Frequent Insertions Research Articles

Alternative interactions between the Tn7 transposase and the Tn7 target DNA binding protein regulate target immunity and transposition.

The Tn7 transposon avoids inserting into a target DNA that contains a pre-existing copy of Tn7. This phenomenon, known as 'target immunity', is established when TnsB, a Tn7 transposase subunit, binds to Tn7 sequences in the target DNA and mediates displacement of TnsC, a critical transposase activator, from the DNA. Paradoxically, TnsB-TnsC interactions are also required to promote transposon insertion. We have probed Tn7 target immunity by isolating TnsB mutants that mediate more frequent insertions into a potentially immune target DNA because they fail to provoke dissociation of TnsC from the DNA. We show that a single region of TnsB mediates the TnsB-TnsC interaction that underlies both target immunity and transposition, but that TnsA, the other transposase subunit, channels the TnsB-TnsC interaction toward transposition.

Extremely complex pattern of microsatellite mutation in the germline of wheat exposed to the post-Chernobyl radioactive contamination

The molecular structure of rare variants at 13 microsatellite loci found in a population of wheat plants grown for one generation in the heavily contaminated 30 km exclusion zone around the Chernobyl Nuclear Power Plant and in a control population was compared. Evidence for rare alterations (variants) was obtained for all 13 loci, including gain and loss of repeats, as well as the complete loss of microsatellite bands. The ratio between gains and losses among variants in the control group was similar to that in the exposed group. Sequencing of variants at six microsatellite loci found in the exposed population revealed extremely complex pattern of germline mutations, including complete deletions of loci, a bias towards mutations with gains and losses of multiple repeat units, and relatively frequent insertions of DNA of unknown origin. The occurrence of large deletions at two loci may be attributed to direct and inverted repeats sequences located just upstream and downstream of the array. The results of our study also suggest that the majority of mutations within the studied wheat microsatellite loci are represented by gains and losses of multiple repeat units, implying that a simple model of replication slippage cannot account for mutation events at these loci. Our data also support the conclusion that the spectra of spontaneous and radiation-induced mutation in wheat may be similar.

Mutational Equilibrium Model of Genome Size Evolution

The paper describes a mutational equilibrium model of genome size evolution. This model is different from both adaptive and junk DNA models of genome size evolution in that it does not assume that genome size is maintained either by positive or stabilizing selection for the optimum genome size (as in adaptive theories) or by purifying selection against too much junk DNA (as in junk DNA theories). Instead the genome size is suggested to evolve until the loss of DNA through more frequent small deletions is equal to the rate of DNA gain through more frequent long insertions. The empirical basis for this theory is the finding of a strong correlation and of a clear power-function relationship between the rate of mutational DNA loss (per bp) through small deletions and genome size in animals. Genome size scales as a negative 1.3 power function of the deletion rate per nucleotide. Such a relationship is not predicted by either adaptive or junk DNA theories. However, if genome size is maintained at equilibrium by the balance of mutational forces, this empirilical relationship can be readily accommodated. Within this framework, this finding would imply that the rate of DNA gain through large insertions scales up a quarter-power function of genome size. On this view, as genome size grows, the rate of growth through large insertions is increasing as a quarter power function of genome size and the rate of DNA loss through small deletions increases linearly, until eventually, at the stable equilibrium genome size value, rates of growth and loss equal each other. The current data also suggest that the long-term variation is genome size in animals is brought about to a significant extent by changes in the intrinsic rates of DNA loss through small deletions. Both the origin of mutational biases and the adaptive consequences of such a mode of evolution of genome size are discussed.

Palindromic repetitive elements in the mitochondrial genome of Volvox

Group I introns were found in the cob and cox I genes of Volvox carteri. These introns contain tandem arrays of short palindromic sequences that are related to each other. Inspection of other regions in the mtDNA revealed that similar palindromic repetitive sequences are dispersed in the non-protein coding regions of the mitochondrial genome. Analysis of the group I intron in the cob gene of another member of Volvocaceae, Volvox aureus, has shown that its sequence is highly homologous to its counterpart in V. carteri with the exception of a cluster of palindromic sequences not found in V. carteri. This indicates that the palindromic clusters were inserted into the introns after divergence of the two species, presumably due to frequent insertions of the palindromic elements during evolution of the Volvocaceae. Possible involvement of the palindromic repetitive elements in the molecular evolution of functional RNAs is discussed.

How Many Mutations in a Cancer?

The role of genomic instability in causing and promoting tumor growth remains controversial. 1,2 Some argue that instability is necessary for tumorigenesis, 3 while others take the view that Darwinian selection is the driving force. 2 It is becoming clear that many cancers harbor multiple mutations, the great majority of which probably have no significant effect on tumor growth. Gross karyotypic abnormalities, 4 widespread microsatellite instability, 5 very frequent allelic loss, 6 and multiple small-scale insertions and deletions 7 have all been found in common types of cancer. Recent estimates have put the total number of mutations in a cancer at more than 10,000. 7 There is, moreover, evidence that some benign 8 or supposedly non-neoplastic 9 lesions harbor many or several mutations, although such findings are by no means universal. The finding of so many mutations has generally been unexpected by most commentators. 10 These data have been taken as evidence to support the central, and perhaps even necessary, role of genomic instability in tumorigenesis. 11

Integrated gene and species phylogenies from unaligned whole genome protein sequences.

Most molecular phylogenies are based on sequence alignments. Consequently, they fail to account for modes of sequence evolution that involve frequent insertions or deletions. Here we present a method for generating accurate gene and species phylogenies from whole genome sequence that makes use of short character string matches not placed within explicit alignments. In this work, the singular value decomposition of a sparse tetrapeptide frequency matrix is used to represent the proteins of organisms uniquely and precisely as vectors in a high-dimensional space. Vectors of this kind can be used to calculate pairwise distance values based on the angle separating the vectors, and the resulting distance values can be used to generate phylogenetic trees. Protein trees so derived can be examined directly for homologous sequences. Alternatively, vectors defining each of the proteins within an organism can be summed to provide a vector representation of the organism, which is then used to generate species trees. Using a large mitochondrial genome dataset, we have produced species trees that are largely in agreement with previously published trees based on the analysis of identical datasets using different methods. These trees also agree well with currently accepted phylogenetic theory. In principle, our method could be used to compare much larger bacterial or nuclear genomes in full molecular detail, ultimately allowing accurate gene and species relationships to be derived from a comprehensive comparison of complete genomes. In contrast to phylogenetic methods based on alignments, sequences that evolve by relative insertion or deletion would tend to remain recognizably similar.

Silencing at Drosophila telomeres: nuclear organization and chromatin structure play critical roles.

Transgenes inserted into the telomeric regions of Drosophila melanogaster chromosomes exhibit position effect variegation (PEV), a mosaic silencing characteristic of euchromatic genes brought into juxtaposition with heterochromatin. Telomeric transgenes on the second and third chromosomes are flanked by telomeric associated sequences (TAS), while fourth chromosome telomeric transgenes are most often associated with repetitious transposable elements. Telomeric PEV on the second and third chromosomes is suppressed by mutations in Su(z)2, but not by mutations in Su(var)2-5 (encoding HP1), while the converse is true for telomeric PEV on the fourth chromosome. This genetic distinction allowed for a spatial and molecular analysis of telomeric PEV. Reciprocal translocations between the fourth chromosome telomeric region containing a transgene and a second chromosome telomeric region result in a change in nuclear location of the transgene. While the variegating phenotype of the white transgene is suppressed, sensitivity to a mutation in HP1 is retained. Corresponding changes in the chromatin structure and inducible activity of an associated hsp26 transgene are observed. The data indicate that both nuclear organization and local chromatin structure play a role in this telomeric PEV.

Retroviral insertional activation in a herpesvirus: transcriptional activation of US genes by an integrated long terminal repeat in a Marek's disease virus clone.

Insertional activation of host proto-oncogenes has been recognized as a basic mechanism by which nonacute retroviruses induce cancer. Our previous work has demonstrated that retroviruses can efficiently integrate into DNA virus genomes. Specifically, coinfection of cultured fibroblasts with a chicken herpesvirus, Marek's disease virus (MDV), and a chicken retrovirus results in frequent stable retroviral insertions into the herpesvirus genome. Such insertions could alter the expression of herpesvirus genes, possibly resulting in novel phenotypic properties. In this article, we report the characterization of a replication-competent clone of MDV with integrated retroviral sequences. This virus was isolated from a chicken following injection of fibroblasts coinfected with MDV and the retrovirus, reticuloendotheliosis virus. Transcripts originating from the reticuloendotheliosis virus long terminal repeat promoters were found to encode the adjoining MDV genes, SORF2, US1, and US10. This virus replicates well in culture but has an unusual phenotype in chickens, characterized by an attenuated virulence which produces no nerve lesions but, rather, severe thymic atrophy. While the causal relationship between the insertion and the observed phenotypes remains to be established, our data provide the first evidence of retroviral insertional activation of herpesvirus genes.

A molecular clock based on the expansion of gene families.

There is evidence to suggest that eukaryotic genomes are subject to frequent insertions and deletions of non-coding DNA. This may lead to a gradual increase or decrease in genome size, or to a dynamic equilibrium in which the overall size remains constant. We argue, however, that there is a bias favouring an accumulation of non-coding DNA in the proximity of genes. Such bias causes a progressive change in genome structure regardless of whether the overall genome size increases, decreases or remains constant. We show that this change may serve as a 'molecular clock', supplementing that provided by nucleotide substitution rates.

Bellow’s Hand Writing: The Tactile Imagination

BELLOW’S HAND WRITING: THE TACTILE IMAGINATION MICHAEL GREENSTEIN University of Toronto Three times the concentred self takes hold, three times The thrice concentred self, having possessed The object, grips it in savage scrutiny. Wallace Stevens, “Credences of Summer” (7.8-11) makes Saul Bellow’s fiction so all-encompassing is his blending of details of Chicago’s underworld with the metaphysics of European philosophy. Sudden shifts from highbrow speculative thought to lower com­ edy or speculation on capitalist markets may prove exhilarating or dizzying to the reader, while his frequent insertions of irony render the task of in­ terpretation and evaluation slippery. To mediate between the extremes of abstraction and the concrete, Bellow relies on the sense of touch to examine the many channels and avenues of his characters’ universe. His emphasis on the tactile is a hallmark of his fiction, as characters grope for higher meaning while exploring the sensory pleasures of the quotidian: on the one hand, the higher reaches of thought; on the other, the reality of American streets paved with fools’ gold. It is a fact well known among Bellow aficionados that he places his manuscript on a music stand while typing and imagines that he is conducting an orchestra. Accordingly, the connection between the musical key and the typewriter key, the sublime and the tactile, is built into the very fabric of his work. By extension, Bellow orchestrates the mind-body problem within the individual or between couples embracing the mysteries of modern life. In Saul Bellow’s world one way of seizing the day is to grab the body, for, as Dr. Adler tells his beleaguered son Wilhelm, “the massage does a world of good.” Whereupon the doctor opens his “small hand on the table in a gesture so old and so typical that Wilhelm felt it like an actual touch upon the foundations of his life” (Seize the Day 44). The contiguity between table and personality extends to a Bellovian connection between touching and metaphysics, or between massage and the transcendental. Toward the middle of To Jerusalem and Back Bellow follows Dr. Adler’s advice and takes time off for the here-and-now of one of his sublime rubdowns: “Moshe English Stu d ies in C a n a d a , 2 1, 4, December 1995 W hat physical the masseur is delicate in person; his hands, however, have the strength that purity of purpose can give. ... He seems untouched by life” (64). And, while the author immediately explains the phrase “untouched by life” as pertaining to someone who has lived without cynicism, the reader should also note the contrast between delicacy and strength in the person of the masseur whose hands fulfil the carpe diem of the flesh. Moshe’s delicate strength not only enables him to relieve people of their muscular tensions, but his oxymoronic quality and purity of purpose transcend the anatomical aspects of his vocation: “For me, massage is a personal relationship and kind of an act of love” (65). If there is any doubt about Bellow’s metaphysics of massage in its approximation of Buber’s I-Thou relationship and Wilhelm Reich’s orgone therapy, then we need only turn the page of To Jerusalem and Back, for the next section begins with a reference to Israel losing touch with reality. In Bellow’s vocabulary “touch” is a loaded term connecting physical sensation to other realms of the imagination. While Bellow’s fiction advocates the therapeutic value of the steam bath, “Him with His Foot in His Mouth” reveals the hazards of excessive handling. In a long epistle to Miss Rose, Dr. Harry Shawmut — musicologist, ironist, and metaphysician — explains to the retired librarian why he insulted her thirty-five years earlier. Having wounded her a generation ago, he now suf­ fers not only from remorse but also from hypertension, cardiac disorders, cracked dental roots, a hemorrhoid the size of a walnut, and creeping arthri­ tis of the hands. In British Columbia, Shawmut discovers that something has gone wrong with the middle finger of his right hand: “The hinge had stopped working and the finger was curled like a snail — a painful new af­ fliction. Quite a...

Variability in the length of the amino terminal sequence contributes to the capsid protein diversity among dasheen mosaic potyvirus isolates.

Western blot analysis of several dasheen mosaic virus isolates revealed molecular weight differences among their capsid proteins (CPs). Sequence analysis of the CP gene showed that the CP of isolate TEN was 15 amino acids shorter than that of isolate LA due to a 57-base deletion and a 12-base insertion into the 5' end of the TEN CP gene. Our data suggest that frequent deletions and insertions are responsible for the CP size diversity among DMV isolates.

Retrieval performance versus disc space utilization on WORM optical discs

Steady progress in the development of optical disc technology over the past decade has brought it to the point where it is beginning to compete directly with magnetic disc technology. WORM optical discs in particular, which permanently register information on the disc surface, have significant advantages over magnetic technology for applications that are mainly archival in nature but require the ability to do frequent on-line insertions. In this paper, we propose a class of access methods that use rewritable storage for the temporary buffering of insertions to data sets stored on WORM optical discs and we examine the relationship between the retrieval performance from WORM optical discs and the utilization of disc storage space when one of these organizations is employed. We describe the performance trade off as one of fast sequential retrieval of the contents of a block versus wasted space owing to data replication. A model of a specific instance of such an organization (a buffered hash file scheme) is described that allows for the specification of retrieval performance objectives. Alternative strategies for managing data replication that allow trade offs between higher consumption rates and better average retrieval performance are also described. We then provide an expected value analysis of the amount of disc space that must be consumed on a WORM disc to meet specified performance limits. The analysis is general enough to allow easy extension to other types of buffered files systems for WORM optical discs.

Mouse rDNA: sequences and evolutionary analysis of spacer and mature RNA regions.

Two regions of mouse rDNA were sequenced. One contained the last 323 nucleotides of the external transcribed spacer and the first 595 nucleotides of 18S rRNA; the other spanned the entire internal transcribed spacer and included the 3' end of 18S rRNA, 5.8S rRNA, and the 5' end of 28S rRNA. The mature rRNA sequences are very highly conserved from yeast to mouse (unit evolutionary period, the time required for a 1% divergence of sequence, was 30 X 10(6) to 100 X 10(6) years). In 18S rRNA, at least some of the evolutionary expansion and increase in G + C content is due to a progressive accretion of discrete G + C-rich insertions. Spacer sequence comparisons between mouse and rat rRNA reveal much more extensive and frequent insertions and substitutions of G + C-rich segments. As a result, spacers conserve overall G + C richness but not sequence (UEP, 0.3 X 10(6) years) or specific base-paired stems. Although no stems analogous to those bracketing 16S and 23S rRNA in Escherichia coli pre-rRNA are evident, certain features of the spacer regions flanking eucaryotic mature rRNAs are conserved and could be involved in rRNA processing or ribosome formation. These conserved regions include some short homologous sequence patterns and closely spaced direct repeats.

A cloned immunoglobulin cDNA fragment enhances transposition of IS elements into recombinant plasmids.

Evidence is presented indicating that a novel DNA sequence arrangement generated by in vitro recombination may elicit high frequency transpositions of IS elements. A 109 bp Bam HI fragment of the cDNA for the immunoglobulin kappa light chain from MOPC 321 myeloma was cloned into the Bam HI site of pBR313. The cloned fragment extends from the codon for Gly 57 to the V-J junction. Insertions of IS1 or IS5 were identified in 6 of 50 plasmid DNAs isolated from freshly transformed clones. Additional transposition events were detected after subculturing for several growth cycles. Three independent insertions of IS1 occurred in the promoter region of the TcR operon. All IS5 and the remaining IS1 insertions were located in the TcR region upstream to the cloned DNA sequence. Sequences homologous to the ends of IS1, or corresponding to the consensus sequence at the target site of IS5 are present near the estimated sites of insertion of IS1 or IS5 respectively. Bacteria harboring recombinant plasmids carrying the cloned DNA in either orientation grew at a reduced rate relative to cells harboring pBR313, suggesting that fused gene products made from the two types of plasmid were inhibitory to cell growth. IS insertions, which relieved this inhibitory effect and thereby provided a selective advantage, were found exclusively in plasmids carrying the cloned DNA in only one of the two orientations. The fact that IS elements were not observed in the other type of recombinant plasmid indicates that selective pressure alone is not sufficient to account for the frequent IS insertions observed and that sequences at a distance from the site of IS insertion may be critical in the regulation of transposition frequency.