Site-specific recognition of bacteriophage T4 DNA by T4 type II DNA topoisomerase and Escherichia coli DNA gyrase

The site specificity of bacteriophage T4-induced type II DNA topoisomerase action on double-stranded DNA has been explored by studying the sites where DNA cleavages are induced by the enzyme. Oxolinic acid addition increases the frequency at which phi X174 duplex DNA is cut by the enzyme by about 100-fold, to the point where nearly every topoisomerase molecule causes a double-stranded DNA cleavage event. The effect of oxolinic acid on the enzyme is very similar to its effect on another type II DNA topoisomerase, the Escherichia coli DNA gyrase. A filter-binding method was developed that allows efficient purification of topoisomerase-cleaved DNA fragments by selecting for the covalent attachment of this DNA to the enzyme. Using this method, T4 topoisomerase recognition of mutant cytosine-containing T4 DNA was found to be relatively nonspecific, whereas quite specific recognition sites were observed on native T4 DNA, which contains glucosylated hydroxymethylcytosine residues. The increased specificity of native T4 DNA recognition seems to be due entirely to the glucose modification. In contrast, E. coli DNA gyrase shows a high level of specificity for both the mutant cytosine-containing DNA and native T4 DNA, recognizing about five strong cleavage sites on both substrates. An unexpected feature of DNA recognition by the T4 topoisomerase is that the addition of the cofactor ATP strongly stimulates the topoisomerase-induced cleavage of native T4 DNA, but has only a slight effect on cleavage of cytosine-containing T4 DNA.     

Two-dimensional gel analysis of rolling circle replication in the presence and absence of bacteriophage T4 primase.

The rolling circle DNA replication structures generated by the in vitro phage T4 replication system were analyzed using two-dimensional agarose gels. Replication structures were generated in the presence or absence of T4 primase (gp61), permitting the analysis of replication forks with either duplex or single-stranded tails. A characteristic arc shape was visualized when forks with single-stranded tails were cleaved by a restriction enzyme with the help of an oligonucleotide that anneals to restriction sites in the single-stranded tail. After calibrating the gel system with this well-studied rolling circle replication reaction, we then analyzed the in vivo replication directed by a T4 replication origin cloned within a plasmid. DNA samples were generated from infections with either wild-type or primase-deletion mutant phage. The only replicative arc that could be detected in the wild-type sample corresponded to duplex Y forms, consistent with very efficient lagging strand synthesis. Surprisingly, we obtained evidence for both duplex and single-stranded DNA tails in the samples from the primase-deficient infection. We conclude that a relatively inefficient mechanism primes lagging strand DNA synthesis in vivo when gp61 is absent.     

Repair of Double-Strand Breaks in Bacteriophage T4 by a Mechanism That Involves Extensive DNA Replication

We investigated double-strand break (dsb) repair in bacteriophage T4 using a physical assay that involves a plasmid substrate with two inverted DNA segments. A dsb introduced into one repeat during a T4 infection induces efficient dsb repair using the second repeat as a template. This reaction is characterized by the following interesting features. First, the dsb induces a repair reaction that is directly coupled to extensive plasmid replication; the repaired/replicated product is in the form of long plasmid concatemers. Second, repair of the dsb site is frequently associated with exchange of flanking DNA. Third, the repair reaction is absolutely dependent on the products of genes uvsX, uvsY, 32, 46, and 59, which are also required for phage genomic recombination-dependent DNA replication. Fourth, the coupled repair/replication reaction is only partly dependent on endonuclease VII (gp49), suggesting that either another Holliday-junction-cleaving activity or an alternate resolution pathway is active during T4 infections. Because this repair reaction is directly coupled to extensive replication, it cannot be explained by the SZOSTAK et al. model. We present and discuss a model for the coupled repair/replication reaction, called the extensive chromosome replication model for dsb repair.     

Integration of Plasmids into the Bacteriophage T4 Genome

We have analyzed the integration of plasmids into the bacteriophage T4 genome via homologous recombination. As judged by genetic selection for a plasmid-borne marker, a mutation in phage gene uvsX or uvsY essentially blocked the integration of a plasmid with homology to the T4 genome but no phage replication origin (non-origin plasmid). The strict requirement for these two proteins suggests that plasmid integration can proceed via a strand-invasion reaction similar to that catalyzed in vitro by the T4-encoded strand-exchange protein (UvsX) in concert with UvsY and gp32. In contrast to the results with the non-origin plasmid, a mutation in uvsX or uvsY reduced the integration of a T4 replication origin-containing plasmid by only 3-10-fold. These results suggest that the origin-containing plasmid integrates by both the UvsXY-dependent pathway used by the non-origin plasmid and by a UvsXY-independent pathway. The origin-containing plasmid integrated into the phage genome during a uvsX-or uvsY-mutant infection of a recA-mutant host, and therefore origin-dependent integration can occur in the absence of both phage- and host-encoded strand-exchange proteins (UvsX and RecA, respectively).     

Seed transmission of turnip yellow mosaic virus in winter turnip and winter oilseed rapes

This is the first record of seed transmission of turnip yellow mosaic virus (TYMV) in oilseed and turnip rapes. The seed transmission of TYMV in a naturally infected winter turnip rape (Brassica napus var. silvestris) cultivar Perko PVH was investigated. By ELISA 1.6%, 3.2% and 8.3% seed transmission of the virus was found in seed of plants from three localities. The proportion of infected seeds produced by artificially infected plants of winter oilseed rape (Brassica napus ssp. oleifera) and winter turnip rape cultivars was determined. The virus transmission rate, expressed as the proportion of virus-infected plants which germinated from the seed was for the oilseed rape cvs Jet Neuf 0.1%, Solida 0.4%, Silesia 0.8%, Darmor 1.2%, SL-507 0.2%, SL-509 0.0% and for the winter turnip rape cv. Perko 1.5%. ELISA cannot be used in direct tests on bulk seed lots to estimate proportion of infected seed, but must be used on germinated seedlings.     

Aspects of hadrosaurian cranial anatomy

Supraorbital bones in Saurolophus angustirostris are described, and their presence in all hadrosaurs is suggested. Frontal-nasal and premaxillar-nasal fontanellae are distinguished in hadrosaurs; their presence is explained as connected with growth and considered to he responsible for the variability of crest structures. New data indicating the presence of a cartilaginous diverticulum nasi within the circumnarial depression in Saurobphus ongustirostris are presented. A physiological (respiratory and/or thermoregulatory) function of the nasal diverticulum is proposed.     

Aspiration biopsy cytology, mammography and clinical exploration: a modern set up in diagnosis of tumors of the breast.

Six hundred and two mammary tumors were examined clinically, by mammography and cytology, with a histologic checkup following surgical biopsy. There were 247 cases of malignoma and 355 benign cases. The limited reliability of the individual methods is demonstrated, and it is shown that their combined use can improve the diagnosis. More malignomas are detected, and preoperative diagnosis is made more safely. If all three methods yield identical results, as was the case in 50.2% of the malignant and 32.7% of the benign lesions, the probability of diagnostic error is less than one per cent. With a malignoma thus established, surgical treatment may follow immediately, or irradiation can be started. In benign cases a surgical biopsy may be foregone and further developments may be awaited with due provision for regular control. If the three methods yield conflicting or doubtful results, elucidation by surgical biopsy and histology is indicated.