Sequence-dependent hydrolysis of RNA using modified oligonucleotide splints and RNase H

To cleave RNA molecules using RNase H in a site-specific manner, a short deoxyoligonucleotide (3-5mer) joining with 2'-O-methyl oligonucleotide(s) was designed as a DNA splint to be used. Model experiments were carried out using ribooligonucleotide substrates (9 and 18 mer). It was found that the use of this type of splints (9 mer) causes a unique cleavage by RNase H. For example, when 3'm (GA)d(AGAA)m(GGU)5' was used as a hybridization strand, 32pUCUUUCUUCUUCCAGGAU was cleaved specifically between U11 and C12 to yield 32pUCUUUCUUCUU. This method will have a variety of applications for the study of RNA.     

A new solid-phase synthesis of oligoribonucleotides by the phosphoro-p-anisidate method using tetrahydrofuranyl protection of 2''-hydroxyl groups.

Six nonaribonucleotides containing the 5'-splice site, one complementary nonamer and an octadecamer containing the 3'-splice site have been synthesized on a polymer support using the phosphoro-p-anisidate method. A 5'-linked 2'-O-tetrahydrofuranyl-N-protected nucleoside 3'-(o-chlorophenyl)phosphoro-p-anisidate was used as the starting nucleotide, and the chain elongated in the 3'-direction by removing the p-anisidate protecting group with isoamyl nitrite under neutral conditions. The octadecamer has been synthesized using dinucleotide blocks and a 3'-terminal trinucleotide.     

Intravascular granuloma induced by intravenous inoculation ofCryptococcus neoformans

In rodents an intravenous administration of viableCryptococcus (C.) neoformans cells frequently resulted in attachment of intravascular cryptococcal granulomas to inner walls of the large to medium-sized veins of various organs, including the lungs, liver and spleen. In order to elucidate the pathogenesis of granulomatous changes, the cells composing the intravascular granulomas were observed by electron microscopic peroxidase (PO) cytochemistry. The granuloma composing cells could be divided into the following four types according to the pattern of endogenous peroxidase activity: exudate macrophage (Mφ, type I), PO-negative Mφ (type II), resident Mφ (type III) and other inflammatory cells (type IV). In the intravenous granulomas of the lung, the percentages of composed cells were 39.0% for type I, 57.9% for type II, 0% for type III and 3.1% for type IV. By contrast, in the interstitial granulomas in the lung, type III Mφs, possibly derived from alveolar Mφs, played a significant role in granuloma formation. This may indicate that the intravascular granuloma is almost composed of macrophages derived from monocytes rather than alveolar macrophages. The expression of ICAM-1 on endothelia of the pulmonary veins was examined by immunoelectron microscopy. An immunogold labeling index was significantly augmented on the surface of endothelia in response to intravenous challenge ofC. neoformans. The intravascular granuloma demonstrates that the monocytes develop into the granuloma-composing macrophages and suppress the cryptococcal activities even hi the peripheral blood resulting in an assistance of endothelial functions.     

Pseudomonas putida KF715 bphABCD operon encoding biphenyl and polychlorinated biphenyl degradation: cloning, analysis, and expression in soil bacteria.

We cloned the entire bphABCD genes encoding degradation of biphenyl and polychlorinated biphenyls to benzoate and chlorobenzoates from the chromosomal DNA of Pseudomonas putida KF715. The nucleotide sequence revealed two open reading frames corresponding to the bphC gene encoding 2,3-dihydroxybiphenyl dioxygenase and the bphD gene encoding 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (ring-meta-cleavage compound) hydrolase.     

A rapid assay of acyl-coenzyme A:lysolecithin acyltransferase activity

A simple and rapid procedure for the assay of acyl-coenzyme A:1-acyl-sn-glycero-3-phosphocholine acyltransferase (lysolecithin acyltransferase, LLAT [EC 2.3.1.23]) activity in crude enzyme preparations is described. The incubation system utilizes lysolecithin and [1-14C]-oleoyl-coenzyme A as substrates. Labeled fatty acid released due to accompanying acyl-coenzyme A hydrolase [EC 3.1.2.2]activity is first removed by di-isopropyl ether extraction. The labeled lecithin produced due to LLAT action is then quantitatively recovered by partition of the incubation medium with di-isopropyl ether-n-butanol 60:40 (v/v). Selective extraction of the labeled lecithin formed and avoidance of customary thin-layer chromatographic isolation procedures permits assay of LLAT activity with excellent accuracy at a substantial saving of time. The entire assay can be completed in less than 30 min as compared to 2-3 hrs when following conventional procedures.     

An endonuclease activity in Bacillus subtilis specific for UV-irradiated DNA

An enzyme activity specific for UV-DNA¹ was found in the extract of $\text{Bacillus subtilis}$(Marburg 168). The enzyme preparation obtained from the extract by ammonium sulfate precipitation acts on UV-DNA endonucleolytically and induces single strand breaks. The number of single strand breaks introduced in DNA is proportional to UV dose.     

Purification and Separation of A and B Forms of N-Acetyl-β-D-Hexosaminidase from Human Aorta

Human aorta has been shown to possess multiple forms of N-Acetyl-6-D-hexosaminidase (β-2-acetamido-2-deoxy-D-glucoside-acetamido-deoxyglucohydro-lase, EC 3.2, 1.30). The enzyme was separable, by gel electrophoresis, into 2 enzymatically active bands representing A and B forms. By gel electro-focussing, A and B forms were further subdivided into at least 5 and 8 bands, respectively. The B form consisted of 4 bands (B₁) and 4 bands (B₂), which were not inactivated at 50° for 3 hr. (at pH 4.4) in the presence of serum; whereas, the 5 bands found in A form were completely inactivated. All forms of the enzyme were active towards naphthol-AS-BI-N-acetyl-β-D-glucosaminide and the corresponding galactosaminide (about one-eighth of the hydrolysis rate of the former), suggesting each single enzyme acts on both substrates. The N-acetyl-hexosaminidases of bull epididymis, by comparison, were also found to be active towards both substrates and to possess 13 bands having pis more alkaline than those of the B form of the human enzyme, By heat inactivation we found that the aortic enzyme consisted of 51% of A and 49% of B (B₁ + B₂ .). Neuraminidase had no effect on either form of the aortic preparation. Both forms were partially purified and separated by conventional methods. They required BSA for their maximal activity; the A form being more dependent BSA than the B form, With PNP-N-acetyl-β-D-glucosaminide and the corresponding galactosaminide, Km of 1.04 mH and 0.54 mM, respectively, for A form and of 1.74 and 1.48 mM, respectively, for B form were obtained. While the purified B form was stable and did not transform into other species, the purified A form gradually transformed into B form as well as into other new forms during storage at -20°.