Identification of two new Pmp22 mouse mutants using large-scale mutagenesis and a novel rapid mapping strategy.

Mouse mutants have a key role in discerning mammalian gene function and modelling human disease; however, at present mutants exist for only 1-2% of all mouse genes. In order to address this phenotype gap, we have embarked on a genome-wide, phenotype-driven, large-scale N-ethyl-N--nitrosourea (ENU) mutagenesis screen for dominant mutations of clinical and pharmacological interest in the mouse. Here we describe the identification of two similar neurological phenotypes and determination of the underlying mutations using a novel rapid mapping strategy incorporating speed back-crosses and high throughput genotyping. Two mutant mice were identified with marked resting tremor and further characterized using the SHIRPA behavioural and functional assessment protocol. Back-cross animals were generated using in vitro fertilization and genome scans performed utilizing DNA pools derived from multiple mutant mice. Both mutants were mapped to a region on chromosome 11 containing the peripheral myelin protein 22 gene (Pmp22). Sequence analysis revealed novel point mutations in Pmp22 in both lines. The first mutation, H12R, alters the same amino acid as in the severe human peripheral neuropathy Dejerine Sottas syndrome and Y153TER in the other mutant truncates the Pmp22 protein by seven amino acids. Histological analysis of both lines revealed hypo-myelination of peripheral nerves. This is the first report of the generation of a clinically relevant neurological mutant and its rapid genetic characterization from a large-scale mutagenesis screen for dominant phenotypes in the mouse, and validates the use of large-scale screens to generate desired clinical phenotypes in mice.

Precise binding of single-stranded DNA termini by human RAD52 protein.

The human RAD52 protein, which exhibits a heptameric ring structure, has been shown to bind resected double strand breaks (DSBs), consistent with an early role in meiotic recombination and DSB repair. In this work, we show that RAD52 binds single-stranded and tailed duplex DNA molecules via precise interactions with the terminal base. When probed with hydroxyl radicals, ssDNA-RAD52 complexes exhibit a four-nucleotide repeat hypersensitivity pattern. This unique pattern is due to the interaction of RAD52 with either a 5' or a 3' terminus of the ssDNA, is sequence independent and is phased precisely from the terminal nucleotide. Hypersensitivity is observed over approximately 36 nucleotides, consistent with the length of DNA that is protected by RAD52 in nuclease protection assays. We propose that RAD52 binds DNA breaks via specific interactions with the terminal base, leading to the formation of a precisely organized ssDNA-RAD52 complex in which the DNA lies on an exposed surface of the protein. This protein-DNA arrangement may facilitate the DNA-DNA interactions necessary for RAD52-mediated annealing of complementary DNA strands.

Wavelength modulation detection of water vapor with a vertical cavity surface-emitting laser.

A vertical cavity surface-emitting laser was studied for gas-sensing applications. Properties of the 962-nm laser that were measured include side-mode suppression, wavelength tuning with temperature and current, power versus injection current, and the amplitude noise spectrum. With wavelength modulation spectroscopy, a rms noise level of 2 x 10(-4) absorbance units was achieved. The large current tuning range (25 cm(-1)) and smaller amplitude modulation (11%/cm(-1)) of the vertical cavity laser compare favorably with Fabry-Perot and distributed feedback diode lasers for spectroscopic gas sensing, especially at atmospheric pressure.

Incidence and possible aetiological factors in the development of pelvic insufficiency fractures following radical radiotherapy.

Five patients out of a total of 183 treated with radical radiotherapy for carcinoma of cervix at The Royal Marsden Hospital from 1991 to 1994 inclusive have developed severe pelvic fractures. Two patients had rheumatoid arthritis, one of whom died as a result of the radiation induced damage. This patient developed radiological evidence of radionecrosis within 1 month of completing radiotherapy. There are very few reports in the literature of such a rapid onset. We suggest that the presence of a connective tissue disorder in a patient with other risk factors such as steroid use, old age and osteopenia should alert the clinician to the risk of radionecrosis following radical irradiation.

The E.coli RuvAB proteins branch migrate Holliday junctions through heterologous DNA sequences in a reaction facilitated by SSB.

During genetic recombination a heteroduplex joint is formed between two homologous DNA molecules. The heteroduplex joint plays an important role in recombination since it accommodates sequence heterogeneities (mismatches, insertions or deletions) that lead to genetic variation. Two Escherichia coli proteins, RuvA and RuvB, promote the formation of heteroduplex DNA by catalysing the branch migration of crossovers, or Holliday junctions, which link recombining chromosomes. We show that RuvA and RuvB can promote branch migration through 1800 bp of heterologous DNA, in a reaction facilitated by the presence of E.coli single-stranded DNA binding (SSB) protein. Reaction intermediates, containing unpaired heteroduplex regions bound by SSB, were directly visualized by electron microscopy. In the absence of SSB, or when SSB was replaced by a single-strand binding protein from bacteriophage T4 (gene 32 protein), only limited heterologous branch migration was observed. These results show that the RuvAB proteins, which are induced as part of the SOS response to DNA damage, allow genetic recombination and the recombinational repair of DNA to occur in the presence of extensive lengths of heterology.

Computerized axial tomographic scan measurements as prognostic indicators in patients with cervical carcinoma.

This retrospective study of 56 patients with carcinoma of the uterine cervix treated with radical radiotherapy at the Royal Marsden Hospital, London, examined whether simple measurements of maximum tumour dimension from computerized axial tomographic (CT) scans have any prognostic significance. Our results indicate that tumour depth (i.e. maximum antero-posterior dimension) of 4 cm or more is associated with a statistically significant increased relative risk of death of 2.4 (95% CI 1.1-5.5; p = 0.045), as compared with tumours with a depth of less than 4 cm. In addition, there was a clear correlation between tumour depth and lymph node involvement (r = 0.36; p < 0.01), and tumour depth and width (r = 0.70; p < 0.005). We suggest that a measurement of maximum tumour depth from the staging CT scan in these patients provides valuable additional information about likely occult lymph node metastases and prognosis, over and above that suggested by the FIGO staging system alone.

Structure of a multisubunit complex that promotes DNA branch migration.

The RuvA and RuvB proteins of Escherichia coli, which are induced in response to DNA damage, are important in the formation of heteroduplex DNA during genetic recombination and related recombinational repair processes. In vitro studies show that RuvA binds Holiday junctions and acts as a specificity factor that targets the RuvB ATPase, a hexameric ring protein, to the junction. Together, RuvA and RuvB promote branch migration, an ATP-dependent reaction that increases the length of the heteroduplex DNA. Electron microscopic visualization of RuvAB now provides a new insight into the mechanism of this process. We observe the formation of a tripartite protein complex in which RuvA binds the crossover and is sandwiched between two hexameric rings of RuvB. The Holliday junction within this complex adopts a square-planar structure. We propose a molecular model for branch migration, a unique feature of which is the role played by the two oppositely oriented RuvB ring motors.

Relationship of computed tomography tumour volume to patient survival in carcinoma of the cervix treated by radical radiotherapy.

In this retrospective study tumour volume was calculated by two different methods from the staging computed tomographic scans obtained in 1987 of 20 patients with carcinoma of the cervix. All patients underwent treatment by radical radiotherapy and the survival figures at 5 years were analysed. The aim was to identify the nature of the relationship between the true tumour volume and tumour volume obtained by measuring the maximum dimensions in each plane ("cuboid" volume). Significant correlation between the product of height x width x depth and true tumour volume was demonstrated (r = 0.983). A multivariate analysis of survival demonstrated a significantly increased relative risk for positive nodes (p < 0.03) and tumour depth > 3.8 cm (p < 0.04) or tumour width > 5.0 cm (p < 0.03). A significant difference (p < 0.02) between the median tumour volumes for early and late stage disease was present irrespective of the method used to calculate tumour volume. This study demonstrates that cuboid tumour volume can be a good reflection of the true volume; in addition, positive nodes, tumour depth and tumour width are significant determinants of survival.

Formation of a RuvAB-Holliday junction complex in vitro.

The ruvA and ruvB genes of Escherichia coli encode a novel DNA helicase that interacts with Holliday junctions and promotes branch migration. In this work, we have investigated the protein-DNA complexes formed between RuvA, RuvB and Holliday junctions. As shown previously, RuvA protein binds a synthetic Holliday junction in vitro, to form a specific protein-DNA complex that can be detected by a band-shift assay. We now show that the combined presence of RuvA and RuvB results in a super-shift of this complex indicative of the formation of a RuvAB-Holliday junction complex. In the absence of RuvA, the RuvB protein fails to bind Holliday junctions. The RuvAB-Holliday junction complex was detected by the band-shift assay only under conditions that favoured its stability, e.g. complex formation in the presence of a nucleoside triphosphate that can not be hydrolysed by RuvB (adenosine 5'-[gamma-thio]triphosphate). In contrast, nucleoside triphosphates that can be hydrolysed (ATP, dATP, dCTP or TTP), lead to RuvAB-mediated branch migration of the junction. These results indicate that the formation of a (RuvAB-ATP)-Holliday junction complex represents the first step in the process of branch migration, and that branch migration is dependent upon ATP hydrolysis. In addition, we show that Holliday junction DNA stimulates the ATPase activity of RuvAB to a greater extent than either single-stranded or linear duplex DNA.

Mammographic accuracy and patient age: a study of 297 patients undergoing breast biopsy.

This study was undertaken to compare the diagnostic accuracy of mammography in patients under 50 years with that of patients over 50, and with further subdivisions under 35 and 65 years. A retrospective review of the mammograms of 297 sequential patients undergoing mammography prior to surgical breast biopsy at the Breast Unit, Royal Marsden Hospital, London from 1988-1991 was performed. The films were reviewed blind by a single radiologist and correlated with histological findings at biopsy. The sensitivity and specificity of mammography compared with histology in four age-groups was assessed. There were 144 patients in the under-50 age group (< 35 years = 20, 35-49 years = 124), and 153 patients aged over 50 (50-64 years = 96, > 65 years = 57). Of all patients with histologically malignant biopsies 79% of the under-50s and 76% of the over-50s were diagnosed mammographically. Sensitivity of mammography was therefore 3% greater in the under-50 age-group (95% confidence interval (CI): +16% to -10%). Of patients who had benign biopsies, 76% of the under-50s and 75% of the over-50s were correctly diagnosed by mammography. Mammographic specificity was therefore 1% greater in the over-50 age-group (95% confidence interval: +15% to -14%). Accuracy of mammography was also comparable in the four subdivided age-groups. The results suggest that in patients requiring breast biopsy, contemporary mammography is similar in accuracy in the younger patient when compared to the older post-menopausal patient.

Interaction of Escherichia coli RuvA and RuvB proteins with synthetic Holliday junctions.

The RuvA, RuvB, and RuvC proteins of Escherichia coli are required for the recombinational repair of ultraviolet light- or chemical-induced DNA damage. In vitro, RuvC protein interacts with Holliday junctions in DNA and promotes their resolution by endonucleolytic cleavage. In this paper, we investigate the interaction of RuvA and RuvB proteins with model Holliday junctions. Using band-shift assays, we show that RuvA binds synthetic Holliday structures to form specific protein-DNA complexes. Moreover, in the presence of ATP, the RuvA and RuvB proteins act in concert to promote dissociation of the synthetic Holliday structures. The dissociation reaction requires both RuvA and RuvB and a nucleotide cofactor (ATP or dATP) and is rapid (40% of DNA molecules dissociate within 1 min). The reaction does not occur when ATP is replaced by either ADP or the nonhydrolyzable analog of ATP, adenosine 5'-[gamma-thio]triphosphate. We suggest that the RuvA and RuvB proteins play a specific role in the branch migration of Holliday junctions during postreplication repair of DNA damage in E. coli.

Formation and resolution of recombination intermediates by E. coli RecA and RuvC proteins.

The recombination of DNA molecules has been reconstituted in vitro using two purified enzymes from Escherichia coli. RecA protein catalyses homologous pairing and strand exchange reactions to form intermediate DNA structures that are acted upon by RuvC. The newly identified RuvC protein resolves the intermediates by specific endonucleolytic cleavage to produce recombinant DNA molecules.

Resolution of Holliday junctions in vitro requires the Escherichia coli ruvC gene product.

In previous studies, Holliday junctions generated during RecA-mediated strand-exchange reactions were resolved by fractionated Escherichia coli extracts. We now report the specific binding and cleavage of synthetic Holliday junctions (50 base pairs long) by a fraction purified by chromatography on DEAE-cellulose, phosphocellulose, and single-stranded DNA-cellulose. The cleavage reaction provided a sensitive assay with which to screen extracts prepared from recombination/repair-deficient mutants. Cells with mutations in ruvC lack the nuclease activity that cleaves synthetic Holliday junctions in vitro. This deficiency was restored by a multicopy plasmid carrying a ruvC+ gene that overexpressed junction-resolving activity. The UV sensitivity and deficiency in recombinational repair of DNA exhibited by ruv mutants lead us to suggest that RuvC resolves Holliday junctions in vivo.

Specificity of binding to four-way junctions in DNA by bacteriophage T7 endonuclease I.

T7 endonuclease I binds specifically to four-way junctions in duplex DNA and promotes their resolution into linear duplexes. Under conditions in which the nuclease activity is blocked by the absence of divalent cations, the enzyme forms a distinct protein-DNA complex with the junction, as detected by gel retardation and filter binding assays. The formation of this complex is structure-specific and contrasts with the short-lived binding complexes formed on linear duplex DNA. The binding complex between T7 endonuclease I and a synthetic Holliday junction analog has been probed with hydroxyl radicals. The results indicate that the nuclease binds all four strands about the junction point.

Interaction of a four-way junction in DNA with T4 endonuclease VII.

The binding of a synthetic four-way junction in DNA by T4 endonuclease VII has been studied using gel retardation and footprint analysis. Two specific protein-DNA complexes have been observed, but only one is stable in the presence of moderate concentrations of salt. The footprint of T4 endonuclease VII in the salt-resistant complex has been probed using hydroxyl radicals generated by the reaction of iron(II)/EDTA with hydrogen peroxide. The hydroxyl radical cleavage pattern indicates protection of approximately 5 residues in two strands that are diametrically opposed across the junction point.

The accuracy of mammographic diagnosis in surgically occult breast lesions.

This paper reports a retrospective study of 69 breast biopsies carried out for surgically occult but radiologically apparent lesions. Specimen radiography was required to confirm excision. The group included 17 carcinomas. Review of the pre-operative mammogram reports provided by a 'pool' of general radiologists demonstrated a high sensitivity (88%), but poor specificity (32%), with an overall accuracy of 46%. 'Blind' review of the mammograms by one experienced mammographic radiologist showed high sensitivity (100%), good specificity (73%) and overall accuracy of almost 80%. This result shows the need for the most experienced radiologist available to be involved in deciding which of these difficult lesions require biopsy. This will reduce unnecessary breast surgery and highlights the role of clinical and radiological review instead of biopsy, the need for continuing self audit by radiologists and the need for regular communication between clinicians, radiologists and pathologists.

Cleavage specificity of bacteriophage T4 endonuclease VII and bacteriophage T7 endonuclease I on synthetic branch migratable Holliday junctions.

Holliday junctions are intermediate structures that are formed and resolved during the process of genetic recombination. To investigate the interaction of junction-resolving nucleases with synthetic Holliday junctions that contain homologous arm sequences, we constructed substrates in which the junction point was free to branch migrate through 26 base-pairs of homology. In the absence of divalent cations, we found that both phage T4 endonuclease VII and phage T7 endonuclease I bound the synthetic junctions to form specific protein-DNA complexes. Such complexes were not observed in the presence of Mg2+, since the Holliday junctions were resolved by the introduction of symmetrical cuts in strands of like polarity. The major sites of cleavage were identified and found to occur within the boundaries of homology. T4 endonuclease VII showed a cleavage preference for the 3' side of thymine bases, whereas T7 endonuclease I preferentially cut the DNA between two pyrimidine residues. However, cleavage was not observed at all the available sites, indicating that in addition to their structural requirements, the endonucleases show strong site preferences.

The impact of computed tomography and ultrasonography on the management of patients with carcinoma of the ovary.

We have carried out a prospective study on the impact of computed tomography (CT) and ultrasonography (US) on the management of patients with carcinoma of the ovary. Seventy-eight CT and 88 US scans were performed on 94 patients. Clinicians decided patient management prospectively at the time the CT and/or US was ordered. Clinical assessment differed from the result obtained by CT or US in 45% of cases (35/78 and 40/88, respectively). CT and US altered patient management in only a minority of cases (14/78, 18% and 9/88, 10% respectively). Even when the scan and clinical assessments differed, management was only altered on 14/35 (40%) occasions after CT and on 9/40 (23%) occasions after US, a difference which was not significant. In patients with clinically undetectable disease, management was altered by CT on 17% of occasions and by US on 10%. We conclude that in patients with carcinoma of the ovary CT and US alters patient management in a minority of cases. In view of current financial restrictions in health care, clinicians should be more selective in the use of these imaging techniques. Furthermore, we recommend that similar prospective studies are performed for other clinical situations.

Reformatted computed tomography of the female pelvis: normal anatomy.

Axial computed tomography (CT) of the pelvis provides useful information in patients with gynaecological malignancies, both for initial staging, to follow disease progression and to assess response to treatment. Additional useful information may be available from reformatted coronal and sagittal images. Knowledge of normal anatomy is essential for correct interpretation of pathology. The anatomy and technique used to acquire the images are described in this paper.