Multiple forms of Go alpha mRNA: analysis of the 3'-untranslated regions

Go, a guanine nucleotide binding protein found predominantly in neural tissues, interacts in vitro with rhodopsin, muscarinic, and other receptors and has been implicated in the regulation of ion channels. Despite the virtual identity of reported cDNA sequences for the alpha subunit of Go (Go alpha), multiple molecular weight forms of mRNA have been identified in tissues from all species examined. To investigate the molecular basis for the size heterogeneity of Go alpha mRNAs, four cDNA clones were isolated from the same retinal lambda gt10 cDNA library that was used earlier to isolate lambda GO9, a clone encompassing the complete coding region of Go alpha. These clones were identified as Go alpha clones based on nucleotide sequence identity with lambda GO9 in the coding region; they diverge, however, from lambda GO9 in the 3'-untranslated region 28 nucleotides past the stop codon. An oligonucleotide probe complementary to a portion of the 3'-untranslated region of lambda GO9 that differs from the newly isolated clones hybridized with 3.0- and 4.0-kb mRNAs present in bovine brain and retina whereas a similar probe for the unique region of the new clones hybridized with a 4.0-kb mRNA in both tissues and with a 2.0-kb mRNA found predominantly in retina. A similar hybridization pattern was observed when brain poly(A+) RNA from other species was hybridized with the different 3'-untranslated region probes. It appears that differences in the 3'-untranslated regions could, in part, be the basis for the observed heterogeneity in Go alpha mRNAs.     

The vacuum UV CD bands of repeating DNA sequences are dependent on sequence and conformation

CD spectra were obtained for eight synthetic double-stranded DNA polymers down to at least 175 nm in the vacuum uv. Three sets of sequence isomers were studied: (a) poly[d(A-C).d(G-T)] and poly[d(A-G).d(C-T)], (b) poly[d(A-C-C).d(G-G-T)] and poly[d(A-C-G).d(C-G-T)], and (c) poly[d(A).d(T)], poly[d(A-T).d(A-T)], poly[d(A-A-T).d(A-T-T)], and poly[d(A-A-T-T).d(A-A-T-T)]. There were significant differences in the CD spectra at short wavelengths among each set of sequence isomers. The (G.C)-containing sequences had the largest vacuum uv bands, which were positive and in the wavelength range of 180-191 nm. There were no large negative bands at longer wavelengths, consistent with the polymers all being in right-handed conformations. Among the set of sequences containing only A.T base pairs, poly[d(A).d(T)] had the largest vacuum uv CD band, which was at 190 nm. This CD band was not present in the spectra of the other (A.T)-rich polymers and was absent from two first-neighbor estimations of the poly[d(A).d(T)] spectrum obtained from the other three sequences. We concluded that the sequence dependence of the vacuum uv spectra of the (A.T)-rich polymers was due in part to the fact that poly[d(A).d(T)] exists in a noncanonical B conformation.     

Guanine nucleotide-dependent ADP-ribosylation of soluble rho catalyzed by Clostridium botulinum C3 ADP-ribosyltransferase. Isolation and characterization of a newly recognized form of rhoA

Two C3 ADP-ribosyltransferase substrates with different characteristics were isolated from bovine brain cytosol. Amino acid sequences of tryptic peptides from the two substrates were identical to rhoA and rhoB; hence, the purified proteins are referred to as rhoA* and rhoB*, respectively. Soluble rhoA* exhibits properties different from those previously reported for rho proteins. In contrast to other C3 substrates, rhoA* behaved as a 77-80-kDa protein on gel filtration, although on sodium dodecyl sulfate-polyacrylamide gel electrophoresis the ADP-ribosylated moiety had a mobility consistent with a 21.5-kDa protein. Furthermore, C3-catalyzed ADP-ribosylation of rhoA* was dependent on guanine nucleotides in the presence of 1 mM Mg2+ or 1 mM EDTA (0.19 microM free Mg2+). Half-maximal stimulation by GTP, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), guanylyl-imidodiphosphate (Gpp(NH)p), and GDP was observed at 16, 20, 220, and 380 nM, respectively; guanosine 5'-O-(2-thiodiphosphate), GMP, and adenine nucleotides were ineffective. In the presence of GTP gamma S, the rate and extent of ADP-ribosylation was enhanced by dimyristoylphosphatidylcholine and/or cholate. This increase in ADP-ribosylation was specific for rhoA*; it was not observed with rhoB* and has not been reported for other C3 substrates. These distinct properties suggest that rhoA* is a newly recognized type of C3 substrate, differing from the rhoA-like proteins previously reported. rhoB*, on the other hand, has properties similar to those reported for membrane-associated rhoB and its ADP-ribosylation was independent of guanine nucleotides in the presence of 1 mM Mg2+ and not affected by dimyristoylphosphatidylcholine and/or cholate.     

Reconstitution of lipoproteins. II. Lipid-protein interaction between dimyristoyl-lecithin and dimyristoyl-lecithin:cholesterol vesicles and purified apolipoprotein C-I and C-III2 studied by isomeric spin-labelled lecithins

The reconstitution of purified apolipoprotein C-I and C-III2 with sn-3-dimyristoyl-lecithin and sn-3-dimyristoyl-lecithin:cholesterol (10:1) vesicles was studied by electron spin resonance spectroscopy using isomeric 5'-, 12'-, and 16'-(N-oxyl-4",4"-dimethyloxazolidine)stearoyl spin-labelled lecithin probes. Results obtained from the temperature-induced changes of lipoprotein recombinants showed the hydrophilic nature of the lipid-protein interactions. The temperature-induced phospholipid phase transition, as measured by 5'-(N-oxyl-4",4"-dimethyloxazolidine)stearoyl spin-labelled lecithin probe in recombinants containing apoprotein C-1 or apoprotein C-iii2, is very broad and has a small cooperative unit indicative of extensive lipid-protein interactions occurring at the head group region of the phospholipid bilayer. When 12"- and 16'-(N-oxyl-4",4"-dimethyloxazolidine)stearoyl spin-labelled lecithins are used as probes in the same system, similar sharper and more cooperative lipid phase changes are detected. These results indicate a surface location for both apoprotein C-I and apoprotein C-III2 with respect to the phospholipid bilayer in lipoprotein recombinants with and without cholesterol.     

Ultrastructural localization of elastase-like enzymes in human neutrophils

The thiol ester, N-t-butyloxycarbonyl-L-alanine-p-nitrothiophenyl ester (Boc-Ala-SNp) has been synthesized and applied as an ultrastructural cytochemical substrate for leukocytic elastase-like enzymes. Incubation of fixed human neutrophils with Boc-Ala-SNp in the presence of gold ions generates electron-dense deposits of gold p-nitrothiophenolate in the nuclear membrane, endoplasmic reticulum, Golgi complex, mitochondria, and granules. Deposition of product is inhibited by pretreatment of cells with general and specific chemical inactivators of neutrophil elastase. This substrate appears to have significant potential as a probe for the ultrastructural localization of elastase-like activity.     

The human Ia system: Definition and characterization by xenogeneic antisera

The production of xenogeneic anti-Ia serum against Ia antigens in serum has been previously described in the mouse and we now describe the production of xenogeneic anti-human Ia antisera using similar methods. With an indirect resetting technique, Ia-like antibodies were shown to react with the majority of B cells (95%), a subpopulation of T cells, with carbonyl iron adherent cells, and with some E^–Ig^– null cells, but there was no reaction with red cells and platelets. These reactions were the same as those obtained with DRW antisera using cytotoxicity testing. In addition, antigens detected with xenogeneic antisera were also found in serum, where they were found to exist in a low molecular weight, dialyzable form. By the selective removal of different cell surface markers by cocapping, no association could be found with the specifities detected with the xenogeneic anti-Ia antisera and with surface Ig, ₂-microglobulin, or HLA-A and B specificities. Alloantibodies to DRW specificities (but not HLA-A, B specificities) were able to specifically block the binding of the rabbit anti-Ia antibodies to B cells, and reciprocal blocking of rabbit antisera by DRW antibodies was also observed. Several xenogenic antisera were produced by immunizing rabbits with the serum of different individuals. Each antiserum was shown to contain a number of different specificities, as they gave different reaction patterns with different individuals when testing was done both directly and by absorption. These xenogeneic anti-la sera also segregated in a family with HLA-A and B specificities. The detection of a polymorphic antigenic system segregating with the HLA complex, distinct from HLA-A and B specificities, and whose antigens occur predominantly on B cells is therefore described. Because of the similarity of the reactions of the xenogeneic antisera in man to those found in the mouse, and because of the close relationship to the DRW specificities, the system has been provisionally called the H.Ia system.Abbreviations used in this paper AET 2-aminoethyl isothiouronium bromide - ₂-M -2 microglobulin - BSA Bovine serum albumin - H.Ia Human Ia - HuRBC Human red blood cells - Ig Immunoglobulin - Ir Immune response - MHC Major histocompatibility complex - MLR Mixed lymphocyte reaction - NHS Normal human serum - NMS Normal mouse serum - PBL Peripheral blood lymphocytes - PBS Phosphate-buffered saline - RAHIg Rabbit anti-human immunoglobulin - RASIg Rabbit anti-sheep immunoglobulin - RFC Rosette-forming cells - SAHIg Sheep anti-human immunoglobulin - SARIy Sheep anti-rabbit immunoglobulin - SRBC Sheep red blood cells     

Restoration of fertility in light-deprived female hamsters by chronic melatonin treatment

Summary Blinding young adult female hamsters was followed by functional involution of the ovaries and uteri and by the cessation of cyclic vaginal phenomena. Light deprivation was also accompanied by elevated plasma and pituitary levels of luteinizing hormone and depressed levels of prolactin in both the blood and the pituitary gland. Only one of 15 blinded hamsters became pregnant when they were exposed to fertile males for 30 days. Both pinealectomy or chronic melatonin treatment (1 mg melatonin implanted subcutaneously per week in beeswax) prevented the changes in the reproductive organs and in pituitary hormone levels attendant on light-deprivation. Both treatment also returned vaginal cycles to normal and restored plasma prolactin titers. Unlike hamsters that were blinded only, light deprived hamsters that were either pinealectomized or melatonin treated were capable of reproducing when they were caged with fertile males. The reproductive capability (i.e., percent of animals that become pregnant and the sizes of their litters) of these animals was equivalent to that of the untreated control hamsters. This is the first report that chronic melatonin treatment restores fertility in blinded female hamsters.Supported by Grant GB-43233X from the National Science Foundation     

Interaction of two mechanisms regulating alkali cations in HeLa cells.

The alkali cation content of HeLa cells is independent of culture density and of whether the cells are grown in suspension or attached to the culture vessel. With a cell doubling time of 28 hours, the cell K content turns over approximately once per hour. Following partial blockade of the alkali-cation transport system with ouabain, two distinct but interrelated mechanisms operate in the cellular response: (a) an increase in intracellular Na stimulates the pump so that the short-term alteration in electrolyte compostition is less than would be expected from the fraction of pump sites inhibited, and (b) there is a cycloheximide-sensitive recovery in transport capacity reflecting a restoration of functional transport sites to their normal density on the cell surface. Experimental manipulations that mimic the effect of ouabain lead to a stimulation of transport, but they do not result in an increase in the number of ouabain-binding sites on the surface. The data are consistent with a four-to-six hour turn-over of transport sites at the surface, but there is no evidence for a speicific induction of the transport system within this short-term recovery period.     

Molecular weight and amino acid composition of purified spinach beet phenolase

The purification of spinach beet phenolase has been modified to include equilibration of the crude macerate with 0.5% cetyl-trimethylammonium bromide a     

High resolution gel electrophoresis of chloroplast membrane polypeptides

In the present study we extend previous work from this laboratory on the polypeptide composition of photosynthetic lamellae. Using a high resolution sodium dodecyl sulfate gel electrophoresis technique, we show that both grana and stroma lamellae have qualitatively very similar polypeptide compositions although some clear quantitative differences are demonstrated.