Purifying selection and birth-and-death evolution in the ubiquitin gene family

Ubiquitin is a highly conserved protein that is encoded by a multigene family. It is generally believed that this gene family is subject to concerted evolution, which homogenizes the member genes of the family. However, protein homogeneity can be attained also by strong purifying selection. We therefore studied the proportion (pS) of synonymous nucleotide differences between members of the ubiquitin gene family from 28 species of fungi, plants, and animals. The results have shown that pS is generally very high and is often close to the saturation level, although the protein sequence is virtually identical for all ubiquitins from fungi, plants, and animals. A small proportion of species showed a low level of pS values, but these values appeared to be caused by recent gene duplication. It was also found that the number of repeat copies of the gene family varies considerably with species, and some species harbor pseudogenes. These observations suggest that the members of this gene family evolve almost independently by silent nucleotide substitution and are subjected to birth-and-death evolution at the DNA level.

Stepwise in vitro affinity maturation of Vitaxin, an αvβ3-specific humanized mAb

A protein engineering strategy based on efficient and focused mutagenesis implemented by codon-based mutagenesis was developed. Vitaxin, a humanized version of the antiangiogenic antibody LM609 directed against a conformational epitope of the αvβ3 integrin complex, was used as a model system. Specifically, focused mutagenesis was used in a stepwise fashion to rapidly improve the affinity of the antigen binding fragment by greater than 90-fold. In the complete absence of structural information about the Vitaxin-αvβ3 interaction, phage-expressed antibody libraries for all six Ig heavy and light chain complementarity-determining regions were expressed and screened by a quantitative assay to identify variants with improved binding to αvβ3. The Vitaxin variants in these libraries each contained a single mutation, and all 20 amino acids were introduced at each complementarity-determining region residue, resulting in the expression of 2,336 unique clones. Multiple clones displaying 2- to 13-fold improved affinity were identified. Subsequent expression and screening of a library of 256 combinatorial variants of the optimal mutations identified from the primary libraries resulted in the identification of multiple clones displaying greater than 50-fold enhanced affinity. These variants inhibited ligand binding to receptor more potently as demonstrated by inhibition of cell adhesion and ligand competition assays. Because of the limited mutagenesis and combinatorial approach, Vitaxin variants with enhanced affinity were identified rapidly and required the synthesis of only 2,592 unique variants. The use of such small focused libraries obviates the need for phage affinity selection approaches typically used, permitting the use of functional assays and the engineering of proteins expressed in mammalian cell culture.

Backtracking leukemia to birth: Identification of clonotypic gene fusion sequences in neonatal blood spots

Epidemiological evidence has suggested that some pediatric leukemias may be initiated in utero and, for some pairs of identical twins with concordant leukemia, this possibility has been strongly endorsed by molecular studies of clonality. Direct evidence for a prenatal origin can only be derived by prospective or retrospective detection of leukemia-specific molecular abnormalities in fetal or newborn samples. We report a PCR-based method that has been developed to scrutinize neonatal blood spots (Guthrie cards) for the presence of numerically infrequent leukemic cells at birth in individuals who subsequently developed leukemia. We demonstrate that unique or clonotypic MLL-AF4 genomic fusion sequences are present and detectable in neonatal blood spots from individuals who were diagnosed with acute lymphoblastic leukemia at ages 5 months to 2 years and, therefore, have arisen during fetal hematopoiesis in utero. This result provides unequivocal evidence for a prenatal initiation of acute leukemia in young patients. The method should be applicable to other fusion genes in children with common subtypes of leukemia and will be of value in attempts to unravel the natural history and etiology of this major subtype of pediatric cancer.

Numbers of deaths related to intrapartum asphyxia and timing of birth in all Wales perinatal survey, 1993-5

Objectives: To investigate the relation between the timing of birth and the occurrence of death related to an intrapartum event.