Cloning of aroG, the gene coding for phospho-2-keto-3-deoxy-heptonate aldolase(phe), in Escherichia coli K-12, and subcloning of the aroG promoter and operator in a promoter-detecting plasmid

Defective transducing phages carrying aroG, the structural gene for phenylalanine (phe)-inhibitable phospho-2-keto-heptonate aldolase (EC 4.1.2.15; previously known as 3-deoxy-D-arabinoheptulosonate-7-phosphate synthetase[phe]), have been isolated, and DNA from two of these phages has been used to construct a restriction map of the region from att lambda to aroG. A 7.6-kb PstI-HindIII fragment from one of these phages was cloned into pBR322 and shown to contain aroG. The location of aroG within the 7.6 kb was established by subcloning and Tn3 transpositional mutagenesis. A fragment carrying the aroG promoter and operator has been cloned into a high copy number promoter-cloning vector (pMC489), and the resulting aroGpo-LacZ' (alpha) fusion subcloned in a low copy number vector. Strains with this fusion on the low copy number vector exhibit negative regulation of beta-galactosidase expression by both phenylalanine and tryptophan and positive regulation by tyrosine in a tyrR+ background.     

Poly-N-Acetyllactosamine Glycans of Cellular Glycoproteins: Predominance of Linear Chains in Mouse Neuroblastoma and Rat Pheochromocytoma Cell Lines

To study the properties of protein-bound oligosaccharides in neuronally differentiating cells, two model systems were used: murine N1E-115 and N-18 neuroblastoma cells inducible by serum starvation and rat PC12 pheochromocytoma cells inducible by nerve growth factor. Glycopeptides were prepared from cells metabolically labeled with [3H]glucosamine and analyzed by gel filtration. The properties of the high-molecular-weight glycopeptides were studied using enzymatic digestion with neuraminidase and endo-beta-galactosidase. In contrast to other cell lines analyzed, the neuroblastoma and pheochromocytoma lines contained predominantly glycopeptides completely cleavable with endo-beta-galactosidase, which indicated that they were linear-type poly-N-acetyllactosamine glycans. The proportion of these linear chains in the high-molecular-weight fraction increased during neuronal differentiation in both cell systems. The linear nature of the glycans was also correlated with positive anti-i and negative anti-I reactivity of the cells in immunofluorescence microscopy. Specific cell surface labeling for poly-N-acetyllactosamine glycans and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed several glycoprotein components, some of which showed changes during neuronal differentiation. The high proportion of linear poly-N-acetyllactosamine chains in these neuronal cell lines and its increase during neuronal differentiation suggests that these glycans may be a characteristic feature of neuronal or neuronally differentiating cells.     

Unsuccessful induction of low-zone tolerance to bovine serum albumin injected into the subarachnoid space

Introduction of T-dependent antigens into the subarachnoid space (isas) resulted in higher systemic antibody responses in mice than injections into the peritoneal cavity (ip) or other sites commonly used for immunization. Antibody production in isas immunized mice was not increased by treatment with cyclophosphamide (Cy) at doses known to abolish T-suppressor-cell activity, but such treatment increased antibody production in ip immunized mice toward the higher level which was observed in the isas immunized animals. Suppressor cell-dependent low zone tolerance (LZT) to TNP-BSA could not be induced by isas injections of deaggregated BSA (d-BSA). Conversely, mice which were unresponsive to ip injected d-BSA showed consistent systemic antibody responses when the antigen was injected isas. These observations indicate that immune responses initiated within the CNS are associated with relatively ineffective induction of systemic suppressor cell activity.     

Structural comparison of the two distinct sugar binding sites in wheat germ agglutinin isolectin II

The two unique sugar binding sites in wheat germ agglutinin, located in the subunit/subunit interface of the dimer molecule and termed primary and secondary binding sites, are compared in the light of the newly obtained chemical amino acid sequence and a high-resolution electron density map (1.8 A). Homology was found in the three amino acid residues directly involved in sugar binding: Tyr73II, Ser62II, Glu115I in the primary site, and Tyr159I, Ser148I, Asp29II in the secondary site (subscripts refer to promoters I and II). Thirteen corresponding side-chain atoms of these three homologous residues in the two sites could be superimposed with a root-mean-square difference of 1.39 A. The three sugar binding residues are located in subsite 1 of each extended binding location and contribute to binding of the terminal, non-reducing N-acetyl-D-glucosamine and N-acetyl-D-neuraminic acid residues only, and they provide three hydrogen bonds for complex stabilization. Two hydrogen bonds are made with the carbonyl and amido portions of the N-acetyl group and the third with the C-3 OH group of the sugar ring. It is suggested that small differences in the sugar binding affinities at these two unique sites exist, due to the different numbers of van der Waals' interactions made at these sites, which contribute to stabilizing, for instance, the wheat germ agglutinin/N,N'-diacetyl-chitobiose complex. The single tryptophan residue is located at a distance of approximately 13 A from the primary site and is thought to have no affect on sugar binding. In addition, the disposition of the four saccharide binding sites of the dimer with respect to three local, pseudo 2-fold symmetry axes, relating domains of opposite protomers, is discussed.     

Biochemical and autoradiographic studies of TRH receptors in sections of rabbit spinal cord

Receptors for thyrotropin-releasing hormone (pGlu-His-Pro-NH2, TRH) on thaw-mounted sections of rabbit spinal cord have been identified biochemically and visualized by light microscopic autoradiography. Binding of [3H] [3-Me-His2]TRH to 20 microns sections exhibited high apparent affinity and a pharmacological specificity almost identical to that previously demonstrated for spinal TRH receptors in membranes. In autoradiograms, the highest density of TRH receptors appeared in the substantia gelatinosa of the dorsal gray and around the central canal, with intermediate levels in the ventral gray.     

The Total Chemical Synthesis of Monocyte Chemotactic Protein-1 (MCP-1)

The affinity-based N^α-amino protecting group tetrabenzo [a,c,g,i]fluorenyl-17-methoxycarbonyl (Tbfmoc) has been utilized as a hydrophobic probe to allow the simple, quick and highly effective isolation of a 76 residue cysteine-containing protein (MCP-1). The base-labile Tbfmoc group can be removed under very mild conditions, which preserve the thiol-con taining protein in the reduced state. Oxidative folding was then used to furnish the biologically active β-chemokine MCP-1.     

Autoradiography of nucleoside uptake into the retina

A selective uptake mechanism for some nucleosides and related substances was found in retinae of light adapted rabbits and fish. After the intravitreal injection in vivo of [³H]adenosine, [³H]inosine, [³H]guanosine and certain related compounds, the distribution of radioactivity was studied by autoradiography. Retinae were also incubated in [³H]adenosine and [³H]inosine and then were similarly processed.In rabbits, the accumulation of radioactivity from [³H]adenosine and [³H]guanosine was predominantly into glial cells, but also into neurons. [³H]Inosine labelled glia almost exclusively. However, the adenosine analog, [³H]methylphenylethyl-adenosine, resulted in well-defined neuronal labelling in this species. In fish, a few photoreceptor cell bodies exhibited strong radioactivity with the nucleosides, presumably representing incorporation into nucleic acids of replicating cells. Labelling was also seen in horizontal cells, amacrine cells and ganglion cells after the injection of either [³H]adenosine, [³H]guanosine or [³H]inosine.To some extent, the selective accumulation of radioactivity is likely to be due to cell replication, but in most neurons, other factors must be responsible. Judging from what is known about the actions of adenosine in central nervous tissue, signal transmission in the retina could be such a factor.     

Colcemid treatment of myeloma prior to cell fusion increases the yield of hybridomas between myeloma and splenocyte

Effect of Colcemid treatment of myeloma (X63-Ag8-6.5.3.) prior to fusion with mouse spleen cell was studied in terms of hybridoma formation. Spleen cells from BALB/c mice immunized with various soluble antigens were fused with the myeloma cells by using polyethylene glycol solution. Colcemid treatment of myeloma cells prior to fusion increased the average number of hybridoma colonies per well by 26-570%. The yield of hybridomas producing antigen-specific antibodies was also higher with the Colcemid treatment. The results suggest that most of the proliferative hybridomas are formed by fusion of cells in the M-phase of the cell cycle.     

Cloning and expression of the phage Mu A gene

We have cloned the phage Mu A gene, with and without the gene ner, under the control of the pL promoter of phage lambda in a multicopy plasmid vector. We demonstrate that plasmid-carrying cells are able to support growth of superinfecting Mu A am phages in a temperature-dependent fashion in a host strain carrying a defective lambda prophage which specifies the cI857-coded lambda repressor. In addition, we show that the presence of the ner gene reduces the efficiency of plating of the superinfecting phage. Analysis of proteins specified by the cloned Mu fragments indicates that two proteins, 70 and 33 kDal, are synthesized. The level of synthesis, compared to that of the vector-encoded beta-lactamase, was found to increase with temperature. This indicates that their transcription is driven by the pL promoter. The Mr of the 70-kDal protein is identical to that previously observed for pA.     

Characteristics of androgen binding in rat adrenal tissue using [3H]methyltrienolone (R1881) as tracer

A high level of binding of [3H]methyltrienolone (R1881 = 17beta-hydroxy-17alpha-methyl-estra-4, 9, 11-trien-3-one) was found in cytosol prepared from adrenals of castrated male rats. Binding of [3H]R1881 was of high affinity (DK = 6.2 nM) and highly specific for androgens. The [3H]R1881 complex migrates at 7-9S on sucrose gradients in low ionic strength buffer and at 4-5S in buffer containing 0.4M KC1. All binding studies have been performed in parallel with rat ventral prostate and adrenal cytosol. The present data suggest the presence of an androgen binding component in rat adrenal tissue.