Assignment of the gene(s) involved in the expression of the proliferation-related Ki-67 antigen to human chromosome 10

Summary The antigen recognized by the monoclonal antibody Ki-67 is a proliferation-related nucleolus-associated constituent used as a marker for cycling cells in tumor diagnosis. Antibody Ki-67 reacts with human proliferating cells, but not with hamster and mouse cells. Expression of the Ki-67 antigen was studied in a panel of human-rodent somatic cell hybrids. The results indicate that a gene involved in the expression of the antigen is located on chromosome 10.     

Cloning and molecular characterization of the chalcone synthase multigene family of Petunia hybrida

Chalcone synthase-encoding genes (chs) in Petunia hybrida comprise a multigene family. Some of the chs genes have been grouped into a subfamily, based upon their strong cross-hybridization and tight genomic linkage. From genomic libraries eight 'complete' chs genes, two chs gene 5'-fragments and two chs gene 3'-fragments have been isolated. The nucleotide sequence of six complete chs genes is presented and discussed in relation to their evolutionary origin and expression in different tissues. Each member of the family consists of two exons separated by an intron of variable size and sequence, which is located at a conserved position. The chs gene fragments represent single exons. Homology between non-linked chs genes is approx. 80% at the DNA level and restricted to protein-coding sequences. Homology between subfamily members (which are tightly linked) is higher (90-99%) and extends into untranslated regions of the gene, strengthening the view that they arose by recent gene duplications. The chsD gene contains a mutated translation stop codon, suggesting that this is an inactive (pseudo)gene. None of the other members of the gene family exhibits characteristics of a pseudogene, indicating that if gene inactivation has occurred during their evolution, it must characteristics of a pseudogene, indicating that if gene inactivation has occurred during their evolution, it must have been a recent event. Homology at the protein level between some (expressed) chs genes is surprisingly low. The possibility that these genes encode proteins with slightly different enzymatic activities is discussed.     

Structure of the bovine eye lens gamma s-crystallin gene (formerly beta s)

The organization of a number of crystallin genes has already been resolved. One of the remaining genes of which the structure was hitherto unknown is the gamma s gene (formerly beta s). We determined the complete sequence of the bovine gamma s-crystallin-coding gene, apart from the middle region of the first intron. Since it contains three exons and two introns, we conclude that the former beta s, also at the gene level is gamma-crystallin-like. However, it is located on chromosome 3, in contrast to other gamma genes which occur in tandem on the human chromosome 2.     

Transport of basic amino acids by membrane vesicles of Lactococcus lactis.

The uptake of the basic amino acids arginine, ornithine, and lysine was studied in membrane vesicles derived from cells of Lactococcus lactis which were fused with liposomes in which beef heart mitochondrial cytochrome c oxidase was incorporated as a proton motive force (PMF)-generating system. In the presence of ascorbate N,N,N'N'-tetramethylphenylenediamine-cytochrome c as the electron donor, these fused membranes accumulated lysine but not ornithine or arginine under aerobic conditions. The mechanism of energy coupling to lysine transport was examined in membrane vesicles of L. lactis subsp. cremoris upon imposition of an artificial electrical potential (delta psi) or pH gradient or both and in fused membranes of these vesicles with cytochrome c oxidase liposomes in which the delta psi and delta pH were manipulated with ionophores. Lysine uptake was shown to be coupled to the PMF and especially to the delta psi, suggesting a proton symport mechanism. The lysine carrier appeared to be specific for L and D isomers of amino acids with a guanidine or NH2 group at the C6 position of the side chain. Uptake of lysine was blocked by p-chloromercuribenzene sulfonic acid but not by maleimides. Counterflow of lysine could not be detected in L. lactis subsp. cremoris, but in the arginine-ornithine antiporter-containing L. lactis subsp. lactis, rapid counterflow occurred. Homologous exchange of lysine and heterologous exchange of arginine and lysine were mediated by this antiporter. PMF-driven lysine transport in these membranes was noncompetitively inhibited by arginine, whereas the uptake of arginine was enhanced by lysine. These observations are compatible with a model in which circulation of lysine via the lysine carrier and the arginine-ornithine antiporter leads to accumulation of arginine.     

Mechanism of L-glutamate transport in membrane vesicles from Bacillus stearothermophilus.

In the presence of electrochemical energy, several branched-chain neutral and acidic amino acids were found to accumulate in membrane vesicles of Bacillus stearothermophilus. The membrane vesicles contained a stereo-specific transport system for the acidic amino acids L-glutamate and L-aspartate, which could not translocate their respective amines, L-glutamine and L-asparagine. The transport system was thermostable (Ti = 70 degrees C) and showed highest activities at elevated temperatures (60 to 65 degrees C). The membrane potential or pH gradient could act as the driving force for L-glutamate uptake, which indicated that the transport process of L-glutamate is electrogenic and that protons are involved in the translocation process. The electrogenic character implies that the anionic L-glutamate is cotransported with at least two monovalent cations. To determine the mechanistic stoichiometry of L-glutamate transport and the nature of the cotranslocated cations, the relationship between the components of the proton motive force and the chemical gradient of L-glutamate was investigated at different external pH values in the absence and presence of ionophores. In the presence of either a membrane potential or a pH gradient, the chemical gradient of L-glutamate was equivalent to that specific gradient at different pH values. These results cannot be explained by cotransport of L-glutamate with two protons, assuming thermodynamic equilibrium between the driving force for uptake and the chemical gradient of the substrate. To determine the character of the cotranslocated cations, L-glutamate uptake was monitored with artificial gradients. It was established that either the membrane potential, pH gradient, or chemical gradient of sodium ions could act as the driving force for L-glutamate uptake, which indicated that L-glutamate most likely is cotranslocated in symport with one proton and on sodium ion.     

Type XII collagen. A large multidomain molecule with partial homology to type IX collagen

Three overlapping cDNAs encoding alpha 1 (XII) collagen have been isolated and sequenced. The DNAs define five sequence domains within the chain. Three domains are nontriple-helical; two are relatively short triple-helical regions. The amino acid sequences of tryptic peptides derived from 16- and 10-kDa pepsin-resistant fragments isolated from tendon extracts are in full agreement with the deduced sequences of the triple-helical regions. Two of the five sequence domains in alpha 1 (XII), one triple-helical and one nontriple-helical, show a high degree of similarity to regions in type IX collagen chains. In addition, examination of seven exons in the alpha 1 (XII) gene shows that the gene is, in part, similar to the structure of type IX collagen genes. Therefore, collagen types IX and XII are partially homologous. The alpha 1 (XII) sequence data predict an asymmetric structure for type XII collagen molecules, fully consistent with the rotary shadowing images. These images show a triple-helical 75-nm tail attached through a central globule to three finger-like structures, each 60 nm long (Dublet, B., Oh, S., Sugrue, S. P., Gordon, M. K., Gerecke, D. R., Olsen, B. R., and van der Rest, M. (1989) J. Biol. Chem. 264, 13150-13156).     

delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase from Aspergillus nidulans. The first enzyme in penicillin biosynthesis is a multifunctional peptide synthetase

A multienzyme catalyzing the formation of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine, the first free intermediate in penicillin biosynthesis, was detected in an assay measuring the formation of tripeptide from L-[U-14C]valine in the presence of L-alpha-aminoadipic acid, L-cysteine, ATP, Mg2+ ions, and dithioerythritol. Enzyme was extracted from dry mycelium using a buffer with a high glycerol concentration and thiol protective agent to stabilize enzyme activity. In five steps the enzyme was purified 118-fold. It catalyzed ATP-pyrophosphate exchange in dependence of all three constituent amino acids, and the enzyme could be amino-acylated with L-[14C]valine. The molecular weight of the protein both native (in gel filtration chromatography) and denatured (polyacrylamide gel electrophoresis) was about 220 kDa. These data suggest that delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase consists of a single polypeptide chain and a multienzyme thiotemplate mechanism for the reaction sequence is postulated.     

Non-carrier-mediated uptake of the mannosidase I inhibitor 1-deoxymannojirimycin by K562 erythroleukemic cells

A 3H label was introduced at the C-1 position of the mannosidase I inhibitor 1-deoxymannojirimycin (dMM) by catalytic hydrogenolysis of benzyl-2,3-O-isopropylidene-5-N-benzyl-6-O-benzyl-alpha-D-mannofurano side with 3H2. 1-[3H]dMM as well as its precursor 1-[3H]2,3-O-isopropylidene-dMM had identical Rf as the nonradioactive compounds on TLC. Furthermore, alpha 1-antitrypsin secreted by HepG2 cells was modified indistinguishably by treatment of the cells with dMM and 1-[3H]dMM. Thus, 1-[3H]dMM had chemical and biological properties identical with authentic dMM. Uptake of [14C]mannose by K562 cells could be inhibited by glucose but not by the mannose analogue dMM. Thus, dMM does not enter the cell through hexose transporter(s). Uptake of 1-[3H]dMM by K562 cells could not be inhibited by increasing concentrations of nonradioactive dMM (from 1-32,000 microM), showing transport of dMM into cells through nonfacilitated diffusion. Furthermore, uptake of 1-[3H]dMM by K562 cells was observed at 0 degrees C.