Pollination-enhanced expression of a receptor-like protein kinase related gene in tobacco styles

We have isolated a putative serine/threonine receptor kinase gene with an expression pattern indicating that it may play a role in the stylar response to pollination. Differential display PCR was used to select tobacco mRNAs with increased accumulation following pollination. NTS16, a cDNA identified by this method, is homologous to a ca. 2.4 kb mRNA primarily expressed in pistil tissues. Levels of this mRNA increase during floral development and are further increased by pollination reaching maximal accumulation 12-18 hours after pollination and then declining. mRNA levels can also be increased by the application of ethylene to unpollinated flowers. A polypeptide encoded by the NTS 16 open reading frame has sequence similarity to the catalytic domain of several receptor protein kinases from plants including the S-receptor kinases implicated in the rejection of self-pollen in Brassica species and the Pto gene product of tomato which confers resistance to a bacterial pathogen.     

Molecular characterization of 5 alpha-reductase type 2 deficiency and fertility in a Swedish family

The molecular background of 5 alpha-reductase type 2 deficiency was investigated in a Swedish family with no known consanguinity and in which the affected males were fertile. The three male siblings were born with ambiguous external genitalia, and the diagnosis of 5 alpha-reductase deficiency was established at the ages of 16, 14, and 10 yr, respectively. All three siblings underwent surgery for hypospadias repair. At least two of the brothers are demonstrably fertile. Molecular analysis showed the three brothers to be compound heterozygotes, carrying two different mutations in exon 4 of the 5 alpha-reductase type 2 gene. The two mutations (G196S and H231R) have been described previously and reported to give rise to partially functioning enzymes, which may explain the milder phenotype and perhaps the fertility in the preset three patients.     

Cloning and characterization of a eukaryotic pantothenate kinase gene (panK) from Aspergillus nidulans

Pantothenate kinase (PanK) is the key regulatory enzyme in the CoA biosynthetic pathway. The PanK gene from Escherichia coli (coaA) has been previously cloned and the enzyme biochemically characterized; highly related genes exist in other prokaryotes. We isolated a PanK cDNA clone from the eukaryotic fungus Aspergillus nidulans by functional complementation of a temperature-sensitive E. coli PanK mutant. The cDNA clone allowed the isolation of the genomic clone and the characterization of the A. nidulans gene designated panK. The panK gene is located on chromosome 3 (linkage group III), is interrupted by three small introns, and is expressed constitutively. The amino acid sequence of A. nidulans PanK (aPanK) predicted a subunit size of 46.9 kDa and bore little resemblance to its bacterial counterpart, whereas a highly related protein was detected in the genome of Saccharomyces cerevisiae. In contrast to E. coli PanK (bPanK), which is regulated by CoA and to a lesser extent by its thioesters, aPanK activity was selectively and potently inhibited by acetyl-CoA. Acetyl-CoA inhibition of aPanK was competitive with respect to ATP. Thus, the eukaryotic PanK has a distinct primary structure and unique regulatory properties that clearly distinguish it from its prokaryotic counterpart.     

Molecular cloning of TER1, a chemokine receptor-like gene expressed by lymphoid tissues

Several chemokine receptors have been cloned and shown to belong to a superfamily of seven transmembrane, G protein-coupled receptors. We report here the molecular cloning of TER1, a novel human chemokine receptor-like gene. The amino acid sequence deduced from the TER1 cDNA shows 43, 40, 40, and 39% identity to CCR4, CCR5, CCR1, and CCR2B beta chemokine receptors, respectively. By the use of fluorescent in situ hybridization, we have mapped the TER1 gene to chromosome 3p21, clustered with other chemokine receptor genes. By Northern blot analysis, TER1 mRNA is found to be expressed in the thymus, spleen, and at barely detectable levels in peripheral blood lymphocytes. Moreover, TER1 message in abundant in the NK cell line NK3.3 and in the T cell line MOLT-4. The restricted TER1 expression in cells and tissues of the lymphoid lineage suggests that this receptor may play a role in regulating immune functions.     

Molecular characterization of an anchor protein (AKAPCE) that binds the RI subunit (RCE) of type I protein kinase A from Caenorhabditis elegans

Classical A kinase anchor proteins (AKAPs) preferentially tether type II protein kinase A (PKAII) isoforms to sites in the cytoskeleton and organelles. It is not known if distinct proteins selectively sequester regulatory (R) subunits of type I PKAs, thereby diversifying functions of these critical enzymes. In Caenorhabditis elegans, a single type I PKA mediates all aspects of cAMP signaling. We have discovered a cDNA that encodes a binding protein (AKAPCE) for the regulatory subunit (RCE) of C. elegans PKAICE. AKAPCE is a novel, highly acidic RING finger protein composed of 1,280 amino acids. It binds RI-like RCE with high affinity and neither RIIalpha nor RIIbeta competitively inhibits formation of AKAPCE.RCE complexes. The RCE-binding site was mapped to a segment of 20 amino acids in an N-terminal region of AKAPCE. Several hydrophobic residues in the binding site align with essential Leu and Ile residues in the RII-selective tethering domain of prototypic mammalian AKAPs. However, the RCE-binding region in AKAPCE diverges sharply from consensus RII-binding sites by inclusion of three aromatic amino acids, exclusion of a highly conserved Leu or Ile at position 8 and replacement of C-terminal hydrophobic amino acids with basic residues. AKAPCE.RCE complexes accumulate in intact cells.     

Molecular screening of the glucokinase gene in familial type 2 (non-insulin-dependent) diabetes mellitus

The question whether penicillin V (pcV) given intermittently upon signs of upper respiratory tract infections (URTI) in otitis-prone children might prevent recurrent bouts of acute purulent otitis media (AOM) is addressed. As compared with continuous long-term antibiotic treatment as prophylaxis in otitis-prone children, intermittent administration would reduce the overall consumption of antibiotics. Seventy-six otitis-prone children less than 18 months of age were included in this double-blind, randomized, placebo-controlled multicentre study. Follow-up was from January till June. One hundred and twenty-three episodes of AOM occurred. The number of AOM episodes was reduced by 50% in the children on pcV during URTI episodes as compared with those on placebo. No obvious ecological drawbacks were noted. Thus, the described mode of pcV administration seems to be a rational and safe way to reduce the number of AOM episodes in otitis-prone children.     

The Lateral suppressor (Ls) gene of tomato encodes a new member of the VHIID protein family

The ability of the shoot apical meristem to multiply and distribute its meristematic potential through the formation of axillary meristems is essential for the diversity of forms and growth habits of higher plants. In the lateral suppressor mutant of tomato the initiation of axillary meristems is prevented, thus offering the unique opportunity to study the molecular mechanisms underlying this important function of the shoot apical meristem. We report here the isolation of the Lateral suppressor gene by positional cloning and show that the mutant phenotype is caused by a complete loss of function of a new member of the VHIID family of plant regulatory proteins.     

Molecular phylogenetic characterization of Streptomyces protease inhibitor family

We previously found that proteinaceous protease inhibitors homologous to Streptomyces subtilisin inhibitor (SSI) are widely produced by various Streptomyces species, and we designated them "SSI-like proteins" (Taguchi S, Kikuchi H, Suzuki M, Kojima S, Terabe M, Miura K, Nakase T, Momose H [1993] Appl Environ Microbiol 59:4338-4341). In this study, SSI-like proteins from five strains of the genus Streptoverticillium were purified and sequenced, and molecular phylogenetic trees were constructed on the basis of the determined amino acid sequences together with those determined previously for Streptomyces species. The phylogenetic trees showed that SSI-like proteins from Streptoverticillium species are phylogenetically included in Streptomyces SSI-like proteins but form a monophyletic group as a distinct lineage within the Streptomyces proteins. This provides an alternative phylogenetic framework to the previous one based on partial small ribosomal RNA sequences, and it may indicate that the phylogenetic affiliation of the genus Streptoverticillium should be revised. The phylogenetic trees also suggested that SSI-like proteins possessing arginine or methionine at the P1 site, the major reactive center site toward target proteases, arose multiple times on independent lineages from ancestral proteins possessing lysine at the P1 site. Most of the codon changes at the P1 site inferred to have occurred during the evolution of SSI-like proteins are consistent with those inferred from the extremely high G + C content of Streptomyces genomes. The inferred minimum number of amino acid replacements at the P1 site was nearly equal to the average number for all the variable sites. It thus appears that positive Darwinian selection, which has been postulated to account for accelerated rates of amino acid replacement at the major reaction center site of mammalian protease inhibitors, may not have dictated the evolution of the bacterial SSI-like proteins.