The design of phenylglycine containing benzamidine carboxamides as potent and selective inhibitors of factor Xa.

Factor Xa, a critical serine protease in the blood coagulation cascade, has become a target for inhibition as a strategy for the invention of novel anti-thrombotic agents. Here we describe the development of phenylglycine containing benzamidine carboxamides as novel, potent and selective inhibitors of factor Xa.

Further characterization of phage T1 DNA clones.

A series of phage T1 DNA clones containing Sau3A fragments of 1 kb or more were tested for their ability to rescue the defects of amber mutations representing all known T1 genes. Rescue was detected by either complementation or recombination. These data have extended the correlation of the T1 genetic and physical maps and provided more conclusive proof that the large map distances at the left end of the genetic map reflect both increased physical separation of the known genetic markers and enhanced recombination and that the right and left chromosomal ends are recombinogenic to the same extent.

Phage T1-mediated transduction of a plasmid containing the T1 pac site.

The T1 pac site has been cloned into a plasmid vector. This recombinant plasmid was tested for T1-mediated transduction efficiency in comparison with a plasmid containing the phage lambda T1-pac-like site esp-lambda, plasmids containing T1 sequences other than the pac site, and plasmids containing neither T1 sequences nor known pac sites. The data obtained indicate that there are at least two distinct mechanisms of T1-mediated plasmid transduction. One requires the presence of any T1 sequence on the plasmid and probably takes place via cointegrate formation with the homologous region of an infecting T1 genome. The other is specifically dependent on the presence of a pac site on the plasmid. Plasmids are packaged as head-to-tail multimers that have one heterogeneous molecular end and the other terminated at pac, and the direction of packaging with respect to the pac site is the same for plasmids as for T1. Possible roles of pac in plasmid packaging and their implications with regard to the packaging of phage DNA are discussed.

In vitro packaging of foreign DNA into heads of bacteriophage T1.

The isolation of a collection of 44 morphologically T1-like phages is described. It is shown that these phages share some similarity with T1 in terms of cross-inactivation with anti-T1 serum, particle proteins and DNA packaging in vitro by the headful process. Virion DNA extracted from these phages was treated with T1 in vitro packaging extracts and the reaction mixtures were tested for the formation of infectious phage particles. The packaging efficiencies observed varied from about 1 to 100% of that of virion T1 DNA. Phage lambda virion DNA was packaged with an efficiency of between 0.01 and 2% (5 X 10(1) to 3 X 10(3) p.f.u./micrograms DNA), the shorter deleted derivative lambda L47 being packaged more efficiently than normal length lambda C1857 DNA. Virion DNA from phages T3 and T7 was also packaged at an efficiency similar to that for lambda. The in vitro packaging of T1 DNA requires the presence of the pac sequence which initiates headful packaging from a concatemeric precursor. The high efficiency of packaging DNA from some of the T1-like phages may indicate the presence of similar packaging sequences. However, in the case of lambda L47, which is known not to contain such a sequence, the in vitro DNA packaging reaction must occur by a secondary pathway unrelated to the headful mechanism.

Correlation of the genetic and physical maps of phage T1.

A series of BglII- and Sau3A-derived fragments of phage T1 DNA were cloned into the plasmid vector pLV59 and the clones were tested for their ability to complement and recombine with infecting genomes of am mutants representing all known T1 essential genes. The resulting data were used to correlate the T1 genetic and physical maps. This analysis led to the following conclusions: the large map distances observed at the left (early) end of the genetic map arise partly from increased genetic recombination at the chromosomal left end and partly from increased physical distances between identified genetic markers in this region, the right chromosomal end is also recombinogenic, genes 3.5 to 18, which occupy the right two-thirds of the DNA molecule, are tightly compacted with little space for additional protein-specifying sequences in this region.

A coupled in vitro system for the formation and packaging of concatemeric phage T1 DNA.

Extracts derived from E. coli cells infected non-permissively with phage T1 amber mutants were used in an in vitro system to investigate the packaging of T1 DNA into phage heads. The standard extract used infections with amber mutants in genes 1 and 2 (g1- g2-) which are defective in T1 DNA synthesis but can synthesis the proteins required for particle morphogenesis. g1- g2- extracts packaged T1+ virion DNA molecules with an efficiency of 3 X 10(5) pfu/micrograms DNA. Extracts from cells infected with phage also defective in DNA synthesis but carrying additional mutations in genes 3.5 or 4 which are required for concatemer formation in vivo (g1- g3.5- and g1- g4- extracts) package T1 virion DNA at substantially lower efficiencies. Analysis of the DNA products from these in vitro reaction showed that concatemeric DNA is formed very efficiently by g1- g2- extracts but not by g1- g3.5- or g1- g4- extracts. These results are interpreted as evidence that the T1 in vitro DNA packaging system primarily operates in a similar manner to the in vivo headful mechanism. This is achieved in vitro by the highly efficient conversion of T1 virion DNA into concatemers which are then packaged with a much lower efficiency into heads to form infectious particles. A secondary pathway for packaging T1 DNA into heads and unrelated to the headful mechanism may also exist.