Colocalization of the gene for nephrogenic diabetes insipidus (DIR) and the vasopressin type 2 receptor gene (AVPR2) in the Xq28 region.

The gene for nephrogenic diabetes insipidus (DIR) and the vasopressin type 2 receptor gene (AVPR2) have both been localized in the Xqter region by genetic mapping and functional expression studies, respectively. In this paper genetic evidence that the DIR locus is localized distal to the DXS305 locus and that the functional gene for the V2 receptor is localized between the markers DXS269 and F8 is presented. These further refinements in the localization of both genes strengthen the assumption that both genes are identical and provide a rationale for cloning the gene by reversed genetics strategies.

In vitro inhibition of HIV-1 proteinase by cerulenin.

Retroviruses encode proteinases necessary for the proteolytic processing of the viral gag and gag-pol precursor proteins. These enzymes have been shown to be structurally and functionally related to aspartyl proteinases such as pepsin and renin. Cerulenin is a naturally occurring antibiotic, commonly used as an inhibitor of fatty acid synthesis. Cerulenin has been observed to inhibit production of Rous sarcoma virus and murine leukaemia virus by infected cells, possibly by interfering with proteolytic processing of viral precursor proteins. We show here that cerulenin inhibits the action of the HIV-1 proteinase in vitro, using 3 substrates: a synthetic heptapeptide (SQNYPIV) which corresponds to the sequence at the HIV-1 gag p17/p24 junction, a bacterially expressed gag precursor, and purified 66 kDa reverse transcriptase. Inhibition of cleavage by HIV-1 proteinase required preincubation with cerulenin. Cerulenin also inactivates endothiapepsin, a well-characterised fungal aspartyl proteinase, suggesting that the action of cerulenin is a function of the common active site structure of the retroviral and aspartic proteinases. Molecular modelling suggests that cerulenin possesses several of the necessary structural features of an inhibitor of aspartyl proteinases and retroviral proteinases. Although cerulenin itself is cytotoxic and inappropriate for clinical use, it may provide leads for the rational design of inhibitors of the HIV proteinase which could have application in the chemotherapy of AIDS.

Synthetic peptides as substrates and inhibitors of human immune deficiency virus-1 protease.

Retroviruses code for a virus-specific protease which is essential for polyprotein processing and viral infectivity. The human immune deficiency virus-1 protease is an aspartic protease of 9 kDa which was synthesized by recombinant DNA technology and arises by autocatalytic processing from a polyprotein precursor which has recently been demonstrated by use of a protease-specific monoclonal antibody. The protease was shown to form dimers. Here we demonstrate that synthetic peptides can be used as both model substrates as well as inhibitors for investigation of the protease. 14 synthetic peptides, 7-18 amino acids in length, containing putative protease cleavage sites of the viral polyprotein gag and pol precursors, have been analyzed with the partially purified protease by the use of high performance liquid chromatography. In seven cases, where cleavage was observed, the length of the peptides did not significantly influence the cleavage efficiencies, heptapeptides being large enough as model substrates. No cleavage was observed with a protein preparation purified in parallel from control bacteria not expressing the human immune deficiency virus-1 protease. The protease was not only able to cut next to a proline but also between other peptides indicating that the proline is not a prerequisite. Three peptides with either reduced bonds at the cleavage site or a substitution by statin were inhibitory while another uncleaved substrate was not. The usefulness of small model substrates for characterization of the protease is further demonstrated by determination of a kinetic optimum pH (3.5-5.5) and incubation temperature (37 degrees C).

The mapping of chromosomes in Saccharomyces cerevisiae. I. A cosmid vector designed to establish, by cloning into cdc-mutants, numerous start loci for chromosome walking in the yeast genome.

A series of vectors for cosmid cloning in yeast has been derived from cosmid pHC79. Vectors pMT4 through pMT6 contain two tandemly arranged cohesive end sites (cos) from the genome of bacteriophage lambda. Their design allows the rapid and simple preparation of cosmid arms by linearizing a vector at the unique PvuII-restriction site located between the two cos-sequences and then cutting the linearized molecule at one of its unique cloning sites for BamHI, ClaI, PvuI, SalI or ScaI. Cosmids generated with arms from the most advanced vector, pMT6, carry the origin of replication (ori) and the ApR gene from pBR322 and the TRP1/ARS1 and URA1 genes from Saccharomyces cerevisiae. A yeast genomic DNA library was established by packaging in vitro, into bacteriophage lambda preheads, of partially restricted yeast DNA fragments ligated to cosmid arms of vector pMT6. About 80% of the clones thus obtained comprise inserts of contiguous genomic DNA over 30 kb in length. Unique DNA probes for the yeast genes CDC10, CDC39, HIS4, LEU2, and PGK1 have successfully been applied when testing for completeness of this library by isolating a series of overlapping cosmid clones that carry the respective genes. The library will thus be useful for the selection of cosmid clones which carry CDC genes from yeast by complementing first, with the vectorial yeast gene URA1, the pyrimidine auxotrophy of most cdc-strains and then, with the respective CDC wild-type genes, of the temperature-sensitive mutant alleles. Most CDC clones thus obtained will provide unique DNA probes which serve as randomly distributed start sequences within the yeast genome for overlap hybridization screening in chromosome mapping studies.