Linked lexitropsins and the in vitro inhibition of HIV-1 reverse transcriptase RNA-directed DNA polymerization: a novel induced-fit of 3,5 m-pyridyl bisdistamycin to enzyme-associated template-primer.

Five classic DNA minor groove-binding drugs and a series of bis-linked lexitropsins based on netropsin and distamycin have been screened for their effectiveness in inhibiting transcription by HIV-1 reverse transcriptase (RT) on a poly(rA).oligo(dT) template-primer (TP). The two most effective drugs, 3,5 m-pyridyl-linked bisdistamycin (MPyr) and trans-vinyl-linked bisdistamycin (TVin), show (1) enhanced inhibition in reactions initiated with pre-incubated enzyme template-primer (ETP) and (2) reduced affinity for a "free" TP analog, when compared with the parent drug distamycin. All three drugs lack the ability to inhibit processive incorporation of nucleotide, suggesting drug intervention instead at initiation or termination of processive cycles. The two bis-linked drugs exhibit different kinetic behavior with reverse transcriptase's two substrates: template-primer and nucleotide. When primer is the variable substrate, TVin is partially noncompetitive and MPyr is dead-end competitive (Ki = 6.5 microM). With nucleotide as substrate, TVin is noncompetitive at low drug concentrations and MPyr is uncompetitive. Gel band mobility shift assays with MPyr indicate that the drug inhibits via entrapment of TP on the enzyme rather than displacement of TP from the enzyme surface. The conformation of nucleic acid is most likely altered upon MPyr binding, enhancing the induced fit of enzyme to hybrid duplex. The relevance of this novel mode of inhibition is considered in relation to enzyme association/dissociation with TP that occurs prior to (-)-DNA strand transfer, and to the structural implications of an enzyme-bound hybrid RNA/DNA nucleic acid.

Design of B-DNA cross-linking and sequence-reading molecules.

We report the design of hybrid molecules to bind in the minor groove of B-DNA, which combine DNA alkylating and cross-linking ability for increased chemotherapeutic efficacy, with sequence specificity, to minimize side effects. Optimal linkage geometries have been determined for the synthesis of bis-anthramycin and anthramycin-netropsin hybrid molecules. Earlier studies on linked drugs have typically been based on molecular mechanics calculations. This work, in contrast, uses the observed crystal structures of a netropsin/DNA complex and a new anthramycin/DNA complex to determine the exact spacing between two individual drugs when bound in the minor groove of B-DNA. Molecular linkers then are designed and tested between these two experimental positions, to form a chimeric or bis-linked compound molecule. A linked anthramycin-netropsin molecule has been designed specifically to target the polypurine tract second-strand primer site of the reverse transcriptase of HIV-1.

Transmembrane domain length variation in the evolution of major histocompatibility complex class I genes.

The fifth exons of major histocompatibility complex (MHC) class I genes encode a transmembrane domain (TM) that is largely responsible for class I antigen cell-surface expression usually through conventional hydrophobic amino acid-membrane interactions or, less often, through phosphatidylinositol linkage. In this report we show that Peromyscus leucopus, a Cricetidae rodent, has MHC class I genes (Pele-A genes) encoding three distinct sizes of TMs. Increases in TM lengths were due to tandem duplications of sequences similar to human hypervariable minisatellite repeats and the lambda chi site. We discerned remnants of a similar duplication event in comparable rodent and primate MHC class I genes. Furthermore, several duplications and deletions appear to have occurred independently in H-2, RT1, Pele-A, and ChLA genes in near-identical positions. Accumulated data suggests that sequences in the fifth exon of MHC class I genes may, therefore, constitute a mutational or recombinational hot spot that is mediated by minisatellite- and chi-like sequences imbedded within the coding region. The MHC class I genes may thus have recruited "selfish" DNA in their evolution to encode cell surface proteins. Expression of Pele-A genes was examined by the polymerase chain reaction (PCR) using oligonucleotide primers specific for exon 4 and 5 sequences. The PCR product sizes indicated that genes encoding each TM domain length are ubiquitously transcribed.

Major histocompatibility complex class I genes of Peromyscus leucopus.

Class I genes of the Peromyscus leucopus major histocompatibility complex (MhcPele) were examined by Southern blot hybridization, genomic cloning, and DNA sequencing. At least three distinct subtypes of Pele class I genes were discerned, which we have designated Pele-A, B, and C. The nucleotide sequences of exon 5-containing regions (encoding the transmembrane domain) suggested that Pele-A genes are homologs of mouse H-2K, D, L, and Q genes and that Pele-B genes correspond to mouse Tla genes. The Pele-C genes appeared similar to mouse M1 genes. The number of unique genes in each subtype cloned from an individual P. leucopus were 20 for Pele-A, 13 for Pele-B, and 2 for Pele-C. Three genomic clones showed cross-hybridization to both Pele-A and Pele-B gene-specific probes. Six genomic clones remained unclassified as they did not cross-hybridize to exon 5-containing probes from Pele-A, B, or C genes. The homology between the transmembrane domains of Pele class I gene subtypes was found to be similar to that observed between the transmembrane domains of H-2 subtypes (or groups). Interspecific similarity of exon 5 was found to be 81%-88% between Pele class I genes and their H-2 counterparts.