Polynucleotide Sequence Research Articles

Polynucleotide sequence relatedness among motileAeromonas species

Relatedness among 55 motileAeromonas strains was assessed by determining the extent of reassociation in heterologous DNA preparations. S1 nuclease and diethylaminoethyl-cellulose filters were used to separate reassociated nucleotide sequences from nonreassociated sequences and to determine the thermal stability of related nucleotide sequences. The motile aeromonads would presently consist of three species:A. hydrophila (type strain ATCC 7966),A. caviae (type strain ATCC 15468), andA. sobria (type strain CIP 7433). These three species are clearly separated on the basis of both biochemical characteristics and similarities in DNA. Each of these three species contains more than one hybridization group: three groups inA. hydrophila; two groups inA. caviae; and at least two groups inA. sobria. DNA hybridization groups are biochemically similar within each species. When additional data is available, these separate DNA hybridization groups may merit designation. Any decision to delineate species in terms of DNA hybridization groups must await a phenotypic basis for their separation.

Evidence for papillomavirus genus-specific antigens and DNA in laryngeal papilloma.

Four juvenile laryngeal papillomas (JLPs) were examined for papillomavirus genus-specific structural antigens and polynucleotide sequences employing reagents derived from bovine papillomavirus type 1 (BPV-1). Three of four JLPs contained intranuclear antigens in the granular layer of differentiating epithelial cells of formalin-fixed, paraffin-embedded papillomas assayed by the peroxidase-antiperoxidase reaction using an antiserum prepared against purified detergent-disrupted BPV-1. Examination of these JLPs for viral sequences indicated two tumor DNA preparations contained polynucleotide sequences which hybridized to an in vitro labeled BPV-1 DNA probe under nonstringent conditions. These sequences were detectable after gel electrophoresis and immobilization of restriction-endonuclease-digested JLP DNAs onto nitrocellulose membranes. Specific hybridization of probe to digested JLP DNA was visualized as bands in autoradiograms coincidental with open circular and linearized papillomavirus DNA. Hybridization was extensively reduced when JLP DNA preparations were prehybridized to an unlabeled papillomavirus DNA heterologous to the probe. These results provide direct evidence for the association of a papillomavirus in JLP.

Binding of microtubule protein to DNA and chromatin: possibility of simultaneous linkage of microtubule to nucleic acid and assembly of the microtubule structure

Microtubule protein binds to DNA through microtubule associated polypeptides (MAPs). Among MAPs there is one high molecular weight polypeptide (MAP2) which interacts with DNA fundamentally through certain polynucleotide sequences. This interaction is not affected by the presence of histones and other chromosomal proteins. DNA can associate to assembled microtubules and when a determinate DNA/protein ratio is reached the nucleic acid behaves as a microtubule associated molecule. The nucleic acid fragments which preferentially bind to microtubules have been isolated and characterized. These fragments contain DNA regions enriched in repetitive sequences that hybridizes preferentially to the pericentromeric zone of metaphase chromosomes. These results give further support to the model of interaction microtubule-chromosome based upon the mediator function of the microtubule associated proteins.

A general function of noncoding polynucleotide sequences. Mass binding of transconformational proteins.

It is proposed that a general function of noncoding DNA and RNA sequences in higher organisms (intergenic and intervening sequences) is to provide multiple binding sites over long stretches of polynucleotide for certain types of regulatory proteins. Through the building up or abolishing of high-order structures, these proteins either sequester sites for the control of, e.g., transcription or make the sites available to local molecular signals. If this is to take place, the existence of a 'c-value paradox' becomes a requirement. Multiple binding sites for a given protein may recur in the form of a sequence 'motif' that is variable within certain limits. Noncoding sequences of the chickens ovalbumin gene furnish an appropriate example of a sequence motif. GAAAATT. Its improbably high frequency and significant periodicity are both absent from the coding sequences of the same gene and from the noncoding sequences of a differently controlled gene in the same organisms, the preproinsulin gene. This distribution of a sequence motif is in keeping with the concepts outlined. Low specificity of sequences that bind protein is likely to be compatible with highly specific conformational changes.

Comments on “growth of a hypercycle” by king (1981)

The notion of hypercycles has been introduced (Eigen, 1971) in order to characterize a functional entity which integrates information stored in individual self-replicating elements. Commonly, these self-replicating elements compete, like polynucleotides do in test tube experiments (Spiegelman, 1971; Biebricher et al., 1981) or, like species do in nature. A single species is selected eventually together with its most frequent mutants and the less efficient competitors disappear. In order to suppress competition, specific coupling terms of second order - these are coupling terms which are proportional to the product of two population numbers or concentration - have to be introduced into the kinetic equations such a link has to extend to all members, requiring the formation of a closed catalytic cycle (Eigen and Schuster, 1979). The copying of polynucleotide sequences, like every transfer of information, can only occur with finite accuracy (Eigen et al., 1981). This physical restriction imposes a limit upon the content of information that can be transmitted. A higher content of information requires more precise replication. A more involved replication machinery, however, presupposes more information to build it. Thus, a single autocatalyst will not be able to increase its content of information over the given threshold. How does one escape from this vicious circle? Hypercycles present a solution to the problem: many information carriers coexist and may cooperate to build a more sophisticated and more precisely copying replication machinery. Optimization of growth rates has never been the goal for which hypercycles were conceived. Any first order process can proceed at a higher rate than a second order process if low enough concentrations are chosen. On the other hand, any realistic hypercycle involves both first and second order terms. At low concentrations first order terms provide for accumulation of material while at higher concentration second order terms stabilize cooperation. At which concentration level such a prevalence changes is a matter of quantitative values of rate parameters rather than of basic principle. An autocatalyst may outgrow a hypercycle under certain conditions but it can never solve the problem of integrating information.

Cloning of human papilloma virus genomic DNAs and analysis of homologous polynucleotide sequences.

The complete DNA genomes of four distinct human papilloma viruses (human papilloma virus subtype 1a [HPV-1a], HPV-1b, HPV-2a, and HPV-4) were molecularly cloned in Escherichia coli, using the certified plasmid vector pBR322. The restriction endonuclease patterns of the cloned HPV-1a and HPV-1b DNAs were similar to those already published for uncloned DNAs. Physical maps were constructed for HPV-2a DNA and HPV-4 DNA, since these viral DNAs had not been previously mapped. By using the cloned DNAs, the genomes of HPV-1a, HPV-2a, and HPV-4 were analyzed for nucleotide sequence homology. Under standard hybridization conditions (Tm = --28 degrees C), no homology was detectable among the genomes of these papilloma viruses, in agreement with previous reports. However, under less stringent conditions (i.e., Tm = --50 degrees C), stable DNA hybrids could be detected between these viral DNAs, indicating homologous segments in the genomes with approximately 30% base mismatch. By using specific DNA fragments immobilized on nitrocellulose filters, these regions of homology were mapped. Hybridization experiments between radiolabeled bovine papilloma virus type 1 (BPV-1) DNA and the unlabeled HPV-1a, HPV-2a, or HPV-4 DNA restriction fragments under low-stringency conditions indicated that the regions of homology among the HPV DNAs are also conserved in the BPV-1 genome with approximately the same degree of base mismatch.

Contribution of DNA-DNA Hybridization to the Transfer of Enterobacter aerogenes to the Genus Klebsiella as K. mobilis

Summary Polynucleotide sequence relatedness reactions were carried out to determine the extent of DNA divergence between labelled DNA of Klebsiella pneumoniae and Enterobacter cloacae and unlabelled DNA of E. aerogenes and K. oxytoca . DNA homology between 14 strains of E. aerogenes and one strain of K. pneumoniae and one strain of E. cloacae was 53% ± 3 and 39% ± 4 respectively. Similarly, unlabelled DNA from 11 strains of K. oxytoca exhibited 55% ± 4 and 40% ± 3 DNA relatedness with labelled DNA of K. pneumoniae and E. cloacae respectively. It is concluded that E. aerogenes should be classified in the genus Klebsiella with the denomination of Klebsiella mobilis as previously proposed by Bascomb et al. (1971).

Characterization of the plasmids comprising the “R Factor” R5 and their relationships to other R plasmids

The “R factor” R5 confers resistance to tetracycline (Tc), streptomycin (Sm) and spectinomycin (Sp), chloramphenicol (Cm), sulfonamides (Su), kanamycin (Km), and mercuric ion (Mer). This phenotype is mediated by the presence of two R plasmids: pMH1 and pMH2, having approximate weights of 18.5 and 62 megadaltons (Mdal), respectively. pMH1 encodes Sm, Su, Cm, Mer, and Km resistance, and is nonconjugative. pMH2 confers Sm, Su, Cm, Mer, and Tc resistance, is conjugative, and belongs to the FII incompatibility group. NR79 is a 63-Mdal R plasmid encoding the same resistances as “R5,” and was derived from the same geographical source. It belongs to the FII incompatibility group and is conjugative. Analysis of restriction endonuclease digestion patterns and polynucleotide sequence homologies indicate that pMH1, pMH2, and NR79 are closely related. In addition, pMH2 and NR79 exhibit nearly complete homology to R100. Restriction endonuclease maps and resistance gene locations for pMH1, pMH2, and NR79 have been derived and a model for the evolutionary relationships of these plasmids is presented.

Polynucleotide sequence relatedness and genome size among Enterobacter intermedium sp. nov. and the species Enterobacter cloacae and Klebsiella pneumoniae

Summary A DNA-DNA hybridization study was carried out on a new group of Enterobacteriaceae (group H1). The highest relative binding ratios were found with the species E. cloacae (37 to 61%) and K. pneumoniae (44 to 60%), The labelling of E. cloacae CUETM 77–120 and K. pneumoniae CUETM 77–169 DNA showed 27 to 32% and 20 to 35%, respectively, of relatedness with the group H1. According to phenotypic data (numerical taxonomy) and genetic data (GC%, DNA/DNA hybridization and relative genome size: K. pneumoniae CUETM 77–169: 100, E. cloacae CUETM 77–120: 80, group H1 CUETM 77–130: 58), the group H1 represents a new species to be called Enterobacter intermedium.

Genetic and molecular characteristics of Vir plasmids of bovine septicemic Escherichia coli.

Three wild strains of bovine septicemic Escherichia coli were selected on the basis of their production of a toxin lethal for mice and chickens and their characteristic surface antigen. The transfer of these virulence (Vir) properties from two of the three to recipient E. coli was detected after mating. One Vir plasmid (pJL1) was derepressed for transfer and associated with mobilization of chromosomal markers. The other, pJL2, was repressed. Both plasmids were tagged with transposon Tn5 (kanamycin resistance), and transfer parameters of the tagged plasmids were studied. The Tn5 insertion in pJL2 usually increased transfer efficiency 100-fold. Plasmid pJL1 was classified as a member of the FIV incompatibility group. A pJL1::Tn5 derivative plasmid was incompatible with ColV1. Plasmid pJL2 behaved as an fi+ plasmid. Both plasmids pJL1 and pJL2 had a molecular weight of 92 x 10(6) and were present at about four copies per chromosome; their deoxyribonucleic acid (DNA) structures were not identical on the basis of restriction enzyme analysis. DNA-DNA hybridization revealed a polynucleotide sequence homology of at least 58% between the two plasmids. No plasmids could be detected in one wild or certain laboratory-derived Vir+ E. coli strains.

Polynucleotide Sequence Divergence Among Some Coagulase-negative Staphylococci

The degree of binding was determined, at both exacting and non-exacting temperatures, between deoxyribonucleic acid (DNA) preparations from coagulase-negative staphylococci and carbon (14C) labelled DNA from nine reference strains, Staphylococcus capitis LK499, S. cohnii GH137, S. epidermidis GH37, S. haemolyticus SM131, S. hominis DM122, S. saprophyticus CCM883, S. simulans SM128, S. warneri AW25 and S. xylosus KL162. The close similarity in nucleotide sequences found between five of the six representatives received as S. capitis, S. hominis and S. warneri indicate that these organisms are separated into too many species. In contrast, little homology was found between the pairs of strains representing S. cohnii and S. haemolyticus, respectively. The divergence indices of the nine reference strains were calculated and the relationship between them expressed in a sorted similarity matrix and dendrogram based upon the average linkage algorithm. The pattern of nucleotide heterogeneity was found by plotting the relative DNA bound at the exacting temperature against the divergence indices. The coagulase-negative staphylococci exhibit extensive genetic diversity due mainly to random mutation.

Messenger RNA coding for phenylalanine ammonia-lyase. Characterization and partial purification from cell suspension cultures of Petroselinum hortense.

The mRNA coding for phenylalanine ammonia-lyase was partially purified from irradiated cell suspension cultures of parsley (Petroselinum hortense). The product of cell-free translation of the mRNA in a reticulocyte lysate was isolated by immunoprecipitation and compared with the native enzyme subunit. Evidence for the identity, or at least a great similarity, of both was provided by tryptic-peptide and gel-electrophoretic analyses. Under partially denaturing conditions, phenylalanine ammonia-lyase mRNA sedimented as a 20--21-S molecule in a sucrose gradient and had an apparent molecular weight of about 1.05 x 10(6) on a polyacrylamide gel. Approximately two-thirds of the polynucleotide sequence of the mRNA were estimated to be required as coding sequence for the enzyme. We suggest that phenylalanine ammonia-lyase mRNA is unlikely to code for more than of three coordinately induced enzymes.

Evolution of multiple-antibiotic-resistance plasmids mediated by transposable plasmid deoxyribonucleic acid sequences

Two plasmid deoxyribonucleic acid sequences mediating multiple antibiotic resistance transposed in vivo between coexisting plasmids in clinical isolates of Serratia marcescens. This event resulted in the evolution of a transferable multiresistance plasmid. Both sequences, designated in Tn1699 and Tn1700, were flanked by inverted deoxyribonucleic acid repetitions and could transpose between replicons independently of the Excherichia coli recA gene function. Tn1699 and Tn1700 mediated ampicillin, carbenicillin, kanamycin, and gentamicin resistance but differed in the type of gentamicin-acetyltransferase enzymes that they encoded. The structural genes for these enzymes share a great deal of polynucleotide sequence similarity despite their phenotypic differences. The transposition of Tn1699 and Tn1700 to coresident transferable plasmids has contributed to the dissemination of antibiotic resistance among other gram-negative bacteria. These organisms have recently caused nosocomial infections in epidemic proportions.

Sequences of vesicular stomatitis virus RNA in the region coding for leader RNA, N protein mRNA, and their junction.

The RNAs extracted from purified preparations of the Indiana and New Jersey serotypes of vesicular stomatitis virus were polyadenylylated in vitro by using polynucleotide phosphorylase and sequence determination was carried out by the dideoxynucleotide method using reverse transcriptase and dT8AC primer. On both virus RNAs a short stretch of adenylic acid residues is present between the regions coding for the leader and N protein mRNAs. Other features of the RNA sequences of the two viruses are compared to each other and to published data.

Conserved polynucleotide sequences among the genomes of papillomaviruses

The DNAs of different members of the Papillomavirus genus of papovaviruses were analyzed for nucleotide sequence homology. Under standard hybridization conditions (Tm - 28 degrees C), no homology was detectable among the genomes of human papillomavirus type 1 (HPV-1), bovine papillomavirus type 2 (BPV-2), or cottontail rabbit (Shope) papillomavirus (CRPV). However, under less stringent conditions (i.e., Tm - 43 degrees C), stable hybrids were formed between radiolabeled DNAs of CRPV, BPV-1, or BPV-2 and the HindIII-HpaI A, B, and C fragments of HPV-1. Under these same conditions, radiolabeled CRPV and HPV-1 DNAs formed stable hybrids with HincII B and C fragments of BPV-2 DNA. These results indicate that there are regions of homology with as much as 70% base match among all these papillomavirus genomes. Furthermore, unlabeled HPV-1 DNA competitively inhibited the specific hybridization of radiolabeled CRPV DNA to bpv-2 DNA fragments, indicating that the homologous DNA segments are common among these remotely related papillomavirus genomes. These conserved sequences are specific for the Papillomavirus genus of papovaviruses as evidenced by the lack of hybridization between HPV-1 DNA and either simian virus 40 or human papovavirus BK DNA under identical conditions. These results indicate a close evolutionary relationship among the papillomaviruses and further establish the papillomaviruses and polyoma viruses as distinct genera.

Comparative Study of Deoxyribonucleic Acid Homology and Physiological Characteristics of Strains of Peptococcus saccharolyticus

Polynucleotide sequence relationships among strains of Peptococcus saccharolyticus were assessed by analysis of deoxyribonucleic acid-deoxyribonucleic acid homo and heteroduplexes with endonuclease S1. The results showed that P. saccharolyticus strains isolated from different subjects form a very tight group, with deoxyribonucleic acid homology levels ranging between 93 and 100%. Physiological tests of 23 strains included 30 different substrates. Results were remarkably uniform. All 23 strains grew better anaerobically with added H2 and CO2 than aerobically. However, colony size was greater on blood agar but not Trypticase soy (BBL Microbiology Systems, Cockeysville, Md.)-yeast extract agar with an atmosphere of 4% O2 with added H2 and CO2 than with anaerobic incubation. The data suggest that these strains are sufficiently closely related to justify their inclusion in a single discrete species, but their appropriate generic classification remains to be resolved.

Molecular nature of two Haemophilus influenzae R factors containing resistances and the multiple integration of drug resistance transposons

The 36-megadalton Haemophilus influenzae R plasmid pHK539 was found to specify resistance to tetracycline (Tc) and ampicillin (Ap). It was shown by molecular hybridization studies and by electron microscopy that the plasmid pHK539 contained the tetracycline translocation deoxyribonucleic acid (DNA)segment (TnTc) as well as the ampicillin translocation segment (TnAp). The TnAp was integrated in the stem of TnTc. The 34-megadalton H. influenzae R plasmid pRI234 carried a translocatable DNA segment which specified both tetracycline and chloramphenicol (Cm) resistance. Self-annealing and DNA-DNA heteroduplex experiments indicated that this transposon is probably composed of TnTc containing an insertion of a chloramphenicol resistance transposon (TnCm). TnCm is inserted into one of the components of the TnTc inverted repetitions and is itself flanked on both sides by long inverted repetitions. The H. influenzae plasmids pHK539 and pRI234 had more than 60% of their polynucleotide sequences in common with all the other 30- to 40-megadalton R factors recently found in H. influenzae isolates from different countries. The tetracycline-chloramphenicol resistance transposon of pRI234 was integrated twice at different sites in the plasmid after its growth in medium containing tetracycline. The presence of the two copies of the transposon was correlated with higher minimum inhibitory concentrations against tetracycline as well as against chloramphenicol. After its growth in medium containing tetracycline, the H. influenzae R plasmid pFR16017 specifying Tc resistance contained one, two, three, or even four copies of TnTc integrated at different sites in the plasmid, or the loop of TnTc was amplified. The heterogeneity of the pFR16017 plasmid was seen in all single-colony isolates and correlated with a higher minimum inhibitory concentration against tetracycline.

Deoxyribonucleic acid relationships among members of the genus Aeromonas

Polynucleotide sequences among 24 motile and 11 non-motile aeromonads were studied by analysis of deoxyribonucleic acid - deoxyribonucleic acid (DNA-DNA) duplexes with endonuclease S1. In addition, DNA base composition (mole % guanine and cytosine (G + C)) and relative genome sizes were determined for selected strains. Large variations in genome size were found and % GC ranged from 57.1 to 62.9%. On the basis of the strains examined, the Genus Aeromonas consists of two genotypically legitimate groups: a diverse group of motile aeromonads, and the genetically more homogeneous non-motile aeromonads, comprising the species Aeromonas salmonicida. Internal homology groups could not be demonstrated within the motile aeromonads, and significant divergence in related sequences was indicated. This diverse motile group forms the single species Aeromonas hydrophila.

Molecular characterization of a small Haemophilus influenzae plasmid specifying beta-lactamase and its relationship to R factors from Neisseria gonorrhoeae.

The ampicillin-resistant Haemophilus influenzae strain Ve445 which caused purulent meningitis and septicaemia in a newborn child in Germany contained a 4.4 megadalton (Mdal) plasmid (pVe445) and produced a TEM type beta-lactamase. The transformation to ampicillin resistance of a sensitive Escherichia coli strain with isolated pVe445 DNA proved that the structural gene for the beta-lactamase resided on this plasmid genome. Molecular DNA-DNA hybridization studies and electron microscope DNA heteroduplex analysis indicated that pVe445 probably contained 38 to 41% of the ampicillin translocation DNA segment (TnA) found on R factors of enteric origin. The TnA fragment present in pVe445 most likely does not contain both of the inverted repeat sequences of TnA. DNA-DNA polynucleotide sequence studies indicated that the 4.4 Mdal plasmid pVe445 was unrelated to the 30 to 38 Mdal H. influenzae R plasmids but was closely related to the 4.1 Mdal ampicillin resistance specifying H. influenzae plasmid RSF0885 isolated in the U.S.A. The H. influenzae plasmid pVe445 shared 91% of its base sequences with the beta-lactamase specifying Neisseria gonorrhoeae plasmid pMR0360 (4.4 Mdal) and had 85% of its base sequences in common with the beta-lactamase specifying N. gonorrhoeae plasmid pMR0200 (3.2 Mdal). All of the four 3.2 to 4.4 Mdal beta-lactamase specifying R plasmids of H. influenzae and N. gonorrhoeae investigated probably have a common evolutionary origin.

Pathogenic Vibrio isolated from cultured eels. IV. Deoxyribonucleic acid studies.

The molecular taxonomic position of a new pathogenic vibrio isolated from cultured eels has been studied. The bacterium had been classified as V. anguillarum Type B, V. anguillarum f. anguillicida, on the basis of phenotypic properties. The percent guanine plus cytosine content of deoxyribonucleic acid (DNA) was 46.3%, which is within or appreciably close to the DNA base composition ranges of the existing Vibrio species. Polynucleotide sequence analysis by means of optical measurement of renatured DNA, however, provided proof that the present vibrio was distinct from the existing species of the genus Vibrio. The isolates from diseased eels represented a homologous group (96-97% homology for reference ET-7617 strain DNA) with the genome size of 2, 000 to 2, 500×106 daltons. On the other hand the reference strain was very distantly related to other Vibrio species: 12-23% related to V. anguillarum; 2-17% to V. fischeri; 14% to V. parahaemolyticus; 12% to V. alginolyticus; 9% to V. cholerae. Therefore the present organism was considered as a new member of the genus Vibrio.