Mapping of QTLs for Oral Alcohol Self-Administration in B6.C and B6.I Quasi-Congenic RQI Strains

One strategy to identify neurochemical pathways of addiction is to map the relevant genes. In the present study we used 43 B6.C and 35 B6.I inbred RQI mouse strains, carrying <3% donor genome on C57BL/6ByJ background, for gene mapping. The strains were phenotyped for consumption of alcohol (12% v/v) in a two-bottle-choice paradigm, and genotyped for 396 microsatellite markers. The current mapping study extends our earlier experiment scanning five mouse chromosomes (Vadasz et al. (2000) Scanning of five chromosomes for alcohol consumption loci. Alcohol 22:25–34) to a whole-genome study, and discusses the differences and limitations. Data were analyzed with composite interval (CIM) and multiple interval (MIM) QTL mapping methods. CIM of B6.C strains detected significant QTLs on chrs. 6 and 12. A suggestive, but not significant, locus was detected in the B6.I strains on chr. 12. The best MIM model for B6.C strains confirmed one QTL on chr. 6 and one QTL on chr. 12, while the MIM model for the B6.I strains confirmed the suggestive locus on chr. 12. Some of the QTLs for alcohol consumption are new, while others confirm previously reported QTLs for alcohol preference, and alcohol acceptance.     

Structural characterization of two lipopolysaccharide O-antigens produced by the endofungal bacterium Burkholderia sp. HKI-402 (B4)

Two different polysaccharides were isolated and identified from the lipopolysaccharide fraction of endofungal bacterium Burkholderia sp. HKI-402 (B4). The complete structure was elucidated by chemical analysis and 2D NMR spectroscopy as the following:     

Progressive introgression between Brassica napus (oilseed rape) and B. rapa

We have earlier shown extensive introgression between oilseed rape (Brassica napus) and B. rapa in a weedy population using AFLP markers specific for the nuclear genomes. In order to describe the progress of this introgression, we examined 117 offspring from 12 maternal plants from the introgressed population with the same AFLP-markers; AFLP data were supported by chromosome counting. We also analysed the offspring with a species-specific chloroplast marker and finally evaluated the reproductive system in selected maternal plants. Our results indicated a high outcrossing rate of the introgressed maternal plants. It seemed that B. rapa most often functioned as the maternal plant in the introgression process and that the amount of oilseed rape DNA was highly diminished in the offspring compared to their introgressed maternal plants. However, our analysis of plants from the weedy population indicated that introgression can lead to both (1) exchange of chloroplast DNA between species producing B. rapa-like plants with B. napus chloroplasts and (2) incorporation of B. napus C-genome DNA into the B. rapa genome. Therefore, we question whether it can be regarded as containment to position transgenes in the chloroplast or in specific parts of the nuclear genome of B. napus.     

Structural analysis of the adenovirus type 5 E1B 55-kilodalton-E4orf6 protein complex.

The adenovirus type 5 (Ad5) early 1B (E1B) 55-kDa (E1B-55kDa)-E4orf6 protein complex has been implicated in the selective modulation of nucleocytoplasmic mRNA transport at late times after infection. Using a combined immunoprecipitation-immunoblotting assay, we mapped the domains in E1B-55kDa required for the interaction with the E4orf6 protein in lytically infected A549 cells. Several domains in the 496-residue 55-kDa polypeptide contributed to a stable association with the E4orf6 protein in E1B mutant virus-infected cells. Linker insertion mutations at amino acids 180 and 224 caused reduced binding of the E4orf6 protein, whereas linker insertion mutations at amino acid 143 and in the central domain of E1B-55kDa eliminated the binding of the E4orf6 protein. Earlier work showing that the central domain of E1B-55kDa is required for binding to p53 and the recent observation that the E4orf6 protein also interacts with the tumor suppressor protein led us to suspect that p53 might play a role in the E1B-E4 protein interaction. However, coimmunoprecipitation assays with extracts prepared from infected p53-negative H1299 cells established that p53 is not needed for the E1B-E4 protein interaction in adenovirus-infected cells. Using two different protein-protein interaction assays, we also mapped the region in the E4orf6 protein required for E1B-55kDa interaction to the amino-terminal 55 amino acid residues. Interestingly, both binding assays established that the same region in the E4orf6/7 protein can potentially interact with E1B-55kDa. Our results demonstrate that two distinct segments in the 55-kDa protein encoding the transformation and late lytic functions independently interact with p53 and the E4orf6 protein in vivo and provide further insight by which the multifunctional 55-kDa EIB protein can exert its multiple activities in lytically infected cells and in adenovirus transformation.     

Rodgers (Rg) and Chido (Ch) determinants on human C4: characterization of two C4 B5 subtypes, one of which contains Rg and Ch determinants

The genetically determined polymorphism of the fourth component of human complement was further extended with the aid of a panel of human allo-anti-C4 sera, anti-Rodgers and anti-Chido. These antisera were found previously to react with the alpha-chains of the C4 molecules controlled by the C4A and C4B loci, respectively. We analyzed a number of new and rare C4 allotypes, and found that they generally followed the expected pattern. Some interesting exceptions, however, were found. The alpha-chain of the allotype C4A1 was found to react with anti-Chido, unlike all other C4A allotypes. Also the C4B5 allotype could be subdivided into two subtypes on the basis of their reaction with anti-Rodgers. They were tentatively named B5Rg+ and B5Rg-. Moreover, the B5Rg+ subtype reacted not only with anti-Rodgers but also with some anti-Chido sera, indicating for the first time that Chido and Rodgers determinants are present on the same allotype.     

Molecular cloning and characterization of two novel retinoic acid-inducible orphan G-protein-coupled receptors (GPRC5B and GPRC5C)

Using homology searching of public databases with a metabotropic glutamate receptor sequence from Caenorhabditis elegans, two novel protein sequences (named RAIG-2 (HGMW-approved symbol GPRC5B) and RAIG-3 (HGMW-approved symbol GPRC5C) were identified containing seven putative transmembrane domains characteristic of G-protein-coupled receptors (GPCRs). RAIG-2 and RAIG-3 encode open reading frames of 403 and 442 amino acid polypeptides, respectively, and show 58% similarity to the recently identified retinoic acid-inducible gene-1 (RAIG-1, HGMW-approved symbol RAI3). Analysis of the three protein sequences places them within the type 3 GPCR family, which includes metabotropic glutamate receptors, GABA(B) receptors, calcium-sensing receptors, and pheromone receptors. However, in contrast to other type 3 GPCRs, RAIG-1, RAIG-2, and RAIG-3 have only short N-terminal domains. RAIG-2 and RAIG-3 cDNA sequences were cloned into the mammalian expression vector pcDNA3 with c-myc or HA epitope tags inserted at their N-termini, respectively. Transient transfection experiments in HEK239T cells using these constructs demonstrated RAIG-2 and RAIG-3 expression at the cell surface. Distribution profiles of mRNA expression obtained by semiquantitative Taq-Man PCR analysis showed RAIG-2 to be predominantly expressed in human brain areas and RAIG-3 to be predominantly expressed in peripheral tissues. In addition, expression of RAIG-2 and RAIG-3 mRNA was increased following treatment with all-trans-retinoic acid in a manner similar to that previously described for RAIG-1. Finally, RAIG-2 was mapped to chromosome 16p12 (D16S405-D16S3045) and RAIG-3 to chromosome 17q25 (D17S1352-D17S785). These results suggest that RAIG-1, RAIG-2, and RAIG-3 represent a novel family of retinoic acid-inducible receptors, most closely related to the type 3 GPCR subfamily, and provide further evidence for a linkage between retinoic acid and G-protein-coupled receptor signal transduction pathways.     

Origin of the different strengths of the (i,i+4) and (i,i+3) leucine pair interactions in helices

Pairs of leucine side chains, spaced either (i,i+3) or (i,i+4), are known to stabilize alanine-based peptide helices, Experiments with new peptide sequences confirm that the (i,i+4) pair interaction is markedly stronger than the (i,i+3) pair interaction. This result is not expected from reported Monte Carlo simulations, which predict that the (i,i+3) interaction is slightly stronger. The interaction strength can be predicted from recently reported measurements of buried non-polar surface area, obtained from structures in the Protein Data Bank: the agreement is reasonable for the (i,i+3) LL interaction but underestimates the (i,i+4) LL interaction. Solvation of peptide groups in the helix backbone may contribute to the different strengths of the two LL pair interactions because different chi(1) leucine rotamers are used and the (i,i+3) pair shields two peptide groups whereas the (i,i+4) pair shields only one. A rough estimate of the backbone solvation effect, based on the difference between the helix propensities of leucine and alanine, agrees with the size of the difference between the (i,i+3) and (i,i+4) leucine pair interactions.     

Influence of N6-isopentenyladenosine (i(6)A) on thermal stability of RNA duplexes.

The thermodynamic stability of self-complementary oligoribonucleotides containing N6-isopentenyladenosine (i(6)A) or N6-isopentanyladenosine (p(6)A) was determined. The base pairs i(6)A.U and p(6)A.U were placed in either an internal (separated and tandem) and a terminal position within the duplex, or unpaired i(6)A and p(6)A as a 3'-dangling ends. The thermal unfolding of the oligomers was determined by means of UV melting profiles and the thermodynamic parameters: enthalpy (DeltaH degrees ), entropy (DeltaS degrees) and free energy (DeltaG degrees (37)) as well as the melting temperature (T(m)) were calculated. Both modified nucleosides destabilized the duplexes, however, the effect depended on the position of the modified adenosine within the duplex. The similarity of the behavior of oligomers containing i(6)A and p(6)A suggests a negligible effect of the double bond on the thermal stability. The largest destabilization was observed when derivatives of adenosine were placed in an internal position. The effect of 3'-dangling ends suggests that the presence of the N6-isopentenyl- or N6-isopentanyl substitutent affects hydrogen bonding rather than stacking within duplex.     

Structural characterization of the dihydroflavonol 4-reductase B (DFR-B) gene in the sweet potato.

A genomic fragment containing the dihydroflavonol 4-reductase B (DFR-B) gene was cloned from the sweet potato (Ipomoea batatas) and its nucleotide sequence was analyzed. The exons and flanking regions were highly homologous to those of previously reported DFR-B genes of the Japanese morning glory, whereas the introns and the intergenic region were less conserved. In addition to the sequences of three miniature inverted-repeat transposable elements (MITEs) and one direct repeat previously reported in the DFR-B gene of Japanese morning glory, two mobile element-like sequences were newly identified in the sweet potato DFR-B gene. At least four allelic sequences were found to exist by amplification of the DFR-B gene from various sweet potato cultivars. One of these allelic sequences had a 2-kb deletion in the intergenic region and was observed in the cultivars with high anthocyanin content in their storage roots.     

Diversity among B6 strains of Agrobacterium tumefaciens.

A total of 20 laboratory substrains of Agrobacterium tumefaciens strain B6 were compared with respect to six characteristics, including 3-ketolactose production, lysogeny, octopine catabolism, tumorigenic host range, and plasmid content. Within this group of strains diversity was found for all characteristics except 3-ketolactose production. Six substrains were lysogenized with an omega-type phage, whereas one substrain appeared neither sensitive to nor lysogenized with this bacteriophage. All but two substrains catabolized octopine and induced tumors on carrot disks. These 18 substrains harbor deoxyribonucleic acid sequences homologous to pTiB6-806. The two substrains unable to catabolize octopine were nontumorigenic and lacked detectable Ti plasmid sequences. Of the 20 substrains, 13 also contained sequences homologous to the cryptic plasmid pAtB6-806; 2 of the 18 substrains tumorigenic on carrots failed to induce tumors on Kalanchoe leaves. Their inability to induced tumors on this host, could not be correlated with lysogeny, with the presence or absence of pAtB6-806, or with the very large cryptic plasmid recently described. The Ti plasmids from these two strains were indistinguishable from pTiB6-806 by restriction enzyme analysis and could genetically convert a cured A. tumefaciens strain to tumorigenicity on both plant species. The results with these two strains suggest that parameters of tumorigenicity, such as host range, may be controlled by the bacterial chromosome.     

Interleukin-5 (IL-5) and IL-6 define two molecularly distinct pathways of B-cell differentiation.

Interleukin-5 (IL-5) and IL-6 have both been reported to act as B-cell differentiation factors by stimulating activated B cells to secrete antibody. However, it has not been possible to directly compare the effects of these two lymphokines because of the lack of a suitable B-cell line capable of responding to both. We have identified a clonal, inducible B-cell lymphoma, CH12, that has this property. Both IL-5 and IL-6 can independently stimulate increases in steady-state levels of immunoglobulin and J-chain mRNA and proteins, and they both induce the differentiation of CH12 into high-rate antibody-secreting cells. Nevertheless, there are significant differences in the activities of these two lymphokines. First, while IL-6 acts only as a differentiation factor, IL-5 also augments the proliferation of CH12 cells. Second, the differentiation stimulated by IL-5 but not by IL-6 is partially inhibited by IL-4. Inhibition of IL-5-induced differentiation was not at the level of IL-5 receptor expression, since IL-4 did not inhibit IL-5-induced proliferation. Third, IL-5 but not IL-6 stimulated increased mouse mammary tumor proviral gene expression in CH12 cells. These results demonstrate that while both IL-5 and IL-6 may act as differentiation factors for B cells, they induce differentiation by using at least partially distinct molecular pathways. Our results also establish that B cells characteristic of a single stage of development can independently respond to IL-4, IL-5, and IL-6.     

Biological and molecular characterization of two tomato strains of potato virus Y (PVY)

Two PVY tomato strains (LYE 84 and LYE 84.2), arising from the same natural isolate, and a strain originating from a wild Solanaceous host, Solanum nigrum (SON 41.2), were compared for host range and symptomatology. All strains induced mosaic without necrosis on tobacco as PVY^O strains. The two tomato strains behaved similarly on pepper, infecting only susceptible pepper cultivars (pathotype 0), whereas SON 41.2 was able to overcome the two alleles of the recessive resistance gene pvr2 (pathotype 1.2). On the other hand, only LYE 84.2 was virulent on tomato and broke the resistance of the wild genitor Lycopersicon hirsutum PI 247087. Sequence determination of the capsid gene and the 3′ non-coding region of LYE 84 and LYE 84.2 showed a total homology at both nucleic acid and amino acid levels. This suggests that LYE 84.2 has probably derived from LYE 84, that both strains have very similar sequences and that the capsid protein does not play a direct role in the resistance-breaking capacity of LYE 84.2.     

Cloning and characterization of the rat cytochrome P450 4F5 (CYP4F5) gene

The analysis of a non-redundant set of human proteins, for which both the crystallographic structures and the corresponding gene sequences are available, show that bases at third codon position are non-uniformly distributed along the coding sequences. Significant compositional differences are found by comparing the gene regions corresponding to the different secondary structures of the proteins. Inter-and intra-structure differences were most pronounced in the GC-richest genes. These results are not compatible with any proposed hypotheses based on a neutral process of formation/maintenance of the high GC₃ levels of the genes localized in the GC-richest isochores of the human genome.     

Isolation and chemical conversion of two novel prostaglandin endoperoxides: 5(6)-epoxy-PGG1 and 5(6)-epoxy-PGH1

The possible molecular heterogeneity of human transferrin receptors was analyzed using two murine monoclonal antibodies, Tü15 and Tü67. Both reagents precipitated from lysates of 125I-labeled HL-60 cells a major component of 88 kDa which could be identified as the transferrin receptor by comparison with the proteins detected by monoclonal antibody OKT9. Although sequential immunoprecipitations appeared to demonstrate molecular heterogeneity of transferrin receptors, since the Tü15-reactive species were fully included in the Tü67-positive population, but not vice versa, the possible association of Tü15-reactive molecules with transferrin receptor is also discussed.