Chromosomal Assignments of Novel Genes Expressed in HL60 Granulocytes

By collecting 3'-directed cDNA sequences called gene signatures(GSs) on a large scale, it is possible to make an expressionprofile of genes in a particular tissue, as well as discoveringa number of novel genes. A total of 305 novel GSs collectedfrom granulocytoid cells derived from HL60, a human promyelocyticleukemia cell line, by exposure to dimethyl sulfoxide were radiolabeledand used for Southern blot analyses to determine the copy numberof the corresponding genes. Of these, 198 GSs identified asrepresenting single-copy genes were then used as probes forhybridization analyses using a monochromosomal hybrid cell DNApanel. Sixty-nine of them were assigned to individual chromosomes.These results demonstrate that the chromosomal distributionof the GSs seems not to be proportional to the cytogenetic lengthof each chromosome.     

Determination of horserdish peroxidase concentration using the chemiluminescence of Cypridina luciferin analogue, 2 methyl-6-(p-methyoxyphenyl)-3,7-dihydroimidazo[1,2-a]pyrazin-3-one

The chemiluminescence of the Cypridina luciferin analogue, 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-a]pyrazin-3-one (MCLA) was observed at 462nm in the presence of horseradish peroxidase (HRP) and the total spectrum of light emitted was found to depend linearly on HRP concentration. Methods for the determination of HRP concentration using the chemiluminescence was investigated. HRP could be detected in the range from 100 pmol/L to 100nmol/L under the optimum condition, H₂O₂ (10mmol/L) and MCLA (10μmol/L) at pH 5.8.     

Structural and biosynthetic studies on linkage region between poly(galactosylglycerol phosphate) and peptidoglycan in Bacillus coagulans

The HF treatment of teichoic acid-glycopeptide complexes isolated from lysozyme digests of Bacillus coagulans AHU 1366 cell walls gave a disaccharide, glucosyl beta (1 leads to 4)N-acetylglucosamine, along with dephosphorylated repeating units of the teichoic acid chain, galactosyl alpha (1 leads to 2) glycerol. Mild alkali treatment of the complexes yielded the disaccharide linked to glycopeptide, whereas direct heating of the cell walls at pH 2.5 yielded the same disaccharide linked to teichoic acid. The Smith degradation of the complexes revealed that the galactose residue is a component of backbone chain. Thus it is concluded that this disaccharide is involved in the linkage region between poly(galactosylglycerol phosphate) and peptidoglycan in cell walls. Membrane-catalyzed synthesis of this disaccharide on a lipid followed by transfer of glycerol phosphate from CDP-glycerol to the disaccharide-linked lipid in the absence or in the presence of UDP-galactose also supports this conclusion.     

Features of ginseng saponin-induced corticosterone secretion.

Ginseng saponin administered intraperitoneally to rats induced a significant rise in plasma corticosterone, while it tended to increase plasma glucose and to decrease plasma immunoreactive insulin. Oral or intraperitoneal administration of ginseng saponin increased plasma corticosterone in unanesthetized, pentobarbital-anesthetized or alloxan-diabetes rats. The histamine-induced rise in plasma corticosterone was suppressed by pretreatment with diphenhydramine, whereas the ginseng-induced rise was not. Ginseng saponin decreased rectal temperature while it increased plasma corticosterone. Ginseng-induced corticosterone secretion was superimposed on the basal levels of plasma corticosterone due to fasting and circadian rhythm. Thus ginseng saponin would be a kind of stressful agent and have different features associated with the stimulation of the pituitary-adrenocortical system from several other chemical agents.     

Relationship between translation-control and reduced polyadenylation of mRNA in elongated roots of pea seedlings

A large portion of newly formed mRNA transported into the cytoplasmwas found as free mRNPs with discrete S-values of 50-55S, 35-38Sand 22-26S in roots of elongated pea seedlings, in contrastto the younger embryo axis, in which most of the newly formedmRNA was associated with the polysomes. Nitta's finding withVicia seedlings, that free mRNPs contain lesser amounts of oligo(dT)-boundpoly(A)-segments (9), was confirmed by analyzing RNAs from thepolysomes and free mRNPs from elongated pea seedling roots thathad been labeled with ³H-adenosine under the selective inhibitionof rRNA synthesis by 5-FU. A clear difference was observed betweenthe young embryo axis and the elongated seedling root in thedistribution of ³H-adenosine-labeled mRNA in the microsomal(P-30) and postmicrosomal (S-30) fractions. The relative ratioof radioactivity associated with the microsomes was lower inseedling roots. The decrease during germination of the percentof the labeled whole cell RNA binding to the Millipore filter,reported in a previous paper, was proved to be the consequenceof reduced polyadenylation of poly(A)-segments after ³H-adenosine-labeledRNA had been treated with RNase A and RNase T₁ and analyzedon polyacrylamide gels. The evidence showed a close correlationbetween the accumulation of free mRNPs and reduced growth ofpoly (A)-segments in the mRNA of seedling roots. The relationshipbetween the ability of mRNPs to associate with the endoplasmicreticulum and the degree of polyadenylation of mRNA is discussed. (Received January 16, 1979; )     

Appearance of newly formed mRNA and rRNA as ribonucleoprotein-particles in the cytoplasmic subribosomal fraction of pea embryos

Incorporation studies with ³H-uridine or ³H-adenosine showedthat germinating pea embryos synthesize all types of poly A(+)RNA, rRNA and 4–5S RNA at the early stage of germination.After the pulse labeling for 30 min, only heterodisperse RNAand 4–5S RNA appeared in the cytoplasm as labeled RNAspecies. At this time the radioactivity was associated withcytoplasmic structures heavier than 80S and RNP particles of68–70S, 52–55S, 36–38S and 20–22S whichare presumed to be free mRNP particles in plants. When the pulse-labeledembryos were incubated for a further 60 min in an isotope-freemedium, the labeled 17S and 25S rRNA emerged in the cytoplasm,together with labeled heterodisperse and 4–5S RNAs. Moreradioactivity accumulated in the regions of the polysome, 62–65Sand 38–42S particles. The results of analysis of RNAsextracted from the whole cytoplasm, polysome or subribosomalfractions indicated that small subunits of newly formed ribosomesappear more rapidly in the cytoplasm than new large subunits,which accumulate for a while as free particles in the cytoplasmthen are incorporated into polysomes. The actino-mycin treatmentwhich caused preferential inhibition of rRNA synthesis reducedthe accumulation of free, newly formed ribosome subunits andpartially permitted detection of the presumed mRNP particlesin the subribosomal region even after the chase treatment. (Received June 28, 1976; )     

Interaction of pneumococcal S-14 polysaccharide with lectins from Ricinus communis,Triticum vulgaris, and bandeiraea simplicifolia

Two purified lectins, namely, wheat-germ agglutinin (from Triticum vulgaris) and the hemagglutinin from Ricinus communis seeds, readily form a precipitate with pneumococcal S-14 polysaccharide, whereas the Bandeiraea simplicifolia lectin (BS 1) does not. Exhaustive periodate oxidation and borohydride reduction of S 14 modifies terminal β-D-galactopyranosyl residues, as well as chain D-glucopyranosyl residues, and abolishes reactivity with both the R. communis lectin and wheat-germ agglutinin. Controlled periodate oxidation followed by Smith degradation cleaves only terminal β-D-galactopyranosyl residues, giving a linear polymer, the structure of which was determined by methylation analysis. This derived polymer, containing (1→6)-linked 2-acetamido-2-deoxy-β-D-glucosyl residues, readily precipitated wheat-germ agglutinin, but not the R. communis lectin.     

1H NMR studies on the conformational characteristics of 2-thiopyrimidine nucleotides found in transfer RNAs.

The molecular conformations of naturally occurring 2-thiopyrimidine nucleosides (5-methylaminomethyl-2-thiouridine, 5-methoxycarbonylmethyl-2-thiouridine and 2-thiocytidine) and 5'-mononucleotides (5-methylaminomethyl-2-thiouridine 5'-monophosphate and 2-thiocytidine 5'-monophosphate) in 2H2O solution were elucidated by analyses of the proton NMR spin-coupling constant, nuclear Overhauser effect, and lanthanide-induced shifts and relaxation enhancements. As monomers, these nucleotides are almost exclusively in the 3E-gg-anti form, even in the absence of ordinary stabilizing factors of this form; i. e., base-stacking and base-pairing interactions with other nucleotide units. This inherent conformational rigidity of the 2-thiopyrimidine units probably contributes to stability of the conformation of tRNA.     

Cytochemical localization of lectin labeled vesicles in GERL region of hepatoma ascites cells

Summary The fate of lectin labeled internalized plasma membrane in the ascites tumor form of the Chang rat hepatoma growing under in vivo and in vitro conditions was investigated cytochemically. Ascites cells were incubated in Concanavalin A (Con A) and horseradish peroxidase (PO), either with or without prior glutaraldehyde fixation and subsequently treated with 3,3-diaminobenzidine. In cells fixed before Con-A-PO labeling the reaction product was localized as a continuous and even layer upon the external surface of the plasma membrane. If unfixed cells were treated with Con A, coupled with PO at 4°C and reinbated in phosphate buffered saline at 37°C for varying periods of time, the Con-A-PO layer was of irregular thickness. In as little as 15 min of reincubation endocytotic vesicles containing PO positive material were closely associated with GERL components of the Golgi Apparatus. Localization of acid phosphatase (ACPase) within GERL vesicles, similar in size and location to those containing Con-A-PO reaction product, indicates that the Con-A-PO labeled vesicles may be a component of the Golgi apparatus in hepatoma cells.Supported by NIH Grant CA 16663.