First Structural View of a Peptide Interacting with the Nucleotide Binding Domain of Heat Shock Protein 90

The involvement of Hsp90 in progression of diseases like cancer, neurological disorders and several pathogen related conditions is well established. Hsp90, therefore, has emerged as an attractive drug target for many of these diseases. Several small molecule inhibitors of Hsp90, such as geldanamycin derivatives, that display antitumor activity, have been developed and are under clinical trials. However, none of these tested inhibitors or drugs are peptide-based compounds. Here we report the first crystal structure of a peptide bound at the ATP binding site of the N-terminal domain of Hsp90. The peptide makes several specific interactions with the binding site residues, which are comparable to those made by the nucleotide and geldanamycin. A modified peptide was designed based on these interactions. Inhibition of ATPase activity of Hsp90 was observed in the presence of the modified peptide. This study provides an alternative approach and a lead peptide molecule for the rational design of effective inhibitors of Hsp90 function.

Mutations in the nucleotide binding and hydrolysis domains of Helicobacter pylori MutS2 lead to altered biochemical activities and inactivation of its in vivo function

Background: Helicobacter pylori MutS2 (HpMutS2), an inhibitor of recombination during transformation is a non-specific nuclease with two catalytic sites, both of which are essential for its anti-recombinase activity. Although HpMutS2 belongs to a highly conserved family of ABC transporter ATPases, the role of its ATP binding and hydrolysis activities remains elusive. Results: To explore the putative role of ATP binding and hydrolysis activities of HpMutS2 we specifically generated point mutations in the nucleotide-binding Walker-A (HpMutS2-G338R) and hydrolysis Walker-B (HpMutS2-E413A) domains of the protein. Compared to wild-type protein, HpMutS2-G338R exhibited similar to 2.5-fold lower affinity for both ATP and ADP while ATP hydrolysis was reduced by similar to 3-fold. Nucleotide binding efficiencies of HpMutS2-E413A were not significantly altered; however the ATP hydrolysis was reduced by similar to 10-fold. Although mutations in the Walker-A and Walker-B motifs of HpMutS2 only partially reduced its ability to bind and hydrolyze ATP, we demonstrate that these mutants not only exhibited alterations in the conformation, DNA binding and nuclease activities of the protein but failed to complement the hyper-recombinant phenotype displayed by mutS2-disrupted strain of H. pylori. In addition, we show that the nucleotide cofactor modulates the conformation, DNA binding and nuclease activities of HpMutS2. Conclusions: These data describe a strong crosstalk between the ATPase, DNA binding, and nuclease activities of HpMutS2. Furthermore these data show that both, ATP binding and hydrolysis activities of HpMutS2 are essential for the in vivo anti-recombinase function of the protein.

Copper homeostasis in Salmonella is atypical and copper-CueP is a major periplasmic metal complex.

Salmonella enterica sv. typhimurium (S. enterica sv. Typhimurium) has two metal-transporting P(1)-type ATPases whose actions largely overlap with respect to growth in elevated copper. Mutants lacking both ATPases over-accumulate copper relative to wild-type or either single mutant. Such duplication of ATPases is unusual in bacterial copper tolerance. Both ATPases are under the control of MerR family metal-responsive transcriptional activators. Analyses of periplasmic copper complexes identified copper-CueP as one of the predominant metal pools. Expression of cueP was recently shown to be controlled by the same metal-responsive activator as one of the P(1)-type ATPase genes (copA), and copper-CueP is a further atypical feature of copper homeostasis in S. enterica sv. Typhimurium. Elevated copper is detected by a reporter construct driven by the promoter of copA in wild-type S. enterica sv. Typhimurium during infection of macrophages. Double mutants missing both ATPases also show reduced survival inside cultured macrophages. It is hypothesized that elevated copper within macrophages may have selected for specialized copper-resistance systems in pathogenic microorganism such as S. enterica sv. Typhimurium.

Applying the Electrochemical Deposition Technology to Construct the Hybrid Devices

Nanowires functionalized by special molecules can be used to as the candidates for biological application in many areas. In this paper, nickel nanowires, which were fabricated by electrochemical deposition and functionalized by biotinylated peptide, were applied to constructing the hybrid device powered by F-1-ATPase motors.

Molecular diagnosis of distal renal tubular acidosis in Tunisian patients: proposed algorithm for Northern Africa populations for the ATP6V1B1, ATP6V0A4 and SCL4A1 genes

Background: Primary distal renal tubular acidosis (dRTA) caused by mutations in the genes that codify for the H+ -ATPase pump subunits is a heterogeneous disease with a poor phenotype-genotype correlation. Up to now, large cohorts of dRTA Tunisian patients have not been analyzed, and molecular defects may differ from those described in other ethnicities. We aim to identify molecular defects present in the ATP6V1B1, ATP6V0A4 and SLC4A1 genes in a Tunisian cohort, according to the following algorithm: first, ATP6V1B1 gene analysis in dRTA patients with sensorineural hearing loss (SNHL) or unknown hearing status. Afterwards, ATP6V0A4 gene study in dRTA patients with normal hearing, and in those without any structural mutation in the ATP6V1B1 gene despite presenting SNHL. Finally, analysis of the SLC4A1 gene in those patients with a negative result for the previous studies. Methods: 25 children (19 boys) with dRTA from 20 families of Tunisian origin were studied. DNAs were extracted by the standard phenol/chloroform method. Molecular analysis was performed by PCR amplification and direct sequencing. Results: In the index cases, ATP6V1B1 gene screening resulted in a mutation detection rate of 81.25%, which increased up to 95% after ATP6V0A4 gene analysis. Three ATP6V1B1 mutations were observed: one frameshift mutation (c.1155dupC; p.Ile386fs), in exon 12; a G to C single nucleotide substitution, on the acceptor splicing site (c.175-1G > C; p.?) in intron 2, and one novel missense mutation (c. 1102G > A; p. Glu368Lys), in exon 11. We also report four mutations in the ATP6V0A4 gene: one single nucleotide deletion in exon 13 (c.1221delG; p. Met408Cysfs* 10); the nonsense c.16C > T; p.Arg6*, in exon 3; and the missense changes c.1739 T > C; p.Met580Thr, in exon 17 and c.2035G > T; p.Asp679Tyr, in exon 19. Conclusion: Molecular diagnosis of ATP6V1B1 and ATP6V0A4 genes was performed in a large Tunisian cohort with dRTA. We identified three different ATP6V1B1 and four different ATP6V0A4 mutations in 25 Tunisian children. One of them, c.1102G > A; p.Glu368Lys in the ATP6V1B1 gene, had not previously been described. Among deaf since childhood patients, 75% had the ATP6V1B1 gene c. 1155dupC mutation in homozygosis. Based on the results, we propose a new diagnostic strategy to facilitate the genetic testing in North Africans with dRTA and SNHL.

Proteomic analysis of the Cdc48/Ubx network identifies a role for Ubx2 in the regulation of lipid biosynthesis

Cdc48/p97 is an essential, highly abundant hexameric member of the AAA (ATPase associated with various cellular activities) family. It has been linked to a variety of processes throughout the cell but it is best known for its role in the ubiquitin proteasome pathway. In this system it is believed that Cdc48 behaves as a segregase, transducing the chemical energy of ATP hydrolysis into mechanical force to separate ubiquitin-conjugated proteins from their tightly-bound partners.

Current models posit that Cdc48 is linked to its substrates through a variety of adaptor proteins, including a family of seven proteins (13 in humans) that contain a Cdc48-binding UBX domain. As such, due to the complexity of the network of adaptor proteins for which it serves as the hub, Cdc48/p97 has the potential to exert a profound influence on the ubiquitin proteasome pathway. However, the number of known substrates of Cdc48/p97 remains relatively small, and smaller still is the number of substrates that have been linked to a specific UBX domain protein. As such, the goal of this dissertation research has been to discover new substrates and better understand the functions of the Cdc48 network. With this objective in mind, we established a proteomic screen to assemble a catalog of candidate substrate/targets of the Ubx adaptor system.

Here we describe the implementation and optimization of a cutting-edge quantitative mass spectrometry method to measure relative changes in the Saccharomyces cerevisiae proteome. Utilizing this technology, and in order to better understand the breadth of function of Cdc48 and its adaptors, we then performed a global screen to identify accumulating ubiquitin conjugates in cdc48-3 and ubxΔ mutants. In this screen different ubx mutants exhibited reproducible patterns of conjugate accumulation that differed greatly from each other, pointing to various unexpected functional specializations of the individual Ubx proteins.

As validation of our mass spectrometry findings, we then examined in detail the endoplasmic-reticulum bound transcription factor Spt23, which we identified as a putative Ubx2 substrate. In these studies ubx2Δ cells were deficient in processing of Spt23 to its active p90 form, and in localizing p90 to the nucleus. Additionally, consistent with reduced processing of Spt23, ubx2Δ cells demonstrated a defect in expression of their target gene OLE1, a fatty acid desaturase. Overall, this work demonstrates the power of proteomics as a tool to identify new targets of various pathways and reveals Ubx2 as a key regulator lipid membrane biosynthesis.

Expanding the repertoire of DNA-mediated signaling in DNA repair

DNA damage is extremely detrimental to the cell and must be repaired to protect the genome. DNA is capable of conducting charge through the overlapping π-orbitals of stacked bases; this phenomenon is extremely sensitive to the integrity of the π-stack, as perturbations attenuate DNA charge transport (CT). Based on the E. coli base excision repair (BER) proteins EndoIII and MutY, it has recently been proposed that redox-active proteins containing metal clusters can utilize DNA CT to signal one another to locate sites of DNA damage.

To expand our repertoire of proteins that utilize DNA-mediated signaling, we measured the DNA-bound redox potential of the nucleotide excision repair (NER) helicase XPD from Sulfolobus acidocaldarius. A midpoint potential of 82 mV versus NHE was observed, resembling that of the previously reported BER proteins. The redox signal increases in intensity with ATP hydrolysis in only the WT protein and mutants that maintain ATPase activity and not for ATPase-deficient mutants. The signal increase correlates directly with ATP activity, suggesting that DNA-mediated signaling may play a general role in protein signaling. Several mutations in human XPD that lead to XP-related diseases have been identified; using SaXPD, we explored how these mutations, which are conserved in the thermophile, affect protein electrochemistry.

To further understand the electrochemical signaling of XPD, we studied the yeast S. cerevisiae Rad3 protein. ScRad3 mutants were incubated on a DNA-modified electrode and exhibited a similar redox potential to SaXPD. We developed a haploid strain of S. cerevisiae that allowed for easy manipulation of Rad3. In a survival assay, the ATPase- and helicase-deficient mutants show little survival, while the two disease-related mutants exhibit survival similar to WT. When both a WT and G47R (ATPase/helicase deficient) strain were challenged with different DNA damaging agents, both exhibited comparable survival in the presence of hydroxyurea, while with methyl methanesulfonate and camptothecin, the G47R strain exhibits a significant change in growth, suggesting that Rad3 is involved in repairing damage beyond traditional NER substrates. Together, these data expand our understanding of redox-active proteins at the interface of DNA repair.

Investigating the Role of the GET3-GET4/GET5 Interaction During Tail-Anchor Protein Targeting

The proper targeting of membrane proteins is essential to the viability of all cells. Tail-anchored (TA) proteins, defined as having a single transmembrane helix at their C-terminus, are post-translationally targeted to the endoplasmic reticulum (ER) membrane by the GET pathway (Guided Entry of TA proteins). In the yeast pathway, the handover of TA substrates is mediated by the heterotetrameric Get4/Get5 (Get4/5) complex, which tethers the co-chaperone Sgt2 to the central targeting factor, the Get3 ATPase. Although binding of Get4/5 to Get3 is critical for efficient TA targeting, the mechanisms by which Get4 regulates Get3 are unknown. To understand the molecular basis of Get4 function, we used a combination of structural biology, biochemistry, and cell biology. Get4/5 binds across the Get3 dimer interface, in an orientation only compatible with a closed Get3, providing insight into the role of nucleotide in complex formation. Additionally, this structure reveals two functionally distinct binding interfaces for anchoring and ATPase regulation, and loss of the regulatory interface leads to strong defects in vitro and in vivo. Additional crystal structures of the Get3-Get4/5 complex give rise to an alternate conformation, which represents an initial binding interaction mediated by electrostatics that facilitates the rate of subsequent inhibited complex formation. This interface is supported by an in-depth kinetic analysis of the Get3-Get4/5 interaction confirming the two-step complex formation. These results allow us to generate a refined model for Get4/5 function in TA targeting.

Lipotoxicidade na leptospirose humana: aspectos prognósticos e potencial efeito benéfico da administração de albumina

A leptospirose humana é uma doença infecciosa aguda de amplo espectro clínico e que cursa com alterações metabólicas e dislipidêmicas envolvendo colesterol total e frações, triglicerídeos e ácidos graxos não esterificados (AGNEs). Dentre os mecanismos celulares envolvidos na sua fisiopatologia encontram-se a inibição da enzima Na, K ATPase pela endotoxina GLP e a lipotoxicidade, ambos agravados pela redução dos níveis circulantes da albumina, molécula que exerce um papel fundamental na adsorção de moléculas lipídicas. Neste estudo observacional, determinamos as concentrações séricas de bilirrubina, creatinina e albumina e, pela técnica de cromatografia líquida de alta performance, a concentração sérica dos AGNEs de cadeia longa (C16: C18) de 27 pacientes com síndrome de Weil durante o período de internação hospitalar, dos quais cinco vieram a falecer. Verificamos correlações significantes (p<0,05) ao longo da internação hospitalar, nas concentrações séricas de marcadores bioquímicos de gravidade da doença (bilirrubina, creatinina e albumina), AGNEs, ácido oléico e ácido linoléico, e relação molar ácido oléico/ albumina, com r (Pearson) de -0,7981, -0,7699, 0,9014, -0,8795 -0,9816, -0,9694, -0,9821, respectivamente. A relação molar ácido oléico/ albumina e ácido oléico+ linoléico/albumina foi significantemente mais elevada nos pacientes que faleceram (p<0,001), retornando aos valores semelhantes aos do grupo controle nos pacientes que evoluíram para a cura. Na análise por Curva Roc, a relação molar ácido oléico/albumina se mostrou um bom teste preditivo, com valor de corte 0,705 associado com maior especificidade e sensibilidade prognóstica. Nossos resultados sugerem que a utilização parenteral da albumina humana em pacientes com leptospirose pode ser uma potente ferramenta terapêutica nos casos mais graves ao interferir positivamente no resgate do equilíbrio bioquímico das relações molares ácido oléico/ albumina e ácido oléico+linoléico/albumina.

A Comparison of the SNP Variation in the Calcification PMCA Gene of Lottia gigantea between a Santa Barbara Population and a More Acidic Monterey Bay Population

As the atmospheric levels of CO2 rise from human activity, the carbonic acid levels of the ocean increase, causing ocean acidification. This increase in acidity breaks down the calcified bodies that many marine organisms depend upon. Upwelling regions such as Monterey Bay in California have pH levels that are not expected to reach the open ocean for a few decades. This study reviews one of the common intertidal animals of the California coast, the Owl Limpet Lottia gigantea, and its genetic variation of the plasma membrane Ca2+ ATPase (PMCA) in relation to the acidity of its environment. The PMCA protein functions in the calcification process of many organisms. Specifically in limpets, this gene functions to form its protective shell. Single-nucleotide polymorphisms (SNPs) were found among five sections of the gene to determine variation between the acidic environment population in Monterey, California and the non-acidic environment population in Santa Barbara, California. While some variation was determined, the Monterey Bay and Santa Barbara Lottia gigantea populations are not significantly distinct at the PMCA gene. Sections B, C, and D were found to be linked. Only one location in Section B was found to have an amino acid change within an exon. Section A has the strongest connection to the sampling location. Monterey individuals were seen to be more genetically recognizable, while Santa Barbara individuals showed slightly more variation. Understanding the trends of ocean acidification, upwelling region activities, and population genetics will assist in determining how the ocean environment will behave in the future.

Investigação de mutações no gene ATP13A2 como um fator de risco para a Doença de Parkinson em pacientes brasileiros

A doença de Parkinson (DP) é a desordem neurodegenerativa motora mais frequente, com uma prevalência de, aproximadamente, 1% entre indivíduos com mais de 60 anos de idade, aumentando para 4 a 5% entre os indivíduos com idade superior a 85 anos. Esta condição é caracterizada pela perda seletiva dos neurônios dopaminérgicos da substância negra e pela presença de inclusões protéicas ricas em α-sinucleína nos neurônios sobreviventes. Pouco se sabe sobre a etiologia e a patogênese da DP. A maioria dos casos aparece esporadicamente, podendo estar associados a diversos fatores de risco ambientais e genéticos. Na última década, estudos de ligação identificaram 15 loci cromossômicos (PARK1 a PARK15) relacionados à DP e, nestes, um novo gene, ATP13A2, tem sido associado a casos de DP de início precoce. Esse gene está situado no 1p36 e codifica a proteína ATPase tipo-P da subfamília P5, de localização lisossômica, que é expressa em diversos tecidos, principalmente no cérebro. Mutações em ATP13A2 levam à formação de proteínas truncadas que ficam retidas no reticulo endoplasmático e posteriormente são degradadas pelo proteossomo, podendo causar a disfunção proteossômica, decorrente da sobrecarga gerada pela proteína mutante, ou causar a disfunção lisossômica, ambas gerando agregação tóxica. Este trabalho tem como objetivo realizar a análise molecular do gene ATP13A2 em uma amostra de 116 pacientes brasileiros com DP, de manifestação precoce (<50 anos), de forma a avaliar se mutações neste gene representam um fator de risco para a DP. O DNA foi extraído a partir de leucócitos do sangue periférico ou de saliva e a análise molecular dos éxons 2, 3, 12, 13, 14, 15, 16, 26 e 27, bem como, dos limites íntronéxons foi realizada por sequenciamento automático dos produtos da PCR. Identificamos oito variantes de sequência: quatro variantes intrônicas (uma no íntron 2, uma no íntron 13 e duas no íntron 27) e quatro variantes silenciosas (uma no éxon 3, 16, 26 e 27). Com base em dados da literatura e através de análises in silico e comparação com amostras controle, classificamos a alteração intrônica c.3084- 3C>T, e as alterações silenciosas c.2970G>A e c.3192C>T como não patogênicas; as alterações intrônicas c.106-30G>T, c.1306+42_1306+43 insC e c.3083+24C>T, e as alterações silenciosas c.132A>G e c.1610G>T foram classificadas como provavelmente não patogênicas. Nosso achados corroboram àqueles encontrados em outras populações e indicam que mutações no gene ATP13A2 não são uma causa comum de DP na amostra de pacientes brasileiros analisados. No entanto, se faz necessário estender nossas análises para outras regiões gênicas, a fim de determinar o real papel deste gene na etiologia da DP em nossa população.

Alcohol-Related Brain Damage in Humans

Chronic excessive alcohol intoxications evoke cumulative damage to tissues and organs. We examined prefrontal cortex (Brodmann's area (BA) 9) from 20 human alcoholics and 20 age, gender, and postmortem delay matched control subjects. H & E staining and light microscopy of prefrontal cortex tissue revealed a reduction in the levels of cytoskeleton surrounding the nuclei of cortical and subcortical neurons, and a disruption of subcortical neuron patterning in alcoholic subjects. BA 9 tissue homogenisation and one dimensional polyacrylamide gel electrophoresis (PAGE) proteomics of cytosolic proteins identified dramatic reductions in the protein levels of spectrin beta II, and alpha- and beta-tubulins in alcoholics, and these were validated and quantitated by Western blotting. We detected a significant increase in a-tubulin acetylation in alcoholics, a non-significant increase in isoaspartate protein damage, but a significant increase in protein isoaspartyl methyltransferase protein levels, the enzyme that triggers isoaspartate damage repair in vivo. There was also a significant reduction in proteasome activity in alcoholics. One dimensional PAGE of membrane-enriched fractions detected a reduction in beta-spectrin protein levels, and a significant increase in transmembranous alpha 3 (catalytic) subunit of the Na+, K+-ATPase in alcoholic subjects. However, control subjects retained stable oligomeric forms of a-subunit that were diminished in alcoholics. In alcoholics, significant loss of cytosolic alpha-and beta-tubulins were also seen in caudate nucleus, hippocampus and cerebellum, but to different levels, indicative of brain regional susceptibility to alcohol-related damage. Collectively, these protein changes provide a molecular basis for some of the neuronal and behavioural abnormalities attributed to alcoholics

Mechanisms of Toxicity of Ag Nanoparticles in Comparison to Bulk and Ionic Ag on Mussel Hemocytes and Gill Cells

Silver nanoparticles (Ag NPs) are increasingly used in many products and are expected to end up in the aquatic environment. Mussels have been proposed as marine model species to evaluate NP toxicity in vitro. The objective of this work was to assess the mechanisms of toxicity of Ag NPs on mussel hemocytes and gill cells, in comparison to ionic and bulk Ag. Firstly, cytotoxicity of commercial and maltose stabilized Ag NPs was screened in parallel with the ionic and bulk forms at a wide range of concentrations in isolated mussel cells using cell viability assays. Toxicity of maltose alone was also tested. LC50 values were calculated and the most toxic Ag NPs tested were selected for a second step where sublethal concentrations of each Ag form were tested using a wide array of mechanistic tests in both cell types. Maltose-stabilized Ag NPs showed size-dependent cytotoxicity, smaller (20 nm) NPs being more toxic than larger (40 and 100 nm) NPs. Maltose alone provoked minor effects on cell viability. Ionic Ag was the most cytotoxic Ag form tested whereas bulk Ag showed similar cytotoxicity to the commercial Ag NPs. Main mechanisms of action of Ag NPs involved oxidative stress and genotoxicity in the two cell types, activation of lysosomal AcP activity, disruption of actin cytoskeleton and stimulation of phagocytosis in hemocytes and increase of MXR transport activity and inhibition of Na-K-ATPase in gill cells. Similar effects were observed after exposure to ionic and bulk Ag in the two cell types, although generally effects were more marked for the ionic form. In conclusion, results suggest that most observed responses were due at least in part to dissolved Ag.

镧与钙的关系及对根系生理的影响

本研究通过探讨毛乌素沙地主要早生植物和中生植物的水分关系。对“早生植物”的概念作了进一步的分析的阐述，探讨了蒸腾作用、光合作用的气乳调节与植物叶片水分平衡的相互关系，并提出了以蒸腾“午体”为基础的关于划分植物水分生态类型的新概念。上述问题的探讨可作为对中国毛鸟素沙化化草地植物环境系统优化模式的水平衡假说的难证。探讨了为合理确定在研究地区种草造林的最佳密度所需进一步研究的内容：水分亏缺对植物量累积的抑制作用。并对该地区主要植物种的季节蒸腾耗水量作了估算。研究了依赖于主导环境因子的沙生植物蒸腾速率的回归模型。得出结论认为：对于蒸腾速率和叶面积指数，建立多元非线性回归可以取得较好结果。最后，对几个重要的蒸散预测模型和土壤水分运动的物理模型进行了评述，并探讨了这些模型对于干旱区蒸散预测研究的可行性，作为本研究的未来探讨的课题。

分子标记技术在甘兰型杂交油菜育种上的研究与应用

本文应用RAPD分标记技术对我国重要的油料作物“杂油59”杂种种子的Fl代杂种的纯度进了技术鉴定，并完善了这一技术，摸索出这一适合于目前生产应用的实用方法，填补了这一技术在油菜作物应用上的空白。用RFLP技术对我国重要的雄性不育材料“陕2A”细胞质进行了分子水平的鉴定，为证明“陕2A”是一类新型的雄性不育材料提供了重要的实验证据。 对甘蓝型油菜采用DNA快速提取法、酚仿法和CTAB法应用于不同的分子标记分析，实验结果显示： CTAB法适用于样品量大，纯度要术高的RFLP技术，酚仿法适用于引物筛选、DNA模板用量大的PCR反应，而快速提取法特别适合于生产上对种子纯度检测，是生产上推广前景很好的实用技术。 对甘蓝型油菜RAPD技术应用当中PCR体系的建立进行了探讨。实验结果显示：热启动对PCR结果的影响至关重要。而Mg++浓度、dNTP浓度、模板浓度、Tag酶用量对反应结果有不同程度的影响。经过反复实验：当PCR各组分按Mg++，2mM;dNTP，200uM;模板浓度，50ng - lOOng时，PCR的结果最好。PCR反应条件经反复实验后确定为：第一个循环：（热启动）94℃，Imin20sec．OoC 2min循环一次;第二个循环：（解链）94℃ 50sec，（退火）40℃ Imin30sec;（延伸）72℃lmin;循环40次。第三个循环：72℃lOmin，循环1次。反应总体积为20ul时最为适用。 用40个lOmer的RAPD随机引物对“杂油59”的2个亲本“垦C8”和“陕3A” 进行RAPD分析，共出现290条带，分布于3530-220bp之间。引物opA-06、opK-03、opK-13、opj-12出现阳性扩增。经重复实验后确定： opK-03的PCR结果重复性最好，该引物序列为：CCAGCTTAGG。用它对两个亲本进行RAPD分析，PCR结果共出现9条带，其中510bp、260bp为二条特征带。在Fl代中这两条特征带重现性很好。用50个商品用种萌发的F1单株进行验证，检测结果为3个个体没有出现510bp的特征带，4个个体没有出现260bp的特征带，有5个个体出现了其它带，纯度为78%，与生产用种的纯度相符。 通过对“杂优59”不同生育时期及不同取样部位作酯酶同工酶电泳方法与RAPD方法相比较，结果显示：RAPD方法可以弥补同工酶方法的缺限。由于它是基于基因水平的分析技术，可以不受环境条件、发育时期、取材部位等客观条件的限制，并具有取样量小、易操作、费用低、灵敏度高、可以检测出亲缘关系相当近的种闾或种内的材料，具有独到的优点。是值得今后在生产上推广的新技术。 用6个雄性不育材料线粒体的特异探针：ALXR 18（线粒体ATPaseα亚基）;COB 640(脱辅基细胞色素-b);COX -I（细胞色素氧化酶亚基-I）;COX -Ⅱ（细胞色素氧化酶亚基-II;PDC - 12（胡萝卜线粒体随机片断）;C2（玉米线粒随机片断），对“陕2A”，Hybrides Polima，Ogura NSL 94/96, Ogura MLCH036, Ogura NSL, Polima, Fu27,Fu38, Anand等9个材料进行RFLP分析，结果显示：用限制性内切酶EcoR I消化后的DNA与探针COB 640杂交，“陕2A”材料在4.5 kb处缺失，与ALXR 18探针杂交，在4.4 kb、4.2 kb处也明显缺失，证明“陕2A”显然不同与其它不育材料。用ALXR l8为探针，与用内切酶Nc01的酶切片断作Sourthern杂交，在RFLP谱带上6.1 kb、2.4 kb、2.5 kb处明显缺带，进一步为“陕2A”是一种新型的甘蓝型油莱雄性不育系提供了证据。