The recombinant DNA controversy: twenty years later.
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Animals↗
Biomedical subjects
P Berg
Publications and source records attributed to P Berg.
Complex formation in yeast double-strand break repair: participation of Rad51, Rad52, Rad55, and Rad57 proteins.
The repair of DNA double-strand breaks in Saccharomyces cerevisiae requires genes of the RAD52 epistasis group, of which RAD55 and RAD57 are members. Here, we show that the x-ray sensitivity of rad55 and rad57 mutant strains is suppressible by overexpression of RAD51 or RAD52. Virtually complete suppression is provided by the simultaneous overexpression of RAD51 and RAD52. This suppression occurs at 23 degrees C, where these mutants are more sensitive to x-rays, as well as at 30 degrees C and 36 degrees C. In addition, a recombination defect of rad55 and rad57 mutants is similarly suppressed. Direct in vivo interactions between the Rad51 and Rad55 proteins, and between Rad55 and Rad57, have also been identified by using the two-hybrid system. These results indicate that these four proteins constitute part of a complex, a "recombinosome," to effect the recombinational repair of double-strand breaks.
Adenosine Triphosphatases↗
Maintenance of an extrachromosomal plasmid vector in mouse embryonic stem cells.
We have constructed and characterized a polyoma virus-based plasmid that is maintained as an autonomously replicating extrachromosomal element (episome) in mouse embryonic stem (ES) cells. Plasmid pMGD20neo contains the polyoma origin of replication harboring a mutated enhancer (PyF101), a modified polyoma early region that encodes the large tumor (T) antigen only, and a gene that confers resistance to G418 (neo). After transfection, the plasmid replicates in ES cells and is maintained as an extrachromosomal element in 15% of G418-resistant clones. Integration of the plasmid DNA is undetectable for at least 28 cell generations. In one clone, the transfected DNA persists unaltered as an episome at 10-30 copies per cell for at least 74 cell generations in the presence of G418. Cells that maintain the autonomously replicating plasmid can efficiently replicate and maintain a second plasmid that carries the polyoma origin of replication. Independent vector-containing ES cell lines showed no significant alteration of the karyotype, and two cell lines yielded several chimeric animals when introduced into blastocysts, suggesting that the presence of an episomal element and expression of polyoma large T do not eliminate the ES cells' ability to populate an embryo. This system offers an efficient means for manipulating and analyzing various aspects of gene expression in ES cells.
Animals↗
[Follow-up of cutaneous paradoxical vascular reactions in atopic patients during and after eczema manifestations].
In atopic eczema patients the well-known abnormal cutaneous reactivity of the blood vessels (white dermographism, delayed blanch after acetylcholine, paradoxical blanching after nicotinic acid application and diminished erythema after histamine injection) were observed during acute eczematous episodes and later in an exzema-free state in comparison with controls. In this follow-up study the use of different stimuli allowed us to demonstrate abnormal cutaneous vascular reactions in the patients depending on the severity of their atopic eczema. Severely affected patients showed persistence of the paradoxical vascular reactions even in an eczema-free cutaneous state.
Acetylcholine↗
A novel allele of Saccharomyces cerevisiae RFA1 that is deficient in recombination and repair and suppressible by RAD52.
To understand the mechanisms involved in homologous recombination, we have performed a search for Saccharomyces cerevisiae mutants unable to carry out plasmid-to-chromosome gene conversion. For this purpose, we have developed a colony color assay in which recombination is induced by the controlled delivery of double-strand breaks (DSBs). Recombination occurs between a chromosomal mutant ade2 allele and a second plasmid-borne ade2 allele where DSBs are introduced via the site-specific HO endonuclease. Besides isolating a number of new alleles in known rad genes, we identified a novel allele of the RFA1 gene, rfa1-44, which encodes the large subunit of the heterotrimeric yeast single-stranded DNA-binding protein RPA. Characterization of rfa1-44 revealed that it is, like members of the RAD52 epistasis group, sensitive to X rays, high doses of UV, and HO-induced DSBs. In addition, rfa1-44 shows a reduced ability to undergo sporulation and HO-induced gene conversion. The mutation was mapped to a single-base substitution resulting in an aspartate at amino acid residue 77 instead of glycine. Moreover, all radiation sensitivities and repair defects of rfa1-44 are suppressed by RAD52 in a dose-dependent manner, and one RAD52 mutant allele, rad52-34, displays nonallelic noncomplementation when crossed with rfa1-44. Presented is a model accounting for this genetic interaction in which Rfa1, in a complex with Rad52, serves to assemble other proteins of the recombination-repair machinery at the site of DSBs and other kinds of DNA damage. We believe that our findings and those of J. Smith and R. Rothstein (Mol. Cell. Biol. 15:1632-1641, 1995) are the first in vivo demonstrations of the involvement of a eukaryotic single-stranded binding protein in recombination and repair processes.
Alleles↗
Suppression of mutations in two Saccharomyces cerevisiae genes by the adenovirus E1A protein.
The protein products of the adenoviral E1A gene are implicated in a variety of transcriptional and cell cycle events, involving interactions with several proteins present in human cells, including parts of the transcriptional machinery and negative regulators of cell division such as the Rb gene product and p107. To determine if there are functional homologs of E1A in Saccharomyces cerevisiae, we have developed a genetic screen for mutants that depend on E1A for growth. The screen is based on a colony color sectoring assay which allows the identification of mutants dependent on the maintenance and expression of an E1A-containing plasmid. Using this screen, we have isolated five mutants that depend on expression of the 12S or 13S cDNA of E1A for growth. A plasmid shuffle assay confirms that the plasmid-dependent phenotype is due to the presence of either the 12S or the 13S E1A cDNA and that both forms of E1A rescue growth of all mutants equally well. The five mutants fall into two classes that were named web1 and web2 (for "wants E1A badly"). Plasmid shuffle assays with mutant forms of E1A show that conserved region 1 (CR1) is required for rescue of the growth of the web1 and web2 E1A-dependent yeast mutants, while the N-terminal 22 amino acids are only partially required; conserved region 2 (CR2) and the C terminus are dispensable. The phenotypes of mutants in both the web1 and the web2 groups are due to a single gene defect, and the yeast genes that fully complement the mutant phenotypes of both groups were cloned. The WEB1 gene sequence encodes a 1,273-amino-acid protein that is identical to SEC31, a protein involved in the budding of transport vesicles from the endoplasmic reticulum. The WEB2 gene encodes a 1,522-amino-acid protein with homology to nucleic acid-dependent ATPases. Deletion of either WEB1 or WEB2 is lethal. Expression of E1A is not able to rescue the lethality of either the web1 or the web2 null allele, implying allele-specific mutations that lead to E1A dependence.
Adenovirus E1A Proteins↗
[Evaluation of the teaching of echo-endoscopy. Application to the assessment of invasiveness of cancer of the esophagus and the cardia].
OBJECTIVES: Learning endosonography (EES) is known to be difficult, and the theory must be understood before performing EES routinely. The aim of the study was to evaluate the diagnostic indexes of EES in the staging of cardio-oesophageal cancer after a period of theoretical apprenticeship of EES and a learning period in a centre experienced in EES since 1989. METHODS: Five observers, having never used EES, followed to EES examinations for a 3-6 month period. They then reviewed the standardized records of 29 patients with cardio-oesophageal cancer. They had to evaluate the degree of tumour infiltration within oesophageal wall and the site of metastatic lymph nodes. Results were compared with the diagnosis of 5 experienced senior endoscopists. Interobserver agreement was estimated with kappa statistics and considered excellent for k > or = 0.75, good to moderate if 0.75 > k > or = 0.40, and poor if k < 0.40. RESULTS: Inter-observer agreement was poor for the topographic diagnosis of lymph nodes (kappa index from -0.09 to 0.33), lower to that of the 5 senior observers (0.33 to 0.77). It was satisfactory for degree of tumour infiltration (T1: k = 0.66; T2: k = 0.58; T3: k = 0.56; T4: k = 0.46). The individual sensitivities were weak according to lymph nodes site, but good for presence of lymph nodes (86 to 100%), with a specificity of 40 to 73%. CONCLUSIONS: After theoretical training of EES, agreement and diagnostic performances are good enough to diagnose pathological images (except for T4 tumors), and poor for localizing images in the mediastinum. This emphasizes the difficulties in learning echo-anatomy and gives useful guidelines for training programs in EES.
Cardia↗
Source analysis of median nerve and finger stimulated somatosensory evoked potentials: multichannel simultaneous recording of electric and magnetic fields combined with 3D-MR tomography.
At the current state of technology, multichannel simultaneous recording of combined electric potentials and magnetic fields should constitute the most powerful tool for separation and localization of focal brain activity. We performed an explorative study of multichannel simultaneous electric SEPs and magnetically recorded SEFs. MEG only sees tangentially oriented sources, while EEG signals include the entire activity of the brain. These characteristics were found to be very useful in separating multiple sources with overlap of activity in time. The electrically recorded SEPs were adequately modelled by three equivalent dipoles located: (1) in the region of the brainstem, modelling the P14 peak at the scalp, (2) a tangentially oriented dipole, modelling the N20-P20 and N30-P30 peaks, and part of the P45, and (3) a radially oriented dipole, modelling the P22 peak and part of the P45, both located in the region of the somatosensory cortex. Magnetically recorded SEFs were adequately modelled by a single equivalent dipole, modelling the N20-P20 and N30-P30 peaks, located close to the posterior bank of the central sulcus, in area 3b (mean deviation: 3 mm). The tangential sources in the electrical data were located 6 mm on average from the area 3b. MEG and EEG was able to locate the sources of finger stimulated SEFs in accordance with the somatotopic arrangement along the central fissure. A combined analysis demonstrated that MEG can provide constraints to the orientation and location of sources and helps to stabilize the inverse solution in a multiple-source model of the EEG.
Adult↗
Smooth-pursuit eye movement dysfunction in schizophrenia: the role of attention and general psychomotor dysfunctions.
Smooth-pursuit eye-tracking performance was examined in 100 schizophrenic patients and various control groups under both attention-enhancing and attention-distracting conditions. The level of attentional demand was varied by introducing a secondary reaction time task that directed attention either toward or away from the visual-tracking target. Distraction from the target led to a significant deterioration of tracking performance in all subjects, which was most pronounced in the group of schizophrenic patients. Attention-enhancement, on the other hand, did not normalize performance in this group. In schizophrenic patients, mainly in the distraction condition, there was a moderate association between performance in tracking and tests presumably measuring prefrontal functions. Tracking accuracy from both conditions was related to general motor performance as measured by the Neurological Evaluation Scale. It was concluded that in schizophrenic patients attentional factors (distraction) may contribute to eye-tracking impairment, and that the impairment may be viewed as an aspect of general motor dysfunctions.
Adolescent↗
A multiple source approach to the correction of eye artifacts.
Previously published methods correct eye artifacts by subtracting proportions of the EOG from EEG electrodes. The implicit assumption made by these methods is that the EOG signals are a good measure of eye activity and contain no EEG. In this paper a new multiple source eye correction (MSEC) method of eye artifact treatment based on multiple source analysis is presented, which incorporates a model of brain activity. An accurate, head model-independent estimate of the spatial distribution of eye activity can be obtained empirically from calibration data containing systematic eye movements and blinks. Using the resulting spatial vectors together with the brain model, eye activity in EEG and event-related response data can be estimated in the presence of overlapping brain activity and corrected. A consequence of the MSEC approach is that data at EOG electrodes can be included in analyses of brain activity. In addition, by suitable selection of the spatial vectors, the eye activity can be split into signals which identify vertical and horizontal movements and eyeblinks. Using auditory ERP data sets with and without large eye artifacts, the MSEC method is compared with a "traditional" method in which brain activity is not modelled, particularly with respect to the spatial distribution of the corrected EEG. Traditional eye correction methods are shown to alter the spatial distribution of the EEG, resulting, for example, in changes in location and orientation of modelled equivalent sources. Such distortion is much reduced in the MSEC method, thus enhancing the precision of topographical EEG analyses.
Acoustic Stimulation↗
A fast method for forward computation of multiple-shell spherical head models.
Using a combination of 3 suitably located dipoles in a homogeneous sphere, the scalp potential due to a dipole source in a 4-shell spherical head model can be approximated with a high degree of precision and a more than 30-fold increase in computing speed. Magnitudes and locations of the 3 equivalent dipoles can be fitted in a homogeneous sphere to data generated from a source at one location in a 4-shell head model. The resulting parameters are used to compute scalp potentials for sources at other locations and orientations. Residual variance measures showed close agreement between the new approximation and a standard 4-shell computation method. Further tests of the method used scalp data from 500 randomly selected pairs of sources generated by the standard 4-shell computation and fitted using, for forward computations, the new approximation and the single-shell Ary-corrected head model. Errors with the new approximation were marginally larger than with the standard computation, but sources were located within 0.5 mm and 0.6 degrees of the original position in 99% of the fits. 99% error limits for the Ary model were up to 18 mm and 25 degrees and depended on the head model parameters.
Brain↗
Alpha 2-globin gene mutation Hb G-Waimanalo: occurrence in combination with alpha-thalassemia-1.
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Adult↗
[Retinal vasculitis and antiphospholipid antibodies].
Antiphospholipid antibodies (APAb) are often found in systemic lupus erythematosus (LE) (secondary antiphospholipid syndrome), leading to arterial or venous thrombosis. In primary antiphospholipid syndrome other LE-associated symptoms are not detectable. We present the clinical course and therapy of three patients with this rarely reported disease, discussing immunopathology and therapy. Three young patients (28, 29 and 31 years) were seen at the university eye clinic, presenting severe occlusive vasculitis with vitreal haemorrhages and/or thrombosis. The first patient did not respond to any therapy (various immunosuppressives, plasma-pheresis, laser coagulation) and ended up with defective light perception in both eyes. The second patient also had light perception as final outcome in spite of acetylsalicylic acid, steroids and immunosuppression. The third patient established complete reperfusion on an arteriovenous occlusion after haemodilution and acetylsalicylic acid. Antibodies directed against phospholipids interfere with the blood clotting system in many ways (activation and aggregation of thrombocytes, endothelial function, coagulation cascade). The optimal therapy of this severe disease is unclear, suggesting that acetylsalicylic acid seems to be important, while the effect of immunosuppression or steroids is uncertain.
Adult↗
[Loops and folds of the carotid and vertebral arteries: indications for surgery].
We present our results of surgical correction of non-atheromatous redundant internal carotid arteries. From 1986 until 1992, we performed 1641 endarterectomies of the internal carotid artery, and in the same time we diagnosed 49 elongations in 42 patients. In 25 cases, indication for surgery was given and reconstruction was performed by shortening of the internal carotid artery, using resection and reanastomosis associated in one third of the cases with a patch. Nineteen patients (76%) were symptomatic, four patients (16%) presented a preoperative stroke and 2 patients (8%) were asymptomatic. All the patients were neurologically monitored by the use of somatosensory evoked potentials. One patient needed an intraluminal shunt because of loss of evoked potentials. No patient died in the postoperative period. One stroke occurred, concomitantly to a thrombosis of the internal carotid artery, but with totally reversible clinical symptoms after reoperation. We noted one transient ischaemic attack. We recommend operative treatment in symptomatic patients or when the stenosis is higher than 60%. A stenosis under 60% should be operated in the case of a contralateral occlusion. We propose a classification of redundant arteries based on the importance of the stenosis. The exact assessment of the stenosis is the determining factor in the diagnosis and is of primordial importance when indicating surgery.
Aged↗
Co-chairman's remarks: reverse genetics: directed modification of DNA for functional analysis.
The classic paradigm for identifying the genetic basis of a particular organism's properties proceeds from the phenotype to the gene, and thence to the molecular structures of the corresponding DNA, RNA and protein. 'Positional cloning' of disease genes and the molecular characterization of the responsible mutations (inappropriately referred to as reverse genetics, initially) exemplifies this approach. Now, the ability to clone, modify and test the biological activities of DNA segments provides a new approach, referred to as 'reverse genetics'. This paradigm begins with a segment of DNA whose molecular structure is known, and proceeds to explore the gene's contribution to the organism's phenotype; thus, the experimental path is from the gene as a nucleotide sequence to the corresponding phenotypic characteristic. Such a strategy follows from the ability to modify these sequences in highly directed and nearly unlimited ways, and to assess the phenotypic relevance of such alterations either in vitro, in cultured cells, or even in whole organisms. This approach permits the full panoply of molecular techniques to be used for creating uniquely altered structures and obviates the reliance on chance events as the source of mutations. As a consequence, the range of questions that can be studied is greatly expanded, and the information that is obtained is all the richer.
Animals↗
A mammalian protein complex that repairs double-strand breaks and deletions by recombination.
We have purified a high molecular weight complex (RC-1) from calf thymus nuclei that catalyzes a recombinational repair of double-strand gaps and deletions in DNA by gene conversion as well as cross-over events leading to cointegrant products. These have been detected by polymerase chain reaction analysis using oligonucleotide primer pairs that detect joined sequences originally present on only one or the other of the recombination substrates. RC-1 has an apparent molecular mass of about 550-600 kDa and contains at least five polypeptide chains: molecular masses about 230, 210, 160, 130, and 40 kDa. RC-1 contains a DNA polymerase, identified as DNA polymerase epsilon, that co-purifies with RC-1. A DNA ligase, most likely mammalian DNA ligase III, and a 5'-3' exonuclease also copurify with the RC-1. Most preparations of RC-1 contain low levels of a double-strand endonuclease, 3'-5' exonuclease and single-strand nuclease activities. However, DNA helicase, terminal deoxynucleotidyl transferase, or DNA topoisomerase I and II were not detected in RC-1. The DNA polymerase and DNA ligase in RC-1 can act in concert to repair a multiply gapped DNA to a covalently repaired duplex. The bovine single-strand-binding protein stimulates the formation of the recombination products and the repair reaction mentioned above about 4-fold.
Animals↗
Ocular artifacts in EEG and event-related potentials. I: Scalp topography.
The ocular artifacts that contaminate the EEG derive from the potential difference between the cornea and the fundus of the eye. This corneofundal or corneoretinal potential can be considered as an equivalent dipole with its positive pole directed toward the cornea. The cornea shows a steady DC potential of approximately +13 mV relative to the forehead. Blink potentials are caused by the eyelids sliding down over the positively charged cornea. The artifacts from eye-movements result from changes in orientation of the corneo-fundal potential. The scalp-distribution of the ocular artifacts can be described in terms of propagation factors--the fraction of the EOG signal at periocular electrodes that is recorded at a particular scalp location. These factors vary with the location of the scalp electrode. Propagation factors for blinks and upward eye-movements are significantly different.
Adolescent↗