Production of hepatitis delta virus and suppression of helper hepatitis B virus in a human hepatoma cell line.

The hepatitis delta virus (HDV) is a defective virus with a coat composing of the surface antigen of its helper virus, hepatitis B virus (HBV). Replication of HDV in the absence of HBV has been shown in cell cultures by transient transfection of the HDV plasmid. However, the formation and release of HDV virions have not been observed. In this report, a human hepatoma cell line HuH-7 was transiently cotransfected with HDV and HBV plasmids. The production of monomeric and multimeric antigenomic RNAs of HDV in the transfected cells indicated replication of the HDV genome. The major 3.5- and 2.1-kb RNAs of HBV were also expressed. Virions of both HDV and HBV were released from the cotransfected cells, as shown by the detection of monomeric genomic HDV RNA and partially double-stranded HBV DNA in the culture medium. Thus, this is the first report that describes the assembly and the release of HDV viral particles in an in vitro cell culture. The HDV virions released possessed physicochemical properties identical to those of the HDV virions found in infected human serum. Furthermore, expression of both the 3.5- and 2.1-kb RNAs of HBV was shown to be dramatically decreased by the presence of HDV, indicating suppression of the expression of HBV genes by HDV. The amount of HBV virions released was similarly suppressed by HDV. Cotransfection of HBV with an expression plasmid of the HDV delta antigen remarkably reduced the levels of the 3.5- and 2.1-kb HBV RNAs, indicating that suppression of the expression of HBV RNAs by HDV occurs via the action of the delta antigen. This HBV- and HDV-cotransfected human hepatoma cell line should provide an excellent system for the study of the function of the delta antigen and the interaction between HDV and its helper, HBV.     

Stimulation of n-alkane conversion to dicarboxylic acid by organic-solvent- and detergent-treated microbes

Summary A wild-type strain of Cryptococcus neoformans and Pseudomonas aeruginosa were used to convert n-pentadecane to the corresponding dioic acid, tridecane 1,13-dicarboxylic acid (DC-15). Altering the cell permeability by treating C. neoformans with 1% (v/v) toluene or 7% (v/v) Triton X-100 stimulated production of DC-15 by 1.5-fold and fourfold, respectively. Furthermore, DC-15 productivity was increased from 2.5 mg/l per hour to 18 or 30 mg/l per hour, respectively. If 10% (v/v) hexane was used to treat the yeast culture, stimulation of DC-15 production could reach 200% and more viable cells remained compared to the toluene-treated culture. Data from the organic solvent treatment experiment indicated that the solvent with a higher polarity showed a more adverse effect on DC-15 production. P. aeruginosa was vulnerable to most organic solvents; however, Tween 80 could greatly stimulate the conversion of n-pentadecane to DC-15. Although organic solvents and non-ionic detergents could enhance DC-15 formation by microbial conversion, it was inhibited by elevated levels of DC-15.Offprint requests to: E.-C. Chan     

Combinatorial splicing of exon pairs by two-site binding of U1 small nuclear ribonucleoprotein particle.

A two-site model for the binding of U1 small nuclear ribonucleoprotein particle (U1 snRNP) was tested in order to understand how exon partners are selected in complex pre-mRNAs containing alternative exons. In this model, it is proposed that two U1 snRNPs define a functional unit of splicing by base pairing to the 3' boundary of the downstream exon as well as the 5' boundary of the intron to be spliced. Three-exon substrates contained the alternatively spliced exon 4 (E4) region of the preprotachykinin gene. Combined 5' splice site mutations at neighboring exons demonstrate that weakened binding of U1 snRNP at the downstream site and improved U1 snRNP binding at the upstream site result in the failure to rescue splicing of the intron between the mutations. These results indicate the stringency of the requirement for binding a second U1 snRNP to the downstream 5' splice site for these substrates as opposed to an alternative model in which a certain threshold level of U1 snRNP can be provided at either site. Further support for the two-site model is provided by single-site mutations in the 5' splice site of the third exon, E5, that weaken base complementarity to U1 RNA. These mutations block E5 branchpoint formation and, surprisingly, generate novel branchpoints that are specified chiefly by their proximity to a cryptic 5' splice site located at the 3' terminus of the pre-mRNA. The experiments shown here demonstrate a true stimulation of 3' splice site activity by the downstream binding of U1 snRNP and suggest a possible mechanism by which combinatorial patterns of exon selection are achieved for alternatively spliced pre-mRNAs.     

Crystallization of trimeric recombinant human tumor necrosis factor (cachectin)

Crystals of tumor necrosis factor (TNF) have been obtained in two forms. Rhombohedral crystals grow in 1.8 to 2.0 M ammonium sulfite, pH 7.8 at 21 degrees C, and tetragonal crystals grow in 2.6 M magnesium sulfate, pH 5.5 at 25 degrees C. Analysis of TNF by isoelectric focusing under native and denaturing conditions indicates that TNF molecules exist as trimers in solution. The rhombohedral cachectin crystals belong to space group R3 and have unit cell constants a = b = c = 47.65 A and alpha = beta = gamma = 88.1 degrees. Density determinations and the space group indicate that the unit cell contains one 51,000-dalton trimer. These crystals are stable in the x-ray beam and diffract to at least 1.85 A but are apparently twinned by merohedry. The tetragonal crystals are space group P4(3)2(1)2 or its enantiomorph P4(1)2(1)2 and have unit cell constants a = b = 95.08, c = 117.49. The asymmetric unit contains one trimer; the crystals are stable in the x-ray beam and diffract to beyond 3 A.     

Loss of sigma-factor of RNA polymerase of Xanthomonas campestris pv. oryzae during phage Xp10 infection

Ten min after infection of Xanthomonas campestris pv. oryzae by phage Xp10, a sharp decrease in the activity of the host RNA polymerase was observed. Host RNA polymerase from phage-infected and uninfected cells was purified, and their properties were compared. The enzyme from uninfected cells contained four polypeptides with Mr = 155,000, 155,000, 93,000, and 37,000, respectively, and assembled with a stoichiometry of alpha 2 beta beta' sigma. The enzyme from infected cells lacked the sigma-subunit. The enzyme from uninfected cells utilized Xp10 DNA and poly[d(A-T)] as templates, the enzyme from phage-infected cells failed to transcribe Xp10 DNA, but retained the ability to transcribe poly(A-T). The regions of the Xp10 genome transcribed by the two enzymes were also investigated. The enzyme from uninfected cells transcribed the leftmost 25-30% of the Xp10 genome. The enzyme from phage-infected cells also transcribed the same region, but the enzyme activity was very low. Other properties such as (a) the response to RNA polymerase inhibitors, (b) the effect of N-ethylmaleimide, (c) the requirement of Mg2+ and Mn2+, and (d) the optimum temperature and pH of the two enzymes were very similar.     

DNA amplification in multidrug, cross-resistant Chinese hamster ovary cells: molecular characterization and cytogenetic localization of the amplified DNA

Vincristine-resistant (VCR) Chinese hamster ovary (CHO) cells have been established by stepwise selection in increasing concentrations of vincristine. These cells exhibit multidrug cross-resistance to a number of drugs that have no structural or functional similarities. Cytogenetic analyses of resistant cells revealed the presence of double minutes and expanded chromosomal segments, thus implicating gene amplification as a possible mechanism of resistance. An amplified DNA segment isolated from other multidrug cross-resistant CHO cell lines (Roninson, I. B., H. T. Abelson, D. E. Housman, N. Howell, and A. Varshavsky, 1984, Nature (Lond.), 309:626-628) is also amplified in our VCR lines. This DNA segment was used as a probe to screen a cosmid library of VCR genomic DNA, and overlapping clones were retrieved. All of these segments, totaling approximately 45 kilobases (kb), were amplified in VCR cells. Using in situ hybridization, we localized the amplification domain to the long arm of CHO chromosome 1 or Z1. Northern hybridization analysis revealed that a 4.3-kb mRNA was encoded by this amplified DNA domain and was over-produced in the VCR cells. Suggestions for the involvement of these amplified DNA segments in the acquisition of multidrug cross-resistance in animal cells are also presented.     

Substrate specificity and protonation state of ornithine transcarbamoylase as determined by pH studies

The ornithine transcarbamoylase catalyzed reaction and its inhibition by L-norvaline have been investigated between pH 5.5 and 10.5. The steady-state turnover rate (kcat) of the enzyme from Escherichia coli increases with pH and plateaus above pH 9. Its change with pH conforms to a single protonation process with an apparent pKa of 7.3. The effect of pH on the apparent Michaelis constant (KMapp) of L-ornithine suggests that this diamino acid in its cationic form is not the substrate. Treating only the zwitterions of ornithine as substrate, the pH profile of the pseudo-first-order rate constant (kcat/KMz) of the reaction is a bell-shaped curve characterized by pKa's of 6.2 and 9.1 and asymptotic slopes of +/- 1. Similar pKa's (6.3 and 9.3) are obtained for the pKi profile of zwitterionic L-norvaline, a competitive inhibitor. The pKi profile further indicates that the alpha-amino group of the inhibitor must be charged for binding. Together, these pH profiles provide sufficient information to suggest that only the minor zwitterionic species of ornithine, H2N(CH2)3CH(NH3+)COO-, binds the enzyme productively. The selection of this substrate form by the enzyme leads to a Michaelis complex in which ornithine is poised for nucleophilic attack. Following such binding, the need for deprotonation of the delta-NH3+ group is avoided, and transcarbamoylation becomes energetically more feasible. Reaction schemes accounting for the effects of pH are proposed for the enzymic reaction.     

Monoclonal antibodies to cytochrome c from Paracoccus denitrificans: effects on electron transport reactions

The effect of a monoclonal antibody to a soluble cytochrome c from Paracoccus denitrificans was tested on the membrane-bound electron-transport system of this bacterium. This antibody (F3-10.2) and one previously described (F3-29.4) (Kuo, L.M., Davies, H.C. and Smith, L. (1984) Biochim. Biophys. Acta 766, 472-482) were deduced to bind to the cytochrome c in the area including amino acid residue number 23 on a loop on the side of the heme crevice. In contrast to the observations with the previously tested antibody, the present data show the second antibody to block completely the reaction of the cytochrome c with cytochrome c oxidase but not that with cytochrome c reductase. Neither antibody has an appreciable inhibitory effect on the NADH oxidase of the isolated detergent-treated membranes. The two antibodies bind in different ways, giving insight into the interaction of a soluble protein with membrane-bound enzymes. The data indicate that the reaction sites on the cytochrome c for the oxidase and reductase moieties of P. denitrificans are different. They also argue against the need for a dissociable cytochrome c comparable to that which functions on the mitochondrial inner membrane.     

Plants have isoforms for acyl carrier protein that are expressed differently in different tissues

Two closely related isoforms of acyl carrier protein (I and II) have been purified from spinach leaves. Differences in the N-terminal amino acid sequence and amino acid composition indicate that these proteins are coded by different genes. The two spinach leaf isoforms have been resolved and characterized by ion-exchange high-performance liquid chromatography, by thin layer isoelectric focusing, and by differences in mobility upon polyacrylamide gel electrophoresis. Both isoforms are effectively bound by antibodies raised to acyl carrier protein I. However, in competition experiments isoform II is only about 40% effective in blocking isoform I binding to antibody. Therefore, the isoforms are immunologically related but hold only some antigenic sites in common. Immunoblot analysis ("Western blotting") of crude spinach leaf tissue extracts probed with antibody to acyl carrier protein I reveals both isoforms. In addition, both forms of acyl carrier protein are present in dark-grown leaf tissue and in isolated chloroplasts. However, in spinach seeds and roots only acyl carrier protein II can be detected. Similar results are observed with extracts of castor oil plant leaf and seed. Therefore, the expression of the two acyl carrier protein isoforms is tissue specific.