Simian virus 40 large T antigen DNA helicase. Characterization of the ATPase-dependent DNA unwinding activity and its substrate requirements

The ATPase of SV40 large T antigen (T antigen) which is essential for the replication of SV40 minichromosomes was recently shown to be functionally related to a newly discovered DNA helicase activity. The T antigen helicase unwinds DNA duplices of several kilobase pairs in a reaction depending on the presence of hydrolyzable ribo- or deoxyribonucleoside triphosphates. The in vitro rate of movement through duplex DNA was found to be about 100 base pairs/min at 37 degrees C. For DNA unwinding, T antigen requires a 3'-single strand extension of a partially double-stranded substrate and invades the double strand section processively, in a 3' to 5' direction. The minimum length of the single-stranded tail was determined to be less than 5 nucleotides. Unwinding studies in the presence of the Escherichia coli single strand-specific DNA-binding protein and competition experiments indicate that T antigen helicase binds preferentially at the single-stranded/double-stranded DNA junction. This DNA structure is therefore proposed to serve as an entry site for the T antigen helicase. Previously reported data suggest that T antigen is the replicative helicase of the SV40 minichromosome. The results presented here are consistent with these findings and imply that T antigen migrates actively and processively along the template for the leading strand.     

Cathepsin D is membrane-associated in macrophage endosomes

Previously we identified an acid protease activity which was located in the endosomes of rabbit alveolar macrophages (Diment, S., and Stahl, P.D. (1985) J. Biol. Chem. 260, 15311-15317). In this study, the endosomal protease is identified as cathepsin D by immunoprecipitation with polyclonal antibodies raised against rabbit cathepsin D and by NH2-terminal sequence. In order to elucidate the mechanism for targeting of cathepsin D to endosomes, we first examined the membrane association of cathepsin D with light (rho = 1.05 g/ml) and heavy density (rho = 1.1 g/ml) vesicles from Percoll density gradients. After sequential washes, 8.4 and 21.9% of cathepsin D activity remained associated with heavy and light density vesicles, respectively. This membrane-associated cathepsin D could not be solubilized in either buffer at pH 5.0 containing mannose 6-phosphate and EDTA or in buffer at pH 10.6. Solubilization required the detergent Triton X-100. To determine whether membrane-associated cathepsin D was found in endosomes, the enzyme was radioiodinated within endosomes and lysosomes with internalized lactoperoxidase. The membrane-associated form was detected in endosomes, but much less in lysosomes. Biosynthetic studies combined with the same extraction procedure revealed that macrophage cathepsin D is first synthesized as an inactive membrane-associated precursor. The precursor is processed to an active, membrane-associated form and then to the active soluble form found in lysosomes. Our studies provide evidence that 1) cathepsin D is in endosomes of macrophages; 2) cathepsin D is transported to endosomes as a membrane-associated form; and 3) the membrane-associated form is a biosynthetic precursor for the soluble form found in endosomes and lysosomes.     

Double-strand breaks can initiate meiotic recombination in S. cerevisiae

We investigated the effects of double-strand breaks on meiotic recombination in yeast. A double-strand break was introduced at the MATa locus by sporulation of a MAT alpha inc/MATa diploid under inducing conditions for the HO-encoded endonuclease; 14% of the resulting tetrads had undergone 4 alpha:0a conversion. Conversion at MAT was associated with co-conversion of a closely linked marker and an increased recombination frequency for flanking markers. We also studied the sporulation products of a diploid heterozygous at the HIS4 locus for an insertion of a 100 bp fragment of MATa containing the HO endonuclease cut site. Under inducing conditions, a significant number of tetrads were formed that had undergone gene conversions in favor of the HIS4+ allele. Although double-strand breaks can initiate meiotic recombination in yeast, the data suggest that they do not normally do so.     

The Na,K-ATPase of nervous tissue

The Na,K-ATPase has been only partially purified from nervous tissue, yet it is clear that two forms (and +) of the catalytic subunit are present. is a component subunit of the glial Na,K-ATPase, which has a relatively low affinity for binding cardiac glycosides and + has been identified as a subunit of the Na,K-ATPase which has relatively high affinity for cardiac glycosides. The + form may also be sensitive to indirect modulation by neurotransmitters or hormones. The ratio of + / changes in the nervous system during development, and + appears to be the predominant species in adult neurones. Changes in Na,K-ATPase activity have been associated with several abnormalities in the nervous system, including epilepsy and altered nerve conduction velocity, but a causal relationship has not been definitively established. Although the Na,K-ATPase has a pivotal role in Na⁺ and K⁺ transport in the nervous system, a special role for the glial Na,K-ATPase in clearing extracellular K⁺ remains controversial.     

An asparagine-rich protein from blood stages of Plasmodium falciparum shares determinants with sporozoites.

We describe a cDNA clone derived from mRNA of asexual blood-stages of the malaria parasite Plasmodium falciparum. This clone, designated Ag319, expresses a P.falciparum antigen fused to beta-galactosidase in Escherichia coli. Human antibodies from Papua New Guinea were affinity-purified by adsorption to extracts of Ag319 immobilized on CNBr-Sepharose. The antibodies reacted predominantly with P. falciparum polypeptides of Mr 220,000 and 160,000, and a number of ill-defined lower molecular weight species. Antibodies reacted in indirect immunofluorescence with all asexual blood-stages although the antigen appeared to be most abundance in the schizont. Surprizingly the antibodies also reacted with sporozoites. The amino acid sequence predicted from the complete nucleotide sequence of this clone is remarkable because 40% of the residues are Asn, and so the antigen has been termed the Asparagine-Rich Protein (ARP). Like other P. falciparum antigens, ARP contains tandemly repetitive sequences, based on the tetrapeptide Asn-Asn-Asn-Met and we have confirmed that these represent natural epitopes by reaction of the corresponding synthetic peptides with human antibodies. Surprisingly, ARP is also rich in Asn outside the tandem repeats.     

Extracellular slime associated with Proteus mirabilis during swarming.

Light microscopy, transmission electron microscopy, and scanning electron microscopy were used to visualize the extracellular slime of Proteus mirabilis swarm cells. Slime was observed with phase-contrast microscopy after fixation in hot sulfuric acid-sodium borate. Ruthenium red was used to stain slime for transmission electron microscopy. Copious quantities of extracellular slime were observed surrounding swarm cells; the slime appeared to provide a matrix through which the cells could migrate. Swarm cells were always found embedded in slime. These observations support the argument that swarming of P. mirabilis is associated with the production of large quantities of extracellular slime. Examination of nonswarming mutants of P. mirabilis revealed that a number of morphological changes, including cell elongation and increased flagellum synthesis, were required for swarm cell migration. It is still unclear whether extracellular slime production also is required for migration.     

Confirmation of protoplast fusion-derived linkages in Staphylococcus aureus by transformation with protoplast DNA

Transformation provided definitive evidence for linkage between tyrB282::Tn551 ermB321 and omega (Chr::Tn551)34, and thus between the separate large linkage groups containing these markers, in Staphylococcus aureus NCTC 8325. Transformation also defined the chromosomal loci for the purC193::Tn551 and omega (Chr::Tn551)42 markers and the linkage of a tetracycline resistance marker (tet-3490) with a fusidic acid resistance marker (fus-149). The use of DNA isolated from protoplasts under conditions that reduced hydrodynamic shear greatly facilitated the demonstration of most of these linkages. These results provide direct evidence confirming several of the linkages predicted by a microcomputer-assisted protoplast fusion analysis in a previous study (M. L. Stahl and P. A. Pattee, J. Bacteriol. 154:395-405, 1983); those markers whose predicted linkages were not confirmed by transformation are probably separated by chromosomal distances that exceed the limits of detection by transformation, even with protoplast DNA.     

A specific high-performance liquid chromatography assay for dehydroascorbic acid shows an increased content in CLL lymphocytes

A method for the assay of dehydroascorbic acid using high-performance liquid chromatography with uv detection is described. The dehydroascorbic acid is separated from ascorbic acid and reduced with dithiothreitol, and is then quantitated as ascorbic acid following rechromatography. Since as little as 22 pmol can be detected, sensitivity is at least 40-fold greater than that of other currently available procedures. This method was used to measure the level of dehydroascorbic acid in normal and chronic lymphocytic leukemia lymphocytes. A significantly higher concentration of dehydroascorbic acid was found in leukemic (21.80 +/- 3.55 nmol/10(8) cells, mean +/- SE) than in normal lymphocytes (9.32 +/- 1.15 nmol/10(8) cells) (P less than 0.03). Analysis of extracts from normal B cell lymphocytes revealed comparable dehydroascorbic acid levels to unfractionated lymphocytes, indicating that the elevated level in chronic lymphocytic leukemia was not simply a reflection of the increased percentage of B lymphocytes in this disorder. These studies illustrate that the technique can be used to measure the dehydroascorbic acid content from sources where only scanty material is available or low levels are found.     

“Unblocking” Serum Activity <Emphasis Type="Italic">in vitro</Emphasis> in the Polyoma System may Correlate with Antitumour Effects of Antiserum <Emphasis Type="Italic">in vivo</Emphasis>

In a variety of tumour systems, individuals carrying progressively growing neoplasms have lymphoid cells with a specific cytotoxic effect on cultured tumour cells from the same individual^1–4. Since the sera of tumour-bearing individuals have been shown to prevent tumour cell destruction by immune lymphocytes in vitro^2,5–8 and since this serum blocking activity appears early in primary and transplant tumour development^5,7, it has been suggested that the appearance of this serum blocking activity might be responsible for the progressive growth of tumours in individuals having cytotoxic lymphocytes. Counteraction of this blocking activity would thus be of primary importance in facilitating the function of an already existing or bolstered cell-mediated immunity. The serum blocking activity might be inhibited in various ways, by preventing the formation of blocking antibody or by interfering with its action (“unblocking”), as demonstrated in Moloney sarcoma regressor sera⁹. This type of serum also has a therapeutic effect on Moloney sarcomas in vivo^10,11, which has been tentatively attributed to its unblocking activity^8,9 or, possibly, to a complement-dependent cytotoxicity¹⁰. Tumour growth in the Moloney sarcoma system, however, might be due in part to continuous recruitment of neoplastic cells by virus-induced transformation and so the therapeutic effect could be due to a virus-neutralizing serum activity^9,10.