Genomic imprinting in ruminants: allele-specific gene expression in parthenogenetic sheep

Studies in the mouse have established that both parental genomes are essential for normal embryonic development. Parthenogenetic mouse embryos (which have two maternal genomes and no paternal genome), for example, are growth-retarded and die at early postimplantation stages. The distinct maternal and paternal contributions are mediated by genomic imprinting, an epigenetic mechanism by which the expression of certain genes is dependent on whether they are inherited from mother or father. Although comparative studies have established that many imprinted mouse (and rat) genes are allele-specifically expressed in humans as well (and vice versa), so far imprinting studies have not been performed in other mammalian species. When considering evolutionary theories of genomic imprinting, it would be important to know how widely it is conserved among placental mammals. We have investigated its conservation in a bovid ruminant, the domestic sheep, by comparing parthenogenetic and normal control embryos. Our study establishes that, like in the mouse, parthenogenetic development in sheep is associated with growth-retardation and does not proceed beyond early fetal stages. These developmental abnormalities are most likely caused by imprinted genes. We demonstrate that, indeed, like in mice and humans, the growth-related PEG1/MEST and Insulin-like Growth Factor 2 (IGF2) genes are expressed from the paternal chromosome in sheep. These observations suggest that genomic imprinting is conserved in a third, evolutionarily rather diverged group of placental mammals, the ruminants. Received: 13 May 1998 / Accepted: 16 July 1998     

Circular-dichroism spectra of truncated and other analogs of angiotensin II.

Circular dichroism spectra on angiotensin II and analogs, and its truncated N-terminal and C-terminal peptides were determined in fluroinated alcohols under several conditions in the peptide or aromatic spectral regions. The following conclusions were suggested: (a) evidence for a beta structure for angiotensin II; (b) evidence for a folding at the N-terminal and C-terminal part of the molecule; (c) an interaction involving the C-terminal residue which decreases progressively when phenylalanine is replaced by isoleucine and then by alanine; (d) the N-terminal amino acid seems to play an important role in the overall conformation of the molecule possibly by interacting with the C-terminus, its absence in the 2 -- 8 heptapeptide giving rise to a more pronounced signal than angiotensin II; (e) in trifluoroethanol the conformation of these peptides is well defined and fits well with observed structure-activity relationships and observed binding data. There is a loss of this relationship when these solvents are diluted with water.     

In vitro study of some medicinally important Mannich bases derived from antitubercular agent

Biologically active Mannich bases with heteroaromatic ring system have been synthesised employing Mannich reaction of isonicotinyl hydrazide with various sulphonamides/secondary amines. They were analysed by elemental analysis and characterized by uv, ir, 1H nmr spectroscopic studies. The Mannich bases were screened for their antibacterial activity against various gram positive and gram negative bacteria and were analyzed statistically. The results have shown that the compounds are quiet active against pathogens under study and were nontoxic.     

Comparative protein profiles of Salmonella and Escherichia coli

Protein profiles of selected Salmonella serovars were compared with E. coli to identify genus specific protein(s) for Salmonella. The PDP formed of different Salmonella serovars were compared with E. coli O78 when subjected to SDS-PAGE yielded 11, 15, 15, 11 and 14 bands in S. Bareilly, S. Gallinarum, S. Typhimurium and S. Weltevreden and E. coli O78 respectively. The bands produced were compared with each other. It was found that S. Weltevreden shared 7 bands with E. coli O78, A protein of molecular weight 20.89 kDa was found in all Salmonella serovars, but not in E. coli O78 suggesting its genus specific attribute.     

Isolation and characterization of the epothilone biosynthetic gene cluster from Sorangium cellulosum

The epothilone biosynthetic gene cluster was isolated from Sorangium cellulosum strain SMP44. The gene cluster contains seven genes and spans approx. 56kb. The genes encoding the PKS, epoA, epoC, epoD, epoE, and epoF, are divided into nine modules. The EpoB protein is a non-ribosomal peptide synthetase (NRPS) that catalyzes formation of the thiazole found in the epothilones. EpoK is a P450 enzyme responsible for the epoxidation of epothilones C and D to epothilones A and B, respectively. EpoK was expressed in Escherichia coli, and the purified protein was shown to convert epothilone D to epothilone B in vitro.     

Mechanistic analysis of a type II polyketide synthase. Role of conserved residues in the beta-ketoacyl synthase-chain length factor heterodimer

Type II polyketide synthases (PKSs) are a family of multienzyme systems that catalyze the biosynthesis of polyfunctional aromatic natural products such as actinorhodin, frenolicin, tetracenomycin, and doxorubicin. A central component in each of these systems is the beta-ketoacyl synthase-chain length factor (KS-CLF) heterodimer. In the presence of an acyl carrier protein (ACP) and a malonyl-CoA:ACP malonyl transferase (MAT), this enzyme synthesizes a polyketide chain of defined length from malonyl-CoA. We have investigated the role of the actinorhodin KS-CLF in priming, elongation, and termination of its octaketide product by subjecting the wild-type enzyme and selected mutants to assays that probe key steps in the overall catalytic cycle. Under conditions reflecting steady-state turnover of the PKS, a unique acyl-ACP intermediate is detected that carries a long, possibly full-length, acyl chain. This species cannot be synthesized by the C169S, H309A, K341A, and H346A mutants of the KS, all of which are blocked in early steps in the PKS catalytic cycle. These four residues are universally conserved in all known KSs. Malonyl-ACP alone is sufficient for kinetically and stoichiometrically efficient synthesis of polyketides by the wild-type KS-CLF, but not by heterodimers that carry the mutations listed above. Among these mutants, C169S is an efficient decarboxylase of malonyl-ACP, but the H309A, K341A, and H346A mutants are unable to catalyze decarboxylation. Transfer of label from [(14)C]malonyl-ACP to the nucleophile at position 169 in the KS can be detected for the wild-type enzyme and for the C169S and K341A mutants, but not for the H309A mutant and only very weakly for the H346A mutant. A model is proposed for decarboxylative priming and extension of a polyketide chain by the KS, where C169 and H346 form a catalytic dyad for acyl chain attachment, H309 positions the malonyl-ACP in the active site and supports carbanion formation by interacting with the thioester carbonyl, and K341 enhances the rate of malonyl-ACP decarboxylation via electrostatic interaction. Our data also suggest that the ACP and the KS dissociate after each C-C bond forming event, and that the newly extended acyl chain is transferred back from the ACP pantetheine to the KS cysteine before dissociation can occur. Chain termination is most likely the rate-limiting step in polyketide biosynthesis. Within the act CLF, neither the universally conserved S145 residue nor Q171, which aligns with the active site cysteine of the ketosynthase, is essential for PKS activity. The results described here provide a basis for a better understanding of the catalytic cycle of type II PKSs and fatty acid synthases.     

Angiotensin-(1-7) regulates the levels of angiotensin II receptor subtype AT1 mRNA differentially in a strain-specific fashion

Ang-(1-7) is an effector peptide of the renin-angiotensin system with several distinct actions that are likely mediated by a specific receptor. Regulatory effects of angiotensin (Ang) peptides, Ang-(1-7) and Ang II, on Ang receptor subtype 1 (AT1) mRNA expression were investigated in vascular smooth muscle cells (VSMC) from four University of Akron (Akr) rat strains (WKY, SHR and two backcross consomic lines SHR/y and SHR/a), and in SHR and WKY cells from Charles River Laboratories (Crl). In WKY/Akr and SHR/Akr, Ang-(1-7) treatment increased the levels of AT1 mRNA. This effect was inhibited by the specific Ang-(1-7) antagonist, A-779, in WKY/Akr but not SHR/Akr. Ang II had no effect in Akr cells, but it down-regulated AT1 mRNA in WKY/Crl and SHR/Crl VSMC. Ang-(1-7) did not affect AT1 mRNA levels in Crl lines. In conclusion, Ang-(1-7) regulates the AT1 receptor either directly or indirectly in a strain-specific fashion. The Ang-(1-7) antagonist, A-779, blocks the actions of Ang-(1-7) only in VSMC from WKY/Akr rats, suggesting either that the binding sites for Ang-(1-7) have different properties in SHR/Akr and WKY/Akr cell lines, or that some of the effects of Ang-(1-7) are not receptor mediated. Further, we found differences between Akr cells and Crl cells that are consistent with their genetic heterogeneity.     

Equilibrium and kinetic analysis of the unusual binding behavior of a highly immunogenic gluten peptide to HLA-DQ2

HLA-DQ2 predisposes an individual to celiac sprue by presenting peptides from dietary gluten to intestinal CD4(+) T cells. A selectively deamidated multivalent peptide from gluten (LQLQPFPQPELPYPQPELPYPQPELPYPQPQPF; underlined residues correspond to posttranslational Q --> E alterations) is a potent trigger of DQ2 restricted T cell proliferation. Here we report equilibrium and kinetic measurements of interactions between DQ2 and (i) this highly immunogenic multivalent peptide, (ii) its individual constituent epitopes, (iii) its nondeamidated precursor, and (iv) a reference high-affinity ligand of HLA-DQ2 that is not recognized by gluten-responsive T cells from celiac sprue patients. The deamidated 33-mer peptide efficiently exchanges with a preloaded peptide in the DQ2 ligand-binding groove at pH 5.5 as well as pH 7.3, suggesting that the peptide can be presented to T cells comparably well through the endocytic pathway or via direct loading onto extracellular HLA-DQ2. In contrast, the monovalent peptides, and the nondeamidated precursor, as well as the tight-binding reference peptide show a much poorer ability to exchange with a preloaded peptide in the DQ2 binding pocket, especially at pH 7.3, suggesting that endocytosis of these peptides is a prerequisite for T cell presentation. At pH 5.5 and 7.3, dissociation of the deamidated 33-mer peptide from DQ2 is much slower than dissociation of its constituent monovalent epitopes or the nondeamidated precursor but faster than dissociation of the reference high-affinity peptide. Oligomeric states involving multiple copies of the DQ2 heterodimer bound to a single copy of the multivalent 33-mer peptide are not observed. Together, these results suggest that the remarkable antigenicity of the 33-mer gluten peptide is primarily due to its unusually efficient ability to displace existing ligands in the HLA-DQ2 binding pocket, rather than an extremely low rate of dissociation.     

The effect of the disruption of a gene encoding a PI4 kinase on the developmental defect exhibited by Dictyostelium rasC(-) cells

The disruption of the gene encoding the Dictyostelium Ras subfamily protein, RasC results in a strain that fails to aggregate with defects in both cAMP signal relay and chemotaxis. Restriction enzyme mediated integration disruption of a second gene in the rasC(-) strain resulted in cells that were capable of forming multicellular structures in plaques on bacterial lawns. The disrupted gene, designated pikD(1), encodes a member of the phosphatidyl-inositol-4-kinase beta subfamily. Although the rasC(-)/pikD(1) cells were capable of progressing through early development, when starved on a plastic surface under submerged conditions, they did not form aggregation streams or exhibit pulsatile motion. The rasC(-)/pikD(1) cells were extremely efficient in their ability to chemotax to cAMP in a spatial gradient, although the reduced phosphorylation of PKB in response to cAMP observed in rasC(-) cells, was unchanged. In addition, the activation of adenylyl cyclase, which was greatly reduced in the rasC(-) cells, was only minimally increased in the rasC(-)/pikD(1) strain. Thus, although the rasC(-)/pikD(-) cells were capable of associating to form multicellular structures, normal cell signaling was clearly not restored. The disruption of the pikD gene in a wild type background resulted in a strain that was delayed in aggregation and formed large aggregation streams, when starved on a plastic surface under submerged conditions. This strain also exhibited a slight defect in terminal development. In conclusion, disruption of the pikD gene in a rasC(-) strain resulted in cells that were capable of forming multicellular structures, but which did so in the absence of normal signaling and aggregation stream formation.     

The cdk5 homologue,crp, regulates endocytosis and secretion in dictyostelium and is necessary for optimum growth and differentiation

Dictyostelium Crp is a member of the cyclin-dependent kinase (Cdk) family of proteins. It is most related in sequence to mammalian Cdk5, which unlike other members of the family, has functions that are unrelated to the cell cycle. In order to better understand the function of Crp in Dictyostelium, we overexpressed a dominant negative form, Crp-D144N, under the control of the actin 15 promoter. Cells overexpressing Crp-D144N exhibit a reduced growth rate in suspension culture and reduced rates of fluid-phase endocytosis and phagocytosis. There is no reduction in Cdc2 kinase activity in extracts from cells overexpressing Crp-D144N, suggesting that the growth defect is not due to inhibition of Cdc2. In addition to the growth defect, the act15::crp-D144N transformants aggregate at a slower rate than wild-type cells and form large aggregation streams. These eventually break up to form small aggregates and most of these do not produce mature fruiting bodies. The aggregation defect is fully reversed in the presence of wild-type cells but terminal differentiation is only partially rescued. In act15::crp-D144N transformants, the countin component of the counting factor, a secreted protein complex that regulates the breakup of streams, mostly appears outside the cell as degradation products and the reduced level of the intact protein may at least partially account for the initial formation of the large aggregation streams. Our observations indicate that Crp is important for both endocytosis and efflux and that defects in these functions lead to reduced growth and aberrant development.