Gene for the catalytic subunit of the human DNA-activated protein kinase maps to the site of the XRCC7 gene on chromosome 8.

The DNA-activated serine/threonine protein kinase (DNA-PK) is composed of a large (approximately 460 kDa) catalytic polypeptide (DNA-PKcs) and Ku, a heterodimeric DNA-binding component (p70/p80) that targets DNA-PKcs to DNA. A 41-kbp segment of the DNA-PKcs gene was isolated, and a 7902-bp segment was sequenced. The sequence contains a polymorphic Pvu II restriction enzyme site, and comparing the sequence with that of the cDNA revealed the positions of nine exons. The DNA-PKcs gene was mapped to band q11 of chromosome 8 by in situ hybridization. This location is coincident with that of XRCC7, the gene that complements the DNA double-strand break repair and V(D)J recombination defects (where V is variable, D is diversity, and J is joining) of hamster V3 and murine severe combined immunodeficient (scid) cells.

Mutational analysis of the conserved region 2 site of adenovirus E1A and its effect on binding to the retinoblastoma gene product: use of the "double-tagging" assay.

We have explored the feasibility of using a "double-tagging" assay for assessing which amino acids of a protein are responsible for its binding to another protein. We have chosen the adenovirus E1A-retinoblastoma gene product (pRB) proteins for a model system, and we focused on the high-affinity conserved region 2 of adenovirus E1A (CR2). We used site-specific mutagenesis to generate a mutant E1A gene with a lysine instead of an aspartic acid at position 121 within the CR2 site. We demonstrated that this mutant exhibited little binding to pRB by the double-tagging assay. We also have shown that this lack of binding is not due to any significant decrease in the level of expression of the beta-galactosidase-E1A fusion protein. We then created a "library" of phage expressing beta-galactosidase-E1A fusion proteins with a variety of different mutations within CR2. This library of E1A mutations was used in a double-tagging screening to identify mutant clones that bound to pRB. Three classes of phage were identified: the vast majority of clones were negative and exhibited no binding to pRB. Approximately 1 in 10,000 bound to pRB but not to E1A ("true positives"). A variable number of clones appeared to bind equally well to both pRB and E1A ("false positives"). The DNA sequence of 10 true positive clones yielded the following consensus sequence: DLTCXEX, where X = any amino acid. The recovery of positive clones with only one of several allowed amino acids at each position suggests that most, if not all, of the conserved residues play an important role in binding to pRB. On the other hand, the DNA sequence of the negative clones appeared random. These results are consistent with those obtained from other sources. These data suggest that a double-tagging assay can be employed for determining which amino acids of a protein are important for specifying its interaction with another protein if the complex forms within bacteria. This assay is rapid and up to 1 x 10(6) mutations can be screened at one time.

Effects of landscape spatial heterogeneity on dryland restoration success. The combined role of site conditions and reforestation techniques in southeastern Spain

In occidental Europe, Spain is one of countries the most severely affected by desertification (Arnalds & Arsher 2000). Particularly, South-eastern Spain is considered as one of the most threatened areas by desertification in Mediterranean Europe (Vallejo 1997). In 2003, the Valencia Regional Forest Service implemented a restoration demonstration project in this area. The project site is a small catchment (25 ha) located in the Albatera municipality. The catchment is highly heterogeneous, with terraced slopes, south-facing slopes and north-facing slopes. The restoration strategy was based on planting evergreen trees and shrubs which can grow quickly after disturbances, and on field treatments aimed at maximizing water collection (micro-catchments, planting furrows), organic amendment (compost), and conservation (tree shelters, mulching). On south landscape unit, the whole category of restoration treatments was applied: water micro-catchment + Tubex tree shelters + mulching & compost, while on north landscape unit: netting tree shelters + mulching & compost only were applied, while in terrace landscape unit: furrows + netting tree shelters + mulching & compost were applied. Survival and growth of the planted seedlings were used as metrics of restoration success. To assess the effects of the treatments applied for soil conservation, soil loss rates (from 2005 to 2009) were evaluated using the erosion pin method. We conclude that, despite the limiting conditions prevailing on the south unit, this landscape unit showed the highest survival and growth plant rates in the area. The best seedling performances on the south landscape unit were probably due to the highest technical efforts applied, consisting in the water micro-catchment installation and the Tubex plant shelters addition. In addition, soil loss rates followed decreasing trends throughout the assessment period. Soil loss rates were highest on south landscape unit in comparison with the other landscape units, due to the more accentuated relief. North landscape unit and terrace unit showed a net soil mass gain, probably reflecting the trapping of sediments produced by plantation works.