Structure of the gene encoding the exoglucanase of Cellulomonas fimi

In Cellulomonas fimi the cex gene encodes an exoglucanase (Exg) involved in the degradation of cellulose. The gene now has been sequenced as part of a 2.58-kb fragment of C. fimi DNA. The cex coding region of 1452 bp (484 codons) was identified by comparison of the DNA sequence to the N-terminal amino acid (aa) sequence of the Exg purified from C. fimi. The Exg sequence is preceded by a putative signal peptide of 41 aa, a translational initiation codon, and a sequence resembling a ribosome-binding site five nucleotides (nt) before the initiation codon. The nt sequence immediately following the translational stop codon contains four inverted repeats, two of which overlap, and which can be arranged in stable secondary structures. The codon usage in C. fimi appears to be quite different from that of Escherichia coli. A dramatic (98.5%) bias occurs for G or C in the third position for the 35 codons utilized in the cex gene.     

Joining of DNA ends bearing non-matching 3'-overhangs

Using the Xenopus egg extract as an in vitro system for double strand break repair, the joining of DNA ends bearing non-complementary, homopolymeric, 4nt 3'-protruding single strands ("overhangs") was examined. Such 3'-overhangs can not be filled-in and cannot align and anneal by canonical base pair interactions, thus presenting a special challenge to the repair machinery. The results indicate that two such non-matching 3'-overhangs typically overlap by 2nt forming non-canonical base pairs, from which the filling-in of the remaining gaps is primed. The repair reaction is inhibited in Ku-depleted extracts. Unexpectedly, with some of the substrates the predominant repair products were joints with no nucleotide loss, suggesting that the two DNA ends aligned without overlap. However, the additional finding that an activity in the egg extract adds one or a few nucleotides to a fraction of the 3'-ends favors a model in which most or all of the zero-loss joints are the net result of 3'-overhang extension and 2bp overlap formation. The nucleotide addition reaction is stimulated by increasing the concentration of the complementary dNTP or ddNTP in the extract, suggesting a process templated by free nucleotides and the involvement of a DNA polymerase-like activity.     

Systematic Characteristic Exploration of the Chimeras Generated in Multiple Displacement Amplification through Next Generation Sequencing Data Reanalysis

Background

The chimeric sequences produced by phi29 DNA polymerase, which are named as chimeras, influence the performance of the multiple displacement amplification (MDA) and also increase the difficulty of sequence data process. Despite several articles have reported the existence of chimeric sequence, there was only one research focusing on the structure and generation mechanism of chimeras, and it was merely based on hundreds of chimeras found in the sequence data of E. coli genome.

Method

We finished data mining towards a series of Next Generation Sequencing (NGS) reads which were used for whole genome haplotype assembling in a primary study. We established a bioinformatics pipeline based on subsection alignment strategy to discover all the chimeras inside and achieve their structural visualization. Then, we artificially defined two statistical indexes (the chimeric distance and the overlap length), and their regular abundance distribution helped illustrate of the structural characteristics of the chimeras. Finally we analyzed the relationship between the chimera type and the average insertion size, so that illustrate a method to decrease the proportion of wasted data in the procedure of DNA library construction.

Results/Conclusion

131.4 Gb pair-end (PE) sequence data was reanalyzed for the chimeras. Totally, 40,259,438 read pairs (6.19%) with chimerism were discovered among 650,430,811 read pairs. The chimeric sequences are consisted of two or more parts which locate inconsecutively but adjacently on the chromosome. The chimeric distance between the locations of adjacent parts on the chromosome followed an approximate bimodal distribution ranging from 0 to over 5,000 nt, whose peak was at about 250 to 300 nt. The overlap length of adjacent parts followed an approximate Poisson distribution and revealed a peak at 6 nt. Moreover, unmapped chimeras, which were classified as the wasted data, could be reduced by properly increasing the length of the insertion segment size through a linear correlation analysis.

Significance

This study exhibited the profile of the phi29MDA chimeras by tens of millions of chimeric sequences, and helped understand the amplification mechanism of the phi29 DNA polymerase. Our work also illustrated the importance of NGS data reanalysis, not only for the improvement of data utilization efficiency, but also for more potential genomic information.     

Triplex-forming oligonucleotides trigger conformation changes of a target hairpin sequence.

We used a DNA duplex formed between the 5' end of a 69mer (69T) and an 11mer (OL7) as a substrate for BamHI. The former oligonucleotide folds into a hairpin structure, the stem of which contains a stretch of pyrimidines in one strand and consequently a stretch of purines in the other strand. The oligomer 69T was used as a target for complementary oligodeoxypyrimidines made of 10 nt (OL1), 16 nt (OL5) or 26 nt (OL2) which can engage the same 10 pyrimidine-purine-pyrimidine triplets with the 69T hairpin stem. Although the binding site of OL7 did not overlap that of OL1, OL2 or OL5, the BamHI activity on 69T-OL7 complexes was drastically modified in the presence of these triplex-forming oligomers: OL1 abolished the cleavage by BamHI whereas OL5 and OL2 strongly increased it. Using footprinting assays and point-mutated oligonucleotides we demonstrated that these variations were due to different conformations of the 69T-OL7 complex induced by the binding of oligomers OL1, OL2 or OL5. Therefore, oligonucleotides can act as structural switchers, offering one additional mode for modulating gene expression.     

Imperfect DNA mirror repeats in E. coli TnsA and other protein-coding DNA

DNA imperfect mirror repeats (DNA-IMRs) are ubiquitous in protein-coding DNA. However, they overlap and often have different centers of symmetry, making it difficult to evaluate their relationship to each other and to specific DNA and protein motifs and structures. This paper describes a systematic method of determining a hierarchy for DNA-IMRs and evaluates their relationship to protein structural elements (PSEs)--helices, turns and beta-sheets. DNA-IMRs are identifed by two different methods--DNA-IMRs terminated by reverse dinucleotides (rd-IMRs) and DNA-IMRs terminated by a single (mono) matching nucleotide (m-IMRs). Both rd-IMRs and m-IMRs are evaluated in 17 proteins, and illustrated in detail for TnsA. For each of the proteins, Fisher's exact test (FET) is used to measure the coincidence between the terminal dinucleotides of rd-IMRs and the terminal amino acids of individual PSEs. A significant correlation over a span of about 3 nt was found for each protein. The correlation is robust and for most genes, all rd-IMRs16 nt contain approximately 88% of the potential functional motifs. The protein translation of the longest rd- and m-IMRs span sequences important to the protein's structure and function. In all 17 proteins studied, the population of rd-IMRs is substantially less than the expected number and the population of m-IMRs greater than the expected number, indicating strong selective pressures. The association of rd-IMRs with PSEs restricts their spatial distribution, and therefore, their number. The greater than predicted number of m-IMRs indicates that DNA symmetry exists throughout the entire protein-coding region and may stabilize the sequence.     

Ecological separation among browsing ungulates in Tsavo East National Park,Kenya

Summary Data on food habits and habitat preferences of four browsing herbivores (black rhinoceros, giraffe, gerenuk and lesser kudu) were analyzed to assess niche width for each species and niche overlap between pairs of species. All four species depended heavily on woody plants as food, and overlap in the utilization of different plant types (trees and shrubs, herbs, grasses, etc.) was very great in three of six species paris. When individual plant species were considered, markedly less overlap was apparent. Three of the four ungulate species preferred the most densely wooded vegetation type. Overlap in habitat preferences tended to be least in those pairs of species with the greates dietary overlap, which resulted in some degree of ecological separation. This was further increased by differences in browsing level. Seasonal variations in the browsing level of the giraffe had the effect of reducing overlap with the other species in the dry season, when food was in relatively short supply. Whether or not actual competition existed among the four ungulate species could not be established; in any event, it would probably be less important than possible competition exerted by the elephant, the dominant herbivore by far in the ecosystem. The ecological separation evident among the four browsing species probably permitted them to coexist in the area before the elephant reached its present dominant position and started altering the original vegetation.Formerly Tsavo East Research Station, Voi, Kenya     

Site-directed mutagenesis by overlap extension using the polymerase chain reaction

Overlap extension represents a new approach to genetic engineering. Complementary oligodeoxyribonucleotide (oligo) primers and the polymerase chain reaction are used to generate two DNA fragments having overlapping ends. These fragments are combined in a subsequent 'fusion' reaction in which the overlapping ends anneal, allowing the 3' overlap of each strand to serve as a primer for the 3' extension of the complementary strand. The resulting fusion product is amplified further by PCR. Specific alterations in the nucleotide (nt) sequence can be introduced by incorporating nucleotide changes into the overlapping oligo primers. Using this technique of site-directed mutagenesis, three variants of a mouse major histocompatibility complex class-I gene have been generated, cloned and analyzed. Screening of mutant clones revealed at least a 98% efficiency of mutagenesis. All clones sequenced contained the desired mutations, and a low frequency of random substitution estimated to occur at approx. 1 in 4000 nt was detected. This method represents a significant improvement over standard methods of site-directed mutagenesis because it is much faster, simpler and approaches 100% efficiency in the generation of mutant product.     

Evolutionary graph theory: breaking the symmetry between interaction and replacement

We study evolutionary dynamics in a population whose structure is given by two graphs: the interaction graph determines who plays with whom in an evolutionary game; the replacement graph specifies the geometry of evolutionary competition and updating. First, we calculate the fixation probabilities of frequency dependent selection between two strategies or phenotypes. We consider three different update mechanisms: birth-death, death-birth and imitation. Then, as a particular example, we explore the evolution of cooperation. Suppose the interaction graph is a regular graph of degree h, the replacement graph is a regular graph of degree g and the overlap between the two graphs is a regular graph of degree l. We show that cooperation is favored by natural selection if b/c>hg/l. Here, b and c denote the benefit and cost of the altruistic act. This result holds for death-birth updating, weak-selection and large population size. Note that the optimum population structure for cooperators is given by maximum overlap between the interaction and the replacement graph (g=h=l), which means that the two graphs are identical. We also prove that a modified replicator equation can describe how the expected values of the frequencies of an arbitrary number of strategies change on replacement and interaction graphs: the two graphs induce a transformation of the payoff matrix.     

A novel subtractive antibody phage display method to discover disease markers

Today's research demands fast identification of potential diagnostic and therapeutic targets. We describe a novel phage display strategy to identify disease-related proteins that are specifically expressed in a certain (diseased) tissue or cells. Phages displaying antibody fragments are selected on complex protein mixtures in a two-step manner combining subtractive selection in solution with further enrichment of specific phages on two-dimensional Western blots. Targets recognized by the resulting recombinant antibodies are immunoaffinity-purified and identified by mass spectrometry. We used antibody fragment libraries from autoimmune patients to discover apoptosis-specific and disease-related targets. One of the three identified targets is the U1-70K protein, a marker for systemic lupus erythematosus overlap disease. Interestingly the epitope on U1-70K recognized by the selected recombinant antibody was shown to be apoptosis-dependent, and such epitopes are believed to be involved in breaking tolerance to self-antigens. The other two proteins were identified as polypyrimidine tract-binding protein-associated splicing factor (PSF)/nuclear RNA- and DNA-binding protein of 54 kDa (p54nrb) and heterogeneous ribonucleoprotein C.     

The divergent 5' ends of DPM2 mRNAs originate from the alternative splicing of two adjacent introns: characterization of the hamster DPM2 gene

Mammalian dolichol-phosphate-mannose (DPM) synthase has three subunits, DPM1, DPM2, and DPM3. In this report, an analysis of the gene and cDNAs of hamster DPM2 is presented. The CHO DPM2 gene has two special features. First, the initiation codon ATG is separated from the remainder of the coding region by intron sequences. Second, within these intron sequences the DPM2 gene contains an adjacent 3' splice site (acceptor) and a 5' splice site (donor), suggestive of a deleted exon between the first and second codons. In fact, these sites overlap by four nucleotides (nt) of AGGT. Splicing intermediates using both of these alternative splice sites were observed. This latter feature appears unique and is particularly unusual considering the relatively small size of the gene (2.7 kb) and of introns a (123 bp) and b (152 bp).     

Does competition between resources change the competition between their consumers to mutualism? Variations on two themes by Vandermeer

How does competition between resources affect the interaction between consumer species that share those resources? Existing theory suggests that high resource competition can lead to mutualism. However, this is based on an analysis that need only apply near equilibrium, and experimental demonstrations of such mutualism are rare. Two alternative approaches to measuring food web mutualism are examined here. These are based on the population-level effects of adding or removing a consumer species or on the amount of additional mortality that can be applied to one consumer without excluding it. Both measures suggest that mutualism is likely to be confined to two situations: when overlap in resource use by the consumers is very low and when the consumers are inefficient users of their resources. Competition between resources is also likely to increase the occurrence and magnitude of "hypercompetition" between consumers, where the reduction in population size caused by the introduced consumer is greater than that caused by a consumer that is identical to the resident species. Competition between resources can also increase the negative interaction between consumers by destabilizing the dynamics of the system. Such destabilization can cause negative indirect interactions between specialist consumers having no overlap in resource use.     

Multivariate measures of niche overlap using discriminant analysis

Two measures of multivariate niche overlap defined on p resource variables are presented. By measuring the niche overlap on the discriminant variable the multivariate problem is reduced to a univariate problem while preserving the relevant multivariate information. The niche overlap is then calculated by two different techniques. The first technique uses the MacArthur-Levins (Amer. Natur.101, 377–385, 1967) measure for probabilities of joint occurrence, while the second computes the density overlap of two use curves. An illustration of the multivariate approach to actual field data is demonstrated.