We discovered a thermostable enzyme from the archaeon Pyrococcus furiosus (Pfu), which increases yields of PCR product amplified with Pfu DNA polymerase. A high molecular mass (>250 kDa) complex with PCR-enhancing activity was purified from Pfu extracts. The complex is a multimer of two discrete proteins, P45 and P50, with significant similarity to bacterial dCTP deaminase͞dUTPase and DNA flavoprotein, respectively. When tested in PCR, only recombinant P45 exhibited enhancing activity. P45 was shown to function as a dUTPase, converting dUTP to dUMP and inorganic pyrophosphate. Pfu dUTPase improves the yield of products amplified with Pfu DNA polymerase by preventing dUTP incorporation and subsequent inhibition of the polymerase by dU-containing DNA. dUTP was found to accumulate during PCR through dCTP deamination and to limit the efficiency of PCRs carried out with archaeal DNA polymerases. In the absence of dUTP inhibition, the combination of cloned Pfu DNA polymerase and Pfu dUTPase (PfuTurbo DNA polymerase) can amplify longer targets in higher yield than Taq DNA polymerase. In vivo, archaeal dUTPases may play an essential role in preventing dUTP incorporation and inhibition of DNA synthesis by family B DNA polymerases.T he use of high-fidelity DNA polymerases in the polymerase chain reaction (PCR) is important for minimizing amplification errors in products that will be cloned, sequenced, and expressed. Several archaeal DNA polymerases with 3Ј-to 5Ј-exonucleasedependent proofreading activity have been commercialized for high-fidelity PCR amplification. Of the enzymes characterized to date, Pyrococcus furiosus (Pfu) DNA polymerase exhibits the highest fidelity, with an average error rate estimated to be 2-to 60-fold lower than other proofreading enzymes and 6-to 100-fold lower than Taq DNA polymerase (1-4), which lacks proofreading activity.Although inherent high fidelity and other unique biochemical properties of Pfu DNA polymerase have been exploited in numerous molecular biology procedures, including blunt-end cloning (5), amplification of GC-rich targets (6), mutation detection (7), ligation-independent cloning (8), site-directed mutagenesis (9), genotyping (10), and trinucleotide repeat analyses (11), it has been reported that proofreading DNA polymerases tend to perform less reliably than Taq DNA polymerase (4, 12). PCRs conducted with Taq are typically more efficient and require less optimization than reactions carried out with proofreading DNA polymerases. Lower product yields with Pfu DNA polymerase have been attributed to the enzyme's relatively slow polymerization rate (550 vs. 2,800 nucleotides per min for Taq) and interference from its associated 3Ј-to 5Ј-exonuclease activity (13).Improvements in the performance of Pfu DNA polymerase have been achieved by increasing PCR extension times (2 min͞kb of target; Stratagene Pfu DNA polymerase manual), incorporating additives such as DMSO (6,14), and mixing Pfu with Taq DNA polymerase (4). Although DNA polymerase mixtures synthesize higher yields of produ...

Positioned at the intersection of the head, body and forelimb, the pectoral girdle has the potential to function in both feeding and locomotor behaviours-although the latter has been studied far more. In ray-finned fishes, the pectoral girdle attaches directly to the skull and is retracted during suction feeding, enabling the ventral body muscles to power rapid mouth expansion. However, in sharks, the pectoral girdle is displaced caudally and entirely separate from the skull (as in tetrapods), raising the question of whether it is mobile during suction feeding and contributing to suction expansion. We measured three-dimensional kinematics of the pectoral girdle in white-spotted bamboo sharks during suction feeding with X-ray reconstruction of moving morphology, and found the pectoral girdle consistently retracted about 11° by rotating caudoventrally about the dorsal scapular processes. This motion occurred mostly after peak gape, so it likely contributed more to accelerating captured prey through the oral cavity and pharynx, than to prey capture as in ray-finned fishes. Our results emphasize the multiple roles of the pectoral girdle in feeding and locomotion, both of which should be considered in studying the functional and evolutionary morphology of this structure.

Despite the importance of intraoral food transport and swallowing, relatively few studies have examined the biomechanics of these behaviors in non-tetrapods, which lack a muscular tongue. Studies show that elasmobranch and teleost fishes generate water currents as a ‘hydrodynamic tongue’ that presumably transports food towards and into the esophagus. However, it remains largely unknown how specific musculoskeletal motions during transport correspond to food motion. Previous studies of white-spotted bamboo sharks (Chiloscyllium plagiosum) hypothesized that motions of the hyoid, branchial arches, and pectoral girdle, generate caudal motion of the food through the long oropharynx of modern sharks. To test these hypotheses, we measured food and cartilage motion with XROMM during intra-oropharyngeal transport and swallowing (n=3 individuals, 2-3 trials per individual). After entering the mouth, food does not move smoothly toward the esophagus, but rather moves in distinct steps with relatively little retrograde motion. Caudal food motion coincides with hyoid elevation and a closed mouth, supporting earlier studies showing that hyoid motion contributes to intra-oropharyngeal food transport by creating caudally-directed water currents. Little correspondence between pectoral girdle and food motion was found, indicating minimal contribution of pectoral girdle motion. Transport speed was fast as food entered the mouth, slower and step-wise through the pharyngeal region and then fast again as it entered the esophagus. The food's static periods in the step-wise motion and its high velocity during swallowing could not be explained by hyoid or girdle motion, suggesting these sharks may also use the branchial arches for intra-oropharyngeal transport and swallowing.

White-spotted bamboo sharks, Chiloscyllium plagiosum, generate strong suction-feeding pressures that rival the highest levels measured in ray-finned fishes. However, the hyostylic jaw suspension of these sharks is fundamentally different from the actinopterygian mechanism, including more mobile hyomandibulae, with the jaws and ceratohyal suspended from the hyomandibulae. Prior studies have proposed skeletal kinematics during feeding in orectolobid sharks from indirect measurements. Here, we tested these hypotheses using XROMM to measure cartilage motions directly. In agreement with prior hypotheses, we found extremely large retraction and depression of the ceratohyal, facilitated by large protraction and depression of the hyomandibula. Somewhat unexpectedly, XROMM also showed tremendous long-axis rotation (LAR) of both the ceratohyal and hyomandibula. This LAR likely increases the range of motion for the hyoid arch by keeping the elements properly articulated through their large arcs of motion. XROMM also confirmed that upper jaw protraction occurs before peak gape, similarly to actinopterygian suction feeders, but different from most other sharks in which jaw protrusion serves primarily to close the mouth. Early jaw protraction results from decoupling the rotations of the hyomandibula, with much of protraction occurring before peak gape with the other rotations lagging behind. In addition, the magnitudes of retraction and protraction of the hyoid elements are independent of the magnitude of depression, varying the shape of the mouth among feeding strikes. Hence, the large variation in suction-feeding behavior and performance may contribute to the wide dietary breadth of bamboo sharks.