Detection of exogenous gene sequences in dissolved DNA from aquatic environments

A method for the concentration and detection of gene sequences in the dissolved DNA from freshwater and marine environments has been developed. The limit of detection in the dot blot format was 167 fg/ml (100 ml sample) for exogenous herpes simplex thymidine kinase (TK) gene that was added to artificial seawater or river water. This procedure has been used to determine the longevity and monitor progressive changes in molecular weight of a plasmid containing the TK gene added to eutrophic estuarine water. The onset of plasmid degradation as determined by change in molecular weight was rapid (within 5 min). Intact plasmid was detected for at least 4 hours and sequences hybridizable to the TK gene probe were present for up to 24 hours.     

Human ribosomal RNA gene spacer sequences are found interspersed elsewhere in the genome

A cloned EcoRI fragment containing human 18 S rRNA gene sequences was used to screen a gene library to obtain a set of 8 overlapping cloned DNA segments extending into the non-transcribed spacer region of the human ribosomal RNA gene cluster. 19.4 kb of the approx. 43-kb rDNA repeat was obtained in cloned form and mapped with restriction endonucleases. None of the clones obtained extended into 28 S rRNA sequences. A 7-kb region of non-transcribed spacer DNA shared in common between five independent clones was subjected to comparative restriction digests. It was estimated that sequences among the five different spacer isolated varied by not more than 1.0%, if all the observed differences are assumed due to point mutation. HaeII-restriction fragments from within this same 7-kb region contain sequences carried not only within the tandem repeats of the gene cluster but interspersed elsewhere in the genome. Some of these sequences correspond to the Alu family of highly repeated interspersed sequences.     

Extent and variation of phage-borne bacterial 16S rRNA gene sequences in wastewater environments

Phage metagenomes isolated from wastewater over a 12-month period were analyzed. The results suggested that various strains of Proteobacteria, Bacteroidetes, and other phyla are likely to participate in transduction. The patterns of 16S rRNA sequences found in phage metagenomes did not follow changes in the total bacterial community.     

A review of diatoms found in highly acidic environments

A review is presented of 28 studies in the literature of diatoms in environments at pH 3.5, including natural and anthropogenic acid sources. A total of 124 diatom taxa have been reported, but many are likely to have been accidental occurrences, because only a few specimens were found. Approximately 19 taxa abundant in at least one study or common in several studies are considered true inhabitants of highly acidic waters. These include: Achnanthes minutissima, Eunotia exigua, E. tenella, E. septentrionalis, E. osoresanensis, E. arcus, E. glacialis, E. pectinalis, Frustulia rhomboides, F. rhomboides var. saxonica, Nitzschia capitellata/subcapitellata, Nitzschia communis, Nitzschia pusilla, Nitzschia vasta, Pinnularia acoricola, P. obscura, P. braunii var. amphicephala, P. subcapitata and P. terminitina. There are inconsistencies in the taxonomy of several of these species and possible synonymies could lower the number of taxa to less than 9. Compared to diatom species richness in environments at pH 4.5–5.0, there are many fewer taxa in environments pH 3.5, suggesting a threshold between pH 4.5 and 3.5 below which many species are unable to maintain a population.     

Characterization of a rat tRNA gene cluster: comparison of nucleotide sequences of the gene for tRNALeu newly found in repeating units

In the rat, DNA carrying a cluster of the genes for tRNAAsp, tRNAGly, and tRNAGlu, aligned in that order, is repeated about 10 times. Seven DNA clones corresponding to the independent repeating units were isolated from a rat gene library. Nucleotide sequence analysis of these clones revealed the presence of a fourth tRNA gene, the gene for tRNALeu, in the cluster. The tRNALeu gene is located about 600 base pairs (bp) upstream from the tRNAAsp gene and its polarity differs from those of the other three tRNA genes. Among the repeating units, the nucleotide sequence of tRNALeu is conserved to a relatively high degree.     

Interspersed repeats are found predominantly in the "old" alpha satellite families

The biased distribution of dispersed repeat insertions in various types of primate specific α satellites (AS) is being discussed in the literature in relation to the modes of AS evolution and their possible roles in maintenance and disruption of functional centromeres. However, such a bias has not been properly documented on a genome-wide scale so far. In this work, using a representative sample of about 100 insertions we show that the “old” AS contains at least 10 times more dispersed repeats than the “new” one. In the new arrays insertions accumulate mostly in poorly homogenized areas, presumably in the edges, and in the old AS, throughout the whole array length. Dating of L1 insertions in the old AS revealed that their massive accumulation started at or after the time when the new AS emerged and expanded in the genome and the centromere function had shifted to the new AS arrays.     

Nearly identical bacteriophage structural gene sequences are widely distributed in both marine and freshwater environments

Primers were designed to amplify a 592-bp region within a conserved structural gene (g20) found in some cyanophages. The goal was to use this gene as a proxy to infer genetic richness in natural cyanophage communities and to determine if sequences were more similar in similar environments. Gene products were amplified from samples from the Gulf of Mexico, the Arctic, Southern, and Northeast and Southeast Pacific Oceans, an Arctic cyanobacterial mat, a catfish production pond, lakes in Canada and Germany, and a depth of ca. 3,246 m in the Chuckchi Sea. Amplicons were separated by denaturing gradient gel electrophoresis, and selected bands were sequenced. Phylogenetic analysis revealed four previously unknown groups of g20 clusters, two of which were entirely found in freshwater. Also, sequences with >99% identities were recovered from environments that differed greatly in temperature and salinity. For example, nearly identical sequences were recovered from the Gulf of Mexico, the Southern Pacific Ocean, an Arctic freshwater cyanobacterial mat, and Lake Constance, Germany. These results imply that closely related hosts and the viruses infecting them are distributed widely across environments or that horizontal gene exchange occurs among phage communities from very different environments. Moreover, the amplification of g20 products from deep in the cyanobacterium-sparse Chuckchi Sea suggests that this primer set targets bacteriophages other than those infecting cyanobacteria.     

Nanoarchaeal 16S rRNA gene sequences are widely dispersed in hyperthermophilic and mesophilic halophilic environments

The Nanoarchaeota, proposed as the fourth sub-division of the Archaea in 2002, are known from a single isolate, Nanoarchaeum equitans, which exists in a symbiotic association with the hyperthermophilic Crenarchaeote, Ignicoccus. N. equitans fails to amplify with standard archaeal 16S PCR primers and can only be amplified using specifically designed primers. We have designed a new set of universal archaeal primers that amplify the 16S rRNA gene of all four archaeal sub-divisions, and present two new sets of Nanoarchaeota-specific primers based on all known nanoarchaeal 16S rRNA gene sequences. These primers can be used to detect N. equitans and have generated nanoarchaeal amplicons from community DNA extracted from Chinese, New Zealand, Chilean and Tibetan hydrothermal sites. Sequence analysis indicates that these environments harbour novel nanoarchaeal phylotypes, which, however, do not cluster into clear phylogeographical clades. Mesophilic hypersaline environments from Inner Mongolia and South Africa were analysed using the nanoarchaeal-specific primers and found to contain a number of nanoarchaeal phylotypes. These results suggest that nanoarchaeotes are not strictly hyperthermophilic organisms, are not restricted to hyperthermophilic hosts and may be found in a large range of environmental conditions.     

Listeria monocytogenes adheres to many materials found in food-processing environments

AIMS: To investigate the adhesion of Listeria monocytogenes 10403S to 17 different, food-use approved materials representing metals, rubbers and polymers. METHODS AND RESULTS: Adhesion assays were conducted by placing 'coupons' of the materials in planktonic cultures at 30 degrees C, and then immediately withdrawing them ('short contact') or leaving them submerged in the cultures for 2 h. Adherent cells were recovered by sonication. In the short contact experiments, the logarithm of the mean viable counts ranged from 3.67 +/- 0.43 to 4.78 +/- 0.38. After 2 h contact time, the numbers of adherent cells had increased significantly for all materials with the exception of polypropylene. The highest count (6.33 +/- 0.31) recorded was for stainless steel 405. CONCLUSION: Adhesion to a wide range of materials was time-dependent and characterized by reversible and irreversible stages. SIGNIFICANCE AND IMPACT OF THE STUDY: Adhesion test protocols must account for cell carry-over and cells which are only weakly bound. Material selection may only have a limited role in reducing food contamination by listeria.     

Variation in gene flow levels among predominantly self-pollinated plants

Levels of gene flow, Nm, were calculated for 34 predominantly self-pollinated plants, using information on differentiation among populations, G_st. Gene flow levels varied from 0.01 to 6.55, and departed significantly from a uniform distribution. High, medium and low levels of gene flow were found among 15, 38, and 47 percent of the species, respectively. The average heterozygosity (H) and the Nm values showed a positively significant association. These results were compared to gene flow levels obtained with a limited number of predominantly outcrossed plants. Gene flow levels in several self-pollinated species were comparable to those characteristic of some outcrossed species. Pollen flow, combined with long distance dispersal of propagules through various vectors may be responsible for the high levels of gene flow observed in self-pollinated species. High gene flow may provide the genetic flexibility required for successful colonization, which is an essential feature of self-pollinated plants.     

Ecological significance of microdiversity: identical 16S rRNA gene sequences can be found in bacteria with highly divergent genomes and ecophysiologies

A combination of cultivation-based methods with a molecular biological approach was used to investigate whether planktonic bacteria with identical 16S rRNA gene sequences can represent distinct eco- and genotypes. A set of 11 strains of Brevundimonas alba were isolated from a bacterial freshwater community by conventional plating or by using a liquid most-probable-number (MPN) dilution series. These strains had identical 16S rRNA gene sequences and represented the dominant phylotype in the plateable fraction, as well as in the highest positive dilutions of the MPN series. However, internally transcribed spacer and enterobacterial repetitive intergenic consensus PCR fingerprinting analyses, as well as DNA-DNA hybridization analyses, revealed great genetic diversity among the 11 strains. Each strain utilized a specific combination of 59 carbon substrates, and the niche overlap indices were low, suggesting that each strain occupied a different ecological niche. In dialysis cultures incubated in situ, each strain had a different growth rate and cell yield. We thus demonstrated that the B. alba strains represent distinct populations with genetically determined adaptations and probably occupy different ecological niches. Our results have implications for assessment of the diversity and biogeography of bacteria and increase the perception of natural diversity beyond the level of 16S rRNA gene sequences.     

Stochastic gene expression in fluctuating environments

Stochastic mechanisms can cause a group of isogenic bacteria, each subject to identical environmental conditions, to nevertheless exhibit diverse patterns of gene expression. The resulting phenotypic subpopulations will typically have distinct growth rates. This behavior has been observed in several contexts, including sugar metabolism and pili phase variation. Under fixed environmental conditions, the net growth rate of the population is maximized when all cells are of the fastest growing phenotype, so it is unclear what fitness advantage is conferred by population heterogeneity. However, unlike ideal laboratory conditions, natural environments tend to fluctuate, either periodically or randomly. Here we use a stochastic population model to show that, during growth in such fluctuating environments, a dynamically heterogenous bacterial population can sometimes achieve a higher net growth rate than a homogenous one. By using stochastic mechanisms to sample several distinct phenotypes, the bacteria are able to anticipate and take advantage of sudden changes in their environment. However, this heterogeneity is beneficial only if the bacterial response rate is sufficiently low. Our results could be useful in the design of artificial evolution experiments and in the optimization of fermentation processes.     

Stochastic gene expression in switching environments

Organisms are known to adapt to regularly varying environments. However, in most cases, the fluctuations of the environment are irregular and stochastic, alternating between favorable and unfavorable regimes, so that cells must cope with an uncertain future. A possible response is population diversification. We assume here that the cell population is divided into two groups, corresponding to two phenotypes, having distinct growth rates, and that cells can switch randomly their phenotypes. In static environments, the net growth rate is maximized when the population is homogeneously composed of cells having the largest growth rate. In random environments, growth rates fluctuate and observations reveal that sometimes heterogeneous populations have a larger net growth rate than homogeneous ones, a fact illustrated recently through Monte-Carlo simulations based on a birth and migration process in a random environment. We study this process mathematically by focusing on the proportion f(t) of cells having the largest growth rate at time t, and give explicitly the related steady state distribution π. We also prove the convergence of empirical averages along trajectories to the first moment , and provide efficient numerical methods for computing .      

Specific nucleotide sequences in HeLa cell inverted repeated DNA: enrichment for sequences found in double-stranded regions of heterogeneous nuclear RNA

Inverted repeat DNA was isolated from HeLa cell nuclei and transcribed in vitro with Escherichia coli RNA polymerase in the presence of [alpha-³²P]nucleoside triphosphates. The RNA products were digested with T₁ ribonuclease and subjected to separation in two dimensions. The pattern of the prominent oligonucleotides was almost indistinguishable from that seen when the double-stranded regions from ³²P-labeled HeLa cell heterogeneous nuclear RNA were fingerprinted in a similar manner. The sequences of several of the largest prominent T₁ ribonuclease-generated oligonucleotides were determined and were found to agree with those isolated from the double-stranded heterogeneous nuclear RNA that migrated to the same positions in the fingerprints. The most prominent component of the inverted repeat DNA appears to be sequences that are transcribed into double-stranded regions in heterogeneous nuclear RNA molecules.     

HeLa cell cytoplasmic mRNA contains three classes of sequences: predominantly poly(A)-free, predominantly poly(A)-containing and bimorphic

The mRNA species which exist in the HeLa cell polyribisomes in a form devoid of A sequences longer than 8 nucleotides constitute the poly(A)-free class of mRNA. The rapidly labelled component of this mRNA class shares no measurable sequence homology with poly(A)-containing RNA. If poly(A)-free mRNA larger than 12 S labelled for 2 h in vivo is hybridized with total cellular DNA, it hybridizes primarily with single-copy DNA. When a large excess of steady poly(A)-containing RNA is added before hybridization of labelled poly(A)-free RNA, no inhibition of hybridization occurs. This indicates the existence of a class of poly(A)-free mRNA with no poly(A)-containing counterpart. Some mRNA species can exist solely as poly(A)-containing mRNAs. These mRNAs in HeLa cells are found almost exclusively in the mRNA species present only a few times per cell (scarce sequences). Some mRNA species can exist in two forms, poly(A)containing and lacking, as evidenced by the translation data in vitro of Kaufmann et al. [Proc. Natl Acad. Sci. U.S.A. 74, 4801--4805 (1977)]. In addition, if cDNA to total poly(A)-containing mRNA is fractionated into abundant and scarce classes, 47% of the scarce class cDNA can be readily hybridized with poly(A)-free mRNA. 10% of the abundant cDNA to poly(A)-containing mRNA will hybridize with poly(A)-free sequences very rapidly while the other 90% hybridize 160 times more slowly, indicating two very different frequency distributions. The cytoplasmic metabolism of these three distinct mRNA classes is discussed.     

Polymorphism in regulatory gene sequences

The extensive polymorphism revealed in non-coding gene-regulatory sequences, particularly in the immune system, suggests that this type of genetic variation is functionally and evolutionarily far more important than has been suspected, and provides a lead to new therapeutic strategies.     

Polymorphism in regulatory gene sequences

The extensive polymorphism revealed in non-coding gene-regulatory sequences, particularly in the immune system, suggests that this type of genetic variation is functionally and evolutionarily far more important than has been suspected, and provides a lead to new therapeutic strategies.