The "megaprimer" method of site-directed mutagenesis

We describe a simple and efficient method of mutagenesis which we term the "megaprimer" method. The method utilizes three oligonucleotide primers to perform two rounds of polymerase chain reaction. In the method, the product of the first polymerase chain reaction is used as one of the polymerase chain reaction primers (a "megaprimer") for the second polymerase chain reaction. When a phage promoter and a translational initiation signal are attached to the appropriate oligonucleotide primer, the mutant protein can be generated without any in vivo manipulations. To illustrate the method, two mutations in the catalytic domain of the human factor IX gene have been generated. The substitution of megaprimers for oligonucleotide primers may have utility in other polymerase chain reaction-based methods.     

Regeneration of transgenic plants of Prunus armeniaca containing the coat protein gene of Plum Pox Virus

Summary A system was developed which allows the transfer of foreign genes into apricot cultivars. We report the transformation and regeneration of Prunus armeniaca plants with Agrobacterium tumefaciens strain LBA 4404 containing various binary plasmids, pBinGUSint, carrying the marker gene ß-glucuronidase (GUS) and pBinPPVm, carrying the coat protein gene of Plum Pox Virus (PPV). The marker gene GUS was used for optical evaluation of the efficiency of the transformation system. The coat protein gene of PPV was used to introduce coat protein mediated resistance against one of the most important pathogens of stone fruit trees in Europe and the whole Mediterranean area. This is the first report of the successful integration of a viral coat protein gene into a fruit tree species, opening a new perspective on the control of the disease.Abbreviations GUS ß-glucuronidase - PPV Plum Pox Virus - BA 6-benzylaminopurine - NPTII neomycin phosphotransferase II - CP coat protein - CaMV Cauliflower Mosaic Virus - P35S 35S promoter - MS Murashige and Skoog - PCR polymerase chain reaction - P/C/I phenol/chloroform/isoamylalcohol - RNase ribonuclease - dNTP deoxyribonucleosidetriphosphate - DMSO dimethyl sulfoxide     

Dideoxy fingerprinting (ddE): a rapid and efficient screen for the presence of mutations.

We describe dideoxy fingerprinting (ddF), a hybrid between dideoxy sequencing and SSCP that can detect the presence of single base and other sequence changes in PCR-amplified segments. As implemented herein, ddF involves a Sanger sequencing reaction with one dideoxynucleotide followed by nondenaturing gel electrophoresis. When ddF was used to examine segments of the human factor IX gene, 84 of 84 different mutations were detected with a very low rate of false positive signals. The approximate locations of the sequence changes could be determined from the ddF pattern and samples with different sequence changes had different fingerprints. In addition, large segments could be amplified and rapidly screened by ddF in multiple smaller subsegments. The patterns observed with ddF are instructive in that they suggest an inherent limitation in the detection of certain mutations by SSCP.     

Polarography of Polynucleotides: III. Polyadenylic Acid: The Electrode Process and Interaction with Polyamines

Polyadenylic acid (poly A) was studied under various conditions using both DC polarography and phase sensitive AC polarography and by measuring the time-course of the current during the lifetime of a single drop of the dropping mercury electrode. Under certain conditions the current at potentials of the limiting portion of the DC polarographic wave does not reach its limiting value and in extreme situations peak-shaped curves are observed. This phenomenon is explained in terms of desorption and repulsion from the electrode of neutral poly A due to its polyanionic character. Consequently, the suppression of the current can be enhanced by increasing negative potential of the electrode and by exposing the negative charges of phosphate groups, e.g., by increasing pH and temperature and by decreasing ionic strength and buffer capacity; vice versa, the current suppression can be at least partially eliminated by reversing these conditions. Polyamines which seem to shield the phosphate groups through specific interactions are very effective in eliminating the current suppression. The effectiveness of a polyamine is determined by its chain length and by the density of its amino groups and the geometry of their distribution.     

Cardiac muscle

Summary The ultrastructure of chicken and frog cardiac muscle are compared and then contrasted with the ultrastructure of mammalian cardiac muscle. Both chicken and frog cardiac muscle have no transverse tubules, remarkably few nexuses and no prominent M-lines. M-fibers of both animals are small (2–5 ) in diameter and contain dense granules. Chicken cardiac muscle like mammalian cardiac muscle has very well developed sarcoplasmic reticulum and couplings. The latter do not occur in frog cardiac muscle and the former is poorly developed in that muscle. Morphologic evidence is presented in the frog and chicken heart that would tend to attribute to the sarcoplasmic reticulum a transport function for electron-dense material (presumably proteinaceous) the possible significance of which is discussed. Purkinje fibers were identified in the form of a network on the endocardial surface of both atria and ventricles of chicken hearts. The topography of these fibers corresponds to that of a population of fibers in small mammalian hearts that, and unlike ventricular fibers in those animals, does not have transverse tubules.This investigation was presented, in part, at the 2nd Annual Summer Workshop of the Council on Basic Science of the American Heart Association in Mountain View, California, August 5–8, 1968; at the Gordon Conference on Myocardial Contractility in Holderness, New Hampshire, August 12–16, 1968; and at the 8th Annual Meeting of the American Society for Cell Biology in Boston, Massachusetts, November 11–13, 1968. This research was supported by grant No. 66737 from the American Heart Association, Inc. and by grant No. HE 08620 from the NIH.