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

To reveal the mutation effect of low-energy ion implantation on Arabidopsis thaliana in vivo, T80II, a stable dwarf mutant, derived from the seeds irradiated by 30 keV N⁺ with the dose of 80×10¹⁵ 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. thalianain 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.     

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 changes in DNA methylation induced by low-energy ion implantation in Arabidopsis thaliana

This study evaluated changes in DNA methylation in Arabidopsis thaliana plants grown from seeds implanted with low-energy N(+) and Ar(+) ions. Methylation-sensitive amplified polymorphism (MSAP) testing revealed altered DNA methylation patterns after ion implantation at doses of 1 × 10(14) to 1 × 10(16) ions/cm(2). Comparison of the MSAP electrophoretic profiles revealed nine types of polymorphisms in ion-implanted seedlings relative to control seedlings, among which four represented methylation events, three represented demethylation events, and the methylation status of two was uncertain. The diversity of plant DNA methylation was increased by low-energy ion implantation. At the same time, total genomic DNA methylation levels at CCGG sites were unchanged by ion implantation. Moreover, a comparison of polymorphisms seen in N(+) ion-implanted, Ar(+) ion-implanted, and control DNA demonstrated that the species of incident ion influenced the resulting DNA methylation pattern. Sequencing of eight isolated fragments that showed different changing patterns in implanted plants allowed their mapping onto variable regions on one or more of the five Arabidopsis chromosomes; these segments included protein-coding genes, transposon and repeat DNA sequence. A further sodium bisulfite sequencing of three fragments also displayed alterations in methylation among either different types or doses of incident ions. Possible causes for the changes in methylation are discussed.     

RAPD分析氮离子注入甜菊种子后的幼苗基因组DNA变异

应用ＲＡＰＤ 技术检测经低能氮离子注入甜菊纯系种子引起的幼苗基因组ＤＮＡ 变异。筛选出ＯＰＪ系列中的１５ 种引物对实验及对照基因组ＤＮＡ 进行了ＰＣＲ 扩增,共获扩增片段１０３ 条,分子量在０．３ － ３ｋｂ 之间,其中５ 种引物ＯＰＪ－ １ ,７ ,９,１１ ,１２ 扩增出差异片段１２ 条。结果表明,低能氮离子注入甜菊种子可引起体内基因组ＤＮＡ 发生突变;ＲＡＰＤ 技术是检测基因组ＤＮＡ 发生诱变的一种简便、有效方法。本文同时探讨了离子强度和Ｔａｇ ＤＮＡ 聚合酶用量对甜菊ＲＡＰＤ 分析结果的影响,以及氮离子注入诱变效应的可能机制。     

氟氏链霉菌离子束注入突变谱的分析

用低能N⁺离子束注入转谷氨酰胺酶产生菌氟氏链霉菌后,通过试验,初步确定了注入的效应曲线,获得了一系列突变菌株。提取原始菌株和突变菌株的DNA,采用PCR反应分段扩增出转谷氨酰胺酶基因进行单链构象多态性分析(SSCP),并将特异性条带克隆测序进行基因突变型的鉴定,分析离子束注入引起链霉菌基因的基因突变类型及特点。结果显示:碱基变异的类型包括转换、颠换和缺失。在检测到的24个碱基突变中,主要是碱基的置换(87.5%),碱基缺失的比例比较小(12.5%)。在碱基置换中,转换的频率(58.3%)高于颠换的频率(29.2%)。转换主要以C→T,A→G为主,颠换以G→T,C→G为主。此外构成DNA的4种碱基均可以被离子束辐照诱发变异,其中胞嘧啶发生突变的频率较高。     

Biochemistry and Genetic Analysis of Bioeffects of Low Energy N+ Implantation and Y-Radiation on Arabidopsis thaliana

We analyzed the activities of specific enzymes,esterase and peroxidase,and the electrophoresis profiling of isozymes of both the second-generation (MI) and the thirdgeneration (M2) individuals of Arabidopsis thaliana after the parental generation was treated with low-energy N+ implantation and y-radiation.Results indicated that the mutagenic effects of both treatments were inheritable.The obvious difference between N+ implantation and γ-radial on the relationship of doses and effects was also observed.For the correlation between effects and doses,the former presented a particular saddle-like pattern,while the latter showed a pattern of linearity.Random amplification of polymorphic DNA analysis showed that the variant DNA bands were consistent in MI and M2 generations treated with both γ-radiation and ion implantation.However,the ratio of individual plant variation between the two treatments was remarkably different.     

Binding of T and T analogs to CG base pairs in antiparallel triplexes.

The goal of this study was to address antiparallel triplex formation at duplex targets that do not conform to a strict oligopurine.oligopyrimidine motif. We focused on the ability of natural bases and base analogs incorporated into oligonucleotide third strands to bind to so-called CG inversions. These are sites where a cytosine base is present in an otherwise purine-rich strand of a duplex target. Using a 26-base-triplet test system, we found that of the standard bases, only thymine (T) shows substantial binding to CG inversions. This is quantitatively similar to the report of Beal and Dervan [Science (1991), 251, 1360-1363]. Binding to CG inversions was only slightly weaker than binding to AT base pairs. Binding of T to CG inversions was also evaluated in two other sequences, with qualitatively similar results. Six different analogs of thymine were also tested for binding to CG inversions and AT base pairs. Significant changes in affinity were observed. In particular, 5-fluoro-2'-deoxyuridine was found to increase affinity for CG inversions as well as for AT base pairs. Studies with oligonucleotides containing pyridin-2-one or pyridin-4-one suggest that thymine O4 plays a critical role in the T.CG interaction. Possible models to account for these observations are discussed.     

Studies on interaction between histone V (f2c) and deoxyribonucleic acids.

Histone V (2fc) from chick erythroctes was used in the study of its interaction with DNA from various sources. Complexes between this histone and DNA were formed using the procedure of continuous NaCl gradient dialysis in urea. Two physical methods, namely thermal denaturation and circular dichroism (CD), were used as analytical tools. Thermal denaturation of nucleohistone V with chick or calf thymus DNA shows three melting bands: band I at 45-50 degrees corresponds to free base pairs; band II at 75-79 degrees, and band III at 90-93 degrees correspond to histone-bound base pairs. In histone-bound regions, there are 1.5 amino acid residues/nucleotide in nucleohistone V. In contrast, a value between 2.9 and 3.3 was determined for nucleohistone I (fl) (H. J. Li (1973), Biopolymers 12, 287). Similar melting properties have been observed for histone V complexed with bacterial DNA from Micrococcus luteus. Histone V binding to DNA induces a slight transition from a B-type CD spectrum to a C-type spectrum. Trypsin treatment of nucleohistone V reduces melting band III much more effectively than band II. Such a treatment also restores DNA to B conformation in the free state. Reduction of the melting bands of nucleohistone V by polylysine binding follows the order of I greater than II greater than III, accompanied by the increase of a new band at 100 degrees. When two bacterial DNAs of varied A + T (adenine + thymine) content simultaneously compete for the binding of histone V, the more (A " T)-rich DNA is selectively favored. Under experimental conditions described here, Clostridium perfringens DNA with 69% A + T is bound by histone V in preference to chicken DNA with 56% A + T although the latter has natural sequences for histone V binding.     

On the chemical nature of DNA and RNA modification by a hemin model system.

In order to model the interaction of hemin with DNA and other polynucleotides, we have studied the degradation of DNA, RNA, and polynucleotides of defined structure by [meso-tetrakis(N-methyl-4-pyridyl)porphinato]manganese(III) (MnTMPP) + KHSO5. The activated porphyrin was shown to release adenine, thymine, and cytosine from DNA; RNA degradation afforded adenine, uracil, and cytosine. The same products were obtained from single- and double-stranded DNA oligonucleotides of defined sequence, and also from single-stranded DNA and RNA homopolymers. The overall yield of bases from the dode-canucleotide d(CGCT3A3GCG) was equal to 14% of the nucleotides present initially, indicating that each porphyrin catalyzed the release of approximately 4 bases. Although no guanine was detected as a product from any of the substrates studied, the ability of MnTMPP + KHSO5 to degrade guanine nucleotides was verified by the destruction of pGp, and by the appearance of bands corresponding to guanosine cleavage following treatment of 32P end labeled DNA restriction fragments with activated MnTMPP. Inspection of a number of sites of MnTMPP-promoted cleavage indicated that the process was sequence-selective, occurring primarily at G residues that were part of 5'-TG-3' or 5'-AG-3' sequences, or at T residues. Also formed in much greater abundance were alkali-labile lesions; these were formed largely at guanosine residues. Also studied was the degradation of a 47-nucleotide RNA molecule containing two hairpins. Degradation of the 5'-32P end labeled RNA substrate afforded no distinct, individual bands, suggesting that multiple modes of degradation may be operative.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alterations in the adenine-plus-thymine-rich region of CEN3 affect centromere function in Saccharomyces cerevisiae.

Centromere DNA from 11 of the 16 chromosomes of the yeast Saccharomyces cerevisiae have been analyzed and reveal three sequence elements common to each centromere, referred to as conserved centromere DNA elements (CDE). The adenine-plus-thymine (A + T)-rich central core element, CDE II, is flanked by two short conserved sequences, CDE I (8 base pairs [bp]) and CDE III (25 bp). Although no consensus sequence exists among the different CDE II regions, they do have three common features of sequence organization. First, the CDE II regions are similar in length, ranging from 78 to 86 bp measured from CDE I to the left boundary of CDE III. Second, the base composition is always greater than 90% A + T. Finally, the A and T residues in these segments are often arranged in runs of A and runs of T residues, sometimes with six or seven bases in a stretch. We constructed insertion, deletion, and replacement mutations in the CDE II region of the centromere from chromosome III, CEN3, designed to investigate the length and sequence requirements for function of the CDE II region of the centromere. We analyzed the effect of these altered centromeres on plasmid and chromosome segregation in S. cerevisiae. Our results show that increasing the length of CDE II from 84 to 154 bp causes a 100-fold increase in chromosome nondisjunction. Deletion mutations removing segments of the A + T-rich CDE II DNA also cause aberrant segregation. In some cases partial function could be restored by replacing the deleted DNA with fragments whose primary sequence or base composition is very different from that of the wild-type CDE II DNA. In addition, we found that identical mutations introduced into different positions in CDE II have very similar effects.     

Calculation on spectrum of direct DNA damage induced by low-energy electrons including dissociative electron attachment

In this work, direct DNA damage induced by low-energy electrons (sub-keV) is simulated using a Monte Carlo method. The characteristics of the present simulation are to consider the new mechanism of DNA damage due to dissociative electron attachment (DEA) and to allow determining damage to specific bases (i.e., adenine, thymine, guanine, or cytosine). The electron track structure in liquid water is generated, based on the dielectric response model for describing electron inelastic scattering and on a free-parameter theoretical model and the NIST database for calculating electron elastic scattering. Ionization cross sections of DNA bases are used to generate base radicals, and available DEA cross sections of DNA components are applied for determining DNA-strand breaks and base damage induced by sub-ionization electrons. The electron elastic scattering from DNA components is simulated using cross sections from different theoretical calculations. The resulting yields of various strand breaks and base damage in cellular environment are given. Especially, the contributions of sub-ionization electrons to various strand breaks and base damage are quantitatively presented, and the correlation between complex clustered DNA damage and the corresponding damaged bases is explored. This work shows that the contribution of sub-ionization electrons to strand breaks is substantial, up to about 40–70%, and this contribution is mainly focused on single-strand break. In addition, the base damage induced by sub-ionization electrons contributes to about 20–40% of the total base damage, and there is an evident correlation between single-strand break and damaged base pair A–T.     

In vivo restriction. Sequence and structure of endonuclease II-dependent cleavage sites in bacteriophage T4 DNA.

Endonuclease II of bacteriophage T4 is required for in vivo restriction of cytosine-containing DNA from its host, Escherichia coli, (as well as from phage mutants lacking cytosine modification), normally the first step in the reutilization of host DNA nucleotides for synthesis of phage DNA in infected cells. The phage cytosine-DNA is fragmented incompletely to yield genetically defined fragments. This restriction is different from that of type I, II, or III restriction enzymes. We have located seven major endonuclease II-dependent restriction sites in the T4 genome, of which three were analyzed in detail; in addition, abundant sites were cleaved in less than or equal to 5% of all molecules. Sites I, II, and III shared the sequence 5'-CCGNNTTGGC-3' and were cleaved in about 25% (I and III) and 65% (II) of all molecules, predominantly staggered around the first or second of the central unspecified base pairs to yield fragments with one 5' base. The less frequently cleaved sites I and III deviated from site II in predicted helical structure when viewed from the consensus strand, and in sequence when viewed from the opposite strand. Thus, interaction with a particular helical structure as well as recognition of the bases in DNA appears important for efficient cleavage.     

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.     

Strand scission of deoxyribonucleic acid by neocarzinostatin, auromomycin, and bleomycin: studies on base release and nucleotide sequence specificity

The nucleotide sequence specificity of neocarzinostatin (NCS), auromomycin (AUR), bleomycin (Blm), phleomycin (Phlm), and tallysomycin (Tlm) has been determined by using these antibiotics and their associated chromophores to create strand scissions in end-labeled restriction fragments of DNA and then determining the base sequence of the oligonucleotides formed. NCS and the NCS chromophore induce similar patterns of cleavage in DNA fragments labeled at the 5' terminus. The pattern produced by the AUR chromophore also resembles that of its holoantibiotic. Dithiothreitol enhances the rate of cleavage of DNA by the AUR chromophore but does not alter the sequence specificity. The results suggest that the polypeptide component of AUR and NCS serves primarily as a carrier for the chromophore. When tested with a fragment labeled at the 3' terminus, the products of NCS and AUR cleavage do not display the patterns of chemically produced oligonucleotides cleaved at phosphodiester bonds, suggesting that the 5' terminus is modified by a sugar fragment. NCS primarily attacks thymine (75% of the total bases attacked) and, to a lesser extent, adenine (19%) and cytosine (6%). AUR preferentially attacks guanine (67% of total bases), while attacking less often thymine (24%) and adenine (9%). Bleomycin and its analogues preferentially cleave purine--pyrimidine (5' leads to 3') and pyrimidine--pyrimidine (3' leads to 5') sequences. All (5' leads to 3') GT and GC sequences were cleaved. Phlm G and Phlm-Pep are less active than bleomycin toward purines while Tlm was more active. The patterns of cleavage produced by Blm A2 and Blm B6 are similar, while those produced by Phlm-Pep, Phlm G, Blm-B1', and Blm-Pep resemble one another. The cleavage pattern of Tlm shows quantitative differences from the other analogues tested. Differences between bleomycin and its analogues may be related to structural differences in these molecules.     

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.     

The effects of substituted pyrimidines in DNAs on cleavage by sequence-specific endonucleases.

The rates of cleavage of DNAs containing substituents at position 5 of thymine or cytosine have been measured for a variety of sequence-specific endonucleases, so as to determine which features in the DNA sequence are being probed. Phage phi e DNA fully substituted with 5-hydroxymethyluracil is cleaved more slowly by enzymes whose recognition sequences contain A-T base pairs than are DNAs containing thymine, but both types of DNA are cleaved at similar rates by enzymes recognizing sequences composed only of G-C base pairs. Phage PBS2 DNA with uracil completely substituted for thymine is cleaved slowly by several enzymes which recognize sequences containing A-T base pairs (endonucleases Hpa I, HindII, and HindIII), while the rates of cleavage by other enzymes (endonucleases EcoRI and BamHI) are not affected. Phage lambda- and P22 DNAs containing 5-bromouracil are cleaved more slowly by several enzymes (endonucleases HindIII, Hpa I, BamHI) than are thymine-containing DNAs. Enzymes that recognize sequence isomers with the composition G:C:2A:2T (endonucleases EcoRI, Hpa I, HindIII) are not equally affected by substitution at position 5 of thymine, suggesting that they differ in their contacts with A-T base pairs. DNA containing glucosylated 5-hydroxymethylcytosine in place of cytosine is resistant to cleavage by all the endonucleases examined.     

Cloning and characterization of a cDNA encoding the beta subunit of human casein kinase II

Previous studies [Summercorn et al. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 8834-8838; Klarlung & Czech (1988) J. Biol. Chem. 263, 15872-15875] have indicated that Balb/c 3T3 cells and 3T3-L1 adipocytes incubated with insulin show increased casein kinase II activity within minutes, implicating this serine/threonine kinase as an early step in an insulin signaling pathway. We recently reported the isolation of a cDNA encoding an alpha subunit of human casein kinase II [Meisner et al. (1989) Biochemistry 28, 4072-4076] as an initial step toward examining the regulation of this enzyme. We now describe a HepG2 cell casein kinase II beta subunit cDNA of 2.57 kb containing 96 bases of 5' untranslated sequence, 645 bases of open reading frame, and 1832 bases of 3' untranslated sequence with two polyadenylation consensus signal sequences and two poly(A) stretches. The open reading frame of the human beta subunit cDNA was 77% and 87% identical with the Drosophila sequence at the nucleotide and amino acid levels, respectively, and 99% identical with the bovine amino acid sequence. RNA analysis of HepG2 cell RNA utilizing HepG2 beta subunit cDNA fragments as probes revealed one major band migrating at 1.2 kb and two minor bands migrating at 3.0 and 4.2 kb. Results from DNA analysis of HepG2 genomic DNA, consistent with results utilizing Drosophila genomic DNA, suggest the presence of a single gene for the beta subunit of casein kinase II.     

Genomic changes at early stages of formation of allopolyploid Aegilops longissima x Triticum urartu

Using the model of synthetic allopolyploid Aegilops longissima TL05 x Triticum urartu TMU06 of the first generation, the degree and character of changes in subtelomeric, microsatellite and random amplified DNA sequences (RAPD) on early stage of polyploidization was estimated. Study of genome changes was performed by comparing of PCR spectra obtained while amplifying genome DNA of allopolyploid and its parental forms. For analysis of subtelomeric DNA, we used 66 pairs of primers composed of 11 singular primers designed for subtelomere DNA sequences of cereals. RAPD analysis was performed with usage of 38 primers, in microsatellite (SSR) analysis 23 primer pairs were used. RAPD analysis appeared to be a more effective PCR-based method to identify genome changes. Absence of some PCR fragments typical for parental genome in RAPD specters of allopolyploid TL05 x TMU06 was shown using 13 primers of 38 (34%), and with usage of subtelomere primers such changes in PSR specters were shown only for one of 66 pays of primers (1.5%). SSR loci were stable during the polyploidization process. Subsequent analysis of PCR fragments absent in specter of synthetic allopolyploid showed that high level of genome changes in RAPD analysis is probably connected with more effective ability of this method to reveal point mutations. Some data was found suggesting that not all genome changes observed in experimentally synthesized allopolyploids of the first generation are consequences of coadaptation of few genomes in one nucleus.