Energy-independent helicase activity of a viral genome packaging motor

The assembly of complex double-stranded DNA viruses includes a genome packaging step where viral DNA is translocated into the confines of a preformed procapsid shell. In most cases, the preferred packaging substrate is a linear concatemer of viral genomes linked head-to-tail. Viral terminase enzymes are responsible for both excision of an individual genome from the concatemer (DNA maturation) and translocation of the duplex into the capsid (DNA packaging). Bacteriophage λ terminase site-specifically nicks viral DNA at the cos site in a concatemer and then physically separates the nicked, annealed strands to mature the genome in preparation for packaging. Here we present biochemical studies on the so-called helicase activity of λ terminase. Previous studies reported that ATP is required for strand separation, and it has been presumed that ATP hydrolysis is required to drive the reaction. We show that ADP and nonhydrolyzable ATP analogues also support strand separation at low (micromolar) concentrations. In addition, the Escherichia coli integration host factor protein (IHF) strongly stimulates the reaction in a nucleotide-independent manner. Finally, we show that elevated concentrations of nucleotide inhibit both ATP- and IHF-stimulated strand separation by λ terminase. We present a model where nucleotide and IHF interact with the large terminase subunit and viral DNA, respectively, to engender a site-specifically bound, catalytically competent genome maturation complex. In contrast, binding of nucleotide to the low-affinity ATP binding site in the small terminase subunit mediates a conformational switch that down-regulates maturation activities and activates the DNA packaging activity of the enzyme. This affords a motor complex that binds tightly, but nonspecifically, to DNA as it translocates the duplex into the capsid shell. These studies have yielded mechanistic insight into the assembly of the maturation complex on viral DNA and its transition to a mobile packaging motor that may be common to all of the complex double-stranded DNA viruses.     

Interval mapping of quantitative trait loci with selective DNA pooling data

Selective DNA pooling is an efficient method to identify chromosomal regions that harbor quantitative trait loci (QTL) by comparing marker allele frequencies in pooled DNA from phenotypically extreme individuals. Currently used single marker analysis methods can detect linkage of markers to a QTL but do not provide separate estimates of QTL position and effect, nor do they utilize the joint information from multiple markers. In this study, two interval mapping methods for analysis of selective DNA pooling data were developed and evaluated. One was based on least squares regression (LS-pool) and the other on approximate maximum likelihood (ML-pool). Both methods simultaneously utilize information from multiple markers and multiple families and can be applied to different family structures (half-sib, F2 cross and backcross). The results from these two interval mapping methods were compared with results from single marker analysis by simulation. The results indicate that both LS-pool and ML-pool provided greater power to detect the QTL than single marker analysis. They also provide separate estimates of QTL location and effect. With large family sizes, both LS-pool and ML-pool provided similar power and estimates of QTL location and effect as selective genotyping. With small family sizes, however, the LS-pool method resulted in severely biased estimates of QTL location for distal QTL but this bias was reduced with the ML-pool.     

Myoepithelioma within the carpal tunnel: a case report and review of the literature

Myoepitheliomas of the extremity are rare and usually benign, while a minority display malignant features. This case demonstrates the diagnosis and management of myoepithelioma within the carpal tunnel. Clinical and radiological tumour features were evaluated. Hematoxylin and eosin stained tumour sections were examined, and immunohistochemistry was performed. Histology revealed a nodular mass of epithelioid cells in clusters within a myxoid/chondroid stroma. No mitoses were noted. Cytokeratins, neuron-specific enolase, synaptophysin, glial fibrillary acidic protein, and S100 were positive on immunohistochemistry. A literature review revealed very few prior reports of myoepithelioma in the wrist, and limited data concerning any relationship between recurrence and quality of surgical margins. In this case, wide local excision would have significantly compromised dominant hand function, and therefore a marginal excision was deemed appropriate in the context of bland histological features. Surgical margins noted in future case reports will aid clinical decision making.     

Stable isotope evidence of ontogenetic changes in the diet of gizzard shad Dorosoma cepedianum

Stable sulphur isotopic composition (δ³⁴S) of gizzard shad Dorosoma cepedianum was used to investigate the seasonal and ontogenetic variation in the diet of young and adult fish. This study evaluated fish from a hypereutrophic lake that had recently undergone a 40% reduction of large (>300 mm total length, L _T) D. cepedianum biomass as part of a biomanipulation experiment, which aimed at reducing internal nutrient loading. Dorosoma cepedianum δ³⁴S values showed evidence of ontogenetic changes with young fish (<200 mm L _T) depending more on benthic food sources than adults (>200 mm L _T). The δ³⁴S composition of the adult fish suggested an increasing importance of zooplankton in the diet, although benthic food sources remained part of the diet of all D. cepedianum collected in this study. The results indicated that benthic feeding is used by D. cepedianum of all sizes, suggesting that biomanipulation efforts may need to target all sizes of fish to realize benefits.     

Phylogenetic analysis of 73 060 taxa corroborates major eukaryotic groups

Obtaining a well supported schema of phylogenetic relationships among the major groups of living organisms requires considering as much taxonomic diversity as possible, but the computational cost of calculating large phylogenies has so far been a major obstacle. We show here that the parsimony algorithms implemented in TNT can successfully process the largest phylogenetic data set ever analysed, consisting of molecular sequences and morphology for 73 060 eukaryotic taxa. The trees resulting from molecules alone display a high degree of congruence with the major taxonomic groups, with a small proportion of misplaced species; the combined data set retrieves these groups with even higher congruence. This shows that tree-calculation algorithms effectively retrieve phylogenetic history for very large data sets, and at the same time provides strong corroboration for the major eukaryotic lineages long recognized by taxonomists.     

Building a virus from scratch: assembly of an infectious virus using purified components in a rigorously defined biochemical assay system

The assembly of double-stranded DNA (dsDNA) viruses such as poxvirus, the herpesviruses and many bacteriophages is a complex process that requires the coordinated activities of numerous proteins of both viral and host origin. Here, we report the assembly of an infectious wild-type lambda virus using purified proteins and commercially available DNA, and optimization of the assembly reaction in a rigorously defined biochemical system. Seven proteins, purified procapsids and tails, and mature lambda DNA are necessary and sufficient for efficient virus assembly in vitro. Analysis of the reaction suggests that (i) virus assembly in vitro is optimal under conditions that faithfully mimic the intracellular environment within an Escherichia coli cell, (ii) concatemeric DNA is required for the successful completion of virus assembly, (iii) several of the protein components oligomerize concomitant with their step-wise addition to the nascent virus particle and (iv) tail addition is the rate-limiting step in virus assembly. Importantly, the assembled virus may enter either of the developmental pathways (lytic or lysogenic) expected of a lambda virion. Thus, we demonstrate for the first time that a wild-type, complex DNA virus may be assembled from purified components under defined biochemical conditions. This system provides a powerful tool to characterize, at the molecular level, the step-by-step processes required to assemble an infectious virus particle. Given the remarkable similarities between dsDNA bacteriophage and eukaryotic dsDNA viruses, characterization of the lambda system has broad biological implications in our understanding of virus development at a global level.     

Evidence for the Presence of a Ganglioside Transfer Protein in Brain

An extract from rat brain has been shown to catalyze the transfer of ganglioside GM1 from sonicated vesicles to erythrocyte ghosts. It also enhanced the transfer of GM1 to a crude neuronal membrane preparation, whereas myelin took up only a very limited amount. The transfer activity was heat-labile. Similar transfer activities were found in extracts from bovine gray and white matter, that of the former being comparable to rat brain whereas the latter was greater per milligram protein.     

Temperature-sensitive mutants ofStaphylococcus aureus: Isolation and preliminary characterization

Temperature-sensitive (ts) mutants ofStaphylococcus aureus were isolated after mutagenesis with nitrosoguanidine and two cycles of enrichment with Penicillin G and D-Cycloserine. The mutants expressed tight, coasting, and leaky phenotypes on solid media. In broth, however, most exhibited coasting for a limited number of generations. The reversion frequency of selected ts mutants was less than 10^–6. Intraperitoneal (i.p.) immunization with ts mutant G/1/2 conferred significant protection (0 dead/6 total vs. 7/7, immunized vs. control; p=0.0006) from lethal i.p. challenge with the parental wild-type (wt)S. aureus suspended in 5% porcine mucin, performed 28 days after i.p. administration of 10⁸ colony-forming units. Protection induced by mutants of coasting phenotype was higher and lasted longer than that induced by mutants of the tight phenotype. The results of this study demonstrate that ts mutants ofS. aureus can be obtained and that ts mutants are able to induce protective immunity from subsequent challenge with the parental wt strain.     

Kinetic characterization of the GTPase activity of phage lambda terminase: evidence for communication between the two "NTPase" catalytic sites of the enzyme.

The terminase enzyme from bacteriophage lambda is responsible for the insertion of viral DNA into the confined space within the capsid. The enzyme is composed of the virally encoded proteins gpA (73.3 kDa) and gpNu1 (20.4 kDa) isolated as a gpA(1).gpNu1(2) holoenzyme complex. Lambda terminase possesses a site-specific nuclease activity, an ATP-dependent DNA strand-separation activity, and an ATPase activity that must work in concert to effect genome packaging. We have previously characterized the ATPase activity of the holoenzyme and have identified catalytic active sites in each enzyme subunit [Tomka and Catalano (1993) Biochemistry 32, 11992-11997; Hwang et al. (1996) Biochemistry 35, 2796-2803]. We have noted that GTP stimulates the ATPase activity of the enzyme, and terminase-mediated GTP hydrolysis has been observed. The studies presented here describe a kinetic analysis of the GTPase activity of lambda terminase. GTP hydrolysis by the enzyme requires divalent metal, is optimal at alkaline pH, and is strongly inhibited by salt. Interestingly, while GTP can bind to the enzyme in the absence of DNA, GTP hydrolysis is strictly dependent on the presence of polynucleotide. Unlike ATP hydrolysis that occurs at both subunits of the holoenzyme, a single catalytic site is observed in the steady-state kinetic analysis of GTPase activity (k(cat) approximately 37 min(-)(1); K(m) approximately 500 microM). Moreover, while GTP stimulates ATP hydrolysis (apparent K(D) approximately 135 microM for GTP binding), all of the adenosine nucleotides examined strongly inhibit the GTPase activity of the enzyme. The data presented here suggest that the two "NTPase" catalytic sites in terminase holoenzyme communicate, and we propose a model describing allosteric interactions between the two sites. The biological significance of this interaction with respect to the assembly and disassembly of the multiple nucleoprotein packaging complexes required for virus assembly is discussed.