Sequence of the gene encoding the mitochondrial capsule selenoprotein of mouse sperm: identification of three in-phase TGA selenocysteine codons.

The mitochondrial selenoprotein is a major structural protein of the keratinous mitochondrial capsule in mammalian sperm, a structure that functions in shaping mitochondria into the helical sheath surrounding the flagellum. A cDNA clone (Kleene et al., 1990) was isolated previously encoding a protein whose predicted size and amino acid content of > 20% cysteine and proline closely resembled a selenoprotein in the bull mitochondrial capsule. The sequences of additional cDNAs and genomic DNA reported here reveal that the mouse mitochondrial capsule selenoprotein reading frame begins 54 codons further upstream than previously reported. Significantly, these 54 codons contain three in-phase UGA codons, which normally signify stop but encode selenocysteine in bacterial and mammalian selenoproteins. The coding region of the mitochondrial capsule selenoprotein gene is interrupted by a single intron. S1 mapping and primer extension demonstrate that the vast majority of MCS mRNAs are spliced using consensus 5' and 3' slice junctions in mammalian cells. However, two cDNAs have been identified that apparently represent rare mRNA variants produced by use of cryptic splice sites.     

Splicing defect at the ornithine aminotransferase (OAT) locus in gyrate atrophy.

Gyrate atrophy (GA), a recessive eye disease involving progressive vision loss due to chorioretinal degeneration, is associated with the deficiency of the mitochondrial enzyme ornithine aminotransferase (OAT), with consequent hyperornithinemia. We and others have reported a number of missense mutations at the OAT locus which result in GA. Here we report a GA patient of Danish/Swedish ancestry in whom one OAT allele produces an mRNA that is missing a single 96-bp exon relative to the normal mRNA. Polymerase-chain-reaction amplification and sequencing revealed a 9-bp deletion covering the splice acceptor region of exon 5, resulting in the absence of exon 5 sequences from the mRNA with no disruption to the reading frame. This mutation, which was not present in 15 other independent GA patients, adds to the array of allelic heterogeneity observed in GA and represents the first example of a splicing mutation associated with this disorder.     

Effects of exogenous 1,3-diaminopropane and spermidine on senescence of oat leaves : I. Inhibition of protease activity, ethylene production, and chlorophyll loss as related to polyamine content

Excision and dark incubation of oat (Avena sativa L., var. Victory) leaves cause a sharp increase in protease activity, which precedes Chl loss. Both these senescence processes are inhibited by exogenously applied 1,3-diaminopropane (Dap), which occurs naturally in leaf segments. The inhibition of protease activity is much greater in vivo than in vitro, suggesting inhibition of protease synthesis as well as protease action by Dap. Chl breakdown in leaves of radish and broccoli, which also senesce rapidly in the dark, is only slightly inhibited by DaP. These differences between cereal and dicotyledonous plants are correlated with the natural occurrence of Dap in cereals. In the light, Dap promotes, rather than retards, the loss of Chl in oat leaves. This resembles previously described effects of other polyamines. Addition of Mg²⁺ to the medium does not antagonize this effect. In the dark, the accumulated Dap also inhibits ethylene production and decreases titer of other polyamines. Addition of Ca²⁺ to the incubation medium containing Dap competitively reduces the effects of Dap. Thus, Dap, like other polyamines, seems to require an initial attachment to a membrane site shared with Ca²⁺ before exerting its antisenescence action.     

Relation of polyamine synthesis and titer to aging and senescence in oat leaves

Polyamine biosynthesis in senescing leaves of Avena sativa L. was measured by determining the activities of arginine decarboxylase (EC 4.1.1.19), ornithine decarboxylase (EC 4.1.1.17) and S-adenosyl-l-methionine decarboxylase (EC 4.1.1.50). Polyamine content was also estimated by thin layer chromatography and high performance liquid chromatography. Arginine decarboxylase activity decreases progressively in aging attached first leaves and in senescing excised leaves in the dark. Conversely, it increases during light exposure of excised leaves, which retards senescence. Ornithine decarboxylase activity is high and constant in the attached leaf, irrespective of age; it decreases in excised leaves kept in the dark and in the light, irrespective of senescence. S-Adenosyl-l-methionine decarboxylase shows no correlation with age or senescence. Levels of putrescine, diaminopropane, agmatine, and spermidine are high in young leaves and decline with age. The best single indicator of senescence is usually spermidine, which decreases in excised leaves incubated in the dark, but increases in such leaves with time of light exposure. Spermidine generally has a reciprocal relationship with putrescine, indicating that spermidine synthase, which converts putrescine to spermidine, may exert important physiological control. These data support the view that polyamines play an important role in the regulation of plant development.     

Surface antigenic determinants of mammalian "hepadnaviruses" defined by group- and class-specific monoclonal antibodies

The hepatitis B-like viruses (human hepatitis B virus, woodchuck hepatitis virus, ground squirrel hepatitis virus, and duck hepatitis B virus) are hepatotropic DNA viruses which have been referred to collectively as "hepadnaviruses." Using a murine monoclonal antibody (101-2) to the surface antigen of woodchuck hepatitis virus, we have shown that the surface antigens of mammalian hepadnaviruses (HBsAg, WHsAg, and GSHsAg) are antigenically related via a common determinant (HV/101). Furthermore, analysis with other monoclonal antibodies to WHsAg revealed that WHsAg and GHsAg are antigenically distinct, although the antigens had more determinants in common with each other than with HBsAg. The hepadnavirus group-specific antibody (101-2) reacted with HBsAg subtypic variants in a group-specific rather than subtype-specific manner. In conjunction with observations with an HBsAg-specific, group-reactive monoclonal antibody (BX259), the present data suggest that there are at least two group-reactive epitopes of HBsAg: one which is virus specific (HBV/259) and one which is common to two other mammalian hepadnaviruses (HV/101).     

Ornithine ketoacid transaminase deficiency in gyrate atrophy of the choroid and retina.

Gyrate atrophy of the choroid and retina is a chorioretinal degeneration associated with hyperornithinemia with an autosomal recessive mode of inheritance. Cultured skin fibroblasts from five affected patients showed a virtual absence of ornithine ketoacid transaminase (OKT) (L-ornithine:2-oxoacid aminotransferase E.C.2.6.1.13) activity. Fibroblasts from four carrier parents showed a 42%-65% reduction in OKT activity. Increasing the concentration of pyridoxal phosphate (vitamin B6 in the assay media resulted in partial restoration of OKT activity in fibroblasts from one out of five patients studied. We conclude that OKT deficiency is closely associated with the genetic defect in gyrate atrophy of the choroid and retina and that genetic heterogeneity exists in this disease.     

Histidine:pyruvate transamination and phyenylalanine:pyruvate transamination may be catalyzed by the same enzyme.

Some properties of histidine:pyruvate transaminase (HPT) and phenylalanine:pyruvate transaminase (PPT) in the cytosol of rat liver were studied. HPT and PPT activity could not be separated by DEAE-Sephadex A-50 or hydroxylapatite column chromatography, and the ratio of $\text{HPT}\text{PPT}$ activity remained constant during these purification procedures. The two enzyme activities also showed similar heat stability and responses to glucagon injection. Based on these findings, we suggest that a single enzyme may specifically catalyze histidine:pyruvate and phenylalanine:pyruvate transamination.     

Removal of Enteroviruses from Sewage by Bench-Scale Rotary-Tube Trickling Filters

The efficacy of a rotary-tube type of trickling filter for removing coxsackievirus A9, poliovirus 1, and echovirus 12 suspended in raw settled sewage was investigated. At filtration rates equivalent to about 10 MGD (million gallons per day)/acre (ca. 3,785 m3/day per acre), the filters removed 95% of the poliovirus, 83% of echovirus 12, and 94% of coxsackievirus A9. Coliform, fecal streptococci, biochemical oxygen demand, and chemical oxygen demand removals were remarkably similar, averaging 94, 92, 93, and 95%, respectively. At filtration rates equivalent to about 23 MGD/acre, 59% of the poliovirus, 63% of the echovirus 23, and 81% of the coxsackievirus A9 were removed. Coliform, fecal streptococci, biochemical oxygen demand, and chemical oxygen demand removals at this filtration rate were 68, 75, 72, and 56%, respectively. Viruses were assumed to be adsorbed to the biological slime growing in the filters, but attempts to disassociate the viruses from the slime were unsuccessful, indicating that the slime-virus complex is very stable or that the viruses were somehow inactivated. The data indicate that coliform and fecal streptococci reductions in this type sewage treatment process can be used as an index of virus reduction. Disinfection, however, must be used to ensure a virus-free final effluent.     

Effects of chloramine on Bacillus subtilis deoxyribonucleic acid.

The lesions induced in Bacillus subtilis deoxyribonucleic acid (DNA) after treating bacterial cells (in vivo) and bacterial DNA (in vitro) with chloramine were studied biologically and physically. Single-strand breaks and a few double-strand scissions (at higher chloramine doses) accompanied loss of DNA-transforming activity in both kinds of treatments. Chloramine was about three times more efficient in vitro than in vivo in inducing DNA single-strand breaks. DNA was slowly chlorinated; the subsequent efficiency of producing DNA breaks was high. Chlorination of cells also reduced activity of endonucleases in cells; however, chlorinated DNA of both treatments was sensitized to cleavage by endonucleases. The procedure of extracting DNA from cells treated with chloramine induced further DNA degradation. Both treatments introduced a small fraction of alkali-sensitive lesions in DNA. DNA chlorinated in vitro showed further reduction in transforming activity as well as further degradation after incubation at 50 C for 5 h whereas DNA extracted from chloramine-treated cells did not show such a heat sensitivity.     

Identification of proprionylcholine in higher plants

Propionylcholine, a novel analogue of acetylcholine, was identified in green plants by gas chromatography/mass spectrometry. Propionylcholine was found in the leaves of the following species previously shown to contain acetylcholine and cholinesterase activity: Codiaeum variegatum Blume, Phaseolus aureus Roxb. cv. Berken, Plantago rugelli Decne., Populus grandidentata Michx., and Betula pendula Roth. The quantities of propionylcholine ranged from a high of 2.3 nmol (g fresh weight)⁻¹ in C. variegatum to a low of 0.11 nmol (g fresh weight)⁻¹ in P. rugelli . These amounts represented 6 to 8% of the levels of acetylcholine. In contrast to animal tissues which rarely synthesize propionylcholine, this compound was found in all species examined which represented five families of flowering plants.