Application of a charge/size two-dimensional gel electrophoresis system to the analysis of the penicillin-binding proteins of Escherichia coli

Phenylalanine-specific tRNA from yeast was hydrolysed with cobra venom ribonuclease in the double-stranded regions and the fragments isolated. The 'dissected' molecules with nicks in positions 28 and 41 were reconstructed from supplementary fragments and treated with T-4 RNA ligase. A phosphodiester bond between two fragments was formed when the fragment combination (1-28) + (29-76) was used. A strong discrimination in the ligation yield between different nick positions in the same helix is shown.     

The fragments from the 28 S ribosomal RNA of Galleria mellonella with unesterified uridine at the 3'-termini.

The three 3'-ends of the 28 S galleria RNA are all unesterified uridines. The two 18-S products due to the primary nick of the 28 S RNA have a similar 3'-terminal dinucleotide: G-Uoh. All of the seven species of secondary products from the Galleria 28 S RNA were suggested to have unesterified uridines, in common, at the 3'-ends. These results raise the possibility that the enzyme concerned in generating the primary and secondary nicks is a uracil-specific and 5'-phosphate-forming phosphodiesterase. The esterase, in association with the higher structure of the rRNA molecule in the larger subunit of the ribosome, probably determines the sites for these nicks in the 28 S rRNA. It is proposed also that the same enzyme can be responsible for cleaving the 28-S and 5.8-S rRNAs from their immediate precursor molecules.     

Induced transcription-dependent synthesis of mitochondrial reduced nicotinamide-adenine dinucleotide dehydrogenase in Drosophila.

The 23S rRNA of Agrobacterium tumefaciens contains at least two nicks which result in the formation of RNA components with mol.wts. of 0.52 X 10(6) and 0.48 X 10(6). Thus under the usual conditions of extraction and analysis, no 23S rRNA was recovered from the bacterium. The experiments show that 23S rRNA is synthesized as a continuous chain, in which one or two nicks are formed almost immediately near the ends of the molecule and an additional nick in the middle at a later time.     

Mapping of the ribosomal RNA genes on spinach chloroplast DNA.

Spinach chloroplast ribosomal RNAs have been hybridized to restriction endonuclease fragments of spinach chloroplast DNA. All three RNA species (23S, 16S and 5S) hybridized to a single large fragment when the DNA was digested with either Sall or Pstl. Hybridization of 23S RNA to fragments produced by Smal yielded two radioactive bands which corresponded to the bi-molar 2.5 X 10(6) and 1.15 X 10(6) Mr fragments. 16S RNA also hybridized to two, bi-molar Smal fragments (3.4 X 10(6) and 2.5 X 10(6) Mr) and 5S RNA hybridized to the 1.15 X 10(6) Mr bi-molar Smal fragment. The 23S RNA and 16S RNA cistrons were each also shown to contain a single EcoRI site. From the data it was possible to conclude that the ribosomal RNA genes are located on the inverted repeat region of the spinach chloroplast DNA restriction map [1,2], that the sequence of the cistrons is 16S - 23S - 5S and that the size of the spacer between the 16S and 23S RNA cistrons is approximately 0.90 X 10(6) Mr.     

Independent binding sites in mouse 5.8S ribosomal ribonucleic acid for 28S ribosomal ribonucleic acid

Limited digestion of mouse 5.8S ribosomal RNA (rRNA) with RNase T2 generates 5'- and 3'-terminal "half-molecules". These fragments are capable of independently and specifically binding to 28S rRNA, so there exist at least two contacts in the 5.8S rRNA for the 28S rRNA. The dissociation constants for the 5.8S/28S, 5' 5.8S fragment/28S, and 3' 5.8S fragment/28S complexes are 9 x 10(-8) M, 6 x 10(-8) M, and 13 x 10(-8) M, respectively. Thus, each of the fragment binding sites contributes about equally to the overall binding energy of the 5.8S/28S rRNA complex, and the binding sites act independently, rather than cooperatively. The dissociation constants suggest that the 5.8S rRNA termini from short, irregular helices with 28S rRNA. Thermal denaturation data on complexes containing 28S rRNA and each of the half-molecules of 5.8S rRNA indicate that the 5'-terminal binding site(s) exist(s) in a single conformation while the 3'-terminal site exhibits two conformational alternatives. The functional significance of the different conformational states is presently indeterminate, but the possibility they may represent alternative forms of a conformational switch operative during ribosome function is discussed.     

Fragment of protein L18 from the Escherichia coli ribosome that contains the 5S RNA binding site.

A fragment of ribosomal protein L18 was prepared by limited trypsin digestion of a specific complex of L18 and 5S RNA. It was characterised for sequence and the very basic N-terminal region of the protein was found to be absent. No smaller resistant fragments were produced. 5S RNA binding experiments indicated that the basic N-terminal region, from amino acid residues 1 to 17, was not important for the L18-5S RNA association. Under milder trypsin digestion conditions three resistant fragments were produced from the free protein. The largest corresponded to that isolated from the complex. The smaller ones were trimmed slightly further at both N- and C-terminal ends. These smaller fragments did not reassociate with 5S RNA. It was concluded on the basis of the trypsin protection observations and the 5S RNA binding results that the region extending from residues 18 to 117 approximates to the minimum amount of protein required for a specific and stable protein-RNA interaction. The accessibility of the very basic N-terminal region of L18, in the L18-5S RNA complex, suggests that it may be involved, in some way, in the interaction of 5S RNA with 23S RNA.     

Oligonucleotide sequences of pancreatic and T1 ribonuclease digests of 5S ribosomal RNA from mouse cells.

Oligonucleotides produced by complete pancreatic and T1 RNase digestion of 5S ribosomal RNA from a mouse hepatoma, MH 134, have been separated with two-dimensional electrophoresis and their nucleotide sequences determined. Except for the presence of a 5'-terminal diphosphate, these nucleotide sequences were identical with those of KB cells, confirming the identity of the primary structure of 5S RNA between these animals. Both oligonucleotide patterns produced with these enzymes from 5S RNA of the liver were also identical with those of the hepatoma. All these agree with the strong conservation of 5S RNA genes in animal species. However, when 5S ribosomal RNA was extracted from ribosomes which were prepared from microsomal pellet, pancreatic RNase digest contained two trinucleotides (A-G-Cp and G-A-Cp) that were not found in 5S RNA prepared with a one-step procedure. It was concluded that different isolation procedure might indeed cause artifactual fragments on enzymatic digestion due to internal nicks produced during isolation. The significance of 5'-terminal diphosphate in relation to the biosynthesis of 5S ribosomal RNA is also discussed.     

The preparation and characterization of locust vitellogenin messenger RNA and the synthesis of its complementary DNA.

Poly(A)+ (polyadenylated) RNA was isolated from vitellogenic female-locus fat-body by LiCl/urea extraction and poly(U)-Sepharose 4B affinity chromatography. Agarose-gel electrophoresis of this poly(A)+ RNA under denaturing conditions shows the presence of a high-molecular-weight species (greater than 31 S, 7100 nucleotides) as the major species, which is absent from the RNA prepared from male-locust fat-body. Inclusion of this poly(A)+ RNA in a mRNA-dependent reticulocyte-lysate system directs the synthesis of polypeptides that could be immunoprecipitated with monospecific antibodies against locust egg vitellin. DNA complementary (cDNA) to the poly(A)+ RNA was synthesized, and back-hybridization of the cDNA to its template reveals a major abundant species comprising about 45% of the total poly(A)+ RNA hybridizing with R0t 1/2 of 2 x 10(-2) mol . litre-1 . s. Abundant cDNA isolated from the total cDNA hybridizes to poly(A)+ RNA with a R0t 1/2 of 9 x 10(-3) mol . litre-1 . s. There are 9.1 x 10(3) copies of vitellogenin mRNA per cell of vitellogenic female-locust fat-body, comprising 55% of the poly(A)+ RNA and equivalent to 0.7% of total cellular RNA.     

A facile synthesis of D-glucose-type gem-diamine 1-N-iminosugars: a new family of glucosidase inhibitors.

gem-Diamine 1-N iminosugars of D-glucose-type, a new type of glycosidase inhibitors, have been synthesized from siastatin B, isolated from Streptomyces culture. 2-Trifluoroacetamido-1-N-iminosugar, (2S,3R,4R,5R)-2-trifluoroacetamido-5-hydroxymethylpiperidine -3,4-diol was proved to be a potent inhibitor for alpha-D- and beta-D-glucosidases (IC50 1.9x10(-7) and 4.2x10(-7) M, respectively). 2-Acetamido-1-N-iminosugar, (2S,3R,4R,5R)-2-acetamido-5-hydroxymethylpiperidine-3,4-diol also affected these enzymes (IC50 2.9x10(-6) and 5.4x10(-6) M, respectively).     

Isolation and organization of calf ribosomal DNA.

Ribosomal DNA (rDNA) from calf was isolated by three density gradient centrifugations. The first centrifugation in Cs2S04/BAMD was used to obtain partially resolved dG+dC-rich fractions from total DNA. The second and third centrifugations, in Cs2S04/Ag+, led to the isolation of an rDNA fraction characterized by a symmetrical band in CsCl, p = 1.724 g/cm3. This new procedure appears to be generally suitable for the isolation of rDNA and other dG+dC-rich repeated genes. The organization of isolated calf rDNA has been studied by restriction enzyme digestion and by hybridization with cloned rDNA from Xenopus laevis. The repeat unit of calf rDNA has a molecular weight of 21x10(6) and is split by EcoR1 into two fragments, 16x10(6) and 5.0x10(6), and by BamHI into seven fragments. EcoRI and BamHI sites have been mapped. Most of the 18S and 28S RNA genes and the transcribed spacer are contained in the small EcoRI fragment, while the non-transcribed spacer is localized in the large EcoRI fragment. This spacer showed length heterogeneity within a single individual; such heterogeneity is limited to two regions of the spacer.     

How an RNA ligase discriminates RNA versus DNA damage

T4 RNA ligase 2 (Rnl2) exemplifies a family of RNA-joining enzymes that includes protozoan RNA-editing ligases. Rnl2 efficiently seals 3'-OH/5'-PO4 RNA nicks in either a duplex RNA or an RNA:DNA hybrid but cannot seal DNA nicks. RNA specificity arises from a requirement for at least two ribonucleotides immediately flanking the 3'-OH of the nick; the rest of the nicked duplex can be replaced by DNA. The terminal 2'-OH at the nick is important for the attack of the 3'-OH on the 5'-adenylated strand to form a phosphodiester, but dispensable for nick recognition and adenylylation of the 5'-PO4 strand. The penultimate 2'-OH is important for nick recognition. Stable binding of Rnl2 at a nick depends on contacts to both the N-terminal adenylyltransferase domain and its signature C-terminal domain. Nick sensing also requires adenylylation of Rnl2. These results provide insights to the evolution of nucleic acid repair systems.     

The presence of a high-molecular-weight (guanine-plus-cytosine)-rich segment at the 3'' end of rabbit 28S ribosomal ribonucleic acid.

The 3' hydroxyl end of 28S L-rRNA (major RNA species of the larger subribosomal particle) was labelled by coupling its 2-hydroxy-3-naphthoic acid hydrazine with diazotized [3H]aniline. The RNA was hydrolysed partially with ribonuclease T1 and fractionated on Sephadex G-200. The results show that a highly structured segment with 78% G+C content and a number-average molecular weight of at least 1.0x10(5)-1.8x10(5) is located at the 3' hydroxyl end of the 28S rRNA molecule.     

Heterogeneity in Chinese hamster ribosomal DNA

A discrete heterogeneity has been detected in Chinese hamster ribosomal DNA after Eco R1 digestion of total DNA followed by a Southern transfer and hybridization with [¹²⁵I]18S or [¹²⁵I]28S ribosomal RNA. Digestion with Eco R1 produces three fragments, 4.3, 6.0 and 9.5×10⁶ daltons respectively, which hybridize with 18S RNA. The smallest fragment also hybridizes with 28S RNA. Either length heterogeneity or sequence heterogeneity (i.e. presence of an additional Eco R1 site in some of the rDNA molecules) must be invoked to account for the two larger Eco R1 fragments that contain 18S but not 28S sequences. Eco R1 and Hind III maps, consistent with either length or sequence heterogeneity, are presented. The data at this time, however, do not distinguish between the two alternatives.     

The lambda terminase enzyme measures the point of its endonucleolytic attack 47 +/- 2 bp away from its site of specific DNA binding, the R site.

lambda terminase is an ATP-interactive, site-specific endonuclease comprising the products of lambda genes Nu1 and A. Terminase binds to cos, at the junction of two chromosomes in a concatemer, catalyzes cos cleavage and initiates the packaging of lambda DNA into proheads. cos consists of a nicking domain, cosN, where terminase cleaves to regenerate the 12 nucleotide cohesive ends of mature lambda chromosomes and a binding domain, cosB, where terminase binds to 16 bp repeat sequences called R3, R2 and R1. Evidence is presented that terminase is a single-strand endonuclease that can nick DNA by one of two mechanisms, both of which require ATP. (i) When bound to any R site, terminase nicks the strand which, within that R site, is purine-rich; the position of this nick is 47 +/- 2 nucleotides away from the mid-point of that R site, measured in the 3' direction; (ii) enzymes that are not bound to R sites nick DNA within certain specific sequences that resemble cosN half sites. These two modes of action are nicely combined for the R3-bound protomer that nicks the bottom strand at position N1 in cosN since the interval between N1 and the R3 midpoint is 47 nucleotides. Within cosN, the bottom and top strand nicks are generated by a rigid protein couple with a 2-fold rotational symmetry. The location of both of these nicks, however, is gauged asymmetrically from R3, 47 nucleotides away. Again, R1 and R2 are separated by 47 bp and orient bound protomers towards each other but, unless the DNA between these R sites is lengthened, the enzymes do not nick, indicating an inhibitory gpA-gpNu1 apposition.     

Analysis of 5S rDNA changes in synthetic allopolyploids Triticum x Aegilops

By the example of three synthetic allopolyploids: Aegilops sharonensis x Ae. umbellulata (2n =28), Triticum urartu x Ae. tauschii (2n =28), T. dicoccoides x Ae. tauschii (2n =42) the 5S rDNA changes at the early stage of allopolyploidization were investigated. Using fluorescent in situ hybridization (FISH), the quantitative changes affecting the separate loci of one of the parental genomes were revealed in plants of S3 generation of each hybrid combination. Souther hybridization with genomic DNA of allopolyploid T. urartu x Ae. tauschii (TMU38 x TQ27) revealed lower intensity of the fragments from Ae. tauschii compared with the T. urartu fragments. It may be confirmation of the reduction of signal on 1D chromosome that was revealed in this hybrid using FISH. Both appearance of a new 5S rDNA fragments and full disappearance of fragments from parental species were not showed by Southern hybridization, as well as PCR-analysis of 5-15 plants of S2-S3 generations. The changes were not found under comparison of primary structure of nine 5S rDNA sequences of allopolyploid TMU38 x TQ27 with analogous sequences from parental species genomes. The observable similarity by FISH results of one of the studied synthetic allopolyploids with natural allopolyploid of similar genome composition indicates the early formation of unique for each allopolyploid 5S rDNA organization.     

An elongated model of the Xenopus laevis transcription factor IIIA-5S ribosomal RNA complex derived from neutron scattering and hydrodynamic measurements.

The precise molecular composition of the Xenopus laevis TFIIIA-5S ribosomal RNA complex (7S particle) has been established from small angle neutron and dynamic light scattering. The molecular weight of the particle was found to be 95,700 +/- 10,000 and 86,700 +/- 9000 daltons from these two methods respectively. The observed match point of 54.4% D2O obtained from contrast variation experiments indicates a 1:1 molar ratio. It is concluded that only a single molecule of TFIIIA, a zinc-finger protein, and of 5S RNA are present in this complex. At high neutron scattering contrast radius of gyration of 42.3 +/- 2 A was found for the 7S particle. In addition a diffusion coefficient of 4.4 x 10(-11) [m2 s-1] and a sedimentation coefficient of 6.2S were determined. The hydrodynamic radius obtained for the 7S particle is 48 +/- 5 A. A simple elongated cylindrical model with dimensions of 140 A length and 59 A diameter is compatible with the neutron results. A globular model can be excluded by the shallow nature of the neutron scattering curves. It is proposed that the observed difference of 15 A in length between the 7S particle and isolated 5S RNA most likely indicates that part(s) of the protein protrudes from the end(s) of the RNA molecule. There is no biochemical evidence for any gross alteration in 5S RNA conformation upon binding to TFIIIA.     

The regulatory region of MS2 phage RNA replicase cistron. III. Characterization of fragments resulting from S1 nuclease digestion.

The functionally active fragments MS2 R(-53 leads to 6) and MS2 R(-53 leads to 3) comprising the regulatory region for the replicase cistron have been isolated from MS2 RNA-coat protein complex following T1 RNase digestion. In order to obtain shorter fragments, active in coat protein binding and initiation of translation, MS2 R(-53 leads to 6) was cleaved with S1 nuclease. The results indicate that S1 nuclease attacks the most susceptible loop regions of the two hairpin helices of MSZ R(-53) leads to 6). Among the three fragments which have been isolated, only MS2 R(-35/33 leads to 6) containing the intact hairpin (b) region with initiation codon AUG is active in the coat protein binding. Functional activity exerted by another polynucleotide MS R(-17 leads to 6) supports the assumption that specific binding with the coat protein is determined by the hairpin (b) region prior to the replicase cistron.     

Nucleic acids of respiratory syncytial virus.

Analysis of purified respiratory syncytial virus revealed that the virion RNA was composed of 50S, 28S, 18S, and 4S species. The 18S and 28S species were presumed to represent host rRNA since virus grown in actinomycin D-treated cells contained only 50S and 4S RNAs. Actinomycin D treatment stimulated production of infectious respiratory syncytial virus 5- to 10-fold. The 50S virion RNA was shown to hybridize with polyadenylated mRNA's isolated from infected cells, indicating that respiratory syncytial virus RNA is of negative-strand sense. Six mRNA's were identified by polyacrylamide gel electrophoresis.