Release of ovoperoxidase from sea urchin eggs hardens the fertilization membrane with tyrosine crosslinks.

One feature of fertilization is the alteration of the vitelline layer, by components released from the egg, to produce an elevated, covalently crosslinked, hard, insoluble, fertilization membrane. The following evidence indicates that crosslinking and hardening are caused by the production of diand trityrosyl residues, by oxidation of protein-bound tyrosyl residues in the presence of a peroxidase. Hardening of the fertilization membrane, as evidenced by its loss of solubility in 50 mM dithiothreitol, is inhibited by compounds known to inhibit many peroxidases. A peroxidase, here called the ovoperoxidase, is released from eggs at fertilization. This enzyme is inhibited by the same compounds that inhibit hardening and at similar concentrations. Inhibitors of the ovoperoxidase and the hardening reaction include KCN, 3-amino-1,2,4-triazole, NaN(3), phenylhydrazine, K(4)Fe(CN)(6), sodium sulfite, and glycine ethyl ester. In addition, tyramine and N-acetyltyrosine both inhibit hardening, but O-methyltyrosine does not. Dityrosyl and trityrosyl residues are found in acid hydrolysates of isolated, hardened fertilization membranes. These residues have been identified by cellulose phosphate column chromatography, thin-layer chromatography, and amino acid analysis. The amino acid data have been used to estimate that there is one dityrosine crosslink per 55,000 daltons of protein. We suggest that, by catalyzing the crosslinking of tyrosyl residues, the ovoperoxidase leads to the production of a hard fertilization membrane that blocks the entry of additional sperm. Because peroxidases are spermicidal, a secondary function of the enzyme could be to kill sperm in the vicinity of the fertilized egg.


Nucleotide sequences near the origin of replication of bacteriophage f1.

The nucleotide sequence of a region related to the initiation of the reaction in which single-stranded DNA gives rise to the replicative form (complementary strand synthesis) in bacteriophage f1 has been determined. The sequence can be drawn in an extensively base-paired structure, i.e., a single hairpin-helix 55 bases long.


Chemical basis for brain-specific serine transfer RNAs.

Serine tRNA from rat brain can be resolved into six isoaccepting species. Three of these species show the same chromatographic behavior as the seryl tRNAs from other rat organs, whereas the remaining species appear to be specific for brain. The isoacceptor tRNAs were purified to homogeneity by chromatography on benzoylated DEAE-cellulose followed by reversed-phase chromatography. We found that the additional species of serine tRNA in brain differ from their counterparts derived from other rat organs by a lack of a specific guanosine ribose-methylation in the dihydrouridine loop. In addition, when total liver tRNA was compared with total brain tRNA, the same degree of undermethylation with respect to 2'-O-methylguanosine was found as a general phenomenon.


Ionophores stimulate prostaglandin and thromboxane biosynthesis.

The role of calcium in triggering prostaglandin and thromboxane synthesis was studied in several systems with ionophores of different ion specificities. Divalent cationophore A23187 stimulates prostaglandin and thromboxane production by washed human platelets in a concentration-dependent manner (0.3-9 muM). A23187 also induces an antimycin A-insensitive burst in oxygen utilization which is partially blocked by 5 mM aspirin or 10 muM indomethacin. Under our conditions, A23187 (up to 10 muM) does not appear to damage platelet membranes since it does not cause appreciable loss of lactate dehydrogenase or beta-glucuronidase. Mono- and divalent cationophore X537A also stimulates platelet thromboxane B(2) production and oxygen utilization, but monovalent cationophores nigericin, monensin A, A204, and valinomycin have no effect. The synthesis of prostaglandins E(2), D(2), and F(2alpha) by rat renal medulla mince is stimulated by 1 and 5 muM A23187 without changes in tissue ATP content, lactate output, or K(+) efflux. X537A, monensin A, and nigericin (all 5 muM) stimulate both prostaglandin output and K(+) efflux from renal medulla, while 5 muM valinomycin or A204 has no effect on either. None of the ionophores stimulates renomedullary prostaglandin production if calcium is omitted from the incubation medium. A23187 also stimulates prostaglandin production by human lymphoma cells, rat stomach and trachea preparations, and guinea pig polymorphonuclear leukocytes. These observations suggest a major role for Ca(2+) in stimulating prostaglandin and thromboxane biosynthesis, and also indicate that prostaglandin and/or thromboxane release may partially mediate some of the previously described effects of ionophores on cells and tissues.


Structure of a promoter for T7 RNA polymerase.

We have determined the nucleotide sequence of a Hpa II restriction fragment of the phage T7 DNA containing a promoter for the phage-specified RNA polymerase. (Hpa II is a restriction endonuclease from Haemophilus parainfluenzae.) Mapping of the Hpa II restriction fragments on the T7 genome shows this promoter to be the second of tandem promoters separated by approximately 170 base pairs that begin transcription by the T7 RNA polymerase at approximately 15% of the genome. Features of the sequence involved in recognition by the T7 RNA polymerase are discussed and include the following region of hyphenated 2-fold symmetry (boxed regions are related through a 2-fold axis of symmetry at the center of the sequence shown). (See article). This sequence includes the initiation site, since the message transcribed from this fragment begins pppG-G-G-A. Combination of our results with work of others has permitted this fragment to be mapped at the junction of T7 genes 1 and 1.1. The RNA transcribed from this fragment begins within gene 1 and contains the RNase III cleavage site that lies between genes 1 and 1.1. This sequence is compared to other processing sites in T7 early message.


31P nuclear magnetic relaxation studies of phosphocreatine in intact muscle: determination of intracellular free magnesium.

31P nuclear magnetic relaxation rates for phosphocreatine in intact frog gastrocnemius were compared with those observed in model solutions at 4 degrees, a temperature at which muscle maintains its physiological state for at least 5 hr. Both nuclear Overhauser effect and spin-lattice relaxation rate (1/T1) experiments indicate that dipole-dipole interactions from the dominant relaxation path for 31P in intact muscle and model solutions, independent of phosphocreatine and Mg concentrations. Spin-spin relaxation rates (1/T2) measured by modified Carr-Purcell-Melboom-Gill spin-echo experiments suggest the importance of scalar coupling modulated by chemical exchange with free Mg. From these results, we estimate the free intracellular Mg in intact muscle as 4.4 mM and demonstrate that 31P T2 experiments can be used as a tool for studying free Mg levels with minimum disturbance of the intact cell.


In vitro biosynthesis of beta-endorphin, gamma-lipoprotein, and beta-lipotropin by the pars intermedia of beef pituitary glands.

Labeled amino acids were incorporated with proteins during incubations of isolated cells from the pars intermedia of beef pituitary glands. After 3 hr of incubation with methionine and [3H]lysine, approximately equivalent amounts of labeled gamma-lipotropin and beta-endorphins were isolated. They were found to be major synthesis products of the pars intermedia. In contrast, very little labeled beta-lipotropin was recovered. Another major synthesis product of the pars intermedia was also purified. Its partial amino acid sequence and molecular weight were determined and it was concluded that this peptide cannot be identified as any known pituitary hormone or protein fragment.


Amino acid sequence of a myosin fragment that contains SH-1, SH-2, and Ntau-methylhistidine.

A peptide having 92 amino acid residues and a calculated molecular weight of 10,478 was isolated from a cyanogen bromide digest of rabbit skeletal muscle myosin. It contained both proline and Ntau-methylhistidine, indicating that it arose from the portion of the heavy chain that folds to form most of the globular head of the myosin molecule. The amino acid sequence of the peptide included the two sulfhydryl groups whose alkylation modifies myosin's catalytic properties: SH-2 at position 11 in the peptide, and SH-1 at position 21. This proximity in the sequence means that SH-1 and SH-2 must be relatively close together in myosin, and several lines of evidence suggest that this region is near the catalytic or actin binding site(s) of myosin.


Reovirus mRNA can be covalently crosslinked via the 5' cap to proteins in initiation complexes.

Proteins that are located adjacent to the 5' end of mRNA in initiation complexes have been detected by chemical crosslinking. Reovirus mRNA containing radioactivity exclusively in the [3H]methyl-labeled "cap," m7G(5')ppp(5')-Gm, was oxidized with sodium periodate to convert the 2',3'-cis-diol of the 5'-terminal m7G to a reactive dialdehyde. Oxidized mRNA was incubated in cell-free protein-synthesizing systems derived from wheat germ or mammalian cells, and the resulting mRNA-ribosome initiation complexes were reduced with NaBH3CN. By this chemical procedure, putative Schiff bases between mRNA 5'termini and amino groups of neighboring proteins were stabilized by reduction, yielding covalently linked protein-RNA conjugates. Under conditions of ribosome binding, a limited number of polypeptides associated with the mRNA-ribosome complexes were crosslinked, suggesting that these proteins are positioned near and may interact with the 5' end of mRNA during initiation. This method should also be useful for studying the spatial relationships between molecules in other similar nucleoprotein complexes.


Regulation of growth hormone gene expression: synergistic effects of thyroid and glucocorticoid hormones.

Cultured rat pituitary cells (GC) respond to thyroid and glucocorticoid hormones by increases in growth hormone production and growth hormone mRNA. When these cells are transferred from medium containing normal animal serum (with 1.8 mug of thyroxine per dl) to a medium containing serum from a thyroidectomized calf, "hypothyroid medium" (with no detectable thyroid hormone), growth hormone production decreases markedly. In cells maintained for 5 days in hypothyroid medium, triiodothyronine induces within 50 hr a 17-fold increase in growth hormone production whereas glucocorticoids, during the same time, produce a negligible (3-fold or less) stimulation. In combination, the two hormones promote a 45-fold stimulation. In all instances the changes in growth hormone production are paralleled by changes in the levels of growth hormone mRNA as measured by cell-free translation. The transfer to hypothyroid medium and the hormonal induction do not affect the relative activities of other mRNAs whose products are detectable on polyacrylamide gels. These studies indicate that thyroid hormone can be an activator of the expression of the growth hormone gene. The results also show that triiodothyronine controls the magnitude of the effect of glucocorticoids on growth hormone mRNA, and provide a model for "permissive" triiodothyronine action. The synergistic effect of these two classes of hormone suggests that they increase levels of growth hormone mRNA by different mechanisms.


Glycerophospholipid synthesis: improved general method and new analogs containing photoactivable groups.

Current methods for phospholipid synthesis involving acylation of sn-glycero-3-phosphorylcholine, lysolecithins, and related glycerophosphate esters are not satisfactory. With N,N-dimethyl-4-aminopyridine as a catalyst and moderate amounts of fatty acid anhydrides (1.2-1.5 mol equiv per OH group), diacyl or 1,2-mixed diacylphosphatidylcholines, N-protected phosphatidylethanolamines, and phosphatide acids now can be conveniently prepared in high yields (75-90%). New phospholipids containing photoactivable groups, such as trifluorodiazopropionyl, diazirinophenoxy, 2-nitro-4-azidophenoxy, m-azidophenoxy, and alpha, beta-unsaturated keto groups, in the fatty acyl chains have been prepared. These phospholipids are of interest in studies of lipid-lipid and lipid-protein interactions in biological membranes.


Use of molecular hybridization to purify and analyze albumin messenger RNA from rat liver.

A new procedure is described for purification of rat liver albumin mRNA. First a population of RNA molecules is enriched for albumin mRNA by immunoprecipitation of polysomes containing albumin nascent chains. Polyadenylylated RNA is prepared from immunoprecipitates, transcribed into complementary DNA, and shown to be enriched severalfold for a particular RNA frequency component. This enriched RNA component is then purified by molecular hybridization to a limited R(0)t value (product of RNA concentration and incubation time), under conditions in which only the most abundant sequence component is annealed. Potentially, this procedure can be employed for the purification of a wide variety of mRNAs present in lesser amounts in the cell. The isolated RNA appears to be a single frequency component by hybridization to complementary DNA transcribed from itself. This RNA is a 17S species and represents 5-8% of total cytoplasmic polyadenylylated RNA. In vitro translation of the purified RNA has shown that it codes for a single polypeptide that can be identified immunologically as albumin and migrates with rat serum albumin on sodium dodecyl sulfate/polyacrylamide gels. This albumin mRNA was determined to be essentially pure by comparing its kinetics of hybridization to those obtained with rabbit alpha + beta globin mRNA and its DNA complement. The sequence complexity of purified rat albumin mRNA corresponds to 5.9 x 10(5) daltons.


Translational control by protein kinase in Artemia salina and wheat germ.

The catalytic subunit of cyclic 3':5'-AMP-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) inhibits translation in Artemia salina and wheat germ extracts. It acts, as in reticulocyte lysates [Datta, A., de Haro, C., Sierra, J. M. & Ochoa, S. (1977) Proc. Natl. Acad. Sci. USA 74, 1463-1467] by catalyzing the conversion of a proinhibitor to an inhibitor of polypeptide chain initiation. Addition of ATP and either cyclic AMP or catalytic subunit promotes the proinhibitor-inhibitor conversion in crude proinhibitor preparations from A. salina embryos. The effect of cyclic AMP is due to stimulation of cyclic AMP-dependent protein kinase, present in such preparations, and is inhibited by hemin. In similar preparations from wheat germ, addition of ATP and catalytic subunit promoted proinhibitor-inhibitor conversion, but addition of ATP and cyclic AMP has little or no effect. As assayed with histone as substrate, wheat germ preparations exhibit a protein kinase activity that is not stimulated by the addition of cyclic AMP or cyclic GMP. Our results suggest that a translational control system, similar to that existing in rabbit reticulocytes and other mammalian cells, is present in organisms evolutionarily far removed from mammals.


Structural gene identification and mapping by DNA-mRNA hybrid-arrested cell-free translation.

We present a simple method for directly correlating structural gene sequences in DNA with their corresponding mRNAs. This is based upon the fact that mRNA hybridized with its complementary DNA will not direct the cell-free synthesis of a complete polypeptide. Full translational activity of the mRNA is recovered upon the heat melting of the hybrid. Utilizing the rabbit beta globin clone PbetaG1, we demonstrate the application of hybrid-arrested translation for the identification of structural gene sequences within recombinant DNA molecules. In addition, the method is used to locate and order precisely several adenovirus 2 polypeptides within the viral genome.


Interactions of a photoaffinity analog of GTP with the proteins of microtubules.

Tubulin dimers isolated from brain contain two GTP binding sites, a nonexchangeable site and an exchangeable site. To localize the exchangeable site, we used a photoaffinity analog of GTP, 8-azidoguanosine triphosphate (8-N3GTP), which supports tubulin polymerization in the absence of activating light. Photolysis of tubulin polymerized in the presence of 0.01 to 0.1 mM [beta, gamma-32P]8-N3GTP resulted in covalent incorporation of radioactivity only onto the beta monomer. Photolysis with 8-N3GTP also prevented any further repolymerization of the tubulin whereas like treatment in the presence of GTP had no effect. Preincubation of tubulin with GTP prevented photo-incorporation of [beta, gamma-32P]8-N3GTP whereas preincubation with ATP did not.


Isolation of pi-alcohol dehydrogenase of human liver: is it a determinant of alcoholism?

HUMAN LIVER ALCOHOL DEHYDROGENASE (ALCOHOL: NAD(+) oxidoreductase, EC 1.1.1.1), homogeneous by physicochemical criteria, has been available in quantity only recently [Lange, L. G. & Vallee, B. L. (1976) Biochemistry 15, 4681-4686]. Until now, the biochemical basis of human alcohol metabolism had to be extrapolated from the properties and behavior of enzymes from other species, primarily horses and yeast. The biological determinants of human alcoholism have remained obscure, although recent evidence indicates a genetic predisposition, requiring delineation. A functionally distinct form of human liver alcohol dehydrogenase (ADH), which we have designated II-ADH, is provocative since, thus far, it seems to be unique to human beings. It has a high K(m) for ethanol and is remarkably insensitive (apparent K(I), 500 muM) to pyrazole and its derivatives, which are usually potent ADH inhibitors (K(I), 1 muM), a property that is the basis for the isolation of II-ADH. The affinity resin 4-[3-(N-6-aminocaproyl)aminopropyl]pyrazole-Sepharose binds all other known forms of ADH but not II-ADH, thereby separating it selectively by affinity chromatography. In turn, this has led to the establishment of its identity with that enzyme form which was previously known as the anodic band and characterized by a high K(m) for ethanol (20 mM at pH 7.5). The remarkable insensitivity of II-ADH to pyrazole inhibition has also permitted quantitation of its role in hepatic ethanol oxidation. At 5 mM ethanol, a saturating concentration for virtually all other forms of ADH, II-ADH contributes less than 15% to total ethanol oxidation. However, at intoxicating concentrations, e.g., 60 mM, it can account for as much as 40% of the total ethanol oxidation rate of liver, indicating a seemingly unique role for this enzyme form in ethanol elimination. Thus far, we have found the amount of II-ADH varies from liver to liver of individuals and is considerably more labile than the other molecular forms, phenomena whose inter- or independence requires further study. The isolation of human II-ADH advances efforts to recognize and understand biochemical mechanisms that may be biological determinants of alcoholism and alcohol-related disease states, now generally approached and managed largely as psychosocial disorders.


Sequence relationship between long and short repetitive DNA of the rat: a preliminary report.

Long and short repetitive sequences of rat DNA can be isolated and characterized. Long [greater than 1.5 kilobases (kb)] sequences can be separated from short (0.2-0.4 kb) sequences by exclusion chromatography after renaturation of 4-kb DNA fragments to a repetitive Cot and digestion with the single-strand-specific S1 nuclease. (Cot is the initial concentration of DNA in mol of nucleotides/liter multiplied by time in sec.) Long repetitive DNA can be driven by an excess of whole rat DNA can also be used to drive tracer quantities of either long (self-renaturation) or short repetitive DNA. Both the extent and the rate of the renaturations are found to be similar, suggesting that long and short DNA fragments share sequences. When long repetitive DNA is used to drive whole DNA tracers of various lengths, a 3.2-kb interspersion period is found. These data are consistent with the concept that short repetitive sequences are present within long repetitive DNA sequences in the rat genome.


Loop structures in hybrids of early RNA and the separated strands of adenovirus DNA.

Separated strands of adenovirus DNA were annealed with early cytoplasmic RNA and visualized in the electron microscope. DNA-RNA duplex regions within the DNA filaments could be recognized by their heavy contour. This contour was often interrupted at distinct locations by loops of displaced, single-stranded DNA. Loops have been observed and mapped in all four early regions of the genome. The structures appear to signal hitherto unknown mechanisms of eukaryotic gene expression.


Computer analysis of nucleic acid regulatory sequences.

We describe a computer program designed to facilitate the analysis of nucleic acid sequences. The program can search several nucleic acid sequences for oligonucleotides common to all of them. It can examine a DNA or RNA sequence for two kinds of homologous regions--repetitions and dyad symmetries. The homologies need not be perfect: mismatches and "looping out" of nucleotides are allowed. The program also finds (A+T)- and (G+C)-rich regions, locates restriction enzyme recognition sites, determines the distribution of di- and trinucleotides, and performs various other functions. We include two representative applications of the program. All published prokaryotic transcription termination sequences (June 1977) were found to share the following features: (i) a string of at least five T residues, (ii) the sequence CGGGC or a close analog immediately preceding the T cluster, (iii) a region of strong dyad symmetry preceding the Ts and including the CGGGC sequence. A sequence of 221 nucleotides consisting of the Escherichia coli trp promoter, operator, and leader was found to contain two strong dyad symmetries. These homologies both occur at known regulatory sites; no comparable homologies occur in regions without regulatory significance.


Cloning specific segments of the mammalian genome: bacteriophage lambda containing mouse globin and surrounding gene sequences.

We have developed a general approach to the cloning of specific segments of the mammalian genome that involves a two-step purification of EcoRI fragments of mammalian DNA and their in vitro insertion into a suitably constructed EK2 derivative of bacteriophage lambda. The combination of fragment purification, exclusion of parental-type recombinants, and simple phage screening techniques permits the isolation of virtually any gene segment for which there is an identifying hybridization probe. We illustrate the approach by describing the cloning of an approximately 7000-base-long segment of mouse DNA containing globin and surrounding gene sequences.


Comparative rates of transfer of lipid-linked oligosaccharides to endogenous glycoprotein acceptors in vitro.

We have shown previously that particulate preparations of Nil 8 fibroblasts catalyze the synthesis of two oligosaccharide-lipids: one composed of N-acetylglucosamine and mannose residues and the other containing, in addition to mannose and N-acetylglucosamine, one or two glucose residues. These two oligosaccharide-lipids were purified and added to fresh microsomal preparations. In comparative studies, we find that the glucose-containing lipid-linked oligosaccharide is transferred much more rapidly to endogenous protein acceptors than the glucose-free compound. With materials of comparable specific activities, as much as 41% of the glucose-containing oligosaccharide was transferred to protein as compared to 5% for the glucose-free compound. These results suggest that the attachment of glucose to mannosyl lipid-linked oligosaccharide serves an important role in the transfer of these compounds from lipid carrier to protein acceptor.


Induction and decay of human fibroblast interferon mRNA.

Polyadenylylated interferon mRNA, obtained from induced human fibroblasts, was quantitatively assayed by synthesis of biologically active human interferon in Xenopus laevis oocytes. The assay for interferon mRNA was used to distinguish between various hypotheses relating to interferon induction and biosynthesis. The data demonstrate that on induction with poly(I-poly(C) human fibroblasts accumulate interferon mRNA for 1-1.5 hr, after which time the mRNA is rapidly degraded with a half-life (t 1/2) of 18 min. Treatment of cells with cycloheximide prolongs the period of accumulation to 3 hr and decreases the rate of mRNA inactivation (t 1/2 = 49 min). Treatment with actinomycin D decreases the rate of inactivation still further (t 1/2 = 68 min). A comparison of cellular interferon synthesis with the relative amounts of interferon m RNA after simple induction or inductionin the presence of the inhibitors (superinduction) indicated a general correlation. Thus, on induction, the genes for interferon are activated to produce a transcript for a short time. The superinducing treatments prolong the period of accumulation and decrease the rate of degradation of this transcript.


Replication of adenovirus type 2 DNA in vitro.

A soluble replication system that synthesizes full-sized adenovirus DNA molecules has been developed. The extraction of infected HeLa nuclei solubilizes approximately 25% of the viral replicating DNA and leaves the HeLa chromatin associated with the insoluble nuclear material. Both the extracted viral DNA and the DNA product synthesized in vitro are intact and identical to the adenovirus DNA or its replicating intermediates produced in whole infected cells. The direction of elongation and the termini of replication of progeny DNA in vitro are essentially identical to those observed in vivo. Nuclear extract replication is dependent on Mg2+ and the four deoxyribonucleoside triphosphates and partially stimulated by ATP.


Surface antigen in early differentiation.

Addition of Fab fragments from rabbit antiserum to surface antigen F9 to 2-cell stage mouse embryos in culture does not alter cleavage; however, the addition prevents culture does not alter cleavage; however, the addition prevents the formation of compact morulae and blastocysts. A similar effect is observed when Fab fragments are added to already compact 8-cell stage or even older morulae, but disappears at the beginning of blastocoel formation. This effect is reversible: uncompact 30-cell embryos washed free of Fab become compact in a few hours, produce blastocysts, and upon reimplantation into pseudopregnant mothers can produce mice. Development is not altered by divalent anti-F9 antibodies, by Fab fragments from sera directed against other embryo surface antigens, or by succinyl concanavalin A.


Dependence of the differentiated state on the cellular environment: modulation of collagen synthesis in tendon cells.

In an adequate environment, primary avian tendon cells are capable of retaining both the full expression of differentiated function and a correct morphological orientation for 1 week in culture. At high density and in the presence of ascorbate, they are fully stabilized in that they devote 25-30% of their total protein synthesis to collagen, a level comparable to that in tendon cells in ovo. However, either at low density or in medium without ascorbate, they synthesize collagen at only a third of this level. If plated on a collagen matrix, these cells will orient themselves in a manner similar to that of tendon cells in vivo. Furthermore, they are capable of fully modulating the percentage of collagen synthesis upon addition or removal of ascorbate and serum. The variation in the percentage of collagen produced is a result of alterations in collagen synthesis rather than of changes in total protein synthesis or hydroxylation of proline in collagen. Primary avian tendon cells, therefore, provide a suitable model for understanding the stability of the differentiated state, the mechanism of action of ascorbate, and the regulation of collagen biosynthesis.


Microviscosity modulation during the cell cycle of neuroblastoma cells.

Microviscosity (n) of the cell membrane lipid layer was determined in synchronized C1300 mouse neuroblastoma cells (clone Neuro-2A) by fluorescence polarization of 1,6-diphenystratum. The determined n value was maximal in mitosis, decreased markedly in the G1 phase, remained constant at a low level during the S phase, and increased again during the G2 phase. These findings imply a direct role of the cell membrane fluidity in regulation of the cell cycle.


MRNA-directed synthesis of catalytically active mouse beta-glucuronidase in Xenopus oocytes.

Catalytically active mouse beta-glucuronidase (beta-D-glucuronide glucuronosohydrolase, EC 3.2.1.31) is formed when Xenopus oocytes are injected with mouse RNA enriched for poly(A)-containing mRNA sequences. With the RNA from androgen-induced kidneys, the efficiency of translation is comparable to that of endogenous Xenopus messenger, and the fidelity of translation is high. Detection of glucuronidase messenger by formation of a catalytically active product is several orders of magnitude more sensitive than detection by incorporation of isotopically labeled amino acids. As well as providing a sensitive technique for examining the regulation of gene expression, the system makes available an opportunity to study the regulation of post-translational polypeptide processing of a lysosomal enzyme.


Hormonal effects on intracellular and secreted casein in cultures of mouse mammary epithelial cells on floating collagen membranes.

Cultured on floating collagen membranes in the presence of lactogenic hormones, dissociated normal mammary epithelial cells from prelactating mice acquire the ultrastructural and biochemical characteristics of differentiated mammary secretory cells in vivo. The cells on floating collagen membranes in medium containing insulin alone have sparse secretory organelles, and a small amount of casein can be detected in these cells with a sensitive radioimmunoassay. These cells resemble counterpart cells in early-pregnant mice. When the cells are exposed to insulin, cortisol, and prolactin, the secretory apparatus is elaborated and significant increases in intracellular and extracellular casein are observed. In this environment, the intracellular casein content is generally four to eight times greater than in freshly dissociated cells or cells cultured in insulin alone. The amount of casein secreted into the medium by floating-collagen-membrane cultures in the three hormones is from 25 to 200 times greater than that secreted by cultures in insulin alone. Cells cultured on plastic substrates in either hormone combination fail to show any increase in intracellular or extracellular casein. On floating collagen membranes, the cells differentiate in response to hormones as they do in vivo and in organ culture. This cell-culture system provides an opportunity to study direct effects of environmental factors on mammary differentiation at the cellular level.


Explant cultures of catecholamine-containing neurons from rat brain: biochemical, histofluorescence, and electron microscopic studies.

Norepinephrine (NE)-producing cells of the nucleus locus ceruleus and dopamine (DA)-producing cells of the substantia nigra were dissected microscopically from embryonic rat brain, explanted, and maintained in culture for up to 5 weeks. The cultured neurons of both brain regions showed normal maturation of axons and dendrites and formed ultrastructurally defined synaptic contacts. Fluorescence microscopy of cultured neurons from both brain regions showed typical in situ cytological features: long axonal processes with multiple varicosities for locus ceruleus cultures, and smooth, wispy nonvaricose processes in the substantia nigra cultures. All cultures processed for fluorescence microscopy contained specific catecholamine-fluorescent cells. By radioenzyme assay for catecholamines, more than half of the locus ceruleus cultures contained measurable (>10 pg) quantities of NE and DA, but, unlike results on intact brains, DA content exceeded NE content. Cultures of substantia nigra neurons retained no NE and very little DA. Media from substantia nigra and locus ceruleus cultures contained substantial quantities of DA. Addition of reserpine (10 muM) to the medium depleted locus ceruleus neurons of both amines. The long survival time in culture of locus ceruleus cells, the normal appearance of fluorescent cell bodies and processes, the apparent development of morphologically specialized interneuronal connections, and the ability to synthesize and store NE make these cultures ideally suited for neurophysiological recording as well as morphological, biochemical, and pharmacological experiments.


Comparison of contact-mediated communication in normal and transformed human cells in culture.

With an assay that quantitates the transfer of 6-thioguanylic acid from hypoxanthine phosphoribosyltransferase (IMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.8)-positive donor cells to negative recipient cells through gap junctions, differences in contact-mediated communication between normal and transformed human cells in culture have been detected. We have compared cells cultured from human tumors and simian virus 40-transformed cells with the normal human fibroblasts from which they were derived as well as with gap junction-deficient L cells. The communication, which is extensive in normal cells, is significantly reduced when transformed cells are used as either donors or recipients in the contact-feeding assay. Furthermore, the reduction in the transfer of nucleotides is enhanced when transformed cells are used as both donors and recipients, indicating a dosage effect or synergism, independent of enzyme activity. Fetal cells have a contact-feeding phenotype intermediate between that of normal and that of transformed cells. We suggest that the decrease in communication of nucleotides in transformed cells reflects quantitative or qualitative changes in membrane components responsible for gap junction formation.


Induction of DNA synthesis in BALB/c 3T3 cells by serum components: reevaluation of the commitment process.

Serum contains a growth factor derived from platelets and also growth factors derived from platelet-poor plasma. Extracts of heated (100 degrees ) human platelets function synergistically with platelet-poor plasma to induce DNA synthesis in quiescent, density-inhibited BALB/c 3T3 cells. Platelet-poor plasma alone did not induce DNA synthesis. Cells exposed to platelet extracts became competent to enter the cell cycle, but the rate of entry into the S phase depended upon the concentration of platelet-poor plasma. The time required for the induction of this competent state was a function of the concentration of the platelet extract. A 2-hr exposure to 100 mug of the platelet extract at 37 degrees caused the entire cell population to become competent to enter the S phase. At 4 degrees or 25 degrees the cells did not become competent to synthesize DNA. The platelet extract-induced competent state was stable for at least 13 hr after removal of the platelet extract; however, in the absence of platelet-poor plasma, these competent cells did not progress through the cell cycle. The addition of an optimal concentration of platelet-poor plasma (5%) to these competent cells initiated cell cycle traverse with a rapid, first-order entry of cells into the S phase beginning 12 hr after addition of the plasma. The addition of a suboptimal concentration of the plasma (0.25%) did not increase the rate of cell entry into the S phase. Thus, the induction of DNA synthesis in quiescent BALB/c 3T3 cells can be resolved into at least two phases, controlled by different serum components: (i) competence, induced by the platelet-derived growth factor; and (ii) progression of competent cells into the cell cycle, mediated by factors in platelet-poor plasma.


Specific factors influencing histotypic aggregation of chick embryo hepatocytes.

Conditions are described for the reproducible assay of substances affecting the in vitro rate of aggregation of isolated chick embryo hepatocytes. Two low molecular weight (less than 1000) fractions--one that promotes hepatocyte aggregation (HAP) and the other that inhibits this stimulation (HAI)--have been isolated and partially purified from adult chicken liver. One major active component of HAP was identified as taurine (2-aminoethanesulfonate). The presence of HAP during the entire time of assay was required for largest aggregate formation. HAP had no effect on aggregation of chick embryo neural retina, kidney, or heart cells. Our results and the fact that puromycin completely inhibits aggregate formation suggest that HAP and HAI influence the specific synthesis and interaction of membrane macromolecules involved in the aggregation process.


Synthesis and secretion of a high molecular weight form of nerve growth factor by skeletal muscle cells in culture.

Rat skeletal muscle cells and a cloned myogenic cell line synthesize and secrete in culture a molecule that is immunologically and biologically indistinguishable from the active form of nerve growth factor (NGF) from mouse submandibular gland. This protein can be detected in medium conditioned by muscle cells both before and after fusion and in the soluble fraction of muscle cell homogenates. Chromatographic data also reveal that the molecular properties of muscle cell NGF differ from those of the growth factor purified from mouse submandibular glands. Muscle cell NGF has a molecular weight between 140,000 and 160,000, whereas purified mouse gland NGF has a molecular weight of 26,000. The biologic function of muscle cell NGF is not known, although it could be that it plays some role relating to the association of nerves and muscle in vivo.


Cystinotic fibroblasts accumulate cystine from intracellular protein degradation.

Fibroblasts derived from patients with cystinosis, an autosomal recessive condition, accumulate the disulfide amino acid cystine within lysosomes. The metabolic defect leading to the cystine accumulation and the source from which the cystine is derived are unknown. In this report we present data showing that cystine in these cells accumulates from the degradation of endogenous protein. This conclusion is based upon: (i) no demonstrable synthesis of cystine from serine; (ii) no difference in cystine reaccumulation between glutathione-depleted and non-glutathione-depleted cystinotic cells; (iii) recovery of labeled cystine only when the protein pool is labeled; (iv) reversible inhibition of cystine reaccumulation by known inhibitors of lysosomal protein degradation (chloroquine and NH4Cl).


Local control of neurite development by nerve growth factor.

A three-chamber culture system was devised in which neurites growing from small clusters of somas of sympathetic neurons penetrated a virtually fluid-impermeable barrier; thus the local fluid environment of the distal portions of the neurites could be controlled independently of the local fluid environment of the somas and proximal portions of the neurites. Neurites regularly penetrated the barriers if a high concentration of nerve growth factor was present on both sides, but never penetrated into chambers to which no nerve growth factor had been added. After neurites crossed the barrier, local removal of nerve growth factor from the distal portions of the neurites caused the growth of these portions to stop, and they eventually appeared to degenerate even though nerve growth factor was continuously present in the chamber that contained their somas and proximal portions. In contrast, local nerve growth factor was not required at the somas and proximal portions of the neurites; many neurons survived its withdrawal provided their somas were associated with neurite bundles that crossed into a chamber containing nerve growth factor. These results show that the growth, and probably the survival, of neurites depends upon nerve growth factor in their local environment, regardless of the nerve growth factor concentrations to which other portions of the neuron are exposed. This is entirely consistent with the notion that nerve growth factor released by sympathetic target tissues promotes the establishment and maintenance of appropriate neuron-target connections during development.


Evidence from UV transcription mapping in HeLa cells that heterogeneous nuclear RNA is the messenger RNA precursor.

The effects of UV irradiation on the incorporation of [3H]uridine in HeLa (human) cell mRNA, rRNA, heterogeneous nuclear RNA (hnRNA) and early mRNA from adenovirus type 2 have been compared. The UV target size of cell mRNA is at least 3 times larger than the average size of the mRNA itself and larger than the adenovirus-2 early mRNA, which is known to derive from transcription units of about 1.5-5.0 kilobases. The UV target size of hnRNA, in contrast, is about the same as its size determined by sedimentation and overlaps with the target size of mRNA. It is concluded that most mRNA derives from a higher molecular weight hnRNA molecule.


Bioelectricity and regeneration: large currents leave the stumps of regenerating newt limbs.

Electrical currents near regenerating newt limbs were measured with a recently developed vibrating probe. Steady currents with local surface densities of 10 to 100 muA/cm2 or more leave the end of the stump during the first 5-10 days after amputation and are balanced by currents with densities of only 1-3 muA/cm2 that enter the intact skin around the stump. They are immediately dependent upon the entry of sodium ions into this skin and are therefore inferred to be skin-driven. The outward currents are comparable in direction, density, duration, and position to artificially imposed currents previously found sufficient to induce significant regeneration of amputated adult frog limbs. This comparison suggests that the endogenous stump currents play some causal role in initiating regeneration.


Detection of antibody to autologous human leukemia cells by immune adherence assays.

The sera of 21 adult patients with acute leukemia were studied for the presence of antibody reacting with surface antigens of autologous leukemia cells. Sequential serum samples were obtained from patients and were tested on cryopreserved leukemia cells in immune adherence assays. Three patients showed autologous serum reactivity and the serum of one of them was analyzed in detail. This antibody reacted with autologous acute lymphocytic leukemia cells but not with autologous cells obtained from peripheral blood, bone marrow, or spleen during clinical remission. In absorption tests, the antigen could not be detected on normal autologous or allogeneic blood lymphocytes, lymphoblastoid lines of T- or B-cell origin, or cells infected with simian sarcoma virus, baboon C-type virus, or Mason-Pfizer virus. Leukemia cells from two other patients with acute lymphocytic leukemia and one patient with acute nonlymphocytic leukemia absorbed specific reactivity. These studies indicate that certain acute leukemia cells express a common antigen that elicits a humoral immune response in the autologous host.


beta-Endorphin: pituitary and adrenal glands modulate its action.

Hypophysectomized rats are supersensitive to the hypothermic effects of morphine and beta-endorphin injected intraventricularly as early as 1 week after surgery. At 2 weeks after surgery, there is a significant increase in the antinociceptive potency for these opiates. The route of opiate injection must be considered in interpretations of these results. The enhanced opiate potency following subcutaneous morphine injection in hypophysectomized rats may be partially explained by adrenal dysfunction, as demonstrated by a similar sensitization to subcutaneous morphine following adrenalectomy. By contrast, no enhancement of opiate potency was observed upon direct intraventricular injection of morphine or beta-endorphin in adrenalectomized rats. Furthermore, the potency of intravenous beta-endorphin is profoundly enhanced in adrenalectomized mice; five out of nine animals died when injected with a dose of the peptide that produced only mild analgesia in sham controls. The complete reversal of this intravenous beta-endorphin supersensitivity by dexamethasone implies a possible physiological interplay between the peptide and adrenal function.


Erythropoietin: isolation by affinity chromatography with lectin-agarose derivatives.

Affinity chromatography using agarose-bound lectins was used to isolate erythropoietin from crude preparations of sheep plasma and human urinary erythropoietin. On the basis of previous estimates of the sugar content of the hormone, six lectins (wheat germ agglutinin, phytohemagglutinin, Ricinus communis 120, soybean agglutinin, concanavalin A, and limulin) were chosen for study. Only wheat germ agglutinin-agarose and phytohemagglutinin-agarose derivatives had significant affinity for erythropoietin. By use of wheat germ aggutinin-agarose columns, erythropoietin could be separated from over 95% of the initial starting protein, resulting  in an 8-to 100-fold purification and a recovery of at least 40% depending on the source of the hormone. Affinity chromatography with agarose-bound lectins provides a simple rapid method for isolating erythropoietin from crude preparations of the hormone.


Role of high-molecular-weight kininogen in surface-binding and activation of coagulation Factor XI and prekallikrein.

In the contact phase of activation of the kinin-forming, intrinsic clotting, and fibrinolytic systems, high-molecular-weight kininogen acts as a cofactor for the activation of Factor XI, prekallikrein, and Hageman factor. One mechanism by which high-molecular-weight kininogen acts as a cofactor has been studied by using 125I-labeled Factor XI and prekallikrein in kaolin-activated normal human plasma and plasmas deficient in high-molecular-weight kininogen and Hageman factor. High-molecular-weight kininogen was found to be essential for normal binding and cleavage of both Factor XI and prekallikrein on the kaolin surface. Hageman factor was essential for cleavage but not for binding of Factor XI and prekallikrein to kaolin. In normal plasma 80% of the activated Factor XI remained surface-bound, whereas 80% of the kallikrein was not surface-bound. These findings are consistent with the hypothesis that, in the initial phase of contact activation, high-molecular-weight kininogen links both Factor XI and prekallikrein to the exposed surface where they are activated by surface-bound activated Hageman factor. Once activated, the Factor XI molecules remain localized at the site of activation, in contrast to the kallikrein molecules which are found largely in the surrounding plasma.


On the enzymic defects in hereditary tyrosinemia.

The activity of the enzyme porphobilinogen synthase (EC 4.2.1.24) in erythrocytes from patients with hereditary tyrosinemia was less than 5% of that in a control group and the activity in liver tissue was less than 1% of the reported normal activity. Urine from patients with hereditary tyrosinemia contained an inhibitor that was isolated and identified as succinylacetone (4,6-dioxoheptanoic acid) by gas/liquid chromatography-mass spectrometry. Fresh urine samples contained succinylacetoacetate (3,5-dioxooctanedioic acid) as well as succinylacetone. The inhibition of porphobilinogen synthase explains the high excretion of 5-aminolevulinate observed in hereditary tyrosinemia. Succinylacetone and succinylacetoacetate presumably originate from maleylacetoacetate or fumarylacetoacetate, or both, and their accumulation indicates a block at the fumarylacetoacetase (EC 3.7.1.2) step in the degradation of tyrosine. We suggest that the severe liver and kidney damage in hereditary tyrosinemia may be due to the accumulation of these tyrosine metabolites and that the primary enzyme defect in hereditary tyrosinemia may be decreased activity of fumarylacetoacetase.


Affinity alkylation labels two subunits of the reduced acetylcholine receptor from mammalian muscle.

The acetylcholine receptor from denervated rat skeletal muscle was purified by affinity chromatography and, after reduction, was treated with the affinity alkylating agent 4-(N-maleimido)benzyltri[3H]methylammonium iodide. The receptor specifically incorporated approximately 1 mol of alkylating agent per mol of 125I-labeled alpha-bungarotoxin bound. Analysis of the labeled receptor by polyacrylamide gel electrophoresis in sodium dodecyl sulfate showed that two subunits were labeled; their apparent molecular weights were 45,000 and 49,000. These results suggest that the affinity reagent labels a second site for acetylcholine binding in the muscle receptor that is not labeled in receptors from Electrophorus or Torpedo.


Immunological distinction between acetylcholine receptor and the alpha-bungarotoxin-binding component on sympathetic neurons.

Clone PC12 of a rat sympathetic neuron cell line binds alpha-bungarotoxin and exhibits carbamoylcholine-stimulated uptake of sodium ions. Concentrations of alpha-bungarotoxin that saturate the alpha-bungarotoxin-binding site have no effect on agonist-stimulated sodium uptake. Conversely, antibodies against eel acetylcholine receptor block the agonist-induced sodium flux but fail to recognize the alpha-bungarotoxin-binding component. Detergent extracts of the PC12 clone inhibit the ability of antibody to eel acetylcholine receptor to recognize 125I-alpha-bungarotoxin-acetylcholine receptor complexes derived from muscle. These results distinguish between a ganglionic nicotinic acetylcholine receptor and an alpha-bungarotoxin-binding component on these cells, and provide evidence for antigenic similarities between muscle acetylcholine receptor and ganglionic acetylcholine receptor.


Mysterious form of referred sensation in man.

A phenomenon is described in which scratching a small excrescence on the skin on one part of the body is referred to a distant point as a "prick" or a "tingle". "Referral" points are elicited mainly by absent-minded scratching of the skin when attention is not focused on the local sensation produced by the scratch. Location of "referral" points seems to follow definite patterns: in all instances, "referral" points occurred on the same side of the body as the "stimulus" point; and each "referral" point was rostral to its "stimulus" point; and each "stimulus" point was associated with only one "referral"  point. "Stimulus" and "referral" points seem to have a fixed relationship. Pathways from a "stimulus" point to a "referral" point are not known at present. Although parallels can be drawn between this phenomenon and Bender's "double simultaneous stimulation" phenomenon, both remain mysteries.


Location of pyridoxal phosphate in glycogen phosphorylase a.

The pyridoxal 5'-phosphate cofactor of glycogen phosphorylase a (1,4-alpha-D-glucan:orthophosphate alpha-glucosyltransferase, EC2.4.1.1.) has been positioned on the protomer with x-ray diffraction data, chemical markers, and sequence information. The electron density was computed from 3.0-A resolution phases calculated from four heavy-atom derivatives. The cofactor is buried inside the protomer adjacent to the glucose-binding site. The phosphoryl substrates Pi and glucose-1-P each bind at two sites on the protomer. At low concentrations, Pi and glucose-1-P bind in the same location as does the allosteric effector AMP, near the monomer-monomer interface and some 30 A from the glucose site. At high concentrations glucose-1-P also binds strongly at the glucose site, with its phosphate only 7.2 A from that of the cofactor. Inorganic phosphate can also bind at this site. Implications for the participation of the pyridoxal phosphate in the catalytic mechanism are discussed in the light of these structural findings as well as the wealth of indirect evidence in the literature.


Complete amino acid sequence of rabbit muscle glycogen phosphorylase.

The sequence of the 841 amino acid residues in each subunit (molecular weight 97,412) of rabbit muscle glycogen phosphorylase b (1,4-alpha-D-glucan:orthophosphate alpha-glucosyltransferase; EC 2.4.1.1) has been determined. The general strategy was based on limited proteolysis of native phosphorylase b by subtilisin BPN', yielding two large segments (light and heavy) which were fragmented by cleavage at methyonyl-, asparaginyl-glycine, and aspartyl-proline bonds. Analysis of two cyanogen bromide fragments (CB14 and CB17) isolated from the intact molecule yielded the overlap between the light and heavy fragments and the remainder of the sequence. The residues involved in the covalent and allosteric control of the enzyme, and in the binding of the cofactor pyridoxal 5'-phosphate, were identified as serine-14, tyrosine-155, and lysine-679, respectively.


HeLa cell poly(A)- mRNA codes for a subset of poly(A)+ mRNA-directed proteins with an actin as a major product.

Poly(A)+ and poly(A)- mRNA from HeLa cells were separated and translated in heterologous messenger-dependent protein synthesizing systems. Two-dimensional electrophoretic analysis revealed three classes of polypeptides. At the level of detectability in the electropherograms, a small number (about 10) of proteins were detected only among the poly(A)- mRNA products, a larger number (about 40) were produced by both poly(A)- and poly(A)+ mRNA, and a large number of polypeptides were found exclusively in the poly(A)+ mRNA products. The major product of both poly(A)+ and poly(A)- mRNA was shown to be the beta form of actin.Previous cross hybridization measurements suggested little homology between poly(A)+ and poly(A)- mRNA populations. In view of the apparent identity of many poly(A)- products with those of poly(A)+, the homology between poly(A)+ and poly(A)- mRNA sequences was examined in greater detail. cDNA complementary to only the most abundant poly(A)+ message sequences was prepared. About 10% of this cDNA hybridized to abundant sequences in the poly(A)- fraction. This corresponded to only 2% of the total mass of poly(A)+ mRNA and accounted for the failure to detect cross hybridization in previous experiments. Thus, a small number of poly(A)+ sequences appear to be present in relatively high concentration in poly(A)- mRNA as evidenced by both the translation products and the cross hybridization results.


2-Aminopurine-induced mutagenesis in T4 bacteriophage: a model relating mutation frequency to 2-aminopurine incorporation in DNA.

We measured the in vivo incorporation of 2-aminopurine into DNA of T4 bacteriophage allelic for gene 43 (DNA polymerase), mutator (L56), 43+, and antimutator (L141). The magnitude of incorporation (mol/mol of Thy) was 1/1500 in L56, 1/1600 in 43+, and 1/8900 in L141. The incorporation ratio L56:43+:L141 in vivo was equal to that mediated by the purified DNA polymerases of these allelic phages in vitro. A model for 2-aminopurine-induced A-T in equilibrium G-C transitions is discussed. The model is used to predict the magnitudes of replication errors (C mispairing with a template 2-aminopurine) and incorporation errors (2-aminopurine mispairing with a template C) per round of replication and to investigate the asymmetry in 2-aminopurine-induced transitions favoring the A-T leads to G-C pathway over G-C leads to A-T. We suggest that the fidelity of L56 and L141 DNA polymerases exemplifies one-step and two-step editing, respectively.


Nonintercalative binding of ethidium bromide to nucleic acids: crystal structure of an ethidium--tRNA molecular complex.

X-ray diffraction studies at 4.5-A resolution on crystals of a complex of ethidium bromide and yeast phenylalanine tRNA reveal a nonintercalative model of binding of the ethidium within the tertiary structure. This is contrary to the expected interacalative binding to the double-helical regions.


Activation of lipoprotein lipase by native and synthetic fragments of human plasma apolipoprotein C-II.

Apolipoprotein C-II (apoC-II), a protein constituent of human very low density lipoproteins, is the activator for lipoprotein lipase (LPL; triacylglycerol acyl-hydrolase, EC 3.1.1.3). The amino acid sequence of the 78 residues of apoC-II has recently been established in this laboratory. To determine the minimal sequence requirements for activation, we have prepared both native and synthetic fragments of apoC-II and tested them for their ability to activate LPL. Cyanogen bromide fragments of apoC-II corresponding to residues 1--9 and 10--59 had little ability to activate LPL. However, the COOH-terminal cyanogen bromide fragment corresponding to residues 60--78 increased hydrolysis 4-fold compared to an average of 9-fold activation for the same concentration of apoC-II. The synthetic peptide containing residues 60--78 prepared by solid-phase techniques enhanced the lipolysis 3-fold. Addition of five residues produced a synthetic fragment 55--78 that enhanced the release of fatty acid 12-fold compared to 13-fold for intact apoC-II. By contrast, the synthetic peptide containing residues 66--78 did not activate. Removal of the three COOH-terminal residues, Gly-Glu-Glu, from fragment 60--78 decreased the ability to activate LPL by greater than 95%. These studies suggest that the maximal activation of LPL by apoC-II requires a minimal sequence contained within residues 55--78.


Structure of histone H1-DNA complex: effect of histone H1 on DNA condensation.

Large doughnut-shaped complexes were formed when histone H1 was mixed with DNA in amounts that extensively neutralized it. The doughnut shape is the most prevalent from observed by electron microscopy for linear double-stranded DNA independent of the molecular weight of the DNA in the range 1.2 x 10(6) to 25 x 10(6) at an H1/DNA input weight ratio of 1.3, at ionic strength 0.17. Doughnuts were not observed for single-stranded DNA-H1 complexes; instead, the complexes were globular. The circumference of the doughnutshaped particles indicates that much of the rigidity of duplex DNA in the complex has remained. Evidently, the condensation of the nucleohistone is constrained by the rigidity of duplex DNA and, under this constraint, surface contact with water is minimized by adopting a doughnut shape. Histone H5 causes a type of DNA condensation similar to that of H1 at comparable charge ratios. Core histones H2A-H2b, H3, and H4 complex with DNA to form globular aggregates of such small diameter that the duplex DNA in them must be much more tightly folded than is the case with the doughnut-shaped complexes. Because these histones are designed to fold DNA into nucleosomes 100 A wide, they must destroy the rigidity of free duplex DNA, perhaps by forming kinks in the chain.


Proteolytic enzyme activation of rat ovarian adenylate cyclase.

Various serine proteases (e.g., trypsin, alpha-chymotrypsin, Pronase, and subtilisin) stimulate adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] activity in a membrane-enriched fraction of the rat ovary. Maximum stimulation is observed at protease concentrations ranging from 3 to 10 mug/ml. Higher protease concentrations inhibit ovarian adenylate cyclase in a dose-dependent manner. Protease stimulation causes a 6- to 8-fold increase in adenylate cyclase activity, which is comparable to the stimulation observed with human chorionic gonadotropin. Combinations of trypsin plus hormone or trypsin plus NaF stimulate ovarian adenylate cyclase activity to a greater extent than does any one of these alone. The mechanism of protease stimulation of adenylate cyclase involves limited proteolysis because zymogen precursors fail to activate the cyclase as does trypsin pretreated with trypsin inhibitors. Unlike cholera toxin, the serine protease stimulation is immediate (within the first 5 min) and requires no additional factors (e.g., NAD(+)). It is unlikely that protease stimulation of adenylate cyclase results from a proteolytic modification of the hormone receptor on the cell surface, because of the additive effects noted above and because protease stimulation is also observed in ovaries desensitized to hormone that lack this hormone receptor. Results with Lubrol-treated membranes also suggest that proteolytic enzymes do not directly activate the catalytic subunit of the cyclase or unmask new catalytic sites because the protease effect (like hormonal stimulation) is abolished by the detergent, whereas fluoride stimulation is enhanced. Other data suggest that serine protease and chorionic gonadotropin stimulation of adenylate cyclase result from activation of a membrane protease that then regulates adenylate cyclase in the ovary.


Homoserine dehydrogenase: spontaneous reactivation by dissociation of p-mercuribenzoate from an inactive enzyme--p-mercuribenzoate complex.

Incubation of Rhodospirillum rubrum homoserine dehydrogenase (L-homoserine:NAD+ oxidoreductase, EC 1.1.1.3) with p-mercuribenzoate (PMB) in the presence of 0.2 M KCl and 2 mM L-threonine resulted in complete loss of enzyme activity. Upon removal of excess PMB, KCl, and L-threonine, a time-dependent recovery of enzyme activity was observed in 25 mM phosphate/I mM EDTA buffer, pH 7.5. Circular dichroism studies indicated that the transition from inactive to reactivated form of the enzyme was accompanied by a conformational change in the protein. Experiments with [14C]PMB revealed loss of enzyme-bound radioactivity during reactivation. Increase in ionic strength of the phosphate buffer and/or addition of L-threonine, leading to enzyme aggregation, decreased the rate of enzyme reactivation, aggregated enzyme that remained inactive retained [14C]PMB on the enzyme. Sulfhydryl titration of various forms of the enzyme suggested a preferential release of PMB from a sulfhydryl group essential to enzymic activity. We conclude that reactivation of the inactive enzyme is due to dissociation of PMB from an "active-site" sulfhydryl group and that changes in the protein structure influence the rate of dissociation of the enzyme-PMB complex.


Genes transcribed at diverse rates have a similar conformation in chromatin.

We have analyzed the DNA generated upon treatment of oviduct nuclei with pancreatic DNase I (deoxyribonucleate 3'-oligonucleotidohydrolase; EC 3.1.4.6), with cDNA copies of specific mRNA sequences to study the structure and organization of transcriptionally active genes in chromatin. In this report we examine the kinetics of digestion of three classes of genes in the oviduct which are transcribed at significantly different rates. Our results indicate that the ovalbumin genes appear to be organized by chromatin proteins in such a way that they are rendered exceedingly sensitive to digestion by DNase I. This sensitivity is not observed in the liver, a tissue in which these genes are transcriptionally inert. Furthermore, the transcriptionally inactive globin genes in the oviduct are not selectively sensitive to nuclease attack and are digested 5 times more slowly in the ovalbumin genes in this tissue. In addition, we have examined the accessibility of a complex subset of genes that are rarely represented in the mRNA and are likely to be transcribed at a frequency orders of magnitude below that of the ovalbumin gene. Comparison of the accessibility of these sequences with that of the ovalbumin gene indicates that these two subsets of genes are recognized and cleaved by DNase I at similar rates. These results suggest that the maintenance of an active conformation about specific genes does not reflect the polymerase distribution about these genes. This active conformation is therefore not confined to sequences actively engaged in the transcription process and may reflect the structure about a subpopulation of the genome which represents the transcriptional potential of a given cell type.


Selective recognition of nucleic acids by proteins: the specificity of guanine interaction with carboxylate ions.

The interaction of carboxylate ions (acetate, butyrate) with nucleic aicd bases and nucleosides has been investigated by proton magnetic resonance in dimethyl sulfoxide (d6)/H2O mixtures. Carboxylate ions interacted only with guanine derivatives and led to a large downfield shift of the NH2 resonance. A 1:1 stoichiometry was deduced from a study of the concentration dependence of chemical shifts. A study of substituted guanine showed that hydrogen bonding involved N(1)H and NH2(2). An association constant of 110 M-1 was determined. This value is about 30 times higher than the association constant for guanine-cytosine base pair formation under the same experimental conditions. As a matter of fact, carboxylate ions induced a dissociation of guanine-cytosine base pairs. This guanine-carboxylate association is experimental evidence for a highly specific interaction that could play an important role in protein/nucleic acid recognition.


Estradiol receptor of calf uterus: interactions with heparin-agarose and purification.

Heparin attached covalently to agarose beads binds the "native" form of the estradiol receptor with very high affinity. Chondroitin sulfate does not bind to the receptor. When the receptor is complexed with hormone, the affinity is at least 10 times higher. Only the "native" and not the "nuclear" or the "derived" (i.e., after activation by a calcium-dependent enzyme) forms of the estradiol receptor interact with heparin. The "native" estradiol-receptor complex is purified to homogeneity after chromatography on columns of heparin-agarose, Sephadex G-200, and DEAE-cellulose, followed by two more Sephadex G-200 columns. The purified molecule is a single polypeptide of molecular weight 69,000 by polyacrylamide gel electrophoresis in sodium dodecyl sulphate. The sedimentation coefficient on sucrose gradients is 4.3 S, the Stokes radius from gel filtration is 36.5 A, and the isoelectric point is 6.4. The purified [3H]estradiol-receptor complex exchanges the radioactive hormone with estradiol or other estrogenic steroids, but not with testosterone, 5alpha-dihydrotestosterone, or progesterone.


Response of endocytosis to altered fatty acyl composition of macrophage phospholipids.

Mouse peritoneal macrophages incubated in serumless medium containing a 19:0 or trans-18:1 fatty acid complexed to bovine serum albumin incorporate the exogenous fatty acid supplement into cellular phospholipids. Within 8 hr, 25% of the total phospholipid fatty acids are derived from the supplement, with cell viability remaining greater than 95%. The incorporation of either of these supplements increases the saturated/unsaturated fatty acid ratio in the phospholipids 2-fold over that of cells cultured in serum and effects striking changes in endocytic activities. The levels of both fluid-phase pinocytosis and receptor-mediated phagocytosis are decreased at all temperatures examined between 15 degrees and 37 degrees. The increased degree of saturation of cell phospholipids correlates with decreased endocytic rates for both processes and with increased activation energies (Eact) for phagocytosis. The Eact values for phagocytosis, which range from 54 to 90 kcal/mol, depend on the supplementation conditions used. Although the levels of pinocytosis are depressed, the Eact values for pinocytosis (17--25 kcal/mol) are not strikingly affected by saturated fatty acid enrichment. These observations suggest that the degree of lipid fluidity of macrophage membranes influences both phagocytosis and pinocytosis in macrophages.


Sequence of an oligonucleotide derived from the 3' end of each of the four brome mosaic viral RNAs.

A 3'-terminal oligonucleotide fragment, 161 bases long, can be obtained from each of the four brome mosaic virus RNAs by means of nuclease digestion. Like the four intact brome mosaic virus RNAs, each fragment accepts tyrosine in a reaction catalyzed by wheat germ aminoacyl-tRNA synthetase. The complete nucleotide sequence of the RNA 4 fragment has been determined by use of standard radiochemical methods. Comparative data for the fragments from RNAs 1, 2, and 3 show that they have nearly the same sequence as the RNA 4 fragment. The eight bases adjacent to the 3' terminus of the RNA 4 fragment are identical in sequence to the eight terminal bases of tyrosine tRNA from Torula utilis and eleven interior bases are identical in sequence to eleven bases encompassing the anticodon region of tyrosine tRNA from Saccharomyces cerevisiae, T. utilis, and Escherichia coli. Nevertheless, reasonable base-pairing schemes yield, at best, a distorted cloverleaf secondary structure.


31P nuclear magnetic resonance studies of HeLa cells.

A survey of phosphorus compounds present in HeLa cells and their acid extracts has been carried out by (31)P nuclear magnetic resonance spectroscopy at 40 MHz. The proton decoupled (31)P spectrum of the neutralized extract had resolution adequate to enable the identification of the main phosphate compounds. The spectral intensities were converted to concentrations. The lower detection limit with extensive signal averaging was 0.02 mumol for the extract. The composition, listed in order of decreasing concentration, was: inorganic phosphate, ATP, phosphorylcholine, creatine phosphate, UTP, NAD(+), glucose 6-phosphate, beta-D-fructose 1,6-bisphosphate, alpha-D-fructose 1,6-bisphosphate, ADP, alpha-glycerophosphorylcholine, and alpha-glycerophosphorylethanolamine. UTP made up (1/5) of the total nucleotide triphosphate content. The composition was compared to the (31)P spectrum of an extract from a human astrocytoma grown in athymic mice. The signal from P-containing macromolecules such as nucleic acids was not detected in the intact HeLa cell spectrum because of broad lines. Effects of the glycolysis inhibitor iodoacetic acid could be clearly shown in spectra of both the intact cell and the extract as buildup of fructose 1,6-bisphosphate at the expense of ATP, UTP, and creatine phosphate.


RNA polymerase binding sites in lambdaplac5 DNA.

The in vitro binding of the Escherichia coli RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase; EC 2.7.7.6) to fragments of lambdaplac5 DNA generated by restriction endonucleases HindII and HindIII has been studied by a filter binding technique. The results are consistent with RNA polymerase binding at p(R)', the INT promoter (p(I)), several sites in the b2 region, the mis promoter, the oop promoter (or p(O)), and p(rm). Binding was also observed on some fragments that are not known to contain active promoters, including the fragment from the cIII-t(L) region. Some of these binding reactions might also be explained by interaction of RNA polymerase with termination sites. Additional polymerase binding sites have been detected by examining which HindII and HindIII sites were not cleaved when digestion was performed after RNA polymerase had been bound to the DNA. This technique revealed polymerase binding at p(L), at p(R), at a site between R and cos, and at a site at the junction of the gamma and cIII-t(L) fragments. A comparison of the location of polymerase binding fragments with the partial denaturation map of the lambda genome indicates that RNA polymerase binding sites are located within A-T rich regions. It is suggested that RNA polymerase binding is a function both of specific sequences (where recognition occurs) and of the base composition of the surrounding regions (which affects the stability of the helix at the specific site).


Differential effects of cholesterol and lanosterol on artificial membranes.

The effects of cholesterol, 4,4-dimethylcholesterol, and lanosterol (4,4',14alpha-trimethyl-delta8,24-cholestadiene-3beta-ol) on some properties of lecithin vesicles have been compared. Unlike cholesterol, lanosterol retards the exit of trapped glucose from phospholipid vesicles only slightly. The 13C nuclear magnetic resonance spectrum of cholesterol/lecithin vesicles shows no resonances attributable to the sterol. By contrast, several resonances attributable to quaternary carbon atoms or methyl groups are seen in the 13C nuclear magnetic resonance spectrum of lanosterol/lecithin vesicles, indicating that lanosterol is much less immobilized than cholesterol. Because the membrane behavior of 4,4-dimethylcholesterol is closely similar to that of cholesterol, it is concluded that the axial 14-alpha-methyl group is responsible for the lessened membrane immobilization of lanosterol. The results emphasize the importance of a planar sterol alpha-face for interaction with phospholipid acyl chains.


Isolation of a protein scaffold from mitotic HeLa cell chromosomes.

We have recently shown that, after the histones and most of the nonhistone proteins are gently removed from HeLa metaphase chromosomes, the chromosomal DNA is still highly organized and relatively compact. The structure of these histone-depleted chromosomes is due to the presence of a number of nonhistone proteins that form a central scaffold that retains the approximate size and shape of intact chromosomes and to which the DNA is attached, predominantly forming loops. We now demonstrate that the protein scaffold may be isolated independently of the DNA by treating HeLa chromosomes with micrococcal nuclease before removing the histones.The chromosomal scaffolds may be isolated by sucrose density gradient centrifugation as a well-defined peak that is stable in 2 M sodium chloride, but is dissociated by treatment with proteases, 4 M urea, or 0.1% sodium dodecyl sulfate. Polyacrylamide gel electrophoresis reveals that the protein content of scaffold preparations is identical to that of histone-depleted chromosomes. Fluorescence microscopy of purified scaffolds in isolation buffer shows that the particles still possess the familiar chromosome morphology. When the scaffolds are examined in the electron microscope, a fibrous structure with the approximate size and shape of intact, paired chromatids is seen. Less than 0.1% of the chromosomal DNA and virtually no histones are associated with the purified scaffold structures.


Enzymatic formation of glutathione-citryl thioester by a mitochondrial system and its inhibition by (-)erythrofluorocitrate.

A soluble extract of the mitochondrial compartment composed of the inner membrane and matrix catalyzes the enzymatic synthesis and hydrolysis of the 1:1 adduct of citric acid and glutathione. The adduct was identified as the thioester by isolation with single and double isotope labeling ([(14)C]citric acid and [(35)S]glutathione) and by conversion to the monohydroxamate of citric acid and comparison with the synthetic product by thin layer chromatography and high voltage electrophoresis. The enzymatic formation of the thioester (pH optimum 7.39 at 30 degrees ) requires oxidized glutathione and citrate; both substrates exhibit a Michaelis-Menten kinetics. During the enzymatic reaction equimolar quantities of thioester and glutathione sulfinic acid are formed. After gel filtration or salt fractionation the enzyme system requires Mn(2+) (or Mg(2+), which is less effective) for maximal activity. When extracts of mitoplast are tested, the time course of reaction is biphasic due to the rapid synthesis of the product by the thioester-forming system (molecular weight 171,000) followed by its decay by the hydrolase (molecular weight 71,000). The two systems were separated by molecular filtration on Sephadex G-200 and by precipitation with (NH(4))(2)SO(4). The thioester-forming system is inhibited by preincubation with 0.5 mM mersalyl. Other inhibitors are 1,2,3-propane tricarboxylic acid, 10 mM Ca(2+), 200 mM K(+), and the free radical trapping agent, phenazine methosulfate. The citrate-glutathione thioester formation is irreversibly and specifically inhibited by (-)erythrofluorocitrate (50% inhibition at 25 pmol of added fluorocitrate per mg of protein), which forms a trichloroacetic acid-stable adduct with the enzyme protein (at 50% inhibition, 0.8 pmol is bound to 1 mg of protein). Synthesis of malyl-glutathione thioester by inner membrane vesicles is selectively inhibited by (-)erythrofluoromalate.


Candida lipolytica mutants defective in an acyl-coenzyme A synthetase: isolation and fatty acid metabolism.

Mutant strains of Candida lipolytica defective in an acyl-CoA synthetase [acid:CoA ligase (AMP-forming); EC 6.2.1.3]were isolated. The mutant strains apparently exhibited no acyl-CoA synthetase activity in vitro and were, in contrast to the wild-type strain, incapable of growing in the presence of exogenous fatty acid when cellular synthesis de novo of fatty acid was blocked. However, the mutant strains grew on either fatty acid or n-alkane as a sole carbon source at rates comparable to that observed for the wild-type strain. Analysis of the fatty acid composition of the lipids from the mutant cells grown on odd-chain-length fatty acid as well as [14C]oleic acid incorporation studies have shown that the mutant cells, unlike the wild-type cells, cannot incorporate exogenous fatty acid as a whole into cellular lipids, but utilize the fatty acid that is synthesized de novo from acetyl-CoA produced by degradation of exogenous fatty acid. This finding indicates the presence of at least two acyl-CoA synthetases that activate long-chain fatty acid. One, designated acyl-CoA synthetase I, which is absent in the mutant strains, is responsible for the production of acyl-CoA to be utilized for the synthesis of cellular lipids. The other acyl-CoA synthetase provides actyl-CoA that is exclusively degraded via beta-oxidation to yield acetyl-CoA.


Analytical and graphical examination of strong binding by half-of-sites in proteins: illustration with aspartate transcarbamylase.

In multiple binding of ligands to a protein, the binding sites may seem to behave as if they are partitioned equally between two modalities. This paper analyzes three different molecular situations in which two actual assemblages appear: (i) two classes of sites exist at the outset in the ligand-free macromolecule; (ii) all sites are initially identical but after half are occupied, the affinity of the residual ones is altered; (iii) all sites are initially identical but they interact in a pairwise manner. The contours of affinity profiles-graphs of normalized stoichiometric binding constants (iK(i)) versus stoichiometric step number i-are examined for each situation to provide a basis for discriminating among them. Proper procedures for evaluating the site binding constants are then described. To illustrate these procedures, published experimental data for two real systems, binding of substrate or modifier by the enzyme aspartate transcarbamylase (carbamoylphosphate: L-aspartate carbamoyltransferase, EC 2.1.3.2), are scrutinized and the meaning of the calculated binding parameters is examined. The results demonstrate concretely that site binding constants cannot be specified without assuming a particular molecular model, but the stoichiometric constants can be assigned unambiguously without regard to the type of behavior at the individual sites.


Isolation and characterization of the opioid peptides from rat pituitary: beta-endorphin.

beta-Endorphin was isolated from 200 rat pituitaries by means of high-performance column chromatography, using sensitive fluorometric methods and a radioreceptor assay for opioid activity. The beta-endorphin was characterized as to molecular weight, amino acid composition, and mapping of tryptic peptides by a new microtechnique. It was found that rat beta-endorphin is identical to camel and sheep beta-endorphin. Furthermore, alpha-endorphin, Met-enkephalin, a nonpeptide morphine-like compound (MLC), and an additional unidentified opioid compound were detected in the extract of rat pituitary.


Contacts between the lac repressor and the thymines in the lac operator.

We have identified important points of contact between the lac repressor and the lac operator by crosslinking the repressor to bromouracil-substituted operator. We substituted bromouracils for thymines in a 55-base-long restriction fragment containing the lac operator and labeled one or the other 5' end with 32P. Ultraviolet irradiation of this fragment produced single-strand breakds at the bromouracils. We examined breakage at each bromouracil in the sequence by denaturing the DNA and displaying the UV-generated fragments on a polyacrylamide gel. In the presence of lac repressor, UV radiation failed to break at specific sites. We attribute this to a competing reaction in which the DNA crosslinks to the repressor rather than breaking. These crosslinkable sites thus define positions at which the lac repressor protein lies close to the methyl group of a thymine in the major groove of DNA.


Synapse turnover: the formation and termination of transient synapses.

Neurons dissociated from chick embryo retina form synapses with cultured rat striated muscle cells in 35-90 min when neurite extension is uncoupled from later steps in synapse formation. The results suggest that a reaction is required for synapse formation after neurons adhere to muscle cells. All synapses between retina neurons and muscle cells are terminated in 3-10 days depending on the developmental age of the neurons. The half-lives of synapses between muscle cells and retina neurons from 8-, 12-, or 13-day embryos are 36, 26, and 5 hr and mean synapse life-times are 53, 37, and 7.1 hr, respectively. The results show that synapses turn over and that the rate of turnover increases during development. The results suggest that both synapse formation and termination rates are regulated and that the specificity of synaptic connections can be increased by selective termination of synapses.


Fate of lipid vesicles in vivo: a gamma-ray perturbed angular correlation study.

The structural integrity of unilamellar vesicles has been studied in vitro and in vivo by use of gamma-ray perturbed angular correlation techniques. These studies utilize 111In3+ weakly bound to the chelator nitrilotriacetic acid as a probe to monitor the percentage of intact vesicles. When complexes of 111In3+ and nitrilotriacetic acid is encapsulated in vesicles, 111In3+ exhibits a fast tumbling rate. Upon alteration of the membrane, 111In3+ is released from the liposomes and becomes bound to macromolecules, consequently exhibiting a decrease in 111In3+ tumbling rate. The in vitro experiments show that the present technique is capable of determining quantitatively the percentage of material released from the vesicles upon the addition of serum or Triton X-100 to vesicles. The percentage of vesicles remaining intact in vivo can also be monitored continuously by the present technique. In mice, the half-life of dipalmitoyl phosphatidylcholine-cholesterol vesicles after intraperitoneal injection was estimated to be 10-13 hr. The present study suggest that the vesicles remain intact in various tissues for extended periods, thereby allowing a slow release of the encapsulated material at those sites.


Emission Mössbauer study of the stereochemical trigger that initiates cooperative interaction of hemoglobin subunits.

An important feature of Perutz's trigger mechanism for cooperativity in the reversible oxygenation of hemoglobin (Hb) is the tension along the histidine--metal linkage in deoxyHb and deoxycobaltohemoglobin (deoxy CoHb), supposedly due to the pull exerted by the globin on the metal atom. We have attempted to verify the existence of this pull by studying the emission Mössbauer spectra of deoxy 57CoHb and oxy 57 CoHb at different temperatures. The emission Mössbauer spectrum for none of the cobalt Hbs agrees with the absorption spectrum of the corresponding iron analog and, moreover, the spectrum of deoxy 57CoHb is characteristic of the intermediate-spin iron. These observations indicate that the daughter 57Fe atom is "frozen" almost in the same spatial situation as that of the parent 57Co. The protein is apparently holding the cobalt atom in position rather rigidly and, after the electron-capture decay of the 57Co atom, the protein does not permit the daughter 57Fe to move to a position characteristic of the iron atom.


Definition of subclasses of nucleoplasmic RNA.

RNA synthesis in isolated HeLa cell nuclei prepared from cells pretreated with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) is inhibited in a time-dependent manner. After 40-min pretreatment of cells with 60 muM DRB in the presence of actinomycin D (0.04 mug/ml), the rate of RNA synthesis in isolated nuclei, measured by [(3)H]UTP incorporation, is decreased by 63%. The DRB-resistant one-third of heterogeneous nuclear is distributed over the entire size range of heterogeneous nuclear RNA with some enrichment in the 18S range, as was observed earlier by pulse-labeling whole cells. A subclass of nucleoplasmic RNA molecules is defined in the approximate size range 110 to 250 x 10(3) daltons (330-740 nucleotides). By using heparin (2 mg/ml) to block the synthesis of smaller RNA, a peak in the chain-length range 330-740 nucleotides can be clearly resolved on 2.2% polyacrylamide/1% agarose gels in nuclei from control and DRB-treated cells. The synthesis of these molecules is largely ( approximately 90%) resistant to DRB but sensitive to alpha-amanitin at 1 mug/ml. The in vitro synthesis of molecules in the 140-330 residue range is also sensitive to alpha-amanitin at 1 mug/ml, and it is not at all affected by pretreatment of cells with DRB. Although the synthesis of the RNA in both the 330-740 and the 140-330 residue size ranges appears to be catalyzed by RNA polymerase II, the results with heparin suggest that there may be reinitiation of molecules in the 140-330 size range but not in the 330-740 range in vitro. The synthesis of 4.5S RNA ( approximately 100 nucleotides) and 5S RNA (120 nucleotides) is unaffected by pretreatment of cells with DRB and, as previously reported, is catalyzed by RNA polymerase III, with reinitiation occurring in vitro. Addition of DRB directly to isolated HeLa cell nuclei in vitro has no detectable effect on the overall rate of RNA synthesis.


Noncoincidence of histone and DNA synthesis in cleavage cycles of early development.

The times of histone and DNA synthesis do not coincide in early cleavage of the sea urchin embryo. In fact, the production of histones increases during the interval after DNA synthesis (G2). Enucleate merogones, parthenogenetically activated, synthesize histones encoded upon maternal histone messenger RNA. The pattern of protein synthesis changes following fertilization, in part, but not solely, due to the increasing synthesis of histones relative to other proteins. Regulation of histone synthesis and the loading of newly replicated DNA with histones must themselves undergo change at the time of transition from cleavage cycles to cycles more typical of somatic cells.


Stimulus-secretion coupling in chromaffin cells isolated from bovine adrenal medulla.

Bovine adrenal chromaffin cells were isolated by removal of the cortex and sequential collagenase digestion of the medulla. The catecholamine secretory function of these cells was characterized with respect to acetylcholine stimulation, cation requirements, and cytoskeletal elements. The dose-response curve for stimulated release had its half-maximum value at 10(-5) M acetylcholine, and maximum secretion was on the average 7 times that of control basal secretion. The differential release of epinephrine versus norepinephrine after stimulation with 0.1 mM acetylcholine occurred in proportion to their distribution in the cell suspension. The cholinergic receptors were found to be predominantly nicotinic. The kinetics of catecholamine release were rapid, with significant secretion occurring in less than 60 sec and 85% of maximum secretion within 5 min. A critical requirement for calcium in the extracellular medium was demonstrated, and 80% of maximum secretion was achieved at physiologic calcium concentrations. Stimulation by excess potassium (65 mM KCl) also induced catecholamine secretion which differed from acetylcholine stimulation in being less potent, in having a different dependence on calcium concentration, and in its response to the local anesthetic tetracaine. Tetracaine, which is thought to inhibit membrane cation permeability, was able to block acetylcholine-stimulated but not KCl-stimulated secretion. The microtubule disrupting agent vinblastine was able to block catecholamine release whereas the microfilament disrupter cytochalasin B had little effect. The results show the isolated bovine chromaffin cells to be viable, functioning, and available in large quantity. These cells now provide an excellent system for studying cell surface regulation of hormone and neurotransmitter release.


Polypeptide hormone binding sites in vivo: initial localization of 125I-labeled insulin to hepatocyte plasmalemma as visualized by electron microscope radioautography.

The rationale of the specific-binding assay was applied to the detection of the liver insulin receptor in vivo. Quantitative electron microscope radioautography indicated that, 3 min after an intraportal injection, 125I-insulin was exclusively located to the hepatocyte plasmalemma.


Selective loss of wheat germ agglutinin binding to agglutinin-resistant mutants of Chinese hamster ovary cells.

The binding of 125I-labeled wheat germ agglutinin (WGA) to parental and three distinct WGA-resistant Chinese hamster ovary cell lines possessing modified cell surface carbohydrate structures has been examined over a 10(6)-fold range of WGA concentrations. The Scatchard plot for WGA binding to parental cells was complex and exhibited positively cooperative binding at the high affinity sites. One of the WGA-resistant mutants (WgaRIII) was apparently not altered in its WGA-binding ability compared with parental cells. However, two of the WGA-resistant lines (WgaRI and WgaRII) had distinct alterations in their WGA-binding properties specific to certain regions of the binding curve. Neither appeared to be affected in either the highest or lowest affinity regions of the binding curve. Thus, lectin-resistant cell mutants altered in specific lectin-binding sites at the cell surface provide a direct approach to analysis of the complex binding parameters that characterize the interaction of WGA with the plasma membrane.


Luminal membrane retrieved after exocytosis reaches most golgi cisternae in secretory cells.

Dextran was used to trace membrane retrieved from the luminal surface after induced exocytosis in secretory cells of rat lacrimal and parotid glands. Two different approaches were used: (a) isolated acini were incubated in vitro with dextran followed by stimulation with carbamylcholine (lacrimal) or isoproterenol (parotid) and (b) rats were injected with isoproterenol followed by dextran infusion into the parotid duct in vivo. The main findings were the same regardless of the gland source or experimental approach. Dextran was taken up initially via coated pits into smooth-surfaced apical vesicles. Shortly thereafter it was found in multiple cell compartments: within the stacked Golgi cisternae, in condensing vacuoles, and in lysosomes. Uptake was more rapid and uniform in vivo; dextran was seen in multiple cisternae of numerous Golgi complexes within 5 min after infusion. In acini incubated in vitro uptake into Golgi cisternae was more delayed and occurred with increasing frequency up to 60 min; also, more dextran was taken up into lysosomes, which were more numerous in vitro than in vivo. The results demonstrate that, after exocytosis, membrane is removed from the cell surface via vesicles that fuse with multiple cell compartments. The two novel findings are: (a) the demonstration that the tracer can reach most of the Golgi cisternae in a given stack and (b) the demonstration of the rapidity with which the process takes place (i.e., within 5 min). The findings imply that at least some membrane retrieved from the cell surface after exocytosis fuses with the stacked Golgi cisternae.


Hemoglobin switching in sheep and goats: induction of hemoglobin C synthesis in cultures of sheep fetal erythroid cells.

Synthesis of HbF (alpha2gamma2) is replaced by synthesis of Hb A (alpha2betaA2) shortly before birth in sheep homozygous for the betaA globin chain whereas Hb C (alpha2betaC2) is produced transiently during the neonatal period. We have obtained a Hb F-to-Hb C switch by generating erythroid colonies at high erythropoietin concentration in plasma clot cultures of mid-gestation fetal bone marrow or liver. Furthermore, high erythropoietin concentration appeared specifically to activate the gene for betaC and not those for betaA or betaB globin in colonies grown from cells of an animal heterozygous for the betaA and betaB genes. Erythropoietic stress in the form of periodic bleeding or erythropoietin injection in utero did not stimulate production of Hb C (alpha2betaC2) in fetal sheep until shortly before birth, and then only in two of six animals. Thus, factors other than erythropoietin may influence the potential for betaC globin synthesis in vivo in fetal sheep.


Is evolution gradual or rectangular? Evidence from living fishes.

The traditional view that most evolutionary change is gradual and cumulative within lineages (phyletic gradualism) has recently been challenged by the proposition that the majority of evolutionary change is concentrated within speciation events (rectangular evolution). The logical implications of these competing hypotheses for the means and variances of genetic distance among living members of rapidly and slowly speciating phylads are examined. An example of a critical test of gradual versus rectangular evolution is provided by electrophoretic analyses of genic composition in 69 species of North American Cyprinidae (minnows), and 19 species of Centrarchidae (sunfish). Rate of protein evolution appears somewhat decelerated, if anything, in the rapidly speciating minnows. Results are inconsistent with predictions of rectangular evolution, but are not demonstrably incongruent with predictions of phyletic gradualism.


Phylogenetic structure of the prokaryotic domain: the primary kingdoms.

A phylogenetic analysis based upon ribosomal RNA sequence characterization reveals that living systems represent one of three aboriginal lines of descent: (i) the eubacteria, comprising all typical bacteria; (ii) the archaebacteria, containing methanogenic bacteria; and (iii) the urkaryotes, now represented in the cytoplasmic component of eukaryotic cells.


Error estimate for the diffusion approximation of the Wright--Fisher model.

The Wright--Fisher model and its approximating diffusion model are compared in terms of the expected value of a smooth but arbitrary function of nth-generation gene frequency. In the absence of selection, this expectation is shown to differ in the two models by at most a linear combination (with coefficients depending only on the derivatives of the smooth function involved) of the maximum mutation rate and the reciprocal of the population size.


Chloroplast DNA distribution in parasexual hybrids as shown by polypeptide composition of fraction I protein.

Sixteen different mature interspecific parasexual hybrids, produced by fusing leaf protoplasts of Nicotiana glauca (G) and N. langsdorffii (L), were analyzed for fraction I protein (ribulose-1,5-bisphosphate carboxylase/oxygenase) which consists of large subunit polypeptides coded by chloroplast DNA and small subunit polypeptides coded by nuclear DNA. All the hybrids showed the combined small subunits of both parents, thus confirming the hybridity of each of the fusion products. Fourteen of the hybrids displayed the large subunit electrofocusing pattern characteristic of only one parent (eight L and six G). From one hybrid callus, two plants were regenerated, of which one had exclusively L-type large subunit and the other had exclusively G. A single plant retained a mixture of L ang G chloroplast DNA's; this later yielded six F2 progeny from one branch, all of which were G type, and three asexual progeny from another branch, all of which had the L-type pattern. In all, 46 F2 progeny and 8 different F3s were analyzed and each of these, with few if any exceptions, showed the same single subunit type as the F1 and F2 parent hybrid plants. Reasons for the rapid sorting out of the chloroplast types are discussed.


Reduction of syngeneic tumor growth by an anti-I-J-alloantiserum.

Highly significant suppression of the growth of S1509a and Sa-I syngeneic sarcomas was observed in A/J mice following daily intravenous injections of 2 microliter of anti-I-Jk alloantiserum. This effect persisted as long as the anti-I-Jk serum was administered (day 15), In contrast, a control anti-I-Js serum had no discernible effect on the growth of the S1509a tumor. The inhibitory activity of the anti-I-Jk serum on the growth of the tumor was absorbed specifically by B10-BR spleen cells bearing I-Jk determinants. In other experiments, we established that A/J mice treated with anti-I-Jk serum, according to the protocol described above, are no longer a source of tumor-specific suppressor cells for adoptive transfer into immune tumor-bearing recipient mice. We conclude that anti-I-Jk serum inhibits tumor growth in A/J mice by abolishing tumor-specific suppressor activity.


Idiotypic-anti-idiotypic regulation of antibody synthesis in rabbits.

Anti-ribonuclease antibodies (Ab1) of one rabbit were used to prepare in two other rabbits anti-idiotypic antibodies (Ab2) against them. Anti-idiotypic antibodies (Ab3) were prepared against Ab2 of one rabbit in two other rabbits that had the same phenotype as the former three for the allotypic a and b systems. After a rest period of 4 months, the latter two rabbits were immunized against ribonuclease. The antibodies produced (Ab1') crossreacted with the two anti-idiotypic sera (Ab2) against Ab1, showing an idiotypic similarity between anti-ribonuclease Ab1' and anti-ribonuclease Ab1. No such crossreaction was observed between the anti-idiotypic sera (Ab2) against Ab1 and any of the 12 unrelated antisera against ribonuclease that were tested. These results suggest (i) that rabbits, at least with the same allotypes, possess a closely related idiotypic repertoire; and (ii) that the immune system is a network of variable (V)-domains.


Chemical characterization of murine Ia alloantigens determined by the i-E/i-C subregions of the H-2 complex.

Alloantisera directed against the alloantigens determined by the I-E and I-C subregions of the murine major histocompatibility complex precipitate two components that have molecular weights of 35,000 and 29,000. These components, when analyzed by partial NH2-terminal sequencing, show no homology to two components of similar size determined by the I-A subregion. However, the large chain determined by the murine I-E and/or I-C subregion is homologous to the large chain of the human HLA-D region alloantigen, although the small chains isolated from these two species do not display any such homology.