Molecular studies of an fi+ plasmid from strains of Salmonella typhimurium.
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Ampicillin↗
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Results for “Circular code”
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Mitochondrial genomic characteristics and phylogenetic analysis of Cunninghamella elegans (Mucorales: Cunninghamellaceae).
Cunninghamella, a filamentous fungal genus with important biomedical and biochemical value, lacks any fully annotated mitochondrial genome to date. Herein, we presented the first complete mitogenome of Cunninghamella elegans, a circular 41,552 bp molecule (GC 27.86%) encoding 14 conserved protein-coding genes, 2 rRNA genes, 24 tRNA genes, and 6 non-conserved ORFs. Structural comparison with related species (Absidia glauca and Gongronella sp. w5) revealed dynamic evolution in intron and repeat elements. Phylogenetics places C. elegans within Cunninghamellaceae, with Gongronella as its closest relative. This reference mitogenome will underpin future evolutionary and taxonomic investigations of this industrially and medically significant lineage.
Cunninghamella elegans↗
Genes encoding the T-cell antigen receptor.
The search for the elusive and controversial T-cell antigen receptor is over. It is now clear that gene complexes for both alpha and beta chains are distinct from those for immunoglobulin genes. They are, however, related to Ig genes as well as to other class I and class II major histocompatibility complex (MHC) gene products. Therefore, they belong to the immunoglobulin super gene family.
DNA, Circular↗
Absence of circular plasmid deoxyribonucleic acid attributable to a genetic determinant for methicillin resistance in Staphylococcus aureus.
Plasmid deoxyribonucleic acid was not detected by centrifugal analysis of lysates of penicillinase-negative strains of Staphylococcus aureus harboring a determinant of methicillin resistance derived from strain Villaluz. When these strains contained a penicillinase plasmid, the plasmid deoxyribonucleic acid of methicillin-resistant and methicillin-susceptible strains was indistinguishable by the methods employed. The results indicate that the genetic determinant for methicillin resistance in the strains examined was not associated with a circular plasmid comparable to those that have been shown to determine resistance to benzylpenicillin, tetracycline, and chloramphenicol in S. aureus.
Centrifugation, Density Gradient↗
Human papillomavirus type 16 DNA from a vulvar carcinoma in situ is present as head-to-tail dimeric episomes with a deletion in the non-coding region.
A number of genital cancer biopsy samples were screened for the presence of human papillomavirus type 16 (HPV-16) DNA sequences. One of these samples (a vulvar carcinoma in situ) was found to contain more than 100 copies of HPV-16 DNA sequences per cell. Using this tumour DNA, a genomic library was constructed in bacteriophage lambda and the library was screened for recombinant phage containing HPV-16 sequences. Five recombinant phage clones were isolated and their DNA was analysed by restriction endonuclease digestion and blot hybridization. All five recombinants contained two copies of the HPV-16 genome present in a head-to-tail arrangement. The data are consistent with the presence of HPV-16 sequences in the tumour DNA arranged as genomic dimers in a circular episomal configuration. The HPV-16 genomes contained a deletion within the non-coding region, a region which includes the viral origin of DNA replication and transcriptional control sequences. Possible consequences of this deletion for viral replication and transcription are discussed.
Base Sequence↗
Fourteen internal transcribed spacers in the circular ribosomal DNA of Euglena gracilis.
Cytoplasmic ribosomes from Euglena gracilis contain 16 rRNA components. These include the typical 5 S, 5.8 S and 19 S rRNAs that are found in other eukaryotes as well as 13 discrete small RNAs that interact to form the equivalent of eukaryotic 25-28 S rRNA (accompanying paper). We have utilized DNA sequencing techniques to establish that genes for all of these RNAs, with the exception of 5 S rRNA, are encoded by the 11,500 base-pair circular rDNA of E. gracilis. We have determined the relative positions of the coding regions for the 19 S rRNA and the 14 components (including 5.8 S rRNA) of the large subunit rRNA, thereby establishing that the genes for each of these rRNAs are separated by internal transcribed spacers. We conclude that sequences corresponding to these spacers are removed post-transcriptionally from a high molecular weight pre-rRNA, resulting in a multiply fragmented large subunit rRNA. Internal transcribed spacers, in positions analogous to some of these additional Euglena rDNA spacers, have been found in the rDNA of other organisms and organelles. This finding supports the view that at least some internal transcribed spacers may have been present at an early stage in the evolution of rRNA genes.
Animals↗
Coil leads to helix transition in polyadenylic acid induced by the binding of epinephrine, norepinephrine, and isoproterenol: circular dichroism study.
A circular dichroism spectropolarimetric study on the conformation of polyadenylic acid (poly A) in neutral solutions demonstrated a coil leads to helix transition induced by intercalative binding of critical amounts of epinephrine, norepinephrine, and isoproterenol relative to poly A. Theoretical treatment of the experimental data indicated a first-order kinetic transition in poly A. It was possible to measure transition rate constants of the epinephrine-poly A and norepinephrine-poly A systems and to calculate the activation energies. The results indicate a high level of temperature dependence of the rate constants. The effects can be reversed by increasing ionic strength, indicating the significance of the electrostatic interactions. The importance of the results is discussed in terms of the possible role of the catecholamines as control mechanisms for the poly A-regulated translation of the genetic code on mRNA.
Circular Dichroism↗
Analysis of the interaction between DNA and major core protein in adenovirus chromatin by circular dichroism and ultraviolet light induced cross-linking.
Adenovirus chromatin is constituted with three kinds of core proteins, VII, V, and mu, that are coded by the virus genome. Since a hexamer of VII contributes to formation of the nucleosome-like structure of the virion chromatin, we analyzed the interaction between DNA and VII in vitro, by the use of ultraviolet light-induced cross-linking and circular dichroism (CD) spectroscopy. It was observed that DNA and VII in a plain mixture form a structure resembling viral chromatin. The DNA in the virion core or in the simply mixed complex appears to take a tight conformation by superfolding, based on the result that the ellipticity at 275 nm of DNA was reduced to approximately 3,000 degrees, and the wave-length of the positive peak was shifted from 275 to 285 nm. The change in CD spectrum caused by interaction of VII with DNA is similar to that of a protamine rather than that of a histone mixture. The interaction of VII with DNA is preferential, and VII is capable of associating more efficiently with double stranded DNA than with single stranded. The interaction is loosened by salt (0.3 M NaCl) and tightened by magnesium ion. However, the interaction of a precursor core protein pro-VII with DNA was not as tight as that of VII and was not influenced by magnesium ion, presumably because of the existence of a hydrophobic processing sequence in the molecule.
Adenoviruses, Human↗
A fully active variant of dihydrofolate reductase with a circularly permuted sequence.
The amino acid sequence of mouse dihydrofolate reductase was permuted circularly at the level of the gene. By transposing the 3'-terminal half of the coding sequence to its 5' terminus, the naturally adjacent amino and carboxyl termini of the native protein were fused, and one of the flexible peptide loops at the protein surface was cleaved. The steady-state kinetic constants, the dissociation constants of folate analogues, and the degree of activation by both mercurials and salt as well as the resistance toward digestion by trypsin were almost indistinguishable from those of a recombinant wild-type protein. Judged by these criteria, the circularly permuted variant has the same active site and overall structure as the wild-type enzyme. The only significant difference was the lower stability toward guanidinium chloride and the lower solubility of the circularly permuted variant. This behavior may be due to moving a mononucleotide binding fold from the interior of the sequence to the carboxyl terminus. Thus, dihydrofolate reductase requires neither the natural termini nor the cleaved loop for stability, for the conformational changes that accompany catalysis as well as the binding of inhibitors, and for the folding process.
Amino Acid Sequence↗
Leaky transcription termination produces larger and smaller than genome size hepatitis B virus X gene transcripts.
The genomic DNA of hepatitis B virus (HBV) is circular and has only one known transcription termination site. The HBV X protein coding sequence is flanked by this transcription termination site at the 3' end and a promoter element at the 5' end. Transcription initiating from the X promoter and terminating at the termination site would produce a transcript 0.7 kb in length, which we have detected in cell lines that produce HBV particles. Unexpectedly, a 3.9-kb transcript containing two copies of the X gene sequence was also detected in these cell lines. Polymerase chain reaction analysis indicates that this 3.9-kb transcript contains sequences from both upstream and downstream of the termination site. Thus, transcription of this 3.9-kb transcript initiates from the X promoter, reads through the termination site, and terminates the second time it encounters the site. Analysis using an SV40-derived vector indicates that the transcription termination site in the HBV genome is also leaky for X gene transcription when a heterologous promoter initiates the transcription. Based on these results, the mechanism of how the transcription termination of HBV mRNA is regulated is discussed.
Animals↗
Evidence for in vivo trans splicing of pre-mRNAs in tobacco chloroplasts.
The rps12 gene codes for chloroplast ribosomal protein S12. In the tobacco chloroplast genome (156 kbp circular DNA), exons II and III of this gene are separated by an intron of 536 bases and are present in two copies in the inverted repeat region, while exon I is located in the large single copy region at a distance of 90 kb and 126 kb from the two copies of exons II and III. These three exons were artificially combined in cloned DNA fragments and hybridized with tobacco chloroplast RNA. Electron microscopic analysis of RNA-DNA hybrids showed that exon I is transcribed as part of a polycistronic RNA containing upstream and downstream sequences; the same is true for exons II and III. Exon I is shown to be transcribed separately from exons II and III. In the most abundant class of the hybridized RNA molecules, exon I was covalently linked to exon II. In these molecules the sequences downstream of exon I and upstream of exon II are not present. These data indicate that maturation of rps12 pre-mRNAs in chloroplasts of tobacco involves trans splicing.
Chloroplasts↗
Transcription patterns of amplified Dytiscus genes coding for ribosomal RNA after injection into Xenopus oocyte nuclei.
Oocytes of the frog Xenopus were injected with purified circular DNA containing amplified rRNA genes of the water beetle Dytiscus. Nuclear contents of injected oocytes were spread and examined by electron microscopy. Most of the Dytiscus DNA seen in injected nuclei contained regions indensely packed with polymerases and nascent transcripts. Apparently normal, as well as abnormal, patterns of transcription were observed. By this type of experiment, it may become possible to recognize the transcribed regions and immediate transcripts of cloned DNA molecules whose activity cannot be seen by electron microscopy of normal nuclei.
Animals↗
Lack of feedback inhibition of V kappa gene rearrangement by productively rearranged alleles.
Circular DNAs excised by immunoglobulin kappa chain gene rearrangements were cloned and characterized. 16 of 17 clones examined were double recombination products containing a V kappa-J kappa rearrangement (coding joint) as well as the reciprocal element (signal joint) of another V kappa-J kappa rearrangement. These products suggested multiple recombination, primary inversion, and secondary excision. In primary events, 5 of 16 translational reading frames were in-phase. Thus, V kappa gene rearrangement may not be inhibited by the presence of a productively rearranged allele. An unusually large trinucleotide (P) insertion forming a palindrome of 12 nucleotides was also observed in one of the coding joints.
Alleles↗
Biased reading frames of pre-existing DH--JH coding joints and preferential nucleotide insertions at VH--DJH signal joints of excision products of immunoglobulin heavy chain gene rearrangements.
During B cell differentiation immunoglobulin (Ig) DH segments join to JH segments, followed by joining of VH to DJH. Although circular excision products of DH--JH rearrangements have been characterized, excision products of VH to DJH joining have never been isolated. We selectively denatured chromosomal DNA of mouse splenocytes and enriched circular DNA spanning the long distance between VH and DH. Subsequent PCR amplifications allowed the identification of signal joints of VH to DJH. Sequence analysis indicated that preexisting DH--JH coding joints of excision products showed a strong bias for reading frame 1, and the absence of reading frame 2, which would allow the expression of a truncated mu chain called D mu protein. When comparing the joints of the VH--DJH and DH--JH rearrangements we observed N-nucleotide insertions to be abundant at the VH--DH signal joint, but very sparse at the DH--JH signal joint, while the coding joints of both contained abundant N-insertions. These differences in N region insertions at the signal joints suggest a differential control of the D--J and V--DJ rearrangements.
Animals↗
Large repetition in the non-permuted nucleotide sequence of bacteriophage TI DNA.
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Base Sequence↗
Optical properties of specific complexes between complementary oligoribonucleotides.
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Adenine Nucleotides↗
Interactions between phage lambda replication proteins, lambda DNA and minicell membrane.
Gentle methods for minicell lysis and lysate fractionation have been elaborated: lysis by T4 lysozyme without detergents, and fractionation by equilibrium sedimentation in a metrizamide density gradient, both at low ionic strength. In the lysates of phage-lambda-infected minicells the lambda DNA, trapped at a prereplicative step [Witkiewicz, H. and Taylor, K. (1979) Biochim. Biophys. Acta 564, 31-36], appeared in two peaks of different buoyant densities: as a membrane-bound and a free lambda DNA. The covalently-closed-circular form of lambda DNA appeared exclusively in the membrane fraction. The lambda-coded proteins, synthesized in lambda-infected minicells, appeared in two major fractions: as membrane-bound and as free proteins, and in one minor fraction, bound with free lambda DNA. Neither lambda protein engaged in the initiation of DNA replication was present in the fraction of free proteins: the P-gene product was membrane-associated, and the O-gene product formed a complex with free lambda DNA. The effect of high ionic strength (KCl) and of detergents (Triton X-100 and sarcosyl) on the binding of replication proteins with lambda DNA and with the membrane was studied. The non-ionic detergent, Triton X-100 caused displacement of a part of lambda DNA from the membrane to the free lambda DNA peak; both lambda replication proteins were bound with free lambda DNA. The binding of the O protein with lambda DNA was relatively stable, but was destroyed by the ionic detergent, sarcosyl.
Bacteriophage lambda↗
Plasmid transformation of Streptococcus lactis protoplasts: optimization and use in molecular cloning.
The parameters affecting polyethylene glycol-induced plasmid transformation of Streptococcus lactis LM0230 protoplasts were examined to increase the transformation frequency. In contrast to spreading protoplasts over the surface of an agar medium, their incorporation into soft agar overlays enhanced regeneration of protoplasts and eliminated variability in transformation frequencies. Polyethylene glycol with a molecular weight of 3,350 at a final concentration of 22.5% yielded optimal transformation. A 20-min polyethylene glycol treatment of protoplasts in the presence of DNA was necessary for maximal transformation. The number of transformants recovered increased as the protoplast and DNA concentration increased over a range of 3.0 X 10(6) to 3.0 X 10(8) protoplasts and 0.25 to 4.0 micrograms of DNA per assay, respectively. With these parameters, transformation was increased to 5 X 10(3) to 4 X 10(4) transformants per microgram of DNA. Linear and recombinant plasmid DNA transformed, but at frequencies 10- to 100-fold lower than that of covalently closed circular DNA. Transformation of recombinant DNA molecules enabled the cloning of restriction endonuclease fragments coding for lactose metabolism into S. lactis LM0230 with the Streptococcus sanguis cloning vector, pGB301. These results demonstrated that the transformation frequency is sufficient to clone plasmid-coded genes which should prove useful for strain improvement of dairy starter cultures.
Base Sequence↗