Changes in DNA base sequences in the mutant of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> induced by low-energy N<Superscript>+</Superscript> implantation

To reveal the mutation effect of low-energy ion implantation on Ambidopsis thaliana in vivo, T80II, a stable dwarf mutant, derived from the seeds irradiated by 30 keV N⁺ with the dose of 80 X 10¹⁵ ions/cm² was used for Random Amplified Polymorphic DNA (RAPD) and base sequence analysis. The results indicated that among total 397 RAPD bands observed, 52 bands in T80II were different from those of wild type showing a variation frequency 13.1%. In comparison with the sequences of A. thaliana in GenBank, the RAPD fragments in T80II were changed greatly in base sequences with an average rate of one base change per 16.8 bases. The types of base changes included base transition, transversion, deletion and insertion. Among the 275 base changes detected, single base substitutions (97.09%) occurred more frequently than base deletions and insertions (2.91%). And the frequency of base transitions (66.55%) was higher than that of base transversions (30.55%). Adenine, thymine, guanine or cytosine could be replaced by any of other three bases in cloned DNA fragments in T80II. It seems that thymine was more sensitive to the irradiation than other bases. The flanking sequences of the base changes in RAPD fragments in T80II were analyzed and the mutational “hotspot” induced by low-energy ion implantation was discussed.     

Changes in DNA base sequences in the mutant of Arabidopsis thaliana induced by low-energy N+ implantation

Arabidopsis thaliana (L.) Heynh has many advantages for genome analysis, including a short generation time, small size, large number of offspring, and a relatively small nuclear genome in comparison to other angiosperms and contains a low proportion of repetitive DNA comparatively. Furthermore, the analysis of the completed genome sequence of A. thaliana has been reported[1]. Low-energy ion implantation has attracted more and more attention from researchers in China and Japan since recent s…     

离子注入诱变莲花突变体分子机理的初步研究

低能离子注入技术作为生物物理诱变的一种新型技术, 在园艺植物育种方面具有很大的应用潜力, 但其诱变的分子机理目前知之甚少。文章对Fe+ 离子注入诱变的白洋淀红莲(Nelumbium speciosum Willd)突变体及其对照的基因组进行RAPD研究, 并将突变体和对照在辐射敏感位点的条带进行克隆测序及DNA序列分析。在已优化好的RAPD体系下扩增, 从110条随机引物中筛选出了10条可以稳定扩增出显著特异条带的引物, 引物多态性为9.09%。将这10条引物扩增出的辐射敏感位点的条带进行克隆测序, 并进行序列比对。结果显示: 突变体的总碱基突变频率为0.87%, 6个突变体的碱基突变频率存在着差异; 碱基突变类型包括碱基的颠换、转换、缺失、插入, 在检测到的159个碱基突变中, 单碱基置换的频率(61.01%)高于碱基插入或者缺失的频率(38.99%), 在碱基置换中, 转换的频率(44.65%)是颠换频率(16.35%)的2.7倍, 其中C/T之间的转换所占比例最大, A→G和A→T也具有较高的替换频率; 构成DNA的4种碱基均可以被离子束辐照诱变发生变异, 除了没有C→G的置换外, 每一种碱基都可以被其他的几种碱基所置换, 但是胸腺嘧啶(T)具有较高的辐射敏感性。通过对碱基突变位点周边序列的分析发现, 嘌呤突变位点的周围嘌呤碱居多, 嘧啶突变位点的周围嘧啶碱居多。研究结果为揭示低能离子注入诱变作用分子机理提供了依据。     

The point mutation induced by the low-energy N+ ion implantation in Impatiens balsamine genome

To investigate the effect of genome mutations induced by low energy ions implantation in higher plants, genome mutation of Impatiens balsamine mutant induced by low energy N⁺ ion implantation were analyzed by the RAPD, ISSR and genome sequence. Six out of the 121 ISSR primers and 6 out of the 135 RAPD primers showed that polymorphism ratios between mutants and wild type were 4.96% and 2.89%, respectively. Sequence analysis revealed that base deletions, insertions, and substitutions were observed in the mutant genome comparable to wild type. N+ induced point mutations were mostly base substitution (77.4%), no duplication, long fragments insertions and deletions was found. In all point mutation, adenine (A) was most sensitive to the N⁺ ion implantation in impatiens. The transition was mainly A ?? guanine (G) (15.90%) and thymine (T) cytosine (C) (12.55%). Transversion happened in A ? T (16.74%), which much higher than C ? G (5.02%), G ? T (6.69%), A ? C (7.11%) bases. These findings indicate that low energy ions being a useful mutagen were mostly cause the point mutation in impatiens.     

Detection of the Gua/Cyt-to-Cyt/Gua mutation in a Gua/Cyt-lined sequence using Zn2+-cyclen polyacrylamide gel electrophoresis

We previously reported a method for the detection of single-nucleotide polymorphisms by polyacrylamide gel electrophoresis (PAGE) with an additive Zn²⁺–cyclen complex (cyclen = 1,4,7,10-tetraazacyclododecane), called Zn²⁺–cyclen–PAGE. The method is based on the difference in mobility of mutant DNA (in the same length) in PAGE that is due to Zn²⁺–cyclen binding to thymine bases accompanying a total charge decrease and a local conformation change of target DNA. In combination with a heteroduplexing technique, the method is more accurate, as shown by clear gel-shifting bands. However, the question remains as to whether the Gua/Cyt-to-Cyt/Gua mutation, which is far apart from the Thy/Ade base (i.e., in a Gua/Cyt-lined sequence), can be detected by this Thy-dependent method. In this study, we determined the potency of Zn²⁺–cyclen–PAGE for the detection of the Gua/Cyt-to-Cyt/Gua single substitutions in some artificial Gua/Cyt-lined sequences derived from a human cardiac sodium channel gene, SCN5A. All Gua/Cyt-to-Cyt/Gua substitutions in the 28-set samples tested, which are 1 to 10 bases away from the nearest Thy/Ade, were successfully detected by designing DNA fragments of the appropriate length.     

Sequence and conformational effects on imino proton exchange in A.T- and A.U-containing DNA and RNA duplexes

¹H-nmr relaxation has been used to study the effect of sequence and conformation on imino proton exchange in adenine–thymine (A · T) and adenine–uracil (A · U) containing DNA and RNA duplexes. At low temperature, relaxation is caused by dipolar interactions between the imino and the adenine amino and AH2 protons, and at higher temperature, by exchange with the solvent protons. Although room temperature exchange rates vary between 3 and 12s^?1, the exchange activation energies (E_α) are insensitive to changes in the duplex sequence (alternating vs homopolymer duplexes), the conformation (B-form DNA vs A-form RNA), and the identity of the pyrimidine base (thymine vs uracil). The average value of the activation energy for the five duplexes studied, poly[d(A-T)], poly[d(A) · d(T)], poly[d(A-U)], Poly[d(A) · d(U)], and poly[r(A) · r(U)], was 16.8 ± 1.3 kcal/mol. In addition, we find that the average E_α for the A.T base pairs in a 43-base-pair restriction fragment is 16.4 ± 1.0 kcal/mol. This result is to be contrasted with the observation that the E_α of cytosine-containing duplexes depends on the sequence, conformation, and substituent groups on the purine and pyrimidine bases. Taken together, the data indicate that there is a common low-energy pathway for the escape of the thymine (uracil) imino protons from the double helix. The absolute values of the exchange rates in the simple sequence polymers are typically 3–10 times faster than in DNAs containing both A · T and G · C base pairs.     

Sequence comparison of two codominant RAPD markers in Brassica nigra: deletions,substitutions and microsatellites

Summary Two RAPD fragments segregating codominantly were investigated in a F₂ population of Brassica nigra. Southern hybridization of these DNA fragments to genomic B. nigra DNA digested with several endonucleases revealed similar restriction profiles. Sequencing of the two fragments disclosed 93% homology. The differences were due mainly to an internal 41 nucleotide deletion in one of the fragments. Minor deletions of one to three bases, including a microsatellite of CTT motif were also observed. In addition, base substitutions, mostly transitions were detected. These relatively small differences suggested that the two RAPD products were indeed different versions of the same sequence. The larger fragment of 1154 bp was denominated A05¹ whereas the shorter one, denominated A05², had 1116 bp.     

Characterization of native <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> strains by PCR-RAPD based fingerprinting

Seventy isolates of Bacillus thuringiensis were isolated from soil samples collected from cotton fields. These isolates were characterized by randomly amplified poylmorphic DNA (RAPD) markers to determine their genetic diversity pattern based on their source of origin. Different random decamer primers were used for RAPD amplification, which generated a total of 1935 fragments; of these 1865 were polymorphic and 68 monomorphic. The primers OPA03, OPA08, OPD14, OPD19, OPD20, OPE17 and OPD19 produced 100% polymorphic fragments, whereas primers OPC06, OPC20 and OPD17 produced 20, 31 and 17 monomorphic fragments, respectively. When the RAPD banding pattern data was subjected to dendrogram construction, the 70 isolates fell into two separate clusters, cluster I and cluster II, which includes 26 and 44 B. thuringiensis isolates, respectively. These two main clusters were further divided into four subclusters at Eucledian distance of 150 and 80% similarity index. All primers showed amplification and indicated the good diversity of B. thuringiensis isolates. The RAPD pattern showed 4–10 bands per isolate, with MWt in the range of 0.4–3.5 Kb and an average of 193.5 fragments were produced per primer. The primer OPE17 was found to be the most discriminatory as it produced 286 polymorphic bands.     

Sequence analysis of lacZ- mutations induced by ion beam irradiation in double-stranded M13mp18DNA

While M13mp18 double-stranded DNA was irradiated with ion beam, and transfected intoE. coli JM103, a decrease of transfecting activity was discovered. The lacZ^- mutation frequency at 20% survival could reach (3.6–16.8) × 10⁴, about 2, 3–10 times that of unirradiated M13DNA. Altogether, 27 IacZ^-mutants were selected, 10 of which were used for sequencing. 7 of the sequenced mutants show base changes in 250-bp region examined (the remaining 3 mutants probably have base changes outside the regions sequenced). 5 of the base-changed mutants contain more than one mutational base sites (some of them even have 5–6 mutational base sites in 250-bp region examined); this dense distribution of base changes in polysites has seldom been seen in X-rays, Y-rays or UV induced DNA mutations. Our experiments also showed that the types of base changes include transitions(50%), transversions (45%) and deletion (5%); no addition or duplication was observed. The transitions were mainly C→T and A→G; the transversions were mainly C→A and C→G. The mutations involving cytosine residue (in the template strand) constitute about 60% of all the base changes observed. In comparison with the surrounding sequences of mutational base sites, the base located between TG and CT is found to be easily substituted.     

Molecular characterization of RAPD and SCAR markers linked to the Tm-1 locus in tomato

We have cloned and sequenced six RAPD fragments tightly linked to the Tm-1 gene which confers tomato mosaic virus (ToMV) resistance in tomato. The terminal ten bases in each of these clones exactly matched the sequence of the primer for amplifying the corresponding RAPD marker, except for one in which the 5-endmost two nucleotides were different from those of the primer. These RAPD clones did not cross-hybridize with each other, suggesting that they were derived from different loci. From Southern-hybridization experiments, five out of the six RAPD clones were estimated to be derived from middle- or high-repetitive sequences, but not from any parts of the ribosomal RNA genes (rDNA), which are known to be tightly linked with the Tm-1 locus. The remaining clone appeared to be derived from a DNA family consisting of a few copies. These six RAPD fragments were converted to sequence characterized amplified region (SCAR) markers, each of which was detectable using a pair of primers having the same sequence as that at either end of the corresponding RAPD clone. All pairs of SCAR primers amplified distinct single bands whose sizes were the same as those of the RAPD clones. In four cases, the SCAR markers were present in the line with Tm-1 but absent in the line without it, as were the corresponding RAPD markers. In the two other cases, the products of the same size were amplified in both lines. When these SCAR products were digested with different restriction endonucleases which recognize 4-bp sequences, however, polymorphisms in fragment length were found between the two lines. These co-dominant markers are useful for differentiating heterozygotes from both types of homozygote.     

Genetic linkage map of ISSR and RAPD markers in Einkorn wheat in relation to that of RFLP markers

 The potential of PCR-based markers for construction of a genetic linkage map in Einkorn wheat was investigated. From a comparison of polymorphisms between two Einkorn wheats, Triticum monococcum (Mn) and T. boeoticum (Bt), we obtained 49 polymorphic bands produced by 33 primers for inter-simple sequence repeat (ISSR) and 36 polymorphic bands shown by 25 combinations of random amplified polymorphic DNA (RAPD) primers for mapping in 66 individuals in the F₂ population. Although 44 ISSR fragments and 29 RAPD fragments statistically showed a 3 : 1 segregation ratio in the F₂ population, only 9 markers each of the ISSR and RAPD bands were able to be mapped on the RFLP linkage map of Einkorn wheat. ISSR markers were distributed throughout the chromosomes. The mapped positions of the ISSR markers seemed to be similar to those obtained by the RFLP markers. On the other hand, 4 of the 9 RAPD markers could map the RFLP marker-poor region on the short arm of 3A^m, suggesting a potential to map novel regions containing repetitive sequences. Comparisons of the genetic linkage map of Einkorn wheat to the linkage map and cytological map of common wheat revealed that the marker orders between the two maps of Einkorn wheat and common wheat coincided except for 4A, which harbors chromosome rearrangements specific for polyploid wheats, indicating a conservatism between the two genomes. Recombinations in Einkorn wheat chromosomes took place more frequently around the centromere and less at the distal part of chromosomes in comparison to those in common wheat. Nevertheless, recombinations even in Einkorn wheat chromosomes were strongly suppressed around the centromere. In fact, the markers located within 1 cM of the centromere were located almost in the central part of the chromosome arm. Received: 7 June 1997 / Accepted: 17 June 1997     

An FTIR Spectroscopic Study of Calf-thymus DNA Complexation with Al(III) and Ga(III) Cations

Abstract

The interaction of calf-thymus DNA with trivalent Al and Ga cations, in aqueous solution at pH =6–7 with cation/DNA(P) (P=phosphate) molar ratios (r) 1/80, 1/40, 1/20, 1/10, 1/4 and 1/2 was characterized by Fourier Transform infrared (FTIR) difference spectroscopy.

Spectroscopic results show the formation of several types of cation-DNA complexes. At low metal ion concentration (r=l/80, 1/40), both cations bind mainly to the backbone PO₂ group and the guanine N-7 site of the G-C base pairs (chelation). Evidence for cation chelate formation comes from major shifting and intensity increase of the phosphate antisymmetric stretch at 1222 cm-¹ and the mainly guanine band at 1717 cm¹. The perturbations of A-T base pairs occur at high cation concentration with major helix destabilization. Evidence for cation binding to A-T bases comes from major spectral changes of the bands at 1663 and 1609 cm-¹ related mainly to the thymine and adenine in-plane vibrations. A major reduction of the B-DNA structure occurs in favor of A-DNA upon trivalent cation coordination.     

Structural Studies of DNA Fragments: The G·T Wobble Base Pair in A,B and Z DNA; The G·A Base Pair in B-DNA

Abstract

The crystal structures of five double helical DNA fragments containing non-Watson-Crick complementary base pairs are reviewed. They comprise four fragments containing G·T base pairs: two deoxyoctamers d(GGGGCTCC) and d(GGGGTCCC) which crystallise as A type helices; a deoxydodecamer d(CGCGAATTTGCG) which crystallises in the B-DNA conformation; and the deoxyhexamer d(TGCGCG), which crystallises as a Z-DNA helix. In all four duplexes the G and T bases form wobble base pairs, with bases in the major tautomer forms and hydrogen bonds linking N1 of G with 02 of T and 06 of G with N3 of T. The X-ray analyses establish that the G·T wobble base pair can be accommodated in the A, B or Z double helix with minimal distortion of the global conformation. There are, however, changes in base stacking in the neighbourhood of the mismatched bases. The fifth structure, d(CGCGAATTAGCG), contains the purine purine mismatch G·A where G is in the anti and A in the syn conformation. The results represent the first direct structure determinations of base pair mismatches in DNA fragments and are discussed in relation to the fidelity of replication and mismatch recognition.     

Cloning and characterization of RAPD markers of parasitic nematodesTrichinella spiralis andT. pseudospiralis

A RAPD analysis was carried out in parasitic nematodesTrichinella spiralis andT. pseudospiralis, which are among the most common and hazardous agents in human and animal helminthoses. Several RAPD fragments were cloned and some of them sequenced. The fragments were shown to correspond to unique or low-repetitive sequences and to contain ORFs, microsatellites, and regions homologous to various pro- and eukaryotic sequences. Several fragments can be used to distinguish betweenT. spiralis andT. pseudospiralis.     

Mutational specificity of thymine deprivation-induced mutation in the lacI gene of Escherichia coli

LacI⁻ mutants obtained following 2 and 6 h of thymine deprivation were cloned and sequenced. The mutational spectra recovered were dissimilar. After 2 h of starvation the majority of mutations were base substitutions, largely G: C→C: G transversions. Frameshift mutations but not deletions were observed. In contrast, following 6 h of starvation, with the exception of the G: C→C: G transversion, all possible base substitutions were recovered. Moreover, several deletions but no frameshift events were observed. The differences in the mutational spectra recovered after two periods of thymine deprivation highlight the role of altered nucleotide pools and the potential influence of DNA replication mechanisms.     

Relationship among acidophilic bacteria from diverse environments as determined by randomly amplified polymorphic DNA analysis (RAPD)

Summary Random amplification of polymorphic DNA (RAPD), a PCR-based technique was applied to evaluate genomic diversity among three strains of Acidithiobacillus thiooxidans, five strains of Acidithiobacillus ferrooxidans and one acidophilic moderate thermophile strain, using 45 random primers of five different series. More than 2200 bands were observed, with an average of 45 bands per primer. Primer OPC-3 produced the maximum number of fragments whereas minimum numbers of fragments were produced with primer OPA-5. A dendrogram was generated using cluster analysis by the unweighted pair group method of arithmetic means (UPGMA). The dendrogram showed three groups with similarity ranging from 29 to 85%. The maximum similarity (85%) was observed between the strains T.t₁ and T.t₂ of Acidithiobacillus thiooxidans.     

用差异显示反转录PCR银染技术研究植物基因表达的差异

通过调整差异显示反转录PCR(DDRT-PCR)中总RNA、锚定引物、随机引物、cDNA和dNTP等关键试剂的用量,优化了适用于银染检测的DDRT-PCR方法.PCR扩增产物经6%变性聚丙烯酰胺凝胶垂直电泳分离后,银染能检测到多而清晰的条带.泳道中的条带数最少为40个,最多达80个,平均为60个,条带大小分布在100～900 bp范围,灵敏度为5 pg/mm² .此方法操作简便快速,灵敏度高,重复性好.采用这个改良的方法,分析了拟南芥野生型和ast突变型基因表达的差异.从16 000个cDNA扩增产物条带中筛选出28个差异条带.二次PCR扩增后,进一步筛选出13个差异条带,其中7个是野生型特异表达的,6个是突变型特异表达的,为进一步认识ast突变表型的产生机制奠定了基础.     

Distamycin and Penta-N-Methylpyrrolecarboxamide Binding Sites on Native DNA A Comparison of Methidiumproyl-EDTA-Fe(II) Footprinting and DNA Affinity Cleaving

Abstract

Using two direct methods we have studied the binding locations and site sizes of distamycin and penta-N-methylpyrrolecarboxamide on three DNA restriction fragments from pBR322 plasmid. We find that methidiumpropyl-EDTA·Fe(II) footprinting and DNA affinity cleaving methods report common binding locations and site sizes for the tri- and pentapeptides bound to heterogeneous DNA. The tripeptide distamycin binds 5-base-pair sites with a preference for poly(dA)·poly(dT) regions. The pentapeptide binds 6–7-base-pair sites with a preference for poly(dA)·poly(dT) regions. These results are consistent with distamycin binding as an isogeometric helix to the minor groove of DNA with the four carboxamide N-H's hydrogen bonding five A+T base pairs. The data supports a model where each of the carboxamide N-H's can hydrogen bond to two bases, either O(2) of thymine or N(3) of adenine, located on adjacent base pairs on opposite strands of the helix. In most (but not all) cases the tri- and pentapeptide can adopt two orientations at each A+T rich binding site.     

Characterization of Thielaviopsis paradoxa Isolates from Oil Palms in Colombia,Ecuador and Brazil

Ceratocystis paradoxa (Anamorph: Thielaviopsis paradoxa) is parasitic on a range of economic and food crops and is the cause of dry basal rot, a limiting disease in oil palm. The objective of this study was to determinate the pathogenic and genetic diversity of Thielaviopsis isolates from oil palms in Colombia, Ecuador and Brazil. A total of 164 strains of Thielaviopsis paradoxa were characterized using pathogenicity tests, random amplified polymorphic DNA (RAPD) markers and PCR sequencing of the internal transcribed spacer (ITS) region of 5.8 S ribosomal DNA. Oil palm seedlings were inoculated by injecting the base of stems in the seedling stage with a fungal suspension and severity scores of disease reactions were evaluated. PCR amplification of the ITS region resulted in a 590 base pair (bp) product. Digestion of the PCR product with two restriction enzymes produced three restriction patterns, which according to ITS sequences could be classified as T. paradoxa. Six RAPD primers gave polymorphic bands in T. paradoxa. Population structure analyses of the RAPD data suggested that most of the isolates obtained in this study belonged to a single population. The genetic diversity of the isolates from South America was intermediate, and therefore, T. paradoxa is likely to be predominantly clonal compared with Ceratocystis species. Sporadic sexual reproduction may occur for T. paradoxa but is secondary to clonal reproduction. Data on pathogen diversity will provide information on breeding strategies and population structures.     

Zinc(II)–cyclen polyacrylamide gel electrophoresis for detection of mutations in short Ade/Thy-rich DNA fragments

We describe an improved gel-based method with an additive Zn²⁺-cyclen complex (cyclen, 1,4,7,10-tetraazacyclododecane), Zn²⁺-cyclen–PAGE, for mutation detection in DNA fragments by PCR that contain more than 65% Ade/Thy bases and fewer than 100 base pairs (bp). Existing techniques have a problem in analyzing such short Ade/Thy-rich fragments because the duplexes are disrupted and are not detectable due to binding of Zn²⁺-cyclen to Thy bases. In this strategy using a PCR primer with a Gua/Cyt-lined sequence attached at its 5′-end, we successfully detected a mutation in an 86-bp Ade/Thy-rich region of the BRCA1 gene from formalin-fixed paraffin-embedded breast cancer-tissue sections.