Characterization of a Mr = 56,000 polypeptide associated with 10S DNA polymerase alpha purified from calf thymus using monoclonal antibody.

Existence of a Mr = 56,000 polypeptide associated with 10S DNA polymerase alpha was shown by production of a monoclonal anti-calf thymus 10S DNA polymerase alpha antibody secreted from a hybridoma line named 3H1. The antibody bound three polypeptides with Mr = 180,000, 56,000 and 32,000 in hydroxylapatite fraction of 10S DNA polymerase alpha by immunoblot. The antibody co-precipitated the polypeptides with the large polypeptide (Mr = 150,000-140,000) of 10S DNA polymerase alpha with the aid of second antibody. Among three polypeptides, the Mr = 56,000 polypeptide was co-purified with DNA polymerase alpha through DNA-cellulose chromatography and repeated sucrose rate-zonal centrifugations. The Mr = 56,000 polypeptide was still associated with 10S DNA polymerase alpha after second sucrose rate-zonal centrifugation, but the amount of it was reduced. The polypeptide was banded at pH 7.2-8.0 and displayed microheterogeneity in respect of isoelectric point by isoelectrofocusing with 7 M urea, and showed weak DNA-binding property after blotting onto a nitrocellulose. The antibody against the polypeptide precipitated DNA polymerase alpha from human, rat, and mouse, and Mr = 56,000 and 32,000 polypeptides were detected in these DNA polymerase alpha fractions by immunoblot. These results suggest that the polypeptide with Mr = 56,000 may take part in the DNA polymerase reaction.     

Conservation of molecular structure of DNA polymerase beta in vertebrates probed by tryptic peptide mapping

DNA polymerase beta's from mouse myeloma, chick embryo, and cherry salmon testis were all composed of a single polypeptide of about 40K daltons as judged by a sodium dodecyl sulfate-polyacrylamide gel electrophoresis of extensively purified enzyme preparations. Although the enzyme from bullfrog ovary was not fully purified, its molecular weight was estimated to be the same as that of the chick enzyme by immunological detection after electrophoresis. All the enzymes tested cross-reacted immunologically with the antibody against chick DNA polymerase beta, indicating that they have a common molecular structure, at least in part. Two-dimensional maps of radioiodinated tryptic peptides directly showed the presence of highly conserved amino acid sequences among mouse, chick, and cherry salmon enzymes. This conserved structure is thought to be essential for the enzyme activity, which is very similar among all these vertebrates.     

Production of Proteinase on Noncarbohydrate Carbon Sources by Pseudomonas aeruginosa

Proteinase production by Pseudomonas aeruginosa was studied in medium containing noncarbohydrate materials, especially various hydrocarbons, as the sole carbon source. On heavy oil, kerosene, n-paraffinic hydrocarbon of C₁₂, C₁₄, or C₁₆, and propylene glycol, the bacteria grew well and high protinase production was observed. However, production on paraffinic hydrocarbon differed remarkably with strains of varied origins. The elastase-positive strain, IFO 3455, showed abundant growth and high proteinase production on medium containing a paraffin of C₁₂, C₁₄, or C₁₆, whereas the elastase-negative strain, IFO 3080, showed little growth on the same medium. Neither elastase-positive nor elastase-negative strains, however, utilized n-paraffins of C₅ to C₁₀, or various aromatic hydrocarbons such as benzene, naphthalene, phenanthrene, and anthracene. The proteinases produced on the noncarbohydrate medium were identical with those produced in glucose medium.     

Combined method of fluorescence tracer technique and PAP immunohistochemistry for discrimination of the transplanted cells

Summary The retrograde fluorescence tracer, True Blue (TB), was injected into the forebrain septal area of neonatal rats. After 3 to 6 days the brains of these animals were carefully removed and placed in ice-cold sterilized physiological saline containing 1% glucose. Under the surgical microscope, one or two pairs of mesencephalic tissue samples, each containing a dorsal raphe nucleus, were punched out and transplanted into the third ventricle of a 5,6-DHT-pretreated adult rat. One month after transplantation, all animals were perfused and their brains sectioned using a cryostat. The sections were examined using a fluorescence microscope, and then processed for serotonin immunohistochemistry. The grafts were found to be successfully implanted and connected with the middle portion of the third ventricle. Four types of neurons, i.e., TB-labeled, serotonin-labeled, both TB-and serotonin-labeled, and non-labeled neurons, were detected in the grafts. This double-labeling method is considered to be a useful technique in characterizing the neurons in grafts which consist of a heterogeneous cell population.Supporied by grants from the Ministry of Education, Science and Culture of Japan     

Leukotriene A4 hydrolase from guinea pig lung: the presence of two catalytically active forms

Leukotriene A4 hydrolase was purified to apparent homogeneity from the guinea pig lung. The molecular weight was determined to be 70 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme exhibited two active forms with different pI values (5.7 and 5.4) depending on the presence or absence of SH-reducing reagents during purification procedures. No significant differences were observed between both forms of the enzyme as regards the catalytic properties. The N-terminal 20 amino acid sequence (PEVVDTXSLASPATVXRTKH) showed a 90% identity to the human enzyme with a constitutive substitution of Ile-3 and Ser-14 (human) by Val-3 and Thr-14 (guinea pig), respectively.     

Structure of major surface determinants and DNA diagnosis of Pneumocystis carinii

Pneumocystis carinii is a pathogen which causes fatal pneumonia in patients with the acquired immune deficiency syndrome (AIDS). To facilitate the basic study of P. carinii, we have analyzed its major surface proteins by both immunochemical and biochemical methods. The major protein components of both cysts and trophozoites are a group of proteins called "P115" with apparent masses of 105-120 kd. It includes 6 isoelectric variants. A monoclonal antibody raised against cysts recognizes all 6 variants and reacts with epitopes located in the cell wall indicating that P115 is an immunoreactive surface component. The isoelectric variants contain identical or closely related protein components and they are mannose-rich glycoproteins. The isoelectric variation may be due primarily to differences in glycosylation. The majority of sera from humans with diagnosed pneumocystosis that were tested reacted strongly with the P115 proteins. To develop probes for DNA diagnosis and to facilitate molecular studies, a genomic DNA library of P. carinii has been constructed. Some of these clones were used for DNA hybridization analysis of rat and human lungs.