Transport to the cell surface of a peptide sequence attached to the truncated C terminus of an N-terminally anchored integral membrane protein.

Attempts to construct hybrid proteins that are transported to the plasma membrane are frequently unsuccessful because of perturbations in polypeptide folding. In seeking to minimize this problem, we have used the less common type of integral membrane protein, which has an uncleaved signal-anchor domain and an extracellular carboxyl portion, to transport a peptide sequence of interest to the cell surface. A set of plasmids was constructed that contained the gene encoding respiratory syncytial virus glycoprotein G (RSVG) interrupted immediately after one of several proline codons by a synthetic sequence containing unique restriction endonuclease sites and a stop codon. The shortened RSVG gene was flanked by vaccinia virus DNA to permit cloning and expression in a vaccinia virus vector. An open reading frame encoding four copies of the immunodominant repeating epitope of the circumsporozoite protein of Plasmodium falciparum was inserted into the tails of the truncated RSVG genes. Recombinant vaccinia viruses were isolated and shown to express hybrid proteins that reacted with a monoclonal antibody directed to the repeating circumsporozoite epitope. Moreover, immunofluorescence studies indicated that the peptide was on the external cell surface and available to react with antibodies. Expression of the hybrid protein also occurred in rabbits inoculated with the live recombinant vaccinia virus, as demonstrated by the generation of antibodies that bound to P. falciparum sporozoites in vitro.     

Prediction of the secondary structures of bovine blood coagulation factor IX, factor X, and prothrombin

The secondary structures of bovine blood coagulation factors IX and X, as well as that of bovine prothrombin, were predicted on the basis of a computerized combination of the Chou-Fasman and Burgess algorithms. Refinements in the predictions were made after consideration of the content of various secondary structures, as determined by circular dichroism studies of these same proteins. The final turn assignments were in good agreement with those assigned with use of an algorithm involving pattern matching of -turns in proteins of known structure.     

Predicting genotypes at loci for autosomal recessive disorders using linked genetic markers: application to Wilson's disease

Summary Recently, the Wilson's disease locus (WND) has been mapped to the long arm of chromosome 13. We have analyzed segregation of serveral chromosome 13 markers flanking the WND locus and used multipoint linkage analysis to determine the most likely WND genotype of each of 57 unaffected individuals in 5 Wilson's disease families. Approximately 46% of these could be classified as carrier (heterozygote), homozygous normal, or homozygous affected (not yet symptomatic) with a probability of at least 90%, while 77% could be classified with a probability of at least 80%. Our results demonstrate that even though there is a significant decrease on average in serum copper concentration in Wilson's disease heterozygotes compared to normal homozygotes, other sources of variation in serum copper concentration are much greater and preclude use of serum copper to detect heterozygotes for Wilson's disease. Subsequent analyses showed that a familial component, independent of WND genotype, is the major factor accounting for variation in ceruloplasmin levels among unaffected individuals; age is another factor accounting for more variation in copper levels among unaffected individuals than WND genotype.     

Microbial cell responses to a non-ionic surfactant

Summary The effects of a non-ionic surfactant, Pluronic F-68, on growth of microbial cell cultures have been studied. Growth ofSaccharomyces cerevisiae at 30°C or 37°C as measured by viable cell counts was unaffected by culture with pluronic. However, corresponding absorbance measurements forS. cerevisiae incubated with 5–10% pluronic were lower than controls at both temperatures. Absorbance ofE. coli cultures was also significantly reduced by incubation with 5.0–10.0% pluronic at both temperatures although viable counts again revealed no significant inhibition of growth.     

Transport of proteins into chloroplasts

The import of cytoplasmically synthesized proteins into chloroplasts involves an interaction between at least two components; the precursor protein, and the import apparatus in the chloroplast envelope membrane. This review summarizes the information available about each of these components. Precursor proteins consist of an amino terminal transit peptide attached to a passenger protein. Transit peptides from various precurosrs are diverse with respect to length and amino acid sequence; analysis of their sequences has not revealed insight into their mode of action. A variety of foreign passenger proteins can be imported into chloroplasts when a transit peptide is present at the amino terminus. However, foreign passenger proteins are not imported as efficiently as natural passenger proteins, and some chimeric precursor proteins are not imported into chloroplasts at all. Therefore, the passenger protein, as well as the transit peptide, influences the import process. Import begins by binding of the precursor to the chloroplast surface. It has been suggested that this binding is mediated by a receptor, but evidence to support this hypothesis remains incomplete and a receptor protein has not yet been characterized. Protein translocation requires energy derived from ATP hydrolysis, although there are conflicting reports as to where hydrolysis occurs and it is unclear how this energy is utilized. The mechanism(s) whereby proteins are translocated across either the two envelope membranes or the thylakoid membrane is not known.Abbreviations EPSP 5-enolpyruvyulshikimate-3-phosphate - LHCP Chlorophyll a/b binding protein of the light-harvesting complex - NPT-II Neomycin phosphotransferase II - PC Plastocyanin - Pr Precursor - Rubisco Ribulose-1,5,-bisphosphate carboxylase/oxygenase - SS Small subunit of Rubisco     

Immunization with a vaccinia virus recombinant expressing herpes simplex virus type 1 glycoprotein D: long-term protection and effect of revaccination.

Previously we showed that mice immunized with a vaccinia virus vector expressing the herpes simplex virus type 1 (HSV-1) glycoprotein D (gD) gene (vaccinia/gD) were protected against both lethal and latent infections with HSV-1 for at least 6 weeks after immunization (K. J. Cremer, M. Mackett, C. Wohlenberg, A. L. Notkins, and B. Moss, Science 228:737-740, 1985). In the experiments described here, we examined long-term immunity to HSV following vaccinia/gD vaccination, the effect of revaccination with vaccinia/gD, and the impact of previous immunity to vaccinia virus on immunization with the gD recombinant. Mice immunized with vaccinia/gD showed 100, 100, and 80% protection against lethal infection with HSV-1 at 18, 44, and 60 weeks postimmunization, respectively. Protection against latent trigeminal ganglionic infection was 70, 50, and 31% at 6, 41, and 60 weeks postvaccination, respectively. To study the effect of reimmunization on antibody levels, mice vaccinated with vaccinia/gD were given a second immunization (booster dose) 3 months after the first. These mice developed a 10-fold increase in neutralizing-antibody titer (221 to 2,934) and demonstrated a significant increase in protection against lethal HSV-1 challenge compared with animals that received only one dose of vaccinia/gD. To determine whether preexisting immunity to vaccinia virus inhibited the response to vaccination with vaccinia/gD virus, mice were immunized with a recombinant vaccinia virus vector expressing antigens from either influenza A or hepatitis B virus and were then immunized (2 to 3 months later) with vaccinia/gD. These mice showed reduced titers of neutralizing antibody to HSV-1 and decreased protection against both lethal and latent infections with HSV-1 compared with animals vaccinated only with vaccinia/gD. We conclude that vaccination with vaccinia/gD produces immunity against HSV-1 that lasts over 1 year and that this immunity can be increased by a booster but that prior immunization with a vaccinia recombinant virus expressing a non-HSV gene reduces the levels of neutralizing antibody and protective immunity against HSV-1 challenge.     

Basal ppGpp level adjustment shown by new spoT mutants affect steady state growth rates and rrnA ribosomal promoter regulation in Escherichia coli

Summary This work describes an approach towards analyzing the regulatory effects of variation of guanosine 3,5-bispyrophosphate (ppGpp) basal levels in Escherichia coli during steady state growth. A series of strains was derived by mutating the spoT gene (which encodes the major cellular ppGppase) so as to obtain systematic increments in ppGpp basal levels. These strains differ genetically at the spoT locus and, in some cases, also at the relA locus because of the severity of spoT mutant alleles. Measurements of ppGpp revealed a ten-fold range of basal levels during growth on minimal medium. The empirical relationship between ppGpp concentration and growth rate is a simple linear inverse correlation. Tandem rrnA ribosomal RNA promoters, present on a multicopy plasmid, are shown to be differentially regulated over this range of basal levels. The upstream P ₁ promoter activity shows an inverse exponential relation to ppGpp concentration whereas the downstream P ₂ promoter is only weakly affected. We conclude that there are systematic regulatory consequences associated with small changes in ppGpp basal levels during steady state growth that probably are part of a continuum with more dramatic effects observed during the stringent response to amino acid deprivation.     

Allometric shape change and heterochrony in the freeliving coral Trachyphyllia bilobata (Duncan)

Regression analysis has been used to study the relationship between age, size, shape, and surface area in two ancestral-descendant populations of the Neogene Caribbean coral Trachyphyllia bilobata. Analyses of the relationship between size and age show that the relationship is isometric and that little difference occurs between populations in mean corallite length or height and in their rates of growth. Onset of columella growth is significantly earlier, however, in the descendant population. Studies of the relationship between size and shape show that growth is allometric, with shape change occurring in both corallum elongation and pinching of the corallite wall during ontogeny. In the descendant population, pinching and elongation initiate earlier in the ontogeny of the coral. These results suggest that the evolutionary development of the meandroid form in freeliving corals has been accomplished by heterochrony, involving a complex set of disassociated peramorphic changes in ontogeny accompanied by paedomorphic changes in astogeny. Further analyses show that the observed heterochronic changes serve to decrease corallum surface area which may in turn enhance sediment removal and nutrition in unstable habitats.     

Identification and characterization of a novel postlarval hemolymph protein from Manduca sexta

The identification, purification and characterization of a new postlarval specific hemolymph protein from Manduca sexta is described. Incorporation of [³⁵S]methionine into Manduca sexta hemolymph proteins in vivo was investigated as a function of development. A major protein band of M_r ≈ 50,000 was highly labeled during the prepupal and adult stage but not in feeding larvae. This postlarval protein (PLP) was isolated from adult male hemolymph and its chemical and immunological properties determined. PLP is a basic protein (pI ～8.6). Electrophoresis under denaturing conditions reveals a subunit M_r ≈ 50,000 while the native protein has an apparent M_r ～ 85,000 by gel permeation chromatography. Anti-PLP serum recognized PLP but not other hemolymph proteins on immunoblots. In vitro translation of fat body mRNA followed by immunoprecipitation revealed that fat body is the site of PLP synthesis. Quantitation of PLP levels in hemolymph throughout development was performed and suggests PLP may play a role in adult development of M. sexta.     

Human hepatocyte and kidney cell metabolism of 2-acetylaminofluorene and comparison to the respective rat cells

The metabolism and mutagenic activation of 2-acetylaminofluorene by human and rat hepatocytes and kidney cells were measured. High performance liquid chromatography was used to separate the 2-acetylaminofluorene metabolites, and a cell-mediated Salmonella typhimurium mutagenesis assay was used to detect mutagenic intermediates. Rat and human differences were observed with cells from both organs and levels of metabolism and mutagenesis were higher in human cells. Within a species, liver and kidney cell differences were also evident, with levels of hepatocyte-mediated metabolism and mutagenesis being greater than kidney cells. Human inter-individual variation was apparent with cells from both organs, but the variation observed was significantly greater in hepatocytes than kidney cells. A knowledge of such differences, including an understanding that they may vary with the chemical being studied, should be useful in the extrapolation of rodent carcinogenesis data to humans.Abbreviations AAF 2-acetylaminofluorene - AF 2-aminofluorene - DMSO dimethylsulfoxide - HPLC high performance liquid chromatography - N-OH-AAF N-hydroxy-2-acetylaminofluorene - 1-OH-AAF 1-hydroxy-2-acetylaminofluorene - 3-OH-AAF 3-hydroxy-2-acetylaminofluorene - 5/9-OH-AAF a combination of 5 and 9-hydroxy-2-acetylaminofluorene - 7-OH-AAF 7-hydroxy-2-acetylaminofluorene - 8-OH-AAF 8-hydroxy-2-acetylaminofluorene