A high-resolution comparative map of porcine chromosome 4 (SSC4).

We used the IMNpRH2(12,000-rad) RH and IMpRH(7,000-rad) panels to integrate 2019 transcriptome (RNA-seq)-generated contigs with markers from the porcine genetic and radiation hybrid (RH) maps and bacterial artificial chromosome finger-printed contigs, into 1) parallel framework maps (LOD ≥ 10) on both panels for swine chromosome (SSC) 4, and 2) a high-resolution comparative map of SSC4, thus and human chromosomes (HSA) 1 and 8. A total of 573 loci were anchored and ordered on SSC4 closing gaps identified in the porcine sequence assembly Sscrofa9. Alignment of the SSC4 RH with the genetic map identified five microsatellites incorrectly mapped around the centromeric region in the genetic map. Further alignment of the RH and comparative maps with the genome sequence identified four additional regions of discrepancy that are also suggestive of errors in assembly, three of which were resolved through conserved synteny with blocks on HSA1 and HSA8.

Soybean genomic survey: BAC-end sequences near RFLP and SSR markers.

We are building a framework physical infrastructure across the soybean genome by using SSR (simple sequence repeat) and RFLP (restriction fragment length polymorphism) markers to identify BACs (bacterial artificial chromosomes) from two soybean BAC libraries. The libraries were prepared from two genotypes, each digested with a different restriction enzyme. The BACs identified by each marker were grouped into contigs. We have obtained BAC- end sequence from BACs within each contig. The sequences were analyzed by the University of Minnesota Center for Computational Genomics and Bioinformatics using BLAST algorithms to search nucleotide and protein databases. The SSR-identified BACs had a higher percentage of significant BLAST hits than did the RFLP-identified BACs. This difference was due to a higher percentage of hits to repetitive-type sequences for the SSR-identified BACs that was offset in part, however, by a somewhat larger proportion of RFLP-identified significant hits with similarity to experimentally defined genes and soybean ESTs (expressed sequence tags). These genes represented a wide range of metabolic functions. In these analyses, only repetitive sequences from SSR-identified contigs appeared to be clustered. The BAC-end sequences also allowed us to identify microsynteny between soybean and the model plants Arabidopsis thaliana and Medicago truncatula. This map-based approach to genome sampling provides a means of assaying soybean genome structure and organization.

Purification, primary structure characterization, and cellular distribution of two forms of cellular retinol-binding protein, type II from adult rat small intestine.

Cellular retinol-binding protein type II (CRBP(II)) is a major protein in the small intestine, accounting for more than 1% of the soluble protein recovered from rat jejunal mucosa. Two forms of the protein, called CRBP(II)A and CRBP(II)B, were purified from rat small intestine using a three-column procedure. The two forms were present in equal abundance. The primary structures of CRBP(II)A and CRBP(II)B were determined using a combination of techniques including amino acid composition and sequence analyses, and fast atom bombardment and gas chromatography-electron impact mass spectrometry. The primary structures of both proteins were found to be identical, but they differed in their NH2-terminal processing. CRBP(II)B was acetylated at its NH2 terminus, while CRBP(II)A was not. The results also confirmed the amino acid sequence of CRBP(II)A that was deduced from the cDNA sequence by Li et al. (Li, E., Demmer, L. A., Sweetser, D. A., Ong, D. E., and Gordon, J. I. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 5770-5783). Antibodies capable of distinguishing between the two forms of CRBP(II) were used for immunohistochemical studies which indicated that the organ and cellular distributions of the two forms were identical. The 50% acetylation observed here in vivo fits the pattern predicted by recent in vitro studies which described the effect of NH2-terminal sequence on cotranslational NH2-terminal processing of cytosolic proteins (Boissel, J. P., Kasper, T. J., and Bunn, H. F. (1988) J. Biol. Chem. 263, 8443-8449). Our results provide a basis for investigating the possibility of different roles of CRBP(II)A and CRBP(II)B within cells, as well as the importance of acetylation of the amino terminus for these biological functions.

Specificity of cellular retinol-binding protein in the transfer of retinol to nuclei and chromatin.

We have reported previously that cellular retinol-binding protein (CRBP) is able to transfer retinol to specific binding sites in nuclei and chromatin. In this report, we have examined the specificity of the interaction of the protein moiety of retinol-CRBP (R-CRBP) with chromatin and nuclei in the transfer process. We first determined the ability of apo-CRBP, apo-serum retinol-binding protein (RBP), and apo beta-lactoglobulin (BLG), all capable of retinol binding, to compete with R-CRBP in the transfer of retinol to chromatin and nuclei. Apo-CRBP was an effective competitor but apo-RBP and apo-BLG showed no competitive ability. On the other hand, cellular retinol-binding protein type II (CRBP(II], whose amino acid sequence shows a considerable similarity to CRBP, did compete for the transfer of retinol from the R-CRBP complex, but less effectively than CRBP. These results demonstrate that the interaction of the protein moiety of the R-CRBP complex with nuclei and chromatin is quite specific.

Cell-specific immunohistochemical localization of a cellular retinol-binding protein (type two) in the small intestine of rat.

One of us recently has reported the purification of a new retinol-binding protein that is distinctly different from the well-known cellular retinol-binding protein, CRBP. This protein, which we propose to name cellular retinol-binding protein type II [CRBP(II)], was found almost exclusively in the small intestine of the adult rat at levels 1000 times greater than that of CRBP. Here we have determined the cellular location of these two proteins in the small intestine of the rat. By using an immunohistochemical technique, the absorptive cells of the small intestine, from the duodenum to the ileum, were strongly stained when antiserum against CRBP(II) was used. More intense staining was observed in absorptive cells near the tips of the villi than in those located at the base of the villi. However, the proliferative cells in the crypts of Lieberkühn were stained only lightly if at all. In contrast to absorptive cells, goblet cells in the villi did not stain. When tissue sections containing the gastroduodenal junction were examined, no staining was observed in the gastric epithelium, while the epithelium of the most proximal portion of the duodenum showed very strong staining. In tissue sections containing the ileocecal junction, staining terminated abruptly at the end of the distal ileum. No staining was observed in the epithelium of the colon. In contrast, the cellular location of CRBP in the small intestine was quite different from the cellular location of CRBP(II). The epithelial cells of the small intestine showed no staining when affinity-purified anti-CRBP was used. Staining was observed for connective tissue cells in the lamina propria and in cells located within the gut-associated lymphoid tissue. The cell-specific localization pattern determined for these two proteins suggests that CRBP(II), rather than CRBP, is the protein that plays a role in the absorption of retinol.

Radioimmunochemical determination of cellular retinol- and cellular retinoic acid-binding proteins in cytosols of rat tissues.

Radioimmunoassays have been developed for cellular retinol-binding protein and cellular retinoic acid-binding protein, postulated mediators of vitamin A action in nonvisual functions. These assays are considerably more sensitive for detection and quantitation of these proteins than previous methods. The higher sensitivity has permitted the detection of cellular retinoic acid-binding protein in adult rat tissues previously considered negative. These include kidney, lung, and spleen. The only tissues negative for both binding proteins were adrenals, skeletal muscle, ileal mucosa, and serum. Assay of tissue levels of cellular retinol-binding protein and cellular retinoic acid-binding protein reveals them to be widely distributed throughout most organs of the rat. No tissue or sex differences in these proteins were detected in these studies.