Characterization of two new alleles at the goat CSN1S2 locus

Two novel alleles at the goat CSN1S2 locus have been identified: CSN1S2(F) and CSN1S2(D). Sequence analyses revealed that the CSN1S2(F) allele is characterized by a G --> A transition at the 13th nucleotide in exon 3 changing the seventh amino acid of the mature protein from Val to Ile. The CSN1S2(D) allele, apparently associated with a decreased synthesis of alpha s2-casein, is characterized by a 106-bp deletion, involving the last 11 bp of the exon 11 and the first 95 bp of the following intron. Methods (PCR-RFLP and PCR) for identification of carriers of these alleles have been developed.     

Condensin-Dependent Chromatin Compaction Represses Transcription Globally during Quiescence

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Nonstereospecific biosynthesis of 11-cis-retinal in the eye

[3H]-all-trans-Retinol injected intraocularly into rats is processed to [3H]-11-cis-retinal, the visually active retinoid that binds to opsin. After 18 h, virtually all (93%) of the radioactive retinals recovered were in the form of 11-cis-retinal. At earlier times, however, both all-trans- and 13-cis-retinals, the latter being a nonphysiological isomer, were formed. Both of these isomers disappeared concomitant with the formation of 11-cis-retinal. The rise and fall of 13-cis-retinal suggest that this isomer can be converted into 11-cis-retinal either directly or indirectly in vivo and, hence, that the biosynthesis of the latter is nonstereospecific. This hypothesis was verified by showing that in double-labeling experiments [14C]-13-cis-retinol was converted into 11-cis-retinal nearly as well (approximately 70%) as [3H]-all-trans-retinol. These studies show that the biosynthesis of 11-cis-retinal can be nonstereospecific and, hence, that the process may be chemically rather than enzymatically mediated in vivo. In contrast, double-labeling studies with [14C]-9-cis-retinol and [3H]-all-trans-retinol showed that very little, if any, of the 9-cis isomer was processed to 11-cis-retinal in vivo although it did form isorhodopsin. This is consistent with what is known about the relative chemical stabilities of 9-cis-retinoids from model studies. The isomerization of 9-cis-retinoids is much slower than that of their all-trans, 13-cis, or 11-cis congeners. These results are discussed in terms of a possible mechanism for the biosynthesis of 11-cis-retinal in vivo and suggest that the isomerization event need not necessarily be enzyme mediated.     

Human alveolar macrophage fibronectin: synthesis, secretion, and ultrastructural localization during gelatin-coated latex particle binding

Human pulmonary alveolar macrophages synthesized and secreted several characteristic high molecular weight proteins for at least 7 d in vitro. Immunoprecipitates of medium and cell lysates from metabolically labeled cultures with specific anti-human plasma fibronectin IgG contained one major labeled polypeptide of molecular weight 440,000 (unreduced) or 220,000 (reduced). An identical polypeptide in conditioned medium from radiolabeled macrophages bound specifically to gelatin-Sepharose, demonstrating that alveolar macrophages synthesized and secreted a molecule immunologically and functionally similar to fibronectin. Fibronectin was the major newly synthesized and secreted polypeptide of freshly harvested alveolar macrophages. Pulse-chase experiments revealed that newly synthesized fibronectin was rapidly secreted into medium, approximately 50 percent appearing by 1 h and 80 percent by 8 h. Immunoperoxidase staining using antifibronectin F(ab’)(2)-peroxidase conjugates revealed the majority of immunoreactive fibronectin to be intracellular, localized to endoplasmic reticulum and Golgi apparatus. No extracellular matrix fibronectin was visualized, and cell surface staining was rarely seen, usually appearing only at sites where cells were closely apposed and not at sites of macrophage-substrate attachment. Similar immunostaining of fibroblast cultures revealed cell surface-associated fibrillar fibronectin. Ultrastructural localization of fibronectin during binding and phagocytosis of gelatin-coated and plain latex particles revealed fibronectin only on gelatin-latex beads and at their cell binding sites. Neigher plain latex beads nor their cell membrane binding sites stained for fibronectin. These results demonstrate that fibronectin is a major product of human alveolar macrophages, is rapidly secreted, and is localized at cell membrane binding sites for gelatin-coated particles. In view of the known binding properties of fibronectin, it may serve as an endogenous opsonic factor promoting the binding of staphylococcus, denatured collagen, fibrin, or other macromolecules to macrophages in the lower respiratory tract.     

Restriction fragment length polymorphism in the 3′ flanking region of the rabbit β1-globin gene

By Southern blot analysis, a restriction fragment length polymorphism in the 3' flanking region of the rabbit beta 1-globin gene was detected. Two alleles, characterized by 9.7- and 12.4-kb BamHI fragments and by 15.3- and 18.0-kb HindIII fragments, have been detected in a small population of White New Zealand rabbits. The long allele is the most frequent (about 70%). The simultaneous changes in the restriction patterns of the two endonucleases and the constant distance between BamHI and HindIII sites in short and long fragments suggest the possibility that the two alleles arise from a rearrangement phenomenon involving a DNA segment 2.7 kb long. In addition, the presence of the two alleles in individuals genetically unrelated to the White New Zealand breed suggests that this polymorphism is widespread.     

Rapid and slow gating of veratridine-modified sodium channels in frog myelinated nerve

The properties of voltage-dependent Na channels modified by veratridine (VTD) were studied in voltage-clamped nodes of Ranvier of the frog Rana pipiens. Two modes of gating of VTD-modified channels are described. The first, occurring on a time scale of milliseconds, is shown to be the transition of channels between a modified resting state and a modified open state. There are important qualitative and quantitative differences of this gating process in nerve compared with that in muscle (Leibowitz et al., 1986). A second gating process occurring on a time scale of seconds, was originally described as a modified activation process (Ulbricht, 1969). This process is further analyzed here, and a model is presented in which the slow process represents the gating of VTD-modified channels between open and inactivated states. An expanded model is a step in the direction of unifying the known rapid and slow physiologic processes of Na channels modified by VTD and related alkaloid neurotoxins.     

Solubilization and partial purification of retinyl ester synthetase and retinoid isomerase from bovine ocular pigment epithelium

Studies reported previously from this laboratory have demonstrated that membranes from the pigment epithelium of the vertebrate eye can transform free all-trans-retinol to 11-cis-retinol as well as 11-cis- and all trans-retinyl esters (Bernstein, P. S., Law, W. C., and Rando, R. R. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 1849-1853; Bernstein, P. S., Law, W. C., and Rando, R. R. (1987) J. Biol. Chem. 262, 16848-16857; Fulton, B. S., and Rando, R. R. (1987) Biochemistry 26, 7938-7945). The congeneric retinals are also formed under conditions where retinol redox activity is present. Here we report the successful solubilization of both the retinyl ester synthetase and isomerase activities from the pigment epithelium membranes of the bovine eye. The zwitterionic detergent Zwittergent 3-14(N-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate; cmc 0.012%) gave optimal solubilization of both activities. Three initial criteria for successful solubilization were used. First, high speed centrifugation (greater than 150,000 x g) left the activities in the supernatant. Second, the solubilized enzymatic activities were found in the included volume upon gel filtration. Finally, the solubilized activities were quantitatively passed through a 0.22-microns filter. Employing anion exchange and gel filtration chromatography results in a partial purification of the retinyl ester synthetase (approximately 189-fold). The solubilized retinoid isomerase is also partially purified (approximately 10-14-fold) following anion exchange chromatography. It is also shown that the membrane-bound and solubilized ester synthetase catalyzes the esterification of retinol using added lecithins as exogenous acyl donors. In addition, evidence is provided indicating that there is a positional selectivity for the acyl group transfer from the lecithin to retinol. The transfer occurs largely, if not entirely, from the 1-position of the lecithin.