The mouse Chromosome 7 distal imprinting domain maps to G-bands F4/F5

Mouse Chromosome (Chr) 7 distal to band F3 on the physical map is known to be subject to imprinting, maternal duplication (MatDp) of the region leading to a late embryonic lethality, while paternal duplication (PatDp) causes death in utero before 11.5 dpc. Using a new mouse reciprocal translocation T(7;11)65H to produce MatDp for distal Chr 7, we have mapped the region subject to imprinting more precisely to bands 7F4/F5 on the cytogenetic map. Fluorescence in situ hybridization (FISH) studies on mitotic and meiotic chromosomes of a T65H heterozygote show that the imprinted gene Igf2 is located in the same region. This was confirmed by the finding that embryos with MatDp of bands 7F4/F5 did not express Igf2. We suggest that other members of the imprinted domain containing Igf2, namely Mash2, H19, Ins2, and p57 ^K1P2 , are also located in 7F4/F5 and that some or all of these genes may be responsible for the two imprinting lethalities seen with MatDp and PatDp for this region. Received: 13 October 1996 / Accepted: 8 December 1996     

Cloning and characterization of a sialidase from the murine histocompatibility-2 complex: low levels of mRNA and a single amino acid mutation are responsible for reduced sialidase activity in mice carrying the Neula allele

The Neu1 locus, in the S region of the murine histocompatibility-2complex, regulates the sialic acid content of several liverlysosomal enzymes. Three alleles, Neu1^a, Neu1^b, and Neu1^c, havebeen described on the basis of differential sialylation of theenzyme liver acid phosphatase. The Neu1^a allele occurs in asmall number of mouse strains, e.g., SM/J and is associatedwith sialidase deficiency. We recently described G9, a sialidasegene in the human major histocompatibility complex (Milner etal. (1997) J. Biol. Chem., 272, 4549–4558), and we nowreport the characterization of the equivalent gene in mouse.The protein product of the murine G9 gene is 409 amino acidsin length and is 83% identical to its human orthologue. Expressionof the murine G9 protein in insect cells has confirmed thatit is a sialidase, with optimal activity at pH 5. To elucidatethe basis of sialidase deficiency in mouse strains carryingthe Neu1^a allele, we have sequenced the G9 coding regions frommice carrying the three Neu1 alleles and hence defined the aminoacid sequence characteristic of each allotype. Of particularinterest is a Leu-209 to Ile mutation that is unique to theNeu1^a allotype and is associated with reductions in sialidaseactivity of 68% and 88% compared to the Neu1^b and Neu1^c allotypes,respectively, when these three protein variants are expressedin insect cells. Additional factors, such as differential expression,may also influence the activities of the Nen1 allotypes in vivo.We have observed that the level of G9 mRNA is substantiallyreduced in mice carrying the Neu1^a allele compared to the Neu1^b(85–95% reduction) and Neu1^c (70% reduction) alleles. H2 complex MHC Neu1 sialidase     

Lysozyme-promoted association of protein I molecules in the outer membrane of Escherichia coli.

Incubation of whole envelopes prepared from sonically oscillated Escherichia coli K-12 cultures with lysozyme in vitro resulted in the appearance of a protein species with an apparent molecular weight double that of outer membrane protein I. Similar dimers were also detected in purified outer membranes and whole envelopes from lysozyme-induced spheroplasts of E. coli K-12. This was confirmed by two-dimensional electrophoresis in which the dimers were resolved in the second dimension to run as single polypeptides of protein I. Formation of dimers was correlated with peptidoglycan degradation, but the ability of protein I molecules to associate may vary between strains of E. coli, since dimers were found only in outer membranes from E. coli W7. We suggest that extensive degradation of peptidoglycan leads to nonspecific formation of protein I aggregates, but that these aggregates do not occur in vivo.     

Effect of salinity and humidity on δ13C value of halophytes—Evidence for diffusional isotope fractionation determined by the ratio of intercellular/atmospheric partial pressure of CO2 under different environmental conditions

Summary Seedlings of two mangrove species, Avicennia marina and Aegiceras corniculatum, were grown in a range of salinities and humidities in controlled environment chambers, and Phaseolus vulgaris plants were grown in the glasshouse. The fractionation of carbon isotopes in the three species was correlated with the ratio of intercellular and ambient partial pressures of CO₂. The results are consistent with fractionation being due both to diffusion in air and to carboxylation in the leaf. It was concluded that the latter process discriminates against ¹³CO₂ relative to ¹²CO₂ by about 27.     

Identifying sites of simultaneous DNA replication in eukaryotes by N-methyl-N''-nitro-N-nitrosoguanidine multiple mutagenesis.

Summary N-methyl-N-nitro-N-nitrosoguanidine (NG) induces certain classes of multiple mutations in yeast at high frequency. By selecting for mutation at one locus (his4 or leu1) one frequently obtains double mutants where another mutation to temperature sensitivity has also been induced. This multiple mutagenesis exhibits a considerable specificity: for mutation at one particular locus there is a high chance that another mutation will be found in the same cell at one of a restricted number of other loci. For any given locus (e.g. his4) there is a spectrum of sites at which temperature-sensitivity mutations are coinduced. This spectrum differs for different loci, such that the spectrum of sites co-mutating with leul differs completely from that for sites co-mutating with his4. This NG-induced co-mutation is interpreted in terms of NG acting to enhance mutagenesis at sites of simultaneous DNA replication within the cell. The results so obtained indicate a very strict control over the order and timing of gene replication in Saccharomyces cerevisiae, and it is suggested that it is now possible to use NG double mutagenesis to try and locate origins of replication in yeast.     

Water relation parameters of the CAM plant Kalanchoë daigremontiana in relation to diurnal malate oscillations

Summary In the CAM plant Kalanchoë daigremontiana, kept in an environmental rhythm of 12 h L: 12 h D in a growth chamber at 60% relative humidity and well watered in the root medium, decreasing water potentials and osmotic potentials of the leaves are correlated with malate accumulation in the dark. In the light increasing water and osmotic potentials ( _W and _S ) are associated with decreasing malate levels. Transpiratory H₂O loss is high in dark and low in light.In continuous light, the CAM rhythm rapidly disappears in the form of a highly damped endogenous oscillation. Malate levels, and water and osmotic potentials of the leaves remain correlated as described above. However, transpiration is very high as malate levels decrease and water and osmotic potentials increase.It can concluded, that water relation parameters like total water potential ( _W ) and osmotic potential ( _S ) change in close correlation with changes of malic acid levels. As an important osmotically active solute in CAM plants, malic acid appears to affect water relations independently of and in addition to transpiration. The question remains open, whether turgor ( _P ) is involved in CAM regulation in intact plants in a similar way as it determines malate fluxes in leaf slices.Abbreviations CAM Crassulacean Acid Metabolism - L Light - D Dark     

Radioimmunoassay of 2-hydroxyestrone

Under the protection of ascorbic acid a 2-hydroxyestrone bovine serum albumin conjugate was prepared containing intact 2-hydroxyestrone as determined by gas chromatographymass spectrometry. Using this antigen highly specific antibodies were raised in rabbits. Cross-reactivity for 2-hydroxyestradiol and 2-hydroxyestriol was 26 and 4.5%, respectively. An assay procedure of 2-hydroxyestrone in human plasma is described. Using special precautions the assay allows the determination of 2-hydroxyestrone in plasma samples of women (50–95 pg/ml), pregnant women (105–220 pg/ml), men (45–65 pg/ml) and children (20–40 pg/ml).     

Partial cleavage mapping of the cytoskeletal protein vinculin. Antibody and talin binding sites.

Vinculin is a 1066-amino acid protein found at several types of actin-membrane junction. To locate sites of interest in the primary structure, a map was derived using partial cleavage reactions. Of several different types of cleavage tested, the most useful was the 5-5'-dithio-bis-(2-nitrobenzoic acid) (DTNB) reaction which cuts at cysteine residues. About 30 well defined fragments were obtained from vinculin, and several methods were used to locate these products in the sequence. Comparison of the peptides generated from whole vinculin with those from the 90-kDa amino-terminal proteolytic fragment revealed which originated there. The use of [14C]cyanide in conjunction with DTNB showed which peptides contained the original amino terminus. Secondary cleavage with N-chlorosuccinimide, a tryptophan-specific reagent, helped locate fragments, although it led to apparent increases in molecular weight of the products. These experiments revealed the location of 10 of the major DTNB fragments on the sequence. This map was used to locate binding sites. The site of interaction between vinculin and the focal contact protein talin was mapped by binding labeled talin to the separated fragments. The binding site was found to be in the amino-terminal 325 amino acids. The binding site of a commercially obtained monoclonal antivinculin antibody was mapped using Western blotting of cleaved vinculin. It proved to bind in the central area of the molecule between amino acid residues 545 and 737. Thus the cysteine cleavage reaction products provide a map of general utility for locating features on the vinculin molecule.     

Continuous culture system for production of biopolymer levan using erwinia herbicola

The optimal production of the fructan biopolymer levan by the bacterium Erwinia herbicola was investigated, including variations in nitrogen, carbon and phosphorous sources, pH, incubation time, culture yields up to 19% by weight produced based on conversion of sucrose as the carbon source when grown in a continuous culture system and processed by tangential flow filtration. Product identity was confirmed with gas chromatography (GC) and (13)C nuclear magnetic resonance (NMR). Gel permeation chromatography (GPC) and low-angle laser light scattering (LALLS) determination of the molecular weight of the product showed a significant difference in molecular weight values dependent on the method of analysis. Analysis by GPC resulted in molecular weight one order of magnitude lower than LALLS independent of sample, underscoring the unusual nature of this biopolymer.