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Halisphingosines A (1) and B (2), modified long-chain sphingoid bases, from the marine sponge Haliclona tubiferacollected in Brazil, were characterized after conversion to their N-Boc derivatives. The 2R,3R,6Rconfiguration of halisphingosine A, a compound first reported from Haliclonasp. from South Korea, was confirmed using a novel CD approach: deconvolution of exciton coupling from mono- and trinaphthoyl derivatives obtained by derivatization of the natural product. The sensitive CD deconvolution method, applicable to submilligram samples, simultaneously predicted the relative and absolute configuration of three stereocenters in halisphingosine A with precision and accuracy. Halisphingosine B was assigned by correlation to halisphingosine A.

Game‐SET‐match: Pyrrole aminoimidazole alkaloids (PAIs) are metabiosynthesized from chlorinated analogues of oroidin by cell‐free enzyme preparations from PAI‐producing sponges. Evidence and implications for the biosynthesis of PAIs include putative single‐electron transfers (SETs) that promote CC bond‐forming reactions of precursors.

Ethylene is an endogenous plant hormone which influences many aspects of plant growth and development, but its effect upon economic yield of crop plants is not understood well. Our objective was to evaluate the effect of ethylene treatment on sugarbeet (Beta vulgarisL.) roots during the growing period on yield, sucrose content, and purity. Field studies were conducted in 1973, 1974, and 1975 in Colorado, Kansas, Nebraska, Wyoming, Montana, and Ohio. Ethylene was injected 20 cm into the soil between rows at the 11‐leaf‐stage of development. Tests showed that ethylene moves 24 m or more in the soil after injection and so experiments were designed to prevent cross‐contamination of plots. Roots were analyzed for weight, sucrose percent, and clarified juice purity, immediately after harvest in September or October. The effect of ethylene on root and foliage growth was also evaluated in hydroponic studies. Ethylene increased green and dry weight yields of hydroponically grown sugarbeet roots 38.9 and 42.9%, respectively, over nontreated beets. Treated roots had a higher water content as well as a higher dry matter yield than non‐treated beets. Ethylene also stimulated fibrous root formation in hydroponic solution. Ethylene injected at the rate of 0.25 liter/m of travel in field trials increased root yield 8.2% in 13 paired comparisons in 1973 and 1974, and 7.6% in six comparisons in 1975. The treatments, however, gave 3.3 and 1.5% reduction, respectively, in sucrose content in the respective tests so that the ethylene treatment resulted in no significant increase in sugar yield per hectare. Treatment at rates of 0.125 and 0.50 liter ethylene/m showed similar effects upon root yield and sucrose content but were not statistically significant. Hydroponic studies demonstrated that ethylene applied to the sugarbeet roots during the growing period can substantially increase dry matter yield. Economic yield increases — sucrose per hectare — were not, however, obtained with ethylene injection in field tests.

The recovery of sucrose from sugarbeets (Beta vulgarisL) has decreased in recent years while root yield has increased. Our objective was to determine the effect of N application rate and topping method on root yield and on the factors affecting recovery of sucrose‐sucrose content, purity, and storage loss. The effect of five N fertility levels (0, 67, 134, 202, and 269 kg/ha) on sugarbeet root yield, quality (percent sucrose and percent purity) and crown tissue size was investigated from 1975‐1977 in 10 field experiments in Colorado, Kansas, and Nebraska. Crown tissue, the portion of the sugarbeet above the lowest leaf scar, increased from 10.64 to 16.03% of the total root weight as the N application rate increased from 0 to 260 kg/ha. Total root yield increased from 45.2 to 54.4 metric tons/ha over the same N range, but much of the yield increase was due to increase in crown size. Sucrose content and clarified juice purity (CJP) decreased 1.42 and 1.97% units, respectivdy, as the N application went from 0 to 269 kg/ha. The decrease in quality offset the increase in yield so that increasing rates of N had no significant effect upon yield of recoverable sucrose per ha. The extractable sucrose per metric ton of beets was reduced from 144.5 kg/ton for the 67 kg N/ha application to 128.6 kg/ton for the 269 kg N/ha application. Crown removal significantly reduced yield of recoverable sucrose per hectare but only increased extractable sucrose per ton from 136.7 to 140.0 kg/ton. Quality (sucrose and CJP) was improved both by proper topping and by N management, however, N management has a more dramatic effect upon quality. Total sucrose yield is reduced by topping, but not significantly affected by N application rate. Three N fertility levels (0, 134, and 269 kg/ha) and three topping methods (flailed, half‐topped, and topped) were evaluated for their effect on sucrose and quality changes in sugarbeet roots during storage in nine experiments. Failed beets had 27.4 and 25.5% less gross sucrose and recoverable sucrose losses than regular topped beets and 17.3 and 15.7% less gross suaose and recoverable sucrose lass than half‐topped beets. Method of topping had a significant effect upon sucrose losses in beets grown in all Iri levels. Nitrogen application rate had no significant effect upon gross sucrose or recoverable sucrose losses. Beet quality (extractable sucrose per metric ton) was higher in topped beets than flailed beets at harvest, but no difference existed between topping treatments after storage. Sucrose production per acre and beet quality was maximized with the low fertility, flailed treatments before and after storage.

Infection of synchronized bovine fetal spleen cells with bovine parvovirus results in changes in the levels and patterns of DNA polymerases alpha and gamma during the cell cycle. The pattern of DNA polymerase alpha activity closely paralled viral DNA synthesis and the production of progeny virus, and levels, of this enzyme were threefold greater than in mock-infected cells during the period of maximal viral DNA synthesis. DNA polymerase gamma activity remained slightly elevated during viral DNA replication. Levels and patterns of DNA polymerase beta were similar in mock- and virus-infected cells.

We have developed a nuclear lysate system from infected, synchronized cells capable of synthesizing unit-length parvoviral DNA in vitro. It was necessary to supplement the nuclear lysates with the polyamines, spermidine and spermine, to prevent degradation of template and product DNAs. In this system RF, RI, and single-stranded progeny DNAs were synthesized. Label incorporated in viral RF DNA in vivo appeared first in RI DNA and then in single-stranded DNA during incubation in vitro. By sedimentation the product DNAs were identical to those found in infected cells. Their viral identity was confirmed by hybridization. The addition of ribonucleotides, RNase, or alpha-amanitin did not affect parvoviral DNA synthesis in this system. The results with the specific inhibitors of mammalian DNA polymerases, aphidicolin, N-ethylmaleimide, and 2',3'-dideoxythymidine 5'-triphosphate indicated that DNA polymerase alpha was required for synthesis of parvoviral DNA in the nuclear lysates. This requirement was confirmed by experiments with antibody to bovine DNA polymerase alpha.

Transcription of the genome of the nondefective parvovirus BPV was examined in nuclei isolated from synchronized bovine fetal spleen cells. The relative levels of total RNA polymerase and RNA polymerase I, II, and III activities in nuclei isolated from BPV-infected and mock-infected cells were found to be similar throughout the course of infection. Hybridization of RNA synthesized in isolated nuceli indicated that BPV-specific RNA synthesis began during the period of 8 to 12 h postinfection and proceeded linearly until at least 20 h postinfection. By 20 h postinfection, 5% of the total RNA synthesized in nuclei from infected cells was virus specific. BPV-specific RNA synthesis was inhibited by 95% in the presence of 0.1 microgram of alpha-amanitin per ml, suggesting that the viral genome is transcribed by cellular RNA polymerase II.

Since parvoviruses apparently do not possess a DNA polymerase activity, one or more of the host cell DNA polymerases must be responsible for replicating the single-stranded DNA genome. We have focused on determining which polymerase, alpha, beta, or gamma (pol alpha, pol beta, or pol gamma, respectively), is responsible for the first step in bovine parvoviral DNA replication: conversion of the single-stranded DNA genome to a parental replicative form (RF). In this study, we used aphidicolin, a specific inhibitor of DNA pol alpha, to assay for the requirement of pol alpha activity in parental RF formation in vivo. Synchronized cell cultures were infected with bovine parvovirus with or without aphidicolin, and the products of viral replication were separated on agarose gels and identified by Southern blot analysis. We found that complete inhibition of viral DNA synthesis resulted when 20 microM aphidicolin was present throughout the infection. In addition, viral DNA synthesis was inhibited by as little as 1 microM aphidicolin, whereas lower concentrations (0.1 and 0.01 microM) resulted in partial inhibition of the replication process. Using 32P-labeled bovine parvovirus as the input virus we differentiated parental RF from daughter RF and progeny DNA synthesis. We conclude that DNA pol alpha is required for the production of RF during bovine parvovirus replication in vivo and that this requirement is most likely for the conversion of bovine parvovirus input single-stranded DNA to parental RF. These results do not rule out a possible role for DNA pol gamma in the first step, nor do they rule out a role for pol alpha or pol gamma in later stages of the replication cycle.

A phosphorylated protein (NP-1) with an Mr of 28,000 has been detected in nuclei of bovine parvovirus (BPV)-infected cells in association with chromatin. No protein in this size range was detected after infection of appropriate cells with several autonomous rodent parvoviruses although the BPV-specific protein is similar in size to noncapsid proteins associated with rabbit parvovirus or adeno-associated virus infection. Structural homology between NP-1 and a BPV capsid protein could be detected by electrophoretic analysis of the products of proteolysis with chymotrypsin. This protein can be detected after in vitro translation of RNA from BPV-infected cells and BPV-specific RNA. Homology between the in vivo- and in vitro-synthesized species was shown by the similarity of the chymotryptic products.

Sequence analyses of the left and right termini of LuIII virus show they are nonidentical imperfect palindromes of 122 and 211 nucleotides, respectively. The left terminus of the minus strand of LuIII DNA, uniquely in the flip conformation, can assume a T-shaped structure. The right terminus of the minus strand of LuIII DNA can assume a U-shaped structure, and it exists in either the flip or flop conformation. The termini of LuIII shared a high degree of sequence homology and showed conserved secondary structure with those of the rodent parvoviruses MVMp and H-1. LuIII, like adeno-associated virus, encapsidates equal amounts of plus- and minus-strand DNA. However, the sequence data for LuIII virus demonstrate that identical termini are not required for this encapsidation pattern.

Genomic clones of the autonomous parvovirus bovine parvovirus (BPV) were constructed by blunt-end ligation of reannealed virion plus and minus DNA strands into the plasmid pUC8. These clones were stable during propagation in Escherichia coli JM107. All clones tested were found to be infectious by the criteria of plaque titer and progressive cytopathic effect after transfection into bovine fetal lung cells. Sequencing of the recombinant plasmids demonstrated that all of the BPV inserts had left-end (3')-terminal deletions of up to 34 bases. DNA isolated from progeny virions arising from transfected infectious clones was found to be indistinguishable from wild-type DNA by restriction enzyme analysis. Defective genomes could also be detected in the progeny DNA even though the infection was initiated with homogenous, cloned DNA. Full-length genomic clones with 3' flip and 3' flop conformations were constructed and were found to have equal infectivity. Analysis of low-molecular-weight DNA isolated from lysates of cells transfected with these clones demonstrated that rescue and replication of BPV DNA could be detected 3 to 8 days after transfection. Expression of capsid proteins from transfected genomes was demonstrated by hemagglutination, indirect immunofluorescence, and immunoprecipitation of [35S]methionine-labeled cell lysates. Use of appropriate antiserum for immunoprecipitation showed the synthesis of BPV capsid and noncapsid proteins after transfection. Independently, a series of genomic clones with increasingly larger 3'-terminal deletions was prepared from separately subcloned 3'-terminal fragments. Transfection of these clones into bovine fetal lung cells revealed that deletions of up to 34 bases at the 3' end lowered but did not abolish infectivity, while deletions of greater than 52 bases were lethal. End-label analysis showed that the 34-base deletion was repaired to wild-type length in the progeny virus.

Two nonstructural proteins of bovine parvovirus (BPV) with apparent molecular sizes of 75,000 and 83,000 daltons have been detected. The proteins were immunoprecipitated from lung cells infected with various isolates of BPV and from in vitro translations of infected cell mRNA. These proteins were expressed as nuclear phosphoproteins and were synthesized early in infection, before the peak of capsid protein synthesis. Early in infection, the 75-kilodalton-size species could be resolved into two bands of equal intensity, but later in infection, the lower-molecular-size form predominated. Antibodies directed against bacterial fusion proteins encoding amino acid sequences from a highly conserved region of the NS-1 polypeptides of two other parvoviruses, minute virus of mice and the human virus B19, gave specific nuclear fluorescence with BPV-infected cells, although the antibodies failed to immunoprecipitate any viral proteins. The noncapsid proteins appear to be homologous to the previously characterized NS-1 proteins of other autonomous parvoviruses.

The distribution of terminal-sequence orientations in the viral DNA of bovine parvovirus (BPV), an autonomous parvovirus, was studied by end labeling and restriction enzyme digestion and also by cloning. The left (3') end of the minus strand of BPV was found in two alternative sequence orientations (designated as flip and flop, which are reverse complements of each other), with a 10-fold excess of flip. This is in contrast to the autonomous rodent parvoviruses which encapsidate minus-strand DNA with only the flip orientation at this end. The right (5') end of the minus strand of BPV contained both sequence orientations with equal frequencies, as in the rodent parvoviruses. Sequence inversions were also detected at both ends of the plus strand, which makes up about 10% of the encapsidated BPV DNA. Each terminus of BPV DNA had a characteristic ratio of flip to flop forms, and this ratio was restored in the progeny DNA resulting from transfection with genomic clones of different defined terminal conformations. Replicative-form DNA showed the same distribution of terminal-sequence orientations as the reannealed plus and minus virion DNAs, suggesting that the distribution of flip and flop forms observed in virion DNA is not due to selective encapsidation, but rather to the specific distribution of replicative forms. The current replication model for autonomous parvoviruses, which was based on the available data for the rodent parvoviruses, cannot account for the observed distribution of BPV DNA. An alternative model is suggested.

We determined the complete nucleotide sequence of bovine parvovirus (BPV), an autonomous parvovirus. The sequence is 5,491 nucleotides long. The terminal regions contain nonidentical imperfect palindromic sequences of 150 and 121 nucleotides. In the plus strand, there are three large open reading frames (left ORF, mid ORF, and right ORF) with coding capacities of 729, 255, and 685 amino acids, respectively. As with all parvoviruses studied to date, the left ORF of BPV codes for the nonstructural protein NS-1 and the right ORF codes for the major parts of the three capsid proteins. The mid ORF probably encodes the major part of the nonstructural protein NP-1. There are promoterlike sequences at map units 4.5, 12.8, and 38.7 and polyadenylation signals at map units 61.6, 64.6, and 98.5. BPV has little DNA homology with the defective parvovirus AAV, with the human autonomous parvovirus B19, or with the other autonomous parvoviruses sequenced (canine parvovirus, feline panleukopenia virus, H-1, and minute virus of mice). Even though the overall DNA homology of BPV with other parvoviruses is low, several small regions of high homology are observed when the amino acid sequences encoded by the left and right ORFs are compared. From these comparisons, it can be shown that the evolutionary relationship among the parvoviruses is B19 in equilibrium with AAV in equilibrium with BPV in equilibrium with MVM. The highly conserved amino acid sequences observed among all parvoviruses may be useful in the identification and detection of parvoviruses and in the design of a general parvovirus vaccine.

Total cytoplasmic RNA from bovine parvovirus (BPV)-infected cells or BPV-specific RNA selected by hybridization to cloned BPV genomic sequences were translated in a message-dependent rabbit reticulocyte lysate. Immunoprecipitation, using immunoglobulin G from rabbits injected with purified BPV, resulted in the detection of [35S]methionine-labeled polypeptides with MrS of 80,000, 72,000, 62,000, and 60,000. These in vitro translation products had the same mobility on sodium dodecyl sulfate-polyacrylamide gels as that of the four proteins found in purified virions. The three largest polypeptides had amino acid sequence homology, as judged by serological methods and partial proteolysis with Staphylococcus aureus V8 protease. Additional noncapsid proteins with MrS of 25,000, 27,000, and 31,000 were also detected as translation products of these RNAs. All of the above species were immunoprecipitated by immunoglobulin G from a calf which was naturally infected with BPV. All four capsid proteins but only one of the lower-molecular-weight polypeptides were detected after the immunoprecipitation of BPV-infected cells. The results presented here indicate that the BPV genome codes for four capsid proteins and a noncapsid protein which may be structurally related to the capsid proteins.