Trigonal crystals of porcine mitochondrial aspartate aminotransferase.

Crystals suitable for X-ray analysis of porcine mitochondrial aspartate aminotransferase in the closed conformation were obtained after the apoenzyme was reconstituted with N-5'-phosphopyridoxyl-L-aspartate, an inhibitor in which the cofactor is covalently bound to the substrate. This results in a crystal form that has not been encountered previously in studies of aspartate aminotransferases. The crystals belong to the trigonal space group P3121 (or the enantiomeric P3221) with unit cell dimensions alpha = b = 202.0 A, c = 58.0 A, alpha = beta = 90 degrees, gamma = 120 degrees and contain one dimer in the asymmetric unit.

Crystallization and preliminary X-ray analysis of gamma-aminobutyric acid transaminase.

gamma-Aminobutyric acid transaminase from pig liver, an alpha 2 dimeric enzyme of Mr 110,100, has been crystallized by the vapour diffusion method with polyethylene glycol as precipitant. The crystals are monoclinic, space group P2(1), unit cell dimensions a = 82.1 A, b = 230.0 A, c = 70.3 A, beta = 123.9 degrees and diffract to 2.5 A resolution. There are two dimers per asymmetric unit.

Restriction sites as identification tags for lymphocyte cDNAs.

A cDNA library was prepared from BW 5147 murine lymphoma cells in lambda ecc III phage and randomly partitioned into 291 sectors, each with 800-1000 recombinant phage plaques. One sector was chosen for further characterization in terms of sensitivity to restriction endonuclease cutting. Aliquots of DNA preparations from this sector were treated with XhoI, SmaI, NcoI, PvuII, PstI, HindIII, EcoRI, BamHI, and ApaLI before being used as templates in a cell-free expression system. The polypeptide products were separated by two-dimensional (2-D) gel electrophoresis and radiofluorographs of the gels were submitted to computer-aided image analysis. The matched patterns were inspected for the presence or absence of spots upon individual endonuclease treatments. Thereafter the results were integrated in a data matrix which served as a basis to construct "restriction tags" for all spots. These (restriction) tags are binary numbers termed "cut numbers" and are a representation of the set of recognition sequences which are (or are not) part of the coding sequence. From 493 sequences (visualized as 2-D gel spots), 12 were not cut by any of the nine enzymes, while 45 were cut by all of them. The percentages of sequences resistant to enzyme treatment ranged between 17% and 77% for NcoI and XhoI, respectively. The enzyme treatments led to the appearance of a certain portion of "new spots", probably products from truncated sequences. From 512 possible cut numbers, 136 were assigned to the 493 spots. Restriction tags are available to facilitate retrieval of cDNA clones from the (partitioned) cDNA library.

Crystallization and structure solution at 4 A resolution of the recombinant synthase domain of N-(5'-phosphoribosyl)anthranilate isomerase:indole-3-glycerol-phosphate synthase from Escherichia coli complexed to a substrate analogue.

The recombinant synthase domain of the bifunctional enzyme N-(5'-phosphoribosyl)anthranilate isomerase:indole-3-glycerol-phosphate synthase from Escherichia coli has been crystallized, and the structure has been solved at 4 A resolution. Two closely related crystal forms grown from ammonium sulphate diffract to 2 A resolution. One form (space group R32, a = 163 A, alpha = 29.5 degrees) contains the unliganded synthase domain; the second crystal form (space group P6(3)22, a = 144 A, c = 158 A) is co-crystallized with the substrate analogue N-(5'-phosphoribit-1-yl)anthranilate. The structure of the synthase-inhibitor complex has been solved by the molecular replacement method. This achievement represents the first successful use of a (beta alpha)8-barrel monomer as a trial model. The recombinant synthase domain associates as a trimer in the crystal, the molecules being related by a pseudo-crystallographic triad. The interface contacts between the three domains are mediated by those residues that are also involved in the domain interface of the bifunctional enzyme. This system provides a model for an interface which is used in both intermolecular and intramolecular domain contacts.

Global analysis of gene expression in cells of the immune system II. Cell-free translation products and high-density filter hybridization data.

We have developed an experimental system for linking information on cell-free transcription and translation products from cDNA clones with data obtained from hybridization signals from complex probes. The work described in this paper consists of two distinct processes, one being the construction of a system of clonal addresses and the other the identification of expressed genes involved in the studied processes. We describe the use of this system to identify genes involved in thymus development. Complex probes from fetal thymuses (GD15, 17 and newborn) of Balb/c mice were used to identify genes, which are up- or downregulated during the process of differentiation. The full set of information is available in the Clone-base of the Basel Institute for Immunology and will be retrievable from the website of the collaborating laboratories.