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1.
A long (147 base pairs), natural A.T rich polypyrimidine/polypurine tract has been found 55 base pairs downstream of a chicken embryonic myosin heavy chain (MHC) gene. Analysis at the nucleotide level of nicks induced by S1 and Neurospora crassa nucleases indicate that this long interrupted polypyrimidine/polypurine tract exists in an alternate DNA structure in vitro at pH 4.5 and pH 7.5 in both supercoiled and linear plasmid DNA. The polypyrimidine/polypurine tract induces this alternate structure upon at least 200 base pairs of its 5' flanking DNA, and thus extends into the 3' coding and non-coding regions of the neighboring MHC gene. The different nicking patterns induced by the nucleases S1 and N. crassa on each strand of this alternate structure suggests that the polypyrimidine/polypurine tract may form heteronomous DNA. When this long polypyrimidine/polypurine tract is present in a supercoiled plasmid at low pH, a new and as yet undefined S1 hypersensitive DNA alteration was detected near the center of this tract.  相似文献   

2.
Using a "single-fly" nucleic acid hybridization method, we have surveyed a collection of D. melanogaster strains in search of variants which affect DNA complementary to the polypyrimidine sequence corresponding to one strand of the 1.705 satellite. Hybridization of labelled polypyrimidine probe to polypurine sequence in nucleic acid extracts of single flies, followed by thermal chromatography over hydroxyapatite led to the identification of one variant. The strain Cy/M(2)S2(10) produced excess hybrid, much of which had low thermal stability. A developmental analysis of the low-melt hybrid phenotype showed that certain tissues, in particular the ovaries, were affected. In addition to the biochemical phenotype, the break down of nurse cell nuclei in Cy/M(2)S2(10) ovaries during oocyte maturation was abnormal. A genetic analysis demonstrated that both the biochemical and cytological phenotypes were the consequences of a single recessive mutation in the DNase-1 gene on chromosome III. Studies with purified DNA demonstrated that the low-melt hybrid phenotype resulted from the accumulation of low molecular weight DNA complementary to the polypyrimidine probe.  相似文献   

3.
Evolution of Polypyrimidines in Drosophila   总被引:3,自引:0,他引:3  
We surveyed 101 different Drosophila species for the presence of a particular highly repetitive DNA sequence containing long tracts of polypyrimidine/polypurine DNA, first found in D. melanogaster. Out of 55 tested species in the melanogaster group, only the sibling species D. simulans and D. mauritiana, as well as one distant relative in the ananassae subgroup, D. varians, contained the same sequence. All four of these species have long pyrimidine tracts as shown by acid hydrolysis of labelled DNA. All four species have the same sequence, bu the amount of this polypyrimidine/polypurine DNA varies greatly. Four other species in the hydei subgroup were found to contain a polypyrimidine/polpurine sequence, with an oligonucleotide composition different from that of D. melanogaster. This polypyrimidine DNA varies from as much as 10% of the total DNA in D. nigrohydei, to as little as 0.4% in D. neohydei. The long pyrimidine tracts in the hydei subgroup are often more than a thousand nucleotides in length, representing exceedingly homogeneous repetitious sequences.--These results show a rapid but discontinuous pattern of evolution for polypyrimidine/polypurine DNA . These sequences are not species specific, yet closely related species have greatly different amounts of polypyrimidines. Drastic changes occur in the amounts of these satellite type DNA sequences, as if the sequence had no continuous selective advantage in evolution. The implications of these results with regard to the general function and evolution of satellite DNA are discussed.  相似文献   

4.
We have developed and characterized a method for the rapid detection and quantitation of specific DNAs in partially purified extracts of single Drosophila. While the method should be applicable to a number of repetitious DNA sequences, we have used the polypyrimidine DNA sequences (TCTCT)n to develop this technique. Using hydroxyapatite chromatography, we were able to measure the amount of nucleic acid hybrid formed and to obtain a thermal elution profile of the hybrid formed in extracts of single flies. Under a variety of conditions, purified DNA and DNA in partially purified extracts gave essentially identical results. The procedure can be used to detect the presence of rare sequences, or to measure the relative abundance of a prevalent DNA species. 40 different wild type strains of Drosophila melanogaster were examined using this technique and all contain similar amounts of the same polypyrimidine/polypurine sequence. From a small scale screening of different laboratory stocks of D. melanogaster, a variant was found which formed more DNA-DNA hybrid with labelled polypyrimidine tracts than did wild type. The additional hybrid was distinguished by a lower thermal stability than the hybrid formed in wild type.  相似文献   

5.
To study possible involvement of polypurine and polypyrimidine DNA tracts capable of forming triple-stranded structures (the H-form of DNA) in compaction of eukaryotic chromosomes, an in silico search for complementary polypurine and polypyrimidine sequences was carried out within 12 eukaryotic genes. It was shown that, in chromosomal gene loci, 10–11 bp polypurine and polypyrimidine tracts potentially capable of interacting with each other with the formation of triplex structures (“structuring” regions) are located in predominantly in introns and gene-flanking regions. In vivo, such DNA-DNA interactions can result in the chromosomal gene domain folding into several small loops. The character of the DNA triplex-mediated compaction of chromosomal gene loci may be related to gene functioning. A similar analysis of long (LINE) and short (SINE) interspersed repeat sequences, as well as of satellite DNA, showed essential resemblance between the compaction mechanisms of coding and noncoding chromosome regions.  相似文献   

6.
DNA structural transitions within the PKD1 gene.   总被引:7,自引:0,他引:7  
Autosomal dominant polycystic kidney disease (ADPKD) affects over 500 000 Americans. Eighty-five percent of these patients have mutations in the PKD1 gene. The focal nature of cyst formation has recently been attributed to innate instability in the PKD1 gene. Intron 21 of this gene contains the largest polypurine. polypyrimidine tract (2.5 kb) identified to date in the human genome. Polypurine.polypyrimidine mirror repeats form intramolecular triplexes, which may predispose the gene to mutagenesis. A recombinant plasmid containing the entire PKD1 intron 21 was analyzed by two-dimensional gel electrophoresis and it exhibited sharp structural transitions under conditions of negative supercoiling and acidic pH. The superhelical density at which the transition occurred was linearly related to pH, consistent with formation of protonated DNA structures. P1 nuclease mapping studies of a plasmid containing the entire intron 21 identified four single-stranded regions where structural transitions occurred at low superhelical densities. Two-dimensional gel electrophoresis and chemical modification studies of the plasmid containing a 46 bp mirror repeat from one of the four regions demonstrated the formation of an H-y3 triplex structure. In summary, these experiments demonstrate that a 2500 bp polypurine.polypyrimidine tract within the PKD1 gene is capable of forming multiple non-B-DNA structures.  相似文献   

7.
Microsatellite DNA sequences are ubiquitous in the human genome, and mutation rates of these repetitive sequences vary with respect to DNA sequence as well as length. We have analyzed polymerase-DNA interactions as a function of microsatellite sequence, using polypyrimidine/polypurine di- and tetranucleotide alleles representative of those found in the human genome. Using an in vitro primer extension assay and the mammalian DNA polymerase alpha-primase complex, we have observed a polymerase termination profile for each microsatellite that is unique to that allele. Interestingly, a periodic termination profile with an interval size (9-11 nucleotides) unrelated to microsatellite unit length was observed for the [TC](20) and [TTCC](9) templates. In contrast, a unit-punctuated polymerase termination profile was found for the longer polypurine templates. We detected strong polymerase pauses within the [TC](20) allele at low reaction pH which were eliminated by the addition of deaza-dGTP, consistent with these specific pauses being a consequence of triplex DNA formation during DNA synthesis. Quantitatively, a strand bias was observed in the primer extension assay, in that polymerase synthesis termination is more intense when the polypurine sequence serves as the template, relative to its complementary polypyrimidine sequence. The HSV-tk forward mutation assay was utilized to determine the corresponding polymerase alpha-primase error frequencies and specificities at the microsatellite alleles. A higher microsatellite polymerase error frequency (50x10(-4) to 60x10(-4)) was measured when polypurine sequences serve as templates for DNA synthesis, relative to the polypyrimidine template (18x10(-4)). Thus, a positive correlation exists between polymerase alpha-primase pausing and mutagenesis within microsatellite DNA alleles.  相似文献   

8.
UV-absorption spectrophotometry and molecular modeling have been used to study the influence of the chemical nature of sugars (ribose or deoxyribose) on triple helix stability. For the Pyrimidine.purine* Pyrimidine motif, all eight combinations were tested with each of the three strands composed of either DNA or RNA. The chemical nature of sugars has a dramatic influence on triple helix stability. For each double helix composition, a more stable triple helix was formed when the third strand was RNA rather than DNA. No stable triple helix was detected when the polypurine sequence was made of RNA with a third strand made of DNA. Energy minimization studies using the JUMNA program suggested that interactions between the 2'-hydroxyl group of the third strand and the phosphates of the polypurine strand play an important role in determining the relative stabilities of triple-helical structures in which the polypyrimidine third strand is oriented parallel to the polypurine sequence. These interactions are not allowed when the third strand adopts an antiparallel orientation with respect to the target polypurine sequence, as observed when the third strand contains G and A or G and T/U. We show by footprinting and gel retardation experiments that an oligoribonucleotide containing G and A or G and U fails to bind double helical DNA, while the corresponding DNA oligomers form stable triple-helical complexes.  相似文献   

9.
DNA Parallel clamps with a polypurine strand linked to a polypyrimidine Hoogsteen strand containing locked nucleic acids bind their corresponding polypyrimidine targets with high affinity.  相似文献   

10.
The polypurine.polypyrimidine sequence requirements for the formation of sticky DNA were evaluated in Escherichia coli plasmid systems to determine the potential occurrence of this conformation throughout biological systems. A mirror repeat, dinucleotide tract of (GA.TC)(37), which is ubiquitous in eukaryotes, formed sticky DNA, but shorter sequences of 10 or 20 repeats were inert. (GGA.TCC)(n) inserts (where n = 126, 159, and 222 bp) also formed sticky DNA. As shown previously, the control sequence (GAA.TTC)(150) (450 bp) readily adopted the X-shaped sticky structure; however, this structure has never been found for the nonpathogenic (GAAGGA.TCCTTC)(65) of the same approximate length (390 bp). A sequence that is replete with polypurine.polypyrimidine tracts that can form triplexes and slipped structures but lacks long repeating motifs (the 2.5-kbp intron 21 sequence from the polycystic kidney disease gene 1) was also inert. Interestingly, tracts of (GAA.TTC)(n) (where n = 176 or 80) readily formed sticky DNA with (GAAGGA.TCCTTC)(65) cloned into the same plasmid when the pair of inserts was in the direct, but not in the indirect (inverted), orientation. The stabilities of the triple base (Watson-Crick and Hoogsteen) interactions in the DNA/DNA associated triplex region of the sticky conformations account for these observations. Our results have significant chemical and biological implications for the structure and function of this unusual DNA conformation in Friedreich's ataxia.  相似文献   

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