Inheritance of the general shell color in the scallop Argopecten purpuratus (Bivalvia: Pectinidae)

Although some external coloration and pigmentation patterns in molluscan shells may be attributable to environmental factors, most variation in these phenotypic characters depends on uncomplicated genetic mechanisms. Genetic research on inheritance of color variations in the north-Chilean scallop (Argopecten purpuratus) has now been expanded to analyze color segregation in juvenile scallops produced under controlled conditions employing self- and cross-fertilization. Calculations from the results were used for comparison with different numerical models based on Mendelian inheritance, and results were also obtained on the inheritance of a dorsoventral white line often observed on the left (upper) valve in this species. The results confirmed the hereditary basis for color variation in the shell of this scallop, suggesting a simple, dominant model of epistasis to explain the distribution of the different color variants observed (purple, brown, orange, yellow, and white). The presence of the white line may be controlled by a recessive allele with simple Mendelian traits on a locus distinct from those that control color variation.     

Molecular characterization of Bombyx mori cytoplasmic polyhedrosis virus genome segment 4

The complete nucleotide sequence of the genome segment 4 (S4) of Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) was determined. The 3,259-nucleotide sequence contains a single long open reading frame which spans nucleotides 14 to 3187 and which is predicted to encode a protein with a molecular mass of about 130 kDa. Western blot analysis showed that S4 encodes BmCPV protein VP3, which is one of the outer components of the BmCPV virion. Sequence analysis of the deduced amino acid sequence of BmCPV VP3 revealed possible sequence homology with proteins from rice ragged stunt virus (RRSV) S2, Nilaparvata lugens reovirus S4, and Fiji disease fijivirus S4. This may suggest that plant reoviruses originated from insect viruses and that RRSV emerged more recently than other plant reoviruses. A chimeric protein consisting of BmCPV VP3 and green fluorescent protein (GFP) was constructed and expressed with BmCPV polyhedrin using a baculovirus expression vector. The VP3-GFP chimera was incorporated into BmCPV polyhedra and released under alkaline conditions. The results indicate that specific interactions occur between BmCPV polyhedrin and VP3 which might facilitate BmCPV virion occlusion into the polyhedra.     

Cellular motor protein KIF-4 associates with retroviral Gag

Previously we demonstrated that murine retroviral Gag proteins associate with a cellular motor protein, KIF-4. Using the yeast two-hybrid assay, we also found an association of KIF-4 with Gag proteins of Mason-Pfizer monkey virus (MPMV), simian immunodeficiency virus (SIV), and human immunodeficiency virus type 1 (HIV-1). Studies performed with mammalian cell systems confirmed that the HIV-1 Gag protein associates with KIF-4. Soluble cytoplasmic proteins from cells infected with recombinant vaccinia virus expressing the entire Gag-Pol precursor protein of HIV-1 or transfected with HIV-1 molecular clone pNL4-3 were fractionated by sucrose gradient centrifugation and further separated by size-exclusion and anion-exchange chromatographies. KIF-4 and HIV-1 Gag cofractionated in both chromatographic separations. Immunoprecipitation assays have also verified the KIF-4-Gag association. KIF-4 binds mainly to the Gag precursor (Pr55 Gag) and a matrix-capsid processing intermediate (Pr42) but not to other processed Gag products. The binding of Gag is mediated by a domain of KIF-4 proximal to the C terminus. These results, and our previous studies, raise the possibility that KIF-4 may play an important role in retrovirus Gag protein transport.     

Competition of Abeta amyloid peptide and apolipoprotein E for receptor-mediated endocytosis

The genetic polymorphism of apolipoprotein E (apoE) is associated with the age of onset and relative risk of Alzheimer's disease (AD). In contrast to apoE3, the wild type allele, apoE4 confers an increased risk of late-onset AD. We demonstrate that the beta-amyloid peptide isoforms Abeta (1-28), Abeta (1-40), and Abeta (1-43) compete for the cellular metabolism of apoE3 and apoE4 containing beta-very low density lipoproteins. An antibody raised against Abeta (1-28) cross-reacted with recombinant apoE. Epitope mapping revealed positive amino acid clusters as common epitopes of Abeta (13 through 17; HHQKL) and apoE (residues 144 through 148; LRKRL), both regions known to be heparin binding domains. Abeta in which amino acids 13 through 17 (HHQKL) were replaced by glycine (GGQGL) failed to compete with the cellular uptake of apoE enriched betaVLDL.These observations indicate that Abeta and apoE are taken up into cells by a common pathway involving heparan sulfate proteoglycans.     

Mismatch repair is diminished during stationary-phase mutation.

This paper is an invited Response to a recent Commentary [P.L. Foster, Rev. Mut. Res. 436 (1999) 179-184] entitled "Are adaptive mutations due to a decline in mismatch repair? The evidence is lacking". The Commentary argues that no evidence exists supporting the idea that mismatch repair is limiting specifically during stationary-phase mutation. A primary concern of the author is to question the method that we used previously to measure growth-dependent mutation. In this method, mutation rates are calculated using counts of mutant colonies taken at times when those colonies arise, rather than at a predetermined, fixed time. Here we show further data that illustrate why this must be done to ensure accurate mutation measurements. Such accuracy was necessary for our published determination that mismatch repair proteins are not limiting during growth-dependent mutation, but become so during stationary-phase mutation. We review the evidence supporting the idea that stationary-phase reversion of a lac frameshift mutation occurs in an environment of decreased mismatch repair capacity. Those data are substantial. The data presented in the Commentary, in apparent contradiction to this idea, do not justify the conclusion presented there.     

Pa-AGOG, the founding member of a new family of archaeal 8-oxoguanine DNA-glycosylases

Oxidative damage represents a major threat to genomic stability, as the major product of DNA oxidation, 8-oxoguanine (GO), frequently mispairs with adenine during replication. In order to prevent these mutagenic events, organisms have evolved GO-DNA glycosylases that remove this oxidized base from DNA. We were interested to find out how GO is processed in the hyperthermophilic archaeon Pyrobaculum aerophilum, which lives at temperatures around 100°C. To this end, we searched its genome for open reading frames (ORFs) bearing the principal hallmark of GO-DNA glycosylases: a helix–hairpin–helix motif and a glycine/proline-rich sequence followed by an absolutely conserved aspartate (HhH-GPD motif). Interestingly, although the P.aerophilum genome encodes three such ORFs, none of these encodes the potent GO-processing activity detected in P.aerophilum extracts. Fractionation of the extracts, followed by analysis of the active fractions by denaturing polyacrylamide gel electrophoresis, showed that the GO-processing enzyme has a molecular size of ~30 kDa. Mass spectrometric analysis of proteins in this size range identified several peptides originating from P.aerophilum ORF PAE2237. We now show that PAE2237 encodes AGOG (Archaeal GO-Glycosylase), the founding member of a new family of DNA glycosylases, which can remove GO from single- and double-stranded substrates with great efficiency.     

A novel concept for ligand attachment to oligonucleotides via a 2'-succinyl linker

Conjugation of ligands to antisense oligonucleotides is a promising approach for enhancing their effects. In this report, a new method for synthesizing oligonucleotide conjugates is described. 2′-Amino-2′-deoxy-5′-dimethoxytrityl-uridine was select ively acylated with a succinic acid linker at the 2′ position. This compound was incorporated at the 3′ end of an oligonucleotide corresponding to the sequence of Oblimersen. The carboxyl group was protected for oligonucleotide synthesis as a benzyl ester, which could be selectively cleaved at the solid phase by a catalytic phase transfer reaction using palladium nanoparticles as catalyst. An oligonucleotide–fluorescein conjugate was prepared by condensation of aminofluorescein. Circular dichroism spectroscopic experiments showed a B-DNA type structure. The melting temperature of the duplex was only slightly lower than that of Oblimersen. Biological activity measured by western blotting resulted in a Bcl-2 target downregulation nearly identical to that of control Oblimersen on human melanoma cells, proving that this method is attractive for the binding of ligands located in the minor groove.     

Mutations affecting somite formation in the Medaka (Oryzias latipes)

The metameric structure of the vertebrate trunk is generated by repeated formation of somites from the unsegmented presomitic mesoderm (PSM). We report the initial characterization of nine different mutants affecting segmentation that were isolated in a large-scale mutagenesis screen in Medaka (Oryzias latipes). Four mutants were identified that show a complete or partial absence of somites or somite boundaries. In addition, five mutations were found that cause fused somites or somites with irregular sizes and shapes. In situ hybridization analysis using specific markers involved in the segmentation clock and antero-posterior (A-P) polarity of somites revealed that the nine mutants can be compiled into two groups. In group 1, mutants exhibit defects in tailbud formation and PSM prepatterning, whereas A-P identity in the somites is defective in group 2 mutants. Three mutants (planlos, pll; schnelles ende, sne; samidare, sam) have characteristic phenotypes that are similar to those in zebrafish mutants affected in the Delta/Notch signaling pathway. The majority of mutants, however, exhibit somitic phenotypes distinct from those found in zebrafish, such as individually fused somites and irregular somite sizes. Thus, these Medaka mutants can be expected to provide clues to uncovering novel components essential for somitogenesis.