Site-directed mutagenesis of the T4 endonuclease V gene: role of lysine-130

The DNA sequence of the bacteriophage T4 denV gene which encodes the DNA repair enzyme endonuclease V was previously constructed behind the hybrid lambda promoter OLPR in a plasmid vector. The OLPR-denV sequence was subcloned in M13mp18 and used as template to construct site-specific mutations in the denV structural gene in order to investigate structure/function relationships between the primary structure of the protein and its various DNA binding and catalytic activities. The Lys-130 residue of the wild-type endonuclease V has been postulated to be associated with its apurinic endonuclease (AP-endonuclease) activity. The codon for Lys-130 was changed to His-130 or Gly-130, and each denV sequence was subcloned into a pEMBL expression vector. These plasmids were transformed into repair-deficient Escherichia coli (uvrA recA), and the following parameters were examined for cells or cell extracts: expression and accumulation of endonuclease V protein (K-130, H-130, or G-130); survival after UV irradiation; dimer-specific DNA binding; and kinetics of phosphodiester bond scission at pyrimidine dimer sites, dimer-specific N-glycosylase activity, and AP-endonuclease activity. The enzyme's intracellular accumulation was significantly decreased for G-130 and slightly decreased for H-130 despite normal levels of denV-specific mRNA for each mutant. On a molar basis, the endonuclease V gene products generally gave parallel levels of each of the catalytic and binding functions with K-130 greater than H-130 greater than G-130 much greater than control denV-.(ABSTRACT TRUNCATED AT 250 WORDS)     

Efficient screening of recombinant DNA junctions afforded by probing with synthetic "bridge" oligonucleotides

Procedures have been developed which simplify and expedite the screening of recombinant DNA constructions for those which only exhibit the desired DNA-DNA junctions. A synthetic DNA oligonucleotide designed to span (or "bridge") sequences around correct restriction enzyme junctions was used as a hybridization probe for the rapid identification of those sequences in several molecular cloning methodologies. It facilitated analyses of the products of random ligation reactions, as well as constructions harbored in bacteria and bacteriophage. "Bridge" probes, [32P]-end-labeled to very high specific activity, remained useful after several hybridizations and often circumvented lengthy restriction analyses.     

The serratus anterior subslip: anatomy and implications for facial and hand reanimation

The ideal donor muscle for facial and hand reanimation has yet to be found. Donor muscles commonly used today, such as the gracilis and pectoralis minor, are limited by bulkiness and the number of force vectors they can provide. In the authors' study of 50 fresh cadaver serratus anterior muscles, they further describe neurovascular anatomy of the muscle slip (i.e., the portion of the muscle that inserts on a rib) and subslip (superficial or deep subdivision of the slip after division along a loose areolar plane). All 260 slips could be separated into a deep and a superficial subslip, yielding a total of 520 subslips. A branch of the serratus artery (a terminal branch of the thoracodorsal artery serving the lower five to seven slips of the muscle) and a branch of the long thoracic nerve were identified for each of these. Deep subslips were thinner than superficial subslips, both at the origin of the slip on the rib periosteum (2.4 mm versus 3.0 mm, p < 0.0001) and centrally at the serratus artery (3.3 mm versus 4.0 mm, p < 0.0001). In addition, the subslips of the most inferior slip were thinner than those of more superior slips, both at the origin of the slip (2.3 mm versus 2.8 mm, p < 0.0001) and at the serratus artery (3.0 mm versus 3.8 mm, p < 0.0001). Fine anastomosing vessels were present between the slips and the subslips. The average number of anastomosing vessels present between adjacent slips was 1.7, and 2.1 anastomosing vessels were present between the subslips of a given slip. Given the thinness of these vessels (all less than 0.2 mm) compared with those of the vascular pedicle of the subslip (mean, 0.7 mm; all greater than 0.4 mm), the authors believe these can be safely divided without compromising subslip vascularity. After division of these vessels, a mean length of 9.6 +/- 1.5 cm is available to allow independent orientation of each subslip. When the serratus muscle flap is separated into its component subslips, a maximum of 10 possible force vectors may be transferred on a single vascular pedicle. Subslips are significantly thinner than donor muscles commonly used today. These two advantages offer the potential for significant functional and aesthetic improvement when the serratus anterior muscle flap is used for face and hand reanimation. Mimetic muscles such as the orbicularis oculi and orbicularis oris could possibly be reconstructed in their proper anatomical positions.     

WW or WoW: the WW domains in a union of bliss

WW domains are small protein modules that recognize proline-rich peptide motifs or phosphorylated-serine/threonine proline sites in cognate proteins. Within host proteins these modules are joined to other protein domains or to a variety of catalytic domains acting together as adaptors or targeting anchors of enzymes. An important aspect of signaling by WW domains is their ability to recognize their cognate ligands in tandem. Tandem WW domains not only act in a synergistic manner but also appear to chaperone the function of each other. In this review, we focus on structure, function, and mechanism of the tandem WW domains co-operativity as well as independent actions. We emphasize here the implications of tandem arrangement and cooperative function of the domains for signaling pathways.