bond. 7. Circular dichroic spectra of EF-Tu from T. thermophilus and E. coli were very similar. Binding of GDP or GTP caused a similar spectral change in both. T. thermophilus and E. coli EF-Tu. On the other hand, the spectra of T. thermophilus EF-G and E. coli EF-G were significantly different, the content of ordered structure being higher in the former as compared to the latter.     

Enhancing effects of CO2 on chloroplast regeneration in glucose-bleached cells of Chlorella protothecoides I. Role of non-photosynthetic CO2-fixation in chloroplast regeneration

Carbon dioxide enhanced chloroplast regeneration in glucose-bleachedcells of Chlorella protothecoides in the presence of CMU inthe light. Both the formation of chlorophyll and the synthesesof RNA and protein were considerably enhanced. The CO₂ metabolism of algal cells during greening was investigatedusing ¹⁴C-bicarbonate as the tracer. Radiocarbon was largelyincorporated into purine and pyrimidine bases in nucleic acidand the arginine in protein, specifically at the crabon atomsderived from carbamylphosphate. ¹Part of this investigation was reported at the conference onthe "Autonomy and biogenesis of mitochondria and chloroplasts"held at Canberra in 1969 (4). (Received August 19, 1975; )