Modification of specific lysine residues in E. coli methionyl-tRNA synthetase by crosslinking to E. coli formylmethionine tRNA

A protein affinity labeling derivative of E. coli tRNAfMet has been prepared which carries an average of one reactive side chain per molecule, distributed over four structural regions. Each side chain contains a disulfide bond capable of reaction with cysteine residues and an N-hydroxysuccinimide ester group capable of coupling to lysine epsilon-amino groups in proteins. Reaction of the modified tRNA with E. coli methionyl-tRNA synthetase leads to crosslinking only by reaction with lysine residues in the protein. Examination of the tRNA present in the crosslinked complex reveals that the enzyme is coupled to side chains attached to the 5' terminal nucleotide, the dihydrouridine loop, the anticodon and the CCA sequence. Digestion of the crosslinked enzyme with trypsin followed by peptide mapping reveals that the major crosslinking reactions occur at four specific lysine residues, with minor reaction at two additional sites. Native methionyl-tRNA synthetase contains 90 lysine residues, 45 in unique sequences of the dimeric alpha 2 enzyme. Crosslinking of the protein to different regions in tRNAfMet thus occurs with the high degree of selectivity necessary for use in determining the peptide sequences which are near specific nucleotide sequences of tRNA bound to the protein.     

Single-stranded bacteriophage T5 stO DNA as template for in vitro fMet-tRNA binding to ribosomes and protein synthesis.

Good evidence is provided that fMet-tRNA binding and aminoacid incorporation, when single-stranded DNA is used instead of mRNA in an E. coli cell-free system, are strictly dependent on the magnesium concentration. Ten sites homologous to the initiation sites of translation can be detected on denatured T5 stO DNA when using ribosomes and initiation factors from uninfected E. coli F. In S-30 extracts, at high magnesium concentrations and in the presence of neomycin, initiation of the translation of denatured T5 stO DNA begins anywhere on the molecule, and yet high molecular weight polypeptides are synthesized. The template potentiality of the denatured T5 stO DNA decreased when using ribosomes plus initiation factors and crude extracts from T5 stO-infected bacteria. By in vitro formation of initiation complexes sites analogous to initiation sites of translation were localized on T5 stO DNA molecules using single-stranded fragments separated by sedimentation in alkaline sucrose gradient.     

Characterization of intercellular junctions in the preimplantation mouse embryo by freeze-fracture and thin-section electron microscopy

Intercellular junction formation in preimplantation mouse embryos was investigated with thin-section and freeze-fracture electron microscopy. At the four-cell stage, regions of close membrane apposition with focal points of membrane contact and occasional underlying cytoplasmic densities were observed between blastomeres of thin-sectioned embryos. Corresponding intramembrane specializations were not, however, observed in freeze-fractured embryos. At the 8- to 16-cell stage, small gap and macula occludens junctions and complexes of these junctions were observed at all levels between blastomeres of freeze-fractured embryos. As development progressed from the early to mid 8- to 16-cell stage, the size of the occludens/gap junction complexes increased, forming fascia occludens/gap junction complexes. At the morula stage, gap junctions and occludens/gap junction complexes were observed on both presumptive trophoblast and inner cell-mass cells. Zonula occludens junctions were first observed at the morula stage on presumptive trophoblast cells of freeze-fractured embryos. The number of embryos possessing zonula occludens junctions increased at the mid compared to the early morula stage. At the blastocyst stage, junctional complexes consisting of zonula occludens, macula adherens, and gap junctions were observed between trophoblast cells of freeze-fractured and thin-sectioned embryos. Isolated gap and occludens junctions, adherens junctions, and occludens/gap junction complexes were observed on trophoblast and inner cell-mass cells.     

A new approach to the characterization of the B and A forms of DNA by I.R. spectroscopy

The RNA conformational changes of B, A and C forms are reflected in the infrared absorption spectra in the region of 800 cm^?1 to 900 cm^?1 and allow one to investigate unoriented samples. The transition to the A form is characterized by the appearence of bands at about 870 cm^?1 and at 813 cm^?1 whereas the B and the C forms exhibit a band at 837 cm^?1, these bands undoubtedly arise from phosphate diester stretching vibrations and yield information about backbone conformation. The presence of these infrared bands provides a criterion for testing the simultaneous presence of two coexisting forms of DNA. It represents a useful method for structural studies of nucleic acid complexes such as protein-DNA for which it is difficult to obtain orientation.     

Influence of histone hyperacetylation on nucleosomal particles as visualized by electron microscopy

Recently, Bode et al. [J. Bode, M. Gómez-Lira, and H. Schröter (1983)Eur. J. Biochem.130, 437–445] have observed that monomeric nucleosomal particles from butyrate-treated Namalva lymphoma cells display a distinct heterogeneity in their mobilities on a non-denaturing 4% polyacrylamide gel. They have proposed that histone hyperacetylation induces a conformational change in monomers that can be modulated by the presence of HMG $\text{14}\text{17}$. The electron microscopic analyses presented here support these proposals.     

Pepsin inhibition by a high specific activity radioiodinated derivative of pepstatin.

Stop-flow kinetic studies are reported for the reaction of glutathione (GSH) with 2-chloromercuri-4-nitrophenol. The second-order rate constant was found to vary by less than twofold in going from pH 5.9 to 7.3, suggesting that the reaction involves attack of the un-ionized glutathione thiol on the mercurial around pH 7. The rate constant was 1.5 × 10⁷m^?1 s^?1 at pH 7.0 and 8 °C in dilute buffer of ionic strength 0.2. The sensitivity of the kinetics to the nature of the leaving group in the mercurial i.e., to the labile ligand, was investigated in complexes of the mercurial with OH^?, N₃^?, Br^?, Cl^?, CN^?, SCN^?, pyrophosphate, ADP, ATP, EDTA, and adenosine. The addition of most of these substances was observed to lead to rate enhancements or rate retardations, the kinetic effects showing typical binding isotherm behavior when plotted as a function of the added ligand concentration. The kinetic midpoints of such plots showed very good correlation with the independently determined affinities they had toward the mercurial. The results demonstrate that many commonly added compounds of biochemical interest can significantly influence mercurial reactivity toward thiols through rapid exchange at the labile ligand position of the mercurial.     

Cellular migration through the cardiac jelly matrix: A stereoanalysis by high-voltage electron microscopy

Formation and migration of cushion tissue in the developing chick heart was analyzed by scanning and high-voltage electron microscopic stereoanalysis. Two methods of fixation which enhance the preservation of water-soluble components of the extracellular matrix (cardiac jelly) were employed: 1% tannic acid in 3% glutaraldehyde (TAG) and 1% cetylpyridinium chloride (CPC) in 3% glutaraldehyde. Our results indicated that the preservation of the cell: matrix interaction exhibited by endocardial cells and migrating cushion tissue is dependent upon the method of fixation. In TAG-fixed embryos, filopodial extensions from the endocardium as well as filopodia of pioneering cells are most often associated with microfibrillar components of the matrix, whereas in CPC-fixed material these same cellular extensions are found in association with pleomorphic anastomosing strands rich in hyaluronate. Following these initial cell:matrix interactions by both the endocardium and pioneering cells, trailing cells invade the extracellular matrical region and clearly encounter in both types of fixation a different microenvironment in which to engage in cell:matrical associations. These observations support the hypothesis that filopodial probing by endocardial cells and pioneering cells results in macromolecular reorderings of the matrix and thus suggest an additional function for filopodia beyond translocation of the cells.     

Structure of the filamentous bacteriophage,Pf3, by X-ray fiber diffraction

Fiber diffraction data have been obtained for the filamentous bacteriophage Pf3. The virus crystallizes on a hexagonal net with lattice constants a = b = 5.61 nm and c = 7.50 nm at 0% relative humidity and a = 6 = 6.20 nm and c = 7.99 nm at 98% relative humidity. The X-ray diffraction pattern resembles those of two other bacteriophages, Pf 1 and Xf, and therefore belongs to class II. The data are consistent with a 27₅ helix symmetry with an axial rise of 0.277 (dry) to 0.296 (wet) nm.     

Transfection of Vibrio cholerae by bacteriophage phi 149 DNA

DNA isolated from Choleraphage ø149 of Group IV was infectious when mixed with competent $\text{V.}\text{cholerae}$ cells. The cells were competent during mid-log phase of growth. The infectivity of phage DNA was destroyed by deoxyribonuclease but not by ribonuclease or pronase. About 5 min is required for the establishment of the DNase resistant state. The dose response curve for transfection suggested that 2 to 3 molecules of DNA are required to produce one infections center. An infectivity of 5 × 10⁴ infectious center per μg of DNA was obtained.     

Inactivation of Salmonella phosphoribosylpyrophosphate synthetase by specific chemical modification of a lysine residue.

Phosphoribosylpyrophosphate synthetase from Salmonella typhimurium contains nine lysine residues per subunit and can be inactivated by reagents specific for this amino acid. Pyridoxal-P reversibly inhibited the enzyme by about 70% by forming a Schiff base derivative with lysine. Reduction with NaBH₄ made this inactivation irreversible. Kinetic experiments indicated that the failure to inactivate the enzyme completely in a single treatment with pyridoxal-P reflects a reversible equilibrium between inactive Schiff base and a noncovalent complex. Modification of one lysine residue per subunit correlated with apparently total loss of activity. The rate of inactivation of the enzyme was decreased fourfold by saturating concentrations of ATP and was decreased at least 20-fold by formation of a quaternary complex of the enzyme with Mg²⁺, α,β-methylene ATP, and ribose-5-P. Trinitrobenzenesulfonate also irreversibly inactivated the enzyme, but this reagent was less specific in that the loss of activity corresponded to the modification of four to five lysine residues. These results suggest that an essential lysine is near the active site of Phosphoribosylpyrophosphate synthetase.     

Nucleotide sequence of polypyrimidines from cloned mouse DNA as determined by base-specific blockage of exonuclease action

Cloned fragments of mouse DNA have been screened for the presence of long polypyrimidine/polypurine segments. The polypyrimidine portion of one such segment (about 200 nucleotides in length) has been isolated by acidic depurination of the entire cloned fragment and plasmid vector followed by selective precipitation and 5'-32P labeling. This polypyrimidine has been used to demonstrate a new procedure for sequencing. Covalent modification of thymine with a water-soluble carbodiimide, or cytosine with glutaric anhydride, at low levels blocked the action of snake venom exonuclease. After deblocking, separation of the products of digestion by polyacrylamide gel electrophoresis yields a sequence ladder which can be used to determine the position of C and T residues as in other sequencing methods. A sequence of 72 residues adjacent to the 5' end has been established, consisting principally of the repeating tetranucleotide (CCTT)n. A low ratio of endonuclease to exonuclease is essential for application of this method to sequences of this size. Accordingly, a very sensitive modification of a fluorometric endonuclease assay was developed and used to optimize pH and Mg2+ conditions to favor exonuclease activity over the accompanying endonuclease activity. The results clearly indicate that long polypyrimidine tracts can be efficiently prepared and their sequences determined with this method using commercially available exonuclease preparations without additional purification.     

A specific micromethod for determination of acetyl residues in proteins

A method is described for the separation and concentration of rat liver lysosomes from mitochondria in a one-step procedure by zonal centrifugation. Some of the practical problems associated with the use of the B-XX1X rotor are discussed.     

A quantitative radioimmunological screening method for specific gene products

A rapid, sensitive, and quantitative radioimmunoassay for specific translation products was developed using Escherichia coli cells grown in 96-well microtiter plates. A simple and inexpensive apparatus that facilitates the simultaneous transfer of all 96 detergent-lysed cultures to nitrocellulose within 30 s is described. Following this quantitative transfer, selected proteins are screened using specific antisera and ¹²⁵I-Protein A. The technique works with either cytoplasmic or membrane proteins. As little as a two- to threefold increase of a gene product over normal levels can easily be detected.     

Measurement of DNA length by gel electrophoresis. I. Improved accuracy of mobility measurements using a digital microdensitometer and computer processing

The electrophoretic mobilities of DNA polymer fragments in an agarose gel have been measured from a photograph of the gel by different methods and converted to lengths by the reciprocal method. The method of measurement can introduce large errors in the length estimates. The use of a digital microdensitometer to obtain optical density profiles of gel tracks with subsequent computer processing to find peak positions was found to give the most accurate DNA lengths.     

Mapping of mitochondrial 4 S RNA genes in Xenopus laevis by electron microscopy

The distribution of sites hybridizing with mitochondrial 4 S RNA molecules on mitochondrial DNA of Xenopus laevis has been mapped in relation to the ribosomal RNA genes and EcoRI restriction endonuclease sites. RNA molecules linked to ferritin were employed for this purpose. We have obtained evidence for 15 4 S RNA sites on the H-strand and six sites on the L-strand of X. laevis mtDNA^∥. An indication of the possible existence of one additional site on the H-strand and four additional sites on the L-strand has been obtained. One 4 S RNA site is located in the gap between the two rRNA genes, and one site flanks each outside end of the rRNA genes. The other 4 S RNA sites are distributed almost evenly throughout both strands of the mtDNA. A comparison with the map of 4 S RNA sites on the mtDNA of HeLa cells (Angerer et al., 1976) suggests considerable evolutionary conservation of site organization.     

In vitro synthesis of simian virus 40 DNA. I. Synthesis by a soluble extract from infected CV1 cells.

Simian Virus 40 (SV40) DNA replication was studied in vitro using cell free extracts prepared from SV40 infected CV1 cells. The cells were fractionated into a soluble cytoplasmic fraction and nuclei. The nuclei were lysed with high salt and used to prepare a soluble nuclear fraction. Both fractions displayed DNA polymerase activity as measured with activated calf thymus DNA. However, only the cytoplasmic fraction was active when SV40 DNA comonent I molecules were used as template. Under these conditions, the cytoplasmic extract was shown to catalyse the SV40 DNA dependent, in vitro incorporation of the four deoxyribonucleotides into DNA molecules which had, at both neutral and alkaline pH, the same sedimentation behavior as authentic SV40 DNA component I and component II molecules. Optimal Mg++ concentration was 5-8 mM. Incorporation of label into DNA component I molecules showed an initial lag of about 15 min., after which it was linear with time for up to 5 hrs at 32 degrees. Incorporation into DNA component II molecules proceeded without obvious lag and reached a plateau after approximately 2 hrs of incubation. It is concluded that the cytoplasmic extract supports the in vitro synthesis of SV40 DNA and that DNA component II molecules appear to be a precursor to DNA component I molecules in the reaction. Labeling of viral DNA molecules was highly dependent on ATP and on an ATP generating system. In the absence of ATP and of the energy generating system, incorporation occurred but both template and newly synthesized DNA molecules were extensively degraded.     

Suppression of rabbit lymphocyte functions by antibodies specific for allotypic membrane determinants

Regulation of immunoglobulin synthesis and secretion was analyzed by exposing spleen cells of b4b4 rabbits to anti-b4 for 24 hr in culture. As noted previously, no lymphocytes with membrane-bound b4 were found immediately after pulse treatment, but substantial regeneration of membrane Ig (mIg) occurred on further culture in antibody-free medium. Splenocytes cultured either in the presence or absence of anti-b4 showed a marked loss of Ig-secreting cells (ISC) after 24 hr in culture but recovered and exhibited peak numbers of ISC on Day 2. However, ISC formation in cultures of antibody-treated cells was significantly suppressed and thereafter declined at a more rapid rate than in control cultures. Polyclonal B cell activators from Nocardia and from gram-negative bacteria stimulated ISC formation in cultures of normal spleen cells, but responsiveness to these activators was depressed following antibody treatment. Antibody-induced suppression of Ig synthesis was attributed to interference with differentiation of B lymphocytes to the secretory stage.