Exponential Megapriming PCR (EMP) Cloning—Seamless DNA Insertion into Any Target Plasmid without Sequence Constraints

We present a fast, reliable and inexpensive restriction-free cloning method for seamless DNA insertion into any plasmid without sequence limitation. Exponential megapriming PCR (EMP) cloning requires two consecutive PCR steps and can be carried out in one day. We show that EMP cloning has a higher efficiency than restriction-free (RF) cloning, especially for long inserts above 2.5 kb. EMP further enables simultaneous cloning of multiple inserts.     

Isolation and characterization of major intrinsic microsomal membrane proteins.

Treatment of the membrane matrix derived from hepatic microsomes with buffered 1 M urea resulted in the selective extraction of a group of proteins together with a portion of the membrane lipid. Thorough chemical characterization of this fraction has been performed, and the proteins have been fractionated by two different procedures. The first of these, preparative polyacrylamide gel electrophoresis, has produced five highly homogeneous membrane proteins which have been characterized with regard to molecular weight, electrophoretic behavior in five different polyacrylamide systems, NH2 terminus, relative carbohydrate content, isoelectric point, and amino acid composition. The five proteins of this group fell in the molecular weight range of 54,000 to 96,000 and had isoelectric points ranging from pH 4.9 to pH 6.7. Further fractionation of the urea-soluble proteins by gel filtration in a sodium dodecyl sulfate-containing medium resulted in the isolation of four homogeneous molecular weight classes of proteins which have been characterized with respect to various physicochemical parameters. The major membrane glycoprotein (apparent molecular weight, 171,000) was isolated by this procedure and found to contain approximately equal amounts of NH2-terminal glycine and serine. suggesting the presence of at least two polypeptide chains in this molecular weight region. From the urea-insoluble fraction of the membrane comprising approximately 80% of the total protein, five intrinsic polypeptides designated S-5 through S-9 were isolated. S-5 (54,000) and S-6 (49,000) represent the most prominent components in the microsomal membrane, accounting for close to 30% of the total protein. Also isolated and characterized is the smallest membrane protein (S-9), a hydrophobic polypeptide of molecular weight 16,000. All of the urea-insoluble proteins are glycoproteins, and S-7 (35,000) gives the second most intense stain for carbohydrate of all proteins in the microsomal membrane.     

Calculation of accurate small angle X-ray scattering curves from coarse-grained protein models

Background  

Genome sequencing projects have expanded the gap between the amount of known protein sequences and structures. The limitations of current high resolution structure determination methods make it unlikely that this gap will disappear in the near future. Small angle X-ray scattering (SAXS) is an established low resolution method for routinely determining the structure of proteins in solution. The purpose of this study is to develop a method for the efficient calculation of accurate SAXS curves from coarse-grained protein models. Such a method can for example be used to construct a likelihood function, which is paramount for structure determination based on statistical inference.     

Purification of a major membrane protein of Toxoplasma gondii by immunoabsorption with a monoclonal antibody

The principal iodinatable surface protein (P30) of our cloned RH strain of Toxoplasma gondii has an apparent molecular weight of 30,000, as measured by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate under reducing conditions. Monoclonal antibody B specifically immunoprecipitated protein P30 from a detergent extract of surface radioiodinated T. gondii. Monoclonal antibody B in the presence of complement was also parasiticidal for T. gondii, and this parasiticidal effect could be blocked by protein P30. Monoclonal antibody B was purified from mouse ascitic fluid and linked to cyanogen bromide-activated Sepharose. The resulting immunoabsorbent was used to purify 1.7 mg of protein P30 from a large number of parasites. The efficiency of recovery of protein P30 was measured by assays of radioactivity and of parasiticidal blocking activity. Protein P30 represented 3 to 5% of the total protein. It is also present in a recently isolated strain of T. gondii. A convalescent human antitoxoplasma serum immunoprecipitated radiolabeled protein P30. Three convalescent antisera when quantitated by an ELISA test had a high anti-protein P30 titer. Charge shift electrophoresis showed that protein P30 has an extensive hydrophobic region and thus is probably an integral membrane protein. Electrophoresis under nonreducing conditions showed no evidence that protein P30 exists as a disulfide linked homo- or heterodimer, although it probably has intramolecular disulfide bonds.     

PERTICA,A NEW GENUS OF DEVONIAN PLANTS FROM NORTHERN MAINE

A new genus of Devonian age fossil plants is described from the Trout Valley Formation of northern Maine. Abundant compression material permits a rather complete understanding of its morphology. Pertica quadrifaria Kasper and Andrews, gen. et sp. nov., was an erect plant, perhaps a meter tall, with a pseudomonopodial main axis and dichotomous side branches. The side branches were arranged in a clockwise spiral (from base to apex) and were tetrastichous. They dichotomized numerous times, with the intervals between dichotomies decreasing distally. The ultimate branchlets bore numerous sporangia in dense clusters. Other side branches were completely sterile. Pertica quadrifaria is classified in the Subdivision Trimerophytina of Banks. Its evolutionary significance rests in the fact that it is a link in the chain of increasingly complex early vascular land plants.     

Heritabilities,social environment effects and genetic correlations of social behaviours in a cooperatively breeding vertebrate

Social animals interact frequently with conspecifics, and their behaviour is influenced by social context, environmental cues and the behaviours of interaction partners, allowing for adaptive, flexible adjustments to social encounters. This flexibility can be limited by part of the behavioural variation being genetically determined. Furthermore, behaviours can be genetically correlated, potentially constraining independent evolution. Understanding social behaviour thus requires carefully disentangling genetic, environmental, maternal and social sources of variations as well as the correlation structure between behaviours. Here, we assessed heritability, maternal, common environment and social effects of eight social behaviours in Neolamprologus pulcher, a cooperatively breeding cichlid. We bred wild‐caught fish in a paternal half‐sibling design and scored ability to defend a resource against conspecifics, to integrate into a group and the propensity to help defending the group territory (“helping behaviour”). We assessed genetic, social and phenotypic correlations within clusters of behaviours predicted to be functionally related, namely “competition,” “aggression,” “aggression‐sociability,” “integration” and “integration‐help.” Helping behaviour and two affiliative behaviours were heritable, whereas there was little evidence for a genetic basis in all other traits. Phenotypic social effects explained part of the variation in a sociable and a submissive behaviour, but there were no maternal or common environment effects. Genetic and phenotypic correlation within clusters was mostly positive. A group's social environment influenced covariances of social behaviours. Genetic correlations were similar in magnitude but usually exceeding the phenotypic ones, indicating that conclusions about the evolution of social behaviours in this species could be provisionally drawn from phenotypic data in cases where data for genetic analyses are unobtainable.