A-tract polarity dominate the curvature in flanking sequences

It is well-known, but little understood, that the nucleotide sequences between phased A(4-6)-tracts (at 10-11 bp intervals) have only a slight effect on overall curvature. To explore this phenomenon, we have examined the gel-migration properties of sequences containing both A-tracts as well as G-tracts (i.e., sequences of the form G(n)C(m) or C(n)G(m), n + m > 4) in various relative positioning. We show that the composite bend of these sequences depends on their relative arrangement. When G-tracts are placed between two A-tracts, such that both tracts are repeated in phase to themselves (e.g., G(5)A(6)G(5)A(5)), or adjacent to the 3'-side of A-tracts (e.g., A(6)G(5)N(10)), they have minimal influence on the extent of bending of the composite sequence. When G-tracts are placed one helical repeat away from A-tracts (e.g., G(5)N(5)A(6)N(6)), or are adjacent only to the 5'-side of A-tracts (e.g., G(5)A(6)N(10)) their influence on the composite bend is larger. The differential behavior of AG- versus GA-tracts means that A-tracts influence their flanking sequences in a polar manner. Whereas they suppress, or make constant, the intrinsic bending characteristics of any sequence placed immediately 3' to them (and hence by definition any sequence placed between two phased A-tracts), sequences adjoining them on their 5'-side are free to modulate the overall curvature. We interpret these results as evidence for the dominant nature of the unique and nonuniform structure adopted by tracts of four adenines or more. The effects of A-tracts extend at least five base pairs into the adjoining 3' region. This is further evidence for the complexity of DNA structure and the inadequacy of simple nearest-neighbor models to explain all its manifestations.     

Microsatellite repeats are not randomly distributed within Norway spruce (Picea abies K.) expressed sequences.

A Norway spruce (Picea abies K.) cDNA library obtained from vegetative bud tissue was screened for the presence of (AG)n and (AC)n microsatellite repeats. Ten (AG)n and six (AC)n microsatellites were found, with an average length of 25.5 repeat units. Most of the microsatellites are simple perfect repeats. The microsatellite distribution within the clones is clearly non-random, with different classes of repeats lying in different positions relative to the coding region and in a highly conserved orientation. An estimate of the frequency of dinucleotide microsatellites in expressed regions was obtained, showing that SSRs (simple sequence repeats) are found in genes about 20 times less frequently than in random genomic clones, with (AG)n repeats more frequent than (AC)n repeats. Potential applications of these sequences as expressed region-based molecular markers are shown by developing six SSR markers for the detection of natural variation in Norway spruce populations and testing two of them for the identification of illegitimate progenies from a mapping population.     

Pathogenic polyglutamine expansion length correlates with polarity of the flanking sequences

Background

Polyglutamine (polyQ) repeat expansion within coding sequence of a soluble protein is responsible for eight autosomal-dominant genetic neurodegenerative disorders. These disorders affect cerebellum, striatum, basal ganglia and other brain regions. The pathogenic polyQ-expansion threshold in these proteins varies from 32Q to 54Q. Understanding the reasons for variability in pathogenic polyQ threshold may provide insights into pathogenic mechanisms responsible for development of these disorders.

Findings

Here we established a quantitative correlation between the polarity of the flanking sequences and pathogenic polyQ-expansion threshold in this protein family. We introduced an “edge polarity index” (^EPI) to quantify polarity effects of the flanking regions and established a strong correlation between ^EPI index and critical polyQ expansion length in this protein family. Based on this analysis we subdivided polyQ-expanded proteins into 2 groups – with strong and weak dependence of polyQ threshold on ^EPI index. The main difference between members of the first and the second group is a polarity profile of these proteins outside of polyQ and flanking regions. PolyQ proteins are known substrates for proteasome and most likely mechanistic explanation for the observed correlation is that proteasome may have an impaired ability to process continuous non-polar regions of proteins.

Conclusions

The proposed hypothesis provides a quantitative explanation for variability in pathogenic threshold among polyQ-expansion disorders, which we established to correlate with polarity of flanking regions. To explain these results we propose that proteasome is not efficient in processing continuous non-polar regions of proteins, resulting in release of undigested and partially digested fragments. If supported experimentally, our hypothesis may have wide implications for further understanding the pathogensis of polyglutamine expansion disorders.

     

Volume and polarity changes accompanied by amino acid substitutions in protein evolution.

We evaluated the volume and polarity changes accompanied by amino acid substitutions along branches of the phylogenetic trees of cytochrome c, myoglobin and hemoglobin alpha and beta chains. In most cases the volume changes accompanied by the substitutions were found to be much larger than the volume of cavities existing in the interior of X-ray-analysed proteins. This implies that the interior of the proteins is very flexible and the necessary space for a larger amino acid residue substitution can be provided by adjusting nearby structures. Also, the volume and polarity changes are not particularly dependent on whether the substituted site is located in the exterior or interior of the proteins. This result supports the concept of the covarions by Fitch and Markowitz, when combined with the known fact that the exterior sites are more variable than the interior ones during protein evolution.     

Molecular cytogenetic analysis of DNA sequences with flanking telomeric repeats in Triticum aestivum cv. Begra.

A cloned genomic DNA fragment (pTa241) formerly derived from a DNA fraction obtained from isolated nuclei of embryos of a Polish cultivar of wheat (Triticum aestivum cv. Begra) comprises a tandem repeat of the telomeric array CCCTAAA, and hybridizes in situ exclusively to the telomeres of all chromosome arms of the somatic chromosome complement of wheat. A second cloned fragment (pTa637) derived from the same fraction is 637 bp long, flanked by 28 bp of the same telomeric repeat unit, and hybridizes in situ to the entire lengths of all the chromosomes of the complement. The same pattern of hybridization was observed when the flanking telomeric sequences were removed. A third DNA fragment (pTa1439), derived from unfractionated genomic DNA and flanked with 62 bp of the same telomeric unit, showed the same patterns of distribution. Together with additional evidence from Southern analysis, these observations were interpreted to mean that these sequences are associated with mobile DNA elements and are distributed widely throughout the genome. The chromosomal distribution of the non-telomeric parts of the clones is consistent with the dispersed genomic distribution characteristic of transposons and retroelements.     

Variability within the rabbit C repeats and sequences shared with other SINES.

The C family of short, interspersed repeats (SINES) is highly repeated in the rabbit genome, and most members have a structure suggestive of a model for their dispersal via reinsertion of a double-stranded copy of an RNA polymerase III transcribed RNA. We have determined the nucleotide sequence of additional members of the repeat family and have compiled them to obtain an improved consensus sequence. This compilation shows that although most regions of the repeat are well conserved, two regions show high variability. Some individual repeats are truncated, and one truncated repeat retains the characteristic structures of a retroposon. The consensus sequence for C repeats does not match the sequence of any other sequenced mammalian SINE over large regions, but short imperfect matches to several primate and rodent SINES are observed. A sequence similar to the 27 nucleotide consensus sequence TCCCAGCAACCACATGGGAGGCAGAGA was found in all mammalian SINES examined. The 3' portion of this sequence matches a DNA segment found at the replication origins of papovaviruses.     

Host sequences flanking the HIV provirus.

A conserved property of retroviral proviruses is the presence of a direct repeat in the host DNA immediately flanking the viral sequence; each virus generates a repeat with a characteristic length. By sequencing the viral/host DNA junctions from five HIV-1 proviral clones, we have confirmed that integration of HIV results in the generation of a five basepair direct repeat. A target sequence in uninfected host DNA was analyzed to establish that the five basepair sequence flanking the provirus was present only once prior to integration. Of the five proviruses examined, two were found to have integrated in known repetitive sequence elements of the human genome; one in a Line-1 element and a second in satellite DNA.     

High polymorphism level of genomic sequences flanking insertion sites of human endogenous retroviral long terminal repeats

The polymorphism at the multitude of loci adjacent to human endogenous retrovirus long terminal repeats (LTRs) was analyzed by a technique for whole genome differential display based on the PCR suppression effect that provides selective amplification and display of genomic sequences flanking interspersed repeated elements. This strategy is simple, target-specific, requires a small amount of DNA and provides reproducible and highly informative data. The average frequency of polymorphism observed in the vicinity of the LTR insertion sites was found to be about 12%. The high incidence of polymorphism within the LTR flanks together with the frequent location of LTRs near genes makes the LTR loci a useful source of polymorphic markers for gene mapping.     

A developmentally regulated deletion element with long terminal repeats has cis-acting sequences in the flanking DNA

Approximately 6000 specific DNA deletion events occur during development of the somatic macronucleus of the ciliate Tetrahymena. The eliminated Tlr1 element is 13 kb or more in length and has an 825 bp inverted repeat near the rearrangement junctions. A functional analysis of the cis-acting sequences required for Tlr1 rearrangement was performed. A construct consisting of the entire inverted repeat and several hundred base pairs of flanking DNA on each side was rearranged accurately in vivo and displayed junctional variability similar to the chromosomal Tlr1 rearrangement. Thus, 11 kb or more of internal element DNA is not required in cis for DNA rearrangement. A second construct with only 51 bp of Tetrahymena DNA flanking the right junction underwent aberrant rearrangement. Thus, a signal for determination of the Tlr1 junction is located in the flanking DNA, 51 bp or more from the right junction. Within the Tlr1 inverted repeat are 19 bp tandem repeats. A construct with the 19mer repeat region deleted from the right half of the inverted repeat utilized normal rearrangement junctions. Thus, despite its transposon-like structure, Tlr1 is similar to other DNA rearrangements in Tetrahymena in possessing cis-acting sequences outside the deleted DNA.     

Introns and their flanking sequences of Bombyx mori rDNA.

We obtained two different clones (16 kb and 13 kb) of B. mori rDNA with intron sequence within the 28S-rRNA coding region. The sequence surrounding the intron was found to be highly conserved as indicated in several eukaryotes (Tetrahymena, Drosophila and Xenopus). The 28S rRNA-coding sequence of 16 kb and 13 kb clone was interrupted at precisely the same sites as those where the D. melanogaster rDNA interrupted by the type I and type II intron, respectively. The intron sequences of B. mori were different from those of D. melanogaster. In 16 kb clone, the intron was flanked by 14 bp duplication of the junction sequence, which was also present once within the 28S rRNA-coding region of rDNA without intron. This 14 bp sequence was identical with those surrounding the introns of Dipteran rDNAs.     

小鼠基因组中的微卫星重复序列的数量、分布和密度

作者分析了老鼠基因组中各染色体及其内含子、外显子和基因间区上各种类型的微卫星(1-6个碱基的重复序列)的数量及其密度。SSR约占老鼠基因组的2.85%,其中46.2%存在于基因间区,4.75%存在于外显子,49.05%在内含子区域,即非编码区富含微卫星。微卫星的数量与染色体或基因区域的大小有关,但密度与染色体或基因区域的大小的关系并不十分密切。第4染色体的外显子区域中6种类型的SSR含量都比其它区域少。A,T,AC,AG,AT,AAC,AAG,AGG,AAAC,AAAG,AAAT,AACC,AAAAC,AAAAG,AAAAT,AAACC,AAAGG,AAGAG,AAAAAC,AAAAAG,AAAAAT,AAAGAG,ACACAT,ACAGAG,ACAGGC,ACATAT是老鼠基因组中主要的SSR类型,而一些5碱基重复单元的SSR在老鼠基因组的某一条甚至某几条染色体都不存在     

TBP flanking sequences: asymmetry of binding, long-range effects and consensus sequences

We carried out in vitro selection experiments to systematically probe the effects of TATA-box flanking sequences on its interaction with the TATA-box binding protein (TBP). This study validates our previous hypothesis that the effect of the flanking sequences on TBP/TATA-box interactions is much more significant when the TATA box has a context-dependent DNA structure. Several interesting observations, with implications for protein–DNA interactions in general, came out of this study. (i) Selected sequences are selection-method specific and TATA-box dependent. (ii) The variability in binding stability as a function of the flanking sequences for (T-A)₄ boxes is as large as the variability in binding stability as a function of the core TATA box itself. Thus, for (T-A)₄ boxes the flanking sequences completely dominate and determine the binding interaction. (iii) Binding stabilities of all but one of the individual selected sequences of the (T-A)₄form is significantly higher than that of their mononucleotide-based consensus sequence. (iv) Even though the (T-A)₄ sequence is symmetric the flanking sequence pattern is asymmetric. We propose that the plasticity of (T-A)_n sequences increases the number of conformationally distinct TATA boxes without the need to extent the TBP contact region beyond the eight-base-pair long TATA box.