The small nuclear RNAs of Drosophila

We have investigated the sequences of the major small nuclear RNAs of Drosophila cultured cells, with the objective of elucidating phylogenetically conserved primary and secondary structures by comparison of the data with previously determined sequences of these RNAs in vertebrate species. Our results reveal striking degrees of conservation between each Drosophila RNA and its vertebrate cognate, and also demonstrate blocks of homology among the Drosophila small nuclear RNAs, as previously described for vertebrates. The most conserved features include the 5' terminal region of U1 RNA, though to function in pre-mRNA splicing, most of the regions of U4 RNA recently implicated in 3' processing of pre-mRNA, and the major snRNP protein binding site ("domain A") that is also shared by vertebrate U1, U2, U4 and U5 RNAs. Several other conserved features have been revealed, suggesting additional regions of functional significance in these RNAs and also providing further insights into the evolutionary history of the small nuclear RNAs.     

Glutamine synthetases from rice: purification and preliminary characterization of two forms in leaves and one form in roots

A relatively rapid five-step procedure was used in purifying to apparent homogeneity the glutamine synthetase from roots and one form of the enzyme (GS_I) from leaves of rice. The steps were: preparation of crude extracts, ammonium sulfate precipitation, filtration on Sepharose 4B, fractionation on DEAE-Sephadex A25, and affinity chromatography on ADP-Sepharose 4B. The purified protein appeared as a single band on polyacrylamide gel electrophoresis. Leaf GS_I and the second type of leaf glutamine synthetase (GS_II) formed distinct peaks when eluted from DEAE-Sephadex (step 4). The root enzyme and leaf GS_I were similar in all the properties which were examined. Both enzymes bound to ADP-Sepharose, had similar biosynthetic (18 μmol P/_img protein/min) and transferase (1324 and 1156 μmol γ-glutamyl hydroxamate/mg protein/min) activities, and the same or nearly the same K_m values for glutamate (2.17 mm), Mg²⁺ (4.5 and 5.0 mm), ATP (286 μm), NH₄⁺ (210 and 135 μm), and ADP (3.8 and 5.3 μm). In contrast, leaf GS_II did not bind to ADP-Sepharose and had much higher K_m values for glutamate (8.3 mm), Mg²⁺ (15 mm), NH₄⁺ (684 μm), and ADP (33 μm).     

A near-infrared method for studying hydration changes in aqueous solution: Illustration with protease reactions and protein denaturation

Proteins undergoing protease reactions, heat denaturation, or interactions with sodium dodecyl sulfate (SDS) were used to demonstrate the effectiveness of a near-infrared method for the quantitative study of changes in hydration or water binding during such processes. The spectra of different proteins showed that the liberation of $\text{COO}{}^{\mathrm{?}}\text{and NH}{}_3{}^{\mathrm{+}}$ groups during a protease reaction is associated with a large increase in hydration and excluded volume. On the basis of experiments with model compounds, other spectral changes, including development of continuum absorbance between 1.55 and 1.85 μm and a band with a peak near 2.1 μm, were also attributed to the liberation of these groups. After heat denaturation or in the presence of SDS, the rate of proteolytic hydrolysis was markedly increased, consistent with the view that some preliminary denaturation is necessary for protease activity. The validity of the hydration changes calculated for protease reactions was supported by model studies with l-lysine, and with poly-l-lysine before and after hydrolysis. The near-infrared spectrum of the protein substrate with no added protease was largely unaffected by heat treatment alone, indicating that the hydration as such was not changed to a large extent by the structural modifications of denaturation. In contrast to the protease reaction, the interactions between SDS and the proteins resulted in a decrease in hydration. Results of this paper are compared with those obtained from other methods. Some unique advantages of the near-infrared method for the study of hydration changes during reactions in aqueous solution are described.     

In vivo regulation of glutamine synthetase by ammonium in the cyanobacterium Anabaena L-31

In cell-free preparations of NH₄⁺-grown cultures of the cyanobacterium Anabaena L-31 the glutamine synthetase activity is only half as much as in N₂-grown cultures. Using a procedure which enables quantitative purification of the enzyme to homogeneity it has been shown that the decrease in the enzyme activity is caused by NH₄⁺-mediated repression. Glutamine synthetase activity in both N₂-grown and NH₄⁺-grown Anabaena remains stable for more than 24 h in the presence of chloramphenicol suggesting low enzyme turnover and an enzyme half-life greater than the generation time (16–18 h) of the cyanobacterium. In N₂-grown cultures, a drastic decrease in the enzyme activity by exogenous NH₄⁺ can be discerned when fresh protein synthesis is prevented by chloramphenicol. The enzyme purified from such cultures has K_m values for NH₄⁺, glutamate Mg²⁺, and ATP similar to those observed for the enzyme from N₂- and NH₄⁺-grown Anabaena, but shows depression in V for all the substrates, leading to drastic decrease in specific activity. The modified enzyme also shows a sharper thermal denaturation profile. These results indicate that NH₄⁺-mediated modification to a less active form may be a means of regulation of glutamine synthetase in N₂-fixing cultures of Anabaena.     

The mitochondrial genome of the fission yeast Schizosaccharomyces pombe. The cytochrome b gene has an intron closely related to the first two introns in the Saccharomyces cerevisiae cox1 gene

The DNA sequence of the cob region of the Schizosaccharomyces pombe mitochondrial DNA has been determined. The cytochrome b structural gene is interrupted by an intron of 2526 base-pairs, which has an open reading frame of 2421 base-pairs in phase with the upstream exon. The position of the intron differs from those found in the cob genes of Saccharomyces cerevisiae, Aspergillus nidulans or Neurospora crassa. The Sch. pombe cob intron has the potential of assuming an RNA secondary structure almost identical to that proposed for the first two cox1 introns (group II) in S. cerevisiae and the p1-cox1 intron in Podospora anserina. It has most of the consensus nucleotides in the central core structure described for this group of introns and its comparison with other group II introns allows the identification of an additional conserved nucleotide stretch. A comparison of the predicted protein sequences of group II intronic coding regions reveals three highly conserved blocks showing pairwise amino acid identities of 34 to 53%. These regions comprise over 50% of the coding length of the intron but do not include the 5' region, which has strong secondary structural features. In addition to the potential intron folding, long helical structures involving repetitive sequences can be formed in the flanking cob exon regions. A comparison of the Sch. pombe cytochrome b sequence with those available from other organisms indicates that Sch. pombe is evolutionarily distant from both budding yeasts and filamentous fungi. As was seen for the Sch. pombe cox1 gene (Lang, 1984), the cob exons are translated using the universal genetic code and this distinguishes Sch. pombe mitochondria from all other fungal and animal mitochondrial systems.     

Immune responses to hapten--lipopolysaccharide conjugates. 1. Modulation of in vivo antibody responses to the polysaccharide

The immune responses of mice to various lipopolysaccharides (LPS) and hapten-LPS conjugates were compared. We found that some strains of mice ($\text{A}\text{J}$ and BALB/c) produced equivalent amounts of anti-LPS antibody after the injection of either LPS or hapten-LPS conjugates. In contrast, however, other strains of mice (C57BL/6J, C3H/St, DBA/1J, DBA/2J, and Swiss) produced fewer anti-LPS-antibody-secreting cells after stimulation with hapten-LPS conjugates than did mice injected with unsubstituted LPS. The covalent coupling of hapten to LPS changed neither the mitogenic capacity nor the antigenicity of the LPS. The differences in the magnitude of antibody responses to hapten-LPS and LPS in these latter strains of mice occurred in the absence of mature T lymphocytes and was restricted to the primary immune response. Furthermore, these differential responder mice (C57BL/6J) did produce anti-LPS antibody when primed with LPS before challenge with the hapten-LPS conjugate. These data are discussed with respect to both the modulatory capacity of the hapten-LPS in the regulation of the primary immune response to LPS and the biochemical and structural requirements of the hapten-LPS conjugate for immunogenicity.     

Selective inactivation of NADPH-cytochrome P-450 (c) reductase by diazotized 3-aminopyridine adenine dinucleotide phosphate

Cyanogen-bromide cleaved glucagon has been extensively purified in yields of 80–85% by the use of gel filtration and by cation-exchange chromatography at pH 4.5–5.2. This pH range maintains a charge difference between the holohormone and its cleavage product, the truncated homoserine lactone derivative, yet maintains the integrity of the lactone ring. Purity is determined by the lack of methionine and the presence of homoserine following peptide hydrolysis. The homoserine lactone is opened by treatment with 0.2 n triethylamine at pH 9.5. The lactone can be reformed by treatment with trifluoroacetic acid for 1 h at room temperature although protection against photooxidation of tryptophan-25 must be provided. The homoserine lactone form binds less well to glucagon receptors than does the homoserine form. Adenylate cyclase is activated by the lactone to an extent comparable to that obtained by native hormone but at elevated concentrations. The procedures described may be useful for purification of other cyanogen bromide cleavage products and is useful for semisynthetic methods based upon cyanogen bromide-cleaved derivatives of glucagon.     

Determination of protein synthesis initiation factor levels in crude lysates of Escherichia coli by a sensitive radioimmune assay.

Antisera specific for protein synthesis initiation factors IF1, IF2, and IF3 were prepared by immunizing rabbits. When crude cell lysates are analyzed by double immunodiffusion or by immunoelectrophoresis, each antiserum forms a single precipitin line antigenically identical to its cognate factor. The antisera do not crossreact with other initiation factors or with ribosomal proteins. A radioimmune assay was developed for each initiation factor by using the specific antisera and radioactive factors prepared by reductive alkylation with [¹⁴C]formaldehyde. The assays detect as little as 10 to 30 ng of factor. Initiation factor concentrations were measured in crude Escherichin coli MRE600 extracts prepared from cells grown exponentially in a rich medium. The three initiation factors are present in approximately stoichiometric amounts and comprise about 1% of the cell protein. The molar ratio of initiation factors to ribosomes is about 0.15, which corresponds to the concentration of native ribosomal subunits.     

Non-random loss of uninterrupted ribosomal DNA repeating units upon induction of a bobbed mutation

To investigate the physical organization of ribosomal RNA genes of two bobbed (bb) loci carried by the Dp(1;f)122 free duplication, a wild type and a deleted one derived from it, genomic DNAs from XXNO-/Dp122bb+ and XXNO-/Dp122bb adult females were analyzed by restriction enzyme digestions. We found that in the bb mutant there was a loss of uninterrupted genes, while genes interrupted by type I and type II insertions remained apparently unchanged. This is an indication that at least in this wild type bb+ locus, carried by the 122 free duplication, the different repeating units are not distributed randomly. In fact, after digestion of the rDNA carried by the bb+ duplication with the enzyme BamHI that cuts only in type I insertions, we have obtained long uncleaved fragments of DNA containing uninterrupted genes.     

The atypical RNA components of cytoplasmic ribosomes from Crithidia fasciculata

RNA extracted by cold phenol from the large cytoplasmic ribosomal subunit of the trypanosomatid flagellate Crithidia fasciculata and analyzed by polyacrylamide gel electrophoresis at 4 °C consisted of one species with a molecular weight of 1.3 × 10⁶ (relative to ribosomal RNA from E. coli MRE 600). When extracted with hot phenol (65 °C), the large ribosomal subunit gave rise to two components with molecular weights of 0.72 and 0.56 × 10⁶. On heating for 60 s, followed by rapid cooling, the single cold-phenol-extracted 1.30 × 10⁶-dalton species completely dissociated into two components of molecular weights 0.72 and 0.56 × 10⁶, present in equimolar amounts. When analyzed by polyacrylamide-agarose gel electrophoresis in the presence of SDS, RNA extracted by cold phenol from the large cytoplasmic ribosomal subunit consisted of three components of molecular weights 1.3, 0.72, and 0.56 × 10⁶, present in apparently equimolar amounts. RNA from the small cytoplasmic ribosomal subunit consisted of one species with a molecular weight of 0.84 × 10⁶, independent of extraction or analytical conditions. It is proposed that under high salt and low temperature conditions, the large ribosomal RNA molecule is held together by its secondary structure, and that denaturing extraction or analytical conditions reveal an otherwise “hidden” lesion present in the molecule in vivo.     

Characterization and localization of the naturally occurring cross-links in vaccinia virus DNA

The vaccinia virus genome is a single, linear, duplex DNA molecule whose complementary strands are naturally cross-linked. The molecular weight has been determined by contour length measurements from electron micrographs to be 122 ± 2.2 × 10⁶. Denaturation mapping techniques indicate that the nucleotide sequence arrangement of the DNA is unique. Two forms of cross-linked vaccinia DNA were observed in alkaline sucrose gradients. The relative S-values of the two cross-linked species were appropriate for a single-stranded circle and a linear single strand, each with a molecular weight twice that expected for an intact, linear, complementary strand of vaccinia DNA. The fraction of sheared vaccinia DNA able to “snap back” after denaturation suggested a minimum of two crosslinks per molecule. Full-length single-stranded circles were observed in the electron microscope after denaturation of vaccinia DNA. Partial denaturation produced single-stranded loops at the ends of all full-length molecules. Exposure of native vaccinia DNA to a single strand-specific endonuclease isolated from vaccinia virions caused disruption of the cross-links, as assayed by alkaline sedimentation, and produced free single-strand ends when partially denatured DNA was observed in the electron microscope. We conclude that vaccinia DNA contains two cross-links, one at or near (within 50 nucleotides) each end in a region of single-stranded DNA. Two models for the cross-links are presented.     

Isolation and characterization of the hydrophobic COOH-terminal domains of the Sindbis virion glycoproteins

Digestion of intact Sindbis virions with α-chymotrypsin produced a single membrane-associated peptide derived from each of the two virion glycoproteins (referred to as RE1 and RE2, or roots derived from E1 and E2, respectively). Amino acid composition data and NH₂-terminal sequence analysis established their location at the extreme COOH-terminal end of each glycoprotein. RE1 and RE2 are rich in hydrophobia amino acids and insoluble in aqueous solutions in the absence of detergents, and show differential solubility in organic solvent systems designed for the extraction of lipids. Essentially all of the covalently attached palmitic acid associated with E1 and E2 was found to be clustered in their hydrophobic, membrane-associated roots. Beginning six to seven residues from their NH₂ termini, RE1 and RE2 contain uninterrupted sequences of hydrophobic amino acids similar in terms of amino acid composition and length to the transmembrane anchors found in other bitopic integral membrane proteins. By comparing the sequence and composition data obtained here with the sequences of E1 and E2 deduced from complementary DNA sequence analysis (Rice & Strauss, 1981) we can make several observations. First, following their uncharged, putative intramembrane segments (33 and 26 amino acids, respectively), E1 and E2 contain clusters of predominantly basic amino acids. By structural analogy to known transmembrane proteins, E1 probably spans the bilayer but contains only a few residues exposed on the inner face of the virion envelope. In contrast, E2 and PE2 (the precursor to E2), which have been shown to span the bilayer completely, contain an additional 33 COOH-terminal residues, which could be either exposed on the cytoplasmic face of the lipid bilayer or which could loop back into the membrane. This region at the extreme COOH-terminal end of E2, which is protected by the virion envelope from digestion by a-chymotrypsin, contains a second uncharged domain (23 amino acids in length) whose orientation is unknown, but which may be involved in the highly specific interaction of the transmembrane glycoproteins in the plasma membrane with the cytoplasmic nucleocapsid during budding.     

Two-dimensional electrophoretic analysis of major histone species and their variants from somatic and germ-line tissue

A two-dimensional electrophoretic procedure has been developed and applied to the analysis of histones from the mouse thymus, liver, and seminiferous epithelium. The technique uses acetic acid-urea polyacrylamide gel electrophoresis in the first dimension to provide a primary separation of major histone species. Separation of additional histone species and variants is achieved in the second dimension by adding 0.4% of the nonionic detergent Lubrol-WX to the polyacrylamide gel. The procedure is relatively simple and highly reproducible and enables the simultaneous resolution of 9 to 16 protein spots corresponding to the major histone species and their variants.     

A mutant of Anabaena sp. CA with oxygen-sensitive nitrogenase activity.

Studies on the O₂ protection mechanism for nitrogenase in a mutant (PM10) of $\text{Anabaena}$ sp. CA indicated that the ability to protect nitrogenase from O₂ was functionally impaired. Growth rates of PM10 were substantially improved when cells were cultured under microaerobic conditions. Nitrogenase activity was totally inhibited by exposure to O₂ for 30 min; partial restoration of activity was attained when cell suspensions were subsequently made microaerobic. Experiments in which induction of nitrogenase activity was followed indicated that the synthesis of the O₂ protection mechanism was temporally separated from synthesis of heterocysts and nitrogenase.     

Purification of thymidine kinase by affinity chromatography with an enzyme inhibitor as the ligand

An affinity column for the purification of thymidine kinase is described. The ligand in this column is a glycoprotein isolated from rat kidney. This glycoprotein inhibits phosphorylation of thymidine in cultured cells and in a cell-free assay system. With an affinity column containing the glycoprotein as a ligand, a 24-fold purification of thymidine kinase from an ammonium sulfate fraction of a crude tissue extract can be obtained. Thymidine kinase eluted from the affinity column migrates as one major band on polyacrylamide and as one diffuse major band on sodium dodecyl sulfate-polyacrylamide. The affinity column, with thymidine kinase bound to the inhibitor, can also be used as an assay system. When the glycoprotein is covalently attached to Sepharose, it retains its binding capacity for thymidine kinase but has apparently lost its ability to inhibit the enzyme. Thymidine kinase eluted from the affinity column is again sensitive to the glycoprotein. It seems to be a carbohydrate moiety of the glycoprotein that is responsible for the inhibition.     

Determination of 3-methoxy-4-hydroxyphenylglycol in urine by high-performance liquid chromatography with amperometric detection

A reversed-phase high-performance liquid chromatographic method has been used for the quantitative determination of 3-methoxy-4-hydroxyphenylglycol (MHPG) in urine. After incubation with glusulase, free MHPG is extracted into ethyl acetate and further isolated by a combination of thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). The addition of amperometric detection provides increased sensitivity to a highly specific assay.     

Base-pairing between distant regions of the Escherichia coli 16 S ribosomal RNA in solution.

Intramolecular crosslinks have been introduced into Escherichia coli 16 S ribosomal RNA in aqueous solution by irradiation in the presence of hydroxymethyl-trimethylpsoralen. When the crosslinked RNA is denatured and examined in the electron microscope the most striking features are a variety of large open loops. In addition, because the crosslinked molecules are shortened compared to non-crosslinked molecules, there are likely to be small hairpins not resolved by the present technique. The sizes and positions of 11 loop classes have been determined and oriented on the molecule. The frequency of occurrence of the different classes of loops depends on the crosslinking conditions. When the crosslinking is done in solutions containing Mg²⁺, at least four of the loop classes appear with greater frequency than they do in 3.5 mm-NaCl. The loops presumably arise because complementary sequences separated by long intervening regions are being crosslinked. These base-pairing interactions between residues distant in the primary structure appear to be prominent features of the secondary structure of rRNA in solution.     

Analysis of neutron distance data

Techniques are described for processing neutron distance data for the purpose of deriving information about the three-dimensional organization of macromolecular assemblies.