Allele and locus classification in electrophoretic population studies

The electrophoretic separation of protein variants having slightly different mobilities is a basic tool of biochemical population genetics. In certain situations it is difficult to determine how to classify the variants as alleles of a number of genetic loci, that is, as variant subsets within each of which the Mendelian laws hold. In this article, we develop and analyze a series of algorithms for solving various versions and generalizations of this problem of optimal classification.     

Molecular cloning and characterization of prolactin-like protein C complementary deoxyribonucleic acid.

In this report, we describe the isolation and characterization of a full length cDNA clone for rat prolactin-like protein C (PLP-C) and describe the expression of PLP-C mRNA in the developing rat placenta. Nucleotide sequence analysis of the PLP-C cDNA clone predicted a mature protein of 238 amino acids, including a 30-amino acid signal sequence. The predicted PLP-C amino acid sequence contains seven cysteine residues, three tryptophan residues, and two putative N-linked glycosylation sites. Six of the cysteine residues in PLP-C are located in positions homologous to the cysteines of pituitary prolactin (PRL). Additional sequence similarities with pituitary PRL and other members of the rat placental PRL family are evident. The PLP-C gene was localized to rat chromosome 17. Northern blot analysis showed that the PLP-C cDNA clone specifically hybridized to a 1.0-kilobase mRNA. PLP-C mRNA was first detectable between days 13 and 14 of gestation, peaked by day 18 of gestation, and remained elevated until term. In situ hybridization analysis indicated that PLP-C mRNA was specifically expressed by spongiotrophoblast cells and some trophoblast giant cells in the junctional zone region of rat chorioallantoic placenta.     

Cell Surfaces in Plant-Microorganism Interactions : VI. Elicitors of Ethylene from Colletotrichum lagenarium Trigger Chitinase Activity in Melon Plants

Treatment of melon leaves or seedlings with elicitors of Colletotrichum lagenarium, a fungal pathogen of melon, increases chitinase activity. In treated leaves, chitinase is enhanced within the first 6 hours and becomes 2 to 10 times higher than in control leaves after 24 hours. Ethylene is increased simultaneously and is correlated with chitinase elicitation. In the presence of aminoethoxyvinylglycine, an inhibitor of ethylene synthesis, both elicitor-induced ethylene and elicitor-induced chitinase are inhibited. This inhibition is overcome by added exogenous ethylene. On the other hand, 1-aminocyclopropane-1-carboxylic acid the direct precursor of ethylene, triggers chitinase activity. Chitinase elicitation is thought to be a protein synthesis dependent process, as it does not occur in the presence of cycloheximide.     

Identification and characterization of a new member of the prolactin family, placental lactogen-I variant

This report describes the identification and characterization of a new member of the placental prolactin (PRL) family, termed placental lactogen-I variant (PL-Iv). PL-Iv was isolated from medium conditioned by late gestation placental explants. Rat PL-Iv was found to be closely related to rat PL-I. Amino-terminal sequence analysis indicated that PL-Iv shared approximately 88% sequence identity with the amino terminus of PL-I. PL-Iv proteins cross-reacted with antiserum to recombinant mouse PL-I and PL-Iv mRNA hybridized with a PL-I cDNA. Multiple PL-I and PL-Iv species were present in placental cytosol. Despite the structural similarities between PL-I and PL-Iv, distinct differences were also evident. Antibodies generated to the amino-terminal 19 amino acids of PL-Iv specifically recognized PL-Iv, while failing to recognize PL-I. Secreted PL-Iv had an affinity for concanavalin A, whereas secreted PL-I lacked affinity for the lectin. PL-I was predominantly secreted as a 36-40-kDa species and PL-Iv was predominantly secreted as a 33-kDa species. Furthermore, PL-I and PL-Iv were synthesized at different times during gestation and by different cell types. PL-I was synthesized by trophoblast giant cells during the first half of gestation, while PL-Iv was predominantly synthesized by spongiotrophoblast cells during the later stages of gestation. PL-Iv was shown to stimulate the proliferation of rat Nb2 lymphoma cells, an in vitro measure of lactogenic activity. In summary, PL-Iv shares structural similarities with PL-I; however, it shows other structural differences in addition to unique cell- and temporal-specific patterns of expression in the rat chorioallantoic placenta.     

Characterization of phosphoproteins and protein kinase activity of virions, noninfectious enveloped particles, and dense bodies of human cytomegalovirus.

Phosphorylation of the proteins of human cytomegalovirus (CMV) virions, noninfectious enveloped particles (NIEPs), and dense bodies was investigated. Analyses of particles phosphorylated in vivo showed the following. Virions contain three predominant phosphoproteins (i.e., basic phosphoprotein and upper and lower matrix proteins) and at least nine minor phosphorylated species. NIEPs contain all of these and one additional major species, the assembly protein. Dense bodies contain only one (i.e., lower matrix) of the predominant and four of the minor virion phosphoproteins. Two-dimensional (charge-size) separations in denaturing polyacrylamide gels showed that the relative net charges of the predominant phosphorylated species ranged from the basic phosphoprotein to the more neutral upper matrix protein. In vitro assays showed that purified virions of human CMV have an associated protein kinase activity. The activity was detected only after disrupting the envelope; it had a pH optimum of approximately 9 to 9.5 and required a divalent cation, preferring magnesium to manganese. In vitro, this activity catalyzed phosphorylation of the virion proteins observed to be phosphorylated in vivo. Peptide comparisons indicated that the sites phosphorylated in vitro are a subset of those phosphorylated in vivo, underscoring the probable biological relevance of the kinase activity. Casein, phosvitin, and to a minor extent lysine-rich histones served as exogenous phosphate acceptors. Arginine-rich and lysine-rich histones and protamine sulfate, as well as the polyamines spermine and spermidine, stimulated incorporation of phosphate into the endogenous viral proteins. Virions of all human and simian CMV strains tested showed this activity. Analyses of other virus particles, including three intracellular capsid forms (i.e., A, B, and C capsids), NIEPs, and dense bodies, indicated that the active enzyme was not present in the capsid. Rate-velocity sedimentation of disrupted virions separated the protein kinase activity into two fractions: one that phosphorylated exogenous casein and another that phosphorylated primarily the endogenous virion proteins.     

Regulation of protein degradation and protease expression by mannose in maize root tips. Pi sequestration by mannose may hinder the study of its signaling properties

The effects of mannose (Man) and glucose (Glc) on central metabolism, proteolysis, and expression of the root starvation-induced protease (RSIP; F. James, R. Brouquisse, C. Suire, A. Pradet, P. Raymond [1996] Biochem J 320: 283-292) were investigated in maize (Zea mays L. cv DEA) root tips. Changes in metabolite concentrations (sugars, ester-phosphates, adenine nucleotides, and amino acids) were monitored using in vivo and in vitro (13)C- and (31)P-NMR spectroscopy, in parallel with the changes in respiration rates, protein contents, proteolytic activities, and RSIP amounts. The inhibition of proteolysis, the decrease in proteolytic activities, and the repression of RSIP expression triggered by Man, at concentrations usually used to study sugar signaling (2 and 10 mM), were found to be related to a drop of energy metabolism, primarily due to a Man-induced Pi sequestration. However, when supplied at low concentration (2 mM) and with the adequate phosphate concentration (30 mM), energy metabolism was restored and Man repressed proteolysis similarly to Glc, when provided at the same concentration. These results indicate that Man should be used with caution as a Glc analog to study signalization by sugars in plants because possible signaling effects may be hindered by Pi sequestration.     

Sho1 and Pbs2 act as coscaffolds linking components in the yeast high osmolarity MAP kinase pathway

Scaffold proteins mediate efficient and specific signaling in several mitogen-activated protein (MAP) kinase cascades. In the yeast high osmolarity response pathway, the MAP kinase kinase Pbs2 is thought to function as a scaffold, since it binds the osmosensor Sho1, the upstream MAP kinase kinase kinase Ste11, and the downstream MAP kinase Hog1. Nonetheless, previous work has shown that Ste11 can be activated even when Pbs2 is deleted, resulting in inappropriate crosstalk to the mating pathway. We have found a region in the C terminus of Sho1 that binds Ste11 independently of Pbs2 and is required for crosstalk. These data support a model in which Sho1 has at least two separable interaction regions: one that binds Ste11 and mediates its activation, and one that binds Pbs2, directing Ste11 to act on Pbs2. Thus, a network of interactions provided by both Sho1 and Pbs2 appears to direct pathway information flow.     

HLM1, an essential signaling component in the hypersensitive response,is a member of the cyclic nucleotide-gated channel ion channel family

The hypersensitive response (HR) in plants is a programmed cell death that is commonly associated with disease resistance. A novel mutation in Arabidopsis, hlm1, which causes aberrant regulation of cell death, manifested by a lesion-mimic phenotype and an altered HR, segregated as a single recessive allele. Broad-spectrum defense mechanisms remained functional or were constitutive in the mutant plants, which also exhibited increased resistance to a virulent strain of Pseudomonas syringae pv tomato. In response to avirulent strains of the same pathogen, the hlm1 mutant showed differential abilities to restrict bacterial growth, depending on the avirulence gene expressed by the pathogen. The HLM1 gene encodes a cyclic nucleotide-gated channel, CNGC4. Preliminary study of the HLM1/CNGC4 gene pro-duct in Xenopus oocytes (inside-out patch-clamp technique) showed that CNGC4 is permeable to both K(+) and Na(+) and is activated by both cGMP and cAMP. HLM1 gene expression is induced in response to pathogen infection and some pathogen-related signals. Thus, HLM1 might constitute a common downstream component of the signaling pathways leading to HR/resistance.