Cryptic DNA Research Articles

Mediation of serum resistance in Salmonella typhimurium by an 11-kilodalton polypeptide encoded by the cryptic plasmid.

A cosmid bank of the DNA (including cryptic plasmid DNA) of a virulent strain of Salmonella typhimurium was prepared in Escherichia coli K12, and clones that contained cryptic plasmid DNA were detected by probing. Two such clones expressed a new outer membrane protein of 11 kilodaltons (kDa) and were serum resistant (E. coli K12 is serum sensitive). The gene encoding the 11-kDa protein was subcloned in a 2.1-kilobase fragment and shown to mediate serum resistance in both E. coli K12 and a cryptic plasmid-free (serum-sensitive) strain of S. typhimurium. The cryptic plasmid-free S. typhimurium strain did not express normal lipopolysaccharide, but introduction of the 11-kDa protein gene into the strain rendered the strain serum resistant without restoration of normal lipopolysaccharide synthesis. The 11-kDa protein gene was not sufficient to restore either macrophage resistance or virulence to a cryptic plasmid-free strain of S. typhimurium.

Hypermethylation of human DNA sequences in embryonal carcinoma cells and somatic tissues but not in sperm

Certain human DNA sequences are much less methylated at CpG sites in sperm than in various adult somatic tissues. The DNA of term placenta displays intermediate levels of methylation at these sequences (Sp-0.3 sequences). We report here that pluripotent embryonal carcinoma (EC) cells derived from testicular germ cell tumors are hypermethylated at the three previously cloned Sp-0.3 sequences and seven newly isolated sequences that exhibit sperm-specific hypomethylation. In contrast to their hypermethylation in EC cells, the Sp-0.3 sequences are hypomethylated in a line of yolk sac carcinoma cells, which like placenta, represent an extraembryonic lineage. These DNA sequences, therefore, appear to be subject to coordinate changes in their methylation during differentiation, probably early in embryogenesis, despite their diversity in copy number (1 to 10(4] and primary structure. Two of these Sp-0.3 sequences are highly homologous to DNA sequences in human chromosomal regions that might be recombination hotspots, namely, a cryptic satellite DNA sequence at a fragile site and the downstream region of the beta-globin gene cluster.

Origin of small beta-lactamase-specifying plasmids in Haemophilus species and Neisseria gonorrhoeae.

Fifty-nine percent of unselected strains of Haemophilus parainfluenzae were found to carry small, phenotypically cryptic plasmid DNA species. Using filter blot hybridization, we found several plasmids which were homologous to the small beta-lactamase-specifying plasmids pJB1 and pFA7, which were originally isolated from Haemophilus ducreyi and Neisseria gonorrhoeae, respectively. Detailed filter hybridization studies combined with electron microscope heteroduplex analysis suggested that three cryptic plasmids are completely homologous to the non-TnA sequences of pJB1. One cryptic plasmid was found to be highly homologous to pJB603, a small beta-lactamase plasmid previously found in two isolates of H. influenzae. A second group of plasmids were found to carry sequences homologous to pJB1 and other sequences homologous to pJB603. These results strongly suggest that small beta-lactamase plasmids found in Haemophilus species and N. gonorrhoeae may have arisen by insertion of the transposable beta-lactamase-specifying element TnA into small, phenotypically cryptic replicons resident in H. parainfluenzae. Attempts to reproduce such a recombination event in the laboratory were not successful.

Intragenic variation by site-specific recombination in the cryptic plasmid of Neisseria gonorrhoeae.

Cryptic plasmid DNA of Neisseria gonorrhoeae was found integrated into the gonococcal chromosome in both plasmid-bearing strains and plasmid-free strains. At several chromosomal locations only segments of the plasmid were found. However, in at least two strains an intact copy of the plasmid seemed to be present with the joints between the plasmid and the chromosomal DNA being located within the cppB gene of the cryptic plasmid. The cppB gene was shown to undergo a sequence-specific intragenic deletion. The deletion removed 54 base pairs, representing 18 amino acids, and did not affect the reading frame. It is proposed that the cryptic plasmid integrates into the chromosome and other gonococcal plasmids within this site-specific deletion region. Models for the site-specific recombination are presented.

Highly repeated DNA and holocentric chromosomes of the woodrush Luzula flaccida (Juncaceae)

Three highly repeated DNA components were characterized from Luzula flaccida. The 1.683 g/cm3 satellite accounts for 22% of the genome and has a sequence complexity of 127 base pairs (BP). A further 8% of the genome consists of a cryptic satellite DNA with a repeating unit of 190 BP. A minor satellite component of buoyant density 1.681 g/cm3 constitutes less than 2% of the total DNA and has a unit length of 184 BP. The presence of segments of the 1.683 satellite DNA lacking Mbol cleavage sites suggests that amplification of sequence variants may have occurred during the evolution of this satellite. While the distribution of satellite DNA appears to be different in being interspersed along the holocentric chromosomes of L. flaccida, the structure and mode of evolution of highly repeated DNA appears to follow the same pattern as seen in organisms with localized centromeres.Key words: Luzula, holocentry, highly repeated DNA.

Plasmid-mediated biodegradative fate of monohalogenated biphenyls in facultatively anaerobic sediments.

The results of these studies have demonstrated that model PCB substrates can be mineralized by indigenous microbial population in contaminated sediments. This catabolic function can be rate limited at the microenvironmental level by physical-chemical processes such as physical partitioning and accumulation. At the biochemical level, this catabolic function is determined by the existence of plasmid borne genes that, under laboratory conditions, can be maintained and expressed in pure or mixed culture. Numerous limitations are encountered in establishing the significance of these biodegradative bacteria and the catabolic plasmids at the environmental level. Relatively little information is available concerning frequencies and stability of the bacteria or the plasmid encoded genes within the community. There is no information on the incompatibility grouping of the isolated plasmid relative to other plasmids maintained within the populations. Such factors will influence the development of gene screening techniques to monitor gene frequency distributions in the sediment community. Although mineralization of 4CBP was observed under moderately reducing conditions, it remains suspect that transient or trace levels of dissolved oxygen may have permitted conventional aerobic metabolism of the substrate. If this is true, demonstrating anaerobic metabolism of environmental contaminants will require strict and tedious cultivation under highly reduced conditions (approximately-300 mV). Large deletions of cryptic DNA observed under laboratory conditions may affect bacterial survival and gene maintenance and transfer under environmental conditions. Little information exists on regulation of catabolic activity of selective pressures required to maintain the degradative genes under environmental conditions. Such limitation encountered in these studies are shared by virtually all attempts to utilize genetically manipulated bacteria or newly isolated strains and plasmids. Perhaps the fundamental question is whether the catabolic genes are maintained and expressed within the community rather than whether the host bacterium can survive in the environment.

Complex organization of a cryptic satellite DNA in the genome of the marine invertebrate Rapana thomasiana Grosse (Gastropoda)

Isopicnic centrifugation in Cs 2SO 4-Ag + gradients at pH 7.0 reveals that the genome of the marine snail Rapana thomasiana Grosse (Gastropoda) contains an AT-rich satellite fraction comprising 5% of the DNA. Restriction enzyme analysis shows that the satellite DNA is composed of a number of related subsets arranged in tandem arrays. They have evolved from the segmental amplification of an 1460 bp long monomer unit with a complex inner organization. Most probably, the present basic repeat originates from an ancestral 400–500 bp long sequence in which some insertions and/or deletions have occurred.

Sequence of the cryptic satellite DNA from the plant Sinapis alba

A satellite DNA with a buoyant density equal to that of main band DNA in neutral cesium chloride (‘cryptic satellite’) can be isolated from the DNA of mustard ( Sinapis alba) nuclei by Ag +/Cs 2SO 4 density gradient centrifugation. This satellite is cleaved into 172 bp repeat units by HinfI, AluI or HaeIII. The HinfI fragments have been further cleaved by AluI, and seven AluI subfragments have been sequenced. As a result two versions of a basic 172 HinfI repeat have been found, one (A + B) with an additional HinfI site. These two sequences (A + B and C) are the most frequent versions of the basic repeat of mustard satellite DNA. The basic 172 bp unit does not contain subrepeats or palindromic sequences. It is not similar (at a criterion of 15 common bases) with any known satellite sequence. It is not unusually highly methylated in the native state.

Organization of African green monkey DNA at junctions between α-satellite and other DNA sequences

Segments of African green monkey DNA containing sequences of the highly reiterated cryptic satellite DNA called α-satellite were selected from a library in λ bacteriophage. This λ library was constructed to enrich for monkey segments that contain (1) irregular regions of α-satellite and (2) α-satellite linked to other monkey sequences. At least 11 of 15 cloned monkey segments between 13 × 10 3 and 16 × 10 3 base-pairs in length, selected by hybridization to α-satellite, also include other monkey sequences. In general, α-satellite sequences close to the junctions with non-α-satellite DNA contain an abundance of divergent forms compared to the average frequency of such forms within total α-satellite. Many of the cloned segments are missing some of the HinIII sites that occur once in most monomer units of α-satellite, and likewise several of the cloned segments contain restriction sites that rarely occur in α-satellite as a whole. In some segments HinIII sites occur that are spaced at distances other than the basic multiple of 172 base-pairs. At least one of the cloned segments, however, is composed mainly of typical 172 base-pair long α-satellite monomer units. Several of these cloned DNAs have been mapped by restriction endonuclease digestion and Southern blot analysis and the arrangements of α-satellite and non-α-satellite sequences have been determined. In addition to segments that contain a boundary where satellite meets other types of sequence, some contain two such boundaries and thus satellite flanks a non-α-satellite segment. Further, two different types of non-α-satellite sequence appear to be common to more than one phage, perhaps indicating some recurring organization at boundaries.

Isolation and preliminary characterization of cryptic satellite DNA in pea

Optimum conditions have been established for isolation of ‘cryptic’ satellite DNA from the genome of pea ( Pisum sativum), using gradients of CS 2SO 4 containing silver ions. At an Ag + :DNA-P ratio ( R) of 0.1, and at alkaline pH, four fractions are obtained: mainband (buoyant density 1.437 g cm 3; 67% of total DNA), satellite I (buoyant density 1.582 g/cm 3; 7% of total DNA), satellite II (buoyant density 1.520 g/cm 3, 11% of total) and satellite III (buoyant density variable between 1.45 and 1.51 g/cm 3; 15% of total). The reiterated DNA content of these four fractions has been investigated by reassociation experiments conducted over a Cot range of 1 × 10 −5 to 2.0. All four fractions contain at least two kinetic components; each fraction, including the mainband, consists at least partly of highly reiterated DNA. Ribosomal RNA hybridizes only to the mainband.

Satellite Ic: a possible link between the satellite DNAs of D. virilis and D. melanogaster

In this study, we isolated and characterized a previously undetected cryptic satellite DNA comprising 0.1% of the total nuclear genome of D. virilis. This satellite is hidden from detection in neutral CsCl by satellite I and is therefore designated cryptic satellite I or Ic. Sequence analysis reveals that Ic is the repeating heptanucleotide [poly d(AATATAG): d(CTATATT)]. It is more closely related to the three simple sequence satellite DNAs of D. melanogaster, a distantly related species, than it is to any of the major D. virilis satellite DNA sequences. Ic may therefore be a link between the simple sequence satellites of D. virilis and D. melanogaster. As an extension of this theory, we have constructed a “family tree” linking the satellites of D. virilis and D. melanogaster by a series of “simple” operations. Only one intermediate required by this evolutionary scheme has not yet been identified.

Characterization of plasmids isolated from Rhizobium meliloti

The occurrence of endogenous plasmid DNA in Rhizobium meliloti is reported. R. meliloti strains carrying RP 4 were used to work out a suitable plasmid isolation method. By sarcosyl lysis RP 4 plasmid DNA as well as cryptic plasmid DNA was isolated. Cryptic plasmid DNA possesses a buoyant density of 1.717 g cm-3 and shows a heterogeneity in contour length. Large plasmid molecules of 130 μm length were detected in the electron microscope. We also found that RP4 is able to restore the nitrogen fixation capacity of an ineffective R. meliloti strain.

The heterogeneity of B-chromosome DNA: no evidence for a B-chromosome specific repetitive DNA correlated with B-chromosome effects on meiotic pairing in the triticinae

The compositional heterogeneity of DNAs of A (normal) and B (supernumerary) chromosomes of Aegilops speltoides, Ae. mutica and Triticum aestivum has been compared in order to elucidate the mechanism of B-chromosome disruption of meiotic pairing in interspecific hybrids. Comparisons of % heterologous association after DNA/DNA hybridation at C0t 10−2 (highly repetitious DNA) and C0t 100 (moderately repetitious DNA), and comparisons of nucleotide base divergence (ΔTms) and thermal elution profiles of homologous and heterologous duplexes, show that genotypes of Aegilops spp., having large numbers of Bs, do not carry additional families of repetitious DNA exclusive to B-chromosomes. Neither the presence of Bs nor the direction of DNA/DNA hybridisation affect the above parameters. No cryptic DNA satellites were revealed in A- and B-chromosome DNA after sedimentation in actinomycin D-CsCl gradients; and there were no significant differences in buoyant densities of main-band DNA. Mean melting temperatures (Tm); transition temperatures (ΔT) and numbers and positions of peaks of dissociating DNA fractions in profiles of differentiated melting curves of native DNAs were similar in strictly comparable denaturation conditions. One small AT-rich (< 5%) DNA fraction correlated with speltoides Bs was revealed; however, no corresponding fraction is associated with mutica Bs. The overall similarity in numbers and base composition of families of DNA (repetitious and unique) of As and Bs is discussed in relation to the origin of Bs and the origin of the meiotic diploidising system in haploid T. aestivum.

Molecular characterization of a stable F [formula omitted] plasmid

F lac S is a thermostable extrachromosomal element isolated in Salmonella typhimurium which is altered in its replication as compared to its precursor F ts114 lac . Sedimentation of both these plasmids in alkaline sucrose gradients has indicated a difference in their sizes. Contour length measurements of open circular plasmid DNA molecules photographed in the electron microscope have revealed the estimated molecular weight of F ts114 lac to be 81 × 10 6 daltons while that of F lac S is 109 × 10 6 daltons. F lac S may carry a segment of S. typhimurium chromosomal or cryptic plasmid DNA.

Clustering of transfer RNA genes of Xenopus laevis

The arrangement of the reiterated DNA sequences complementary to transfer RNA has been studied in Xenopus laevis. Prehybridization of denatured DNA with an excess of unfractionated tRNA results in a small but well-defined increase in the buoyant density of fragments which contain sequences homologous to tRNA. The density increase is smaller than that found for 5 S DNA, but is the same or nearly so for all tRNA coding sequences examined. These results indicate that the majority of tRNA genes are clustered together with spacer DNA, the average size of which is estimated to be approximately 0.5 × 10 6 daltons (native) DNA. In high molecular weight native DNA preparations, the sequences homologous to unfractionated tRNA, tRNA Val, tRNA 1 Met and tRNA 2 Met band in CsCl at 1.707, 1.702, 1.708 and 1.711 g cm −3, respectively. The mean buoyant densities are constant at all molecular weights examined but they do not correspond to the base compositions of the complementary tRNA species. These results indicate that isocoding genes are linked to spacer DNA in separate and extensive gene clusters, and that the different clusters contain different spacer DNA sequences. These clusters form well-defined cryptic DNA satellites which are potentially separable from each other as well as from other chromosomal DNA.