Photoaffinity labeling of mouse fibroblast enzymes by a base excision repair intermediate. Evidence for the role of poly(ADP-ribose) polymerase-1 in DNA repair

To examine the interaction of mammalian base excision repair (BER) enzymes with DNA intermediates formed during BER, we used a novel photoaffinity labeling probe and mouse embryonic fibroblast cellular extracts. The probe was formed in situ, using an end-labeled oligonucleotide containing a synthetic abasic site; this site was incised by apurinic/apyrimidinic endonuclease creating a nick with 3'-hydroxyl and 5'-reduced sugar phosphate groups at the margins, and then a dNMP carrying a photoreactive adduct was added to the 3'-hydroxyl group. With near-UV light (312 nm) exposure of the extract/probe mixture, six proteins were strongly labeled. Four of these include poly(ADP-ribose) polymerase-1 (PARP-1) and the BER participants flap endonuclease-1, DNA polymerase beta, and apurinic/apyrimidinic endonuclease. The amount of the probe cross-linked to PARP-1 was greater than that cross-linked to the other proteins. The specificity of PARP-1 labeling was examined using various competitor oligonucleotides and DNA probes with alternate structures. PARP-1 labeling was stronger with a DNA representing a BER intermediate than with a nick in double-stranded DNA. These results indicate that proteins interacting preferentially with a photoreactive BER intermediate can be selected from the crude cellular extract.     

COVOL: an interactive program for evaluating second virial coefficients from the triaxial shape or dimensions of rigid macromolecules

An interactive program is described for calculating the second virial coefficient contribution to the thermodynamic nonideality of solutions of rigid macromolecules based on their triaxial dimensions. The FORTRAN-77 program, available in precompiled form for the PC, is based on theory for the covolume of triaxial ellipsoid particles [Rallison, J. M., and S.E Harding. (1985). J. Colloid Interface Sci. 103:284-289]. This covolume has the potential to provide a magnitude for the second virial coefficient of macromolecules bearing no net charge. Allowance for a charge-charge contribution is made via an expression based on Debye-Hückel theory and uniform distribution of the net charge over the surface of a sphere with dimensions governed by the Stokes radius of the macromolecule. Ovalbumin, ribonuclease A, and hemoglobin are used as model systems to illustrate application of the COVOL routine.     

Gene organisation, sequence variation and isochore structure at the centromeric boundary of the human MHC.

We have mapped and sequenced the region immediately centromeric of the human major histocompatibility complex (MHC). A cluster of 13 genes/pseudogenes was identified in a 175 kb PAC linking the TAPASIN locus with the class II region. It includes two novel human genes (BING4 and SACM2L) and a thus far unnoticed human leucocyte antigen (HLA) class II pseudogene, termed HLA-DPA3. Analysis of the G+C content revealed an isochore boundary which, together with the previously reported telomeric boundary, defines the MHC class II region as one of the first completely sequenced isochores in the human genome. Comparison of the sequence with limited sequence from other cell lines shows that the high sequence variation found within the classical class II region extends beyond the identified isochore boundary leading us to propose the concept of an "extended MHC". By comparative analysis, we have precisely identified the mouse/human synteny breakpoint at the centromeric end of the extended MHC class II region between the genes HSET and PHF1.     

Resistance of photosynthesis to high temperature in two bean varieties (Phaseolus vulgaris L.)

The increase in the basal fluorescence (F₀) by increases in temperature has been widely used as a screening technique to assess the temperature at which plant species suffer irreversible damage in their photosynthetic apparatus. This method has been used in this study to assess the resistance to extreme high temperature in two bean varieties, Barbucho (Chilean, non commercially bred) and Blue lake (commercially available in the UK). The results suggested that the two varieties have a similar threshold for high temperature at 42–43 °C, however, at 40 °C, Barbucho maintains its photosynthetic functions for a longer period of time compared to Blue-Lake as observed by mean of simultaneous measurement of gas exchange and room temperature fluorescence. It seems likely that inhibition at 40 °C in Blue-Lake is related to the instability of Photosystem II.     

Developmental expression of the amphioxus <Emphasis Type="Italic">Tbx1</Emphasis>/<Emphasis Type="Italic">10</Emphasis> gene illuminates the evolution of vertebrate branchial arches and sclerotome

We have isolated an amphioxus T-box gene that is orthologous to the two vertebrate genes, Tbx1 and Tbx10, and examined its expression pattern during embryonic and early larval development. AmphiTbx1/10 is first expressed in branchial arch endoderm and mesoderm of developing neurulae, and in a bilateral, segmented pattern in the ventral half of newly formed somites. Branchial expression is restricted to the first three branchial arches, and disappears completely by 4 days post fertilization. Ventral somitic expression is restricted to the first 10–12 somites, and is not observed in early larvae except in the most ventral mesoderm of the first three branchial arches. No expression can be detected by 4 days post fertilization. Integrating functional, phylogenetic and expression data from amphioxus and a variety of vertebrate model organisms, we have reconstructed the early evolutionary history of the Tbx1/10 subfamily of genes within the chordate lineage. We conclude that Tbx1/10-mediated branchial arch endoderm and mesoderm patterning functions predated the origin of neural crest, and that ventral somite specification functions predated the origin of vertebrate sclerotome, but that Tbx1 was later co-opted during the evolution of developmental programs regulating branchial neural crest and sclerotome migration.Edited by M. Akam     

Autoclave method for rapid preparation of bacterial PCR-template DNA

An autoclave method for preparing bacterial DNA for PCR template is presented, it eliminates the use of detergents, organic solvents, and mechanical cellular disruption approaches, thereby significantly reducing processing time and costs while increasing reproducibility. Bacteria are lysed by rapid heating and depressurization in an autoclave. The lysate, cleared by microcentrifugation, was either used directly in the PCR reaction, or concentrated by ultrafiltration. This approach was compared with seven established methods of DNA template preparation from four bacterial sources which included boiling Triton X-100 and SDS, bead beating, lysozyme/proteinase K, and CTAB lysis method components. Bacteria examined were Enterococcus and Escherichia coli, a natural marine bacterial community and an Antarctic cyanobacterial-mat. DNAs were tested for their suitability as PCR templates by repetitive element random amplified polymorphic DNA (RAPD) and denaturing gradient gel electrophoresis (DGGE) analysis. The autoclave method produced PCR amplifiable template comparable or superior to the other methods, with greater reproducibility, much shorter processing time, and at a significantly lower cost.     

Redescription of a rare deep-sea batfish, <Emphasis Type="Italic">Halieutopsis bathyoreos</Emphasis> (Lophiiformes: Ogcocephalidae)

Halieutopsis bathyoreos Bradbury, 1988 (Lophiiformes: Ogcocephalidae), previously described only on the basis of the holotype (62.6mm in standard length) from the central North Pacific, is redescribed on the basis of the holotype and six additional specimens (41.2–68.7mm in standard length) collected from the western South Pacific, off Papua New Guinea, and the western North Pacific, including the Japanese Archipelago. Halieutopsis bathyoreos is distinguished from its congeners by having a shelflike rostrum extending anteriorly well beyond the mouth, a dorsal escal lobe slightly bisected ventrally, an illicial cavity square in outline and completely visible in ventral view, and lacking tubercles on the ventral surface of the disk. The following characters are newly added to the diagnoses of this species: rostrum width 21–29% of head length, tubercles on the dorsal surface of the disk about half the diameter of those on the lateral margin, and 13–15 large lateral-line scales on the tail.