Affinity chromatography—dependent selection （ACDS） of genomic DNA fragments bound specifically to bacterial synthesized Myc／Myn proteins

This paper describes an approach to seek for mouse c-Myc/Myn proteins-bound specific sequences among genomic DNA.cDNA fragment of myn gene was obtained through RT-PCR technique from RNA of NIH3T3 cells.DNA fragments encoding BR/HLH/LZ structure of Myc and Myn proteins were cloned in frame into pGEX-2T vector respectively.Fusion GST-Myc and GST-Myn synthesized in E.coli hosts showed affinity to CACGTG E-box DNA and subsequently interacted with genomic fragments prepared through whole-genome-PCR.A PCR-assisted procedure which combines protein-DNA interaction and affinity chromatography was designed to enrich Myc/Myn bound DNA.At least two genomic DNA fragments obtained exhibit specifical binding capacity to Myc/Myn complex but not to GST alone.Significance of the work and of the technique itself as well asidentification of the DNAs are discussed.     

ISOLATION AND CHARACTERIZATION OF PHASE-SPECIFIC COMPLEMENTARY DNAs FROM SPOROPHYTES AND GAMETOPHYTES OF PORPHYRA PURPUREA (RHODOPHYTA) USING SUBTRACTED COMPLEMENTARY DNA LIBRARIES1

The red alga Porphyra purpurea (Roth) C. Agardh has a life cycle that alternates between shell-boring, filamentous sporophytes and free-living, foliose gametophytes. The significant morphological differences between these two phases suggest that many genes should be developmentally regulated and expressed in a phase-specific manner. In this study, we prepared and screened subtracted complementary DNA (cDNA) libraries specific for the sporophyte and gametophyte of P. purpurea. This involved the construction of cDNA libraries from each phase, followed by the removal of common clones through subtractive hybridization. Sampling of the subtracted libraries indicated that 8–10% of the recombinant colonies in each library were specific for the appropriate phase. Of 20 putative phase-specific cDNAs selected from each subtracted library, eight unique clones were obtained for the sporophyte and seven for the gametophyte. After confirming their phase-specificities by hybridization to gametophyte and sporophyte messenger RNA, these 15 phase-specific cDNAs were sequenced, and the deduced amino acid sequences were used to search protein databanks. Two proteins encoded by the sporophyte-specific cDNAs and two by the gametophyte-specific cDNAs were identified by their similarity to databank entries.     

Construction and characterisation of a yeast artificial chromosome library containing five haploid sugarbeet (Beta vulgaris L.) genome equivalents

A yeast artificial chromosome (YAC) genomic library of Beta vulgaris was constructed in the pYAC4 vector. High-molecular-weight DNA was prepared from agarose-embedded leaf protoplasts from a triploid cultivar. The library was found to contain 33,500 clones in an ordered array of microtiter plates. Mean size of the inserts was estimated to be 135 kb, and the library should therefore represent the equivalent of five haploid genomes. The library was characterised for the presence of highly repetitive, chloroplast and single-copy sequences. In order to isolate single-copy sequences, 18 pools of DNA, each from 1920 individual YAC clones, were prepared for rapid screening of the library by the polymerase chain reaction. The results of these screenings showed that the number of isolated clones was at or near the frequency expected.     

Elymus sibiricus, E. nutans 和 E. burchan-buddae 的形态学鉴定及其染色体组亲缘关系的研究

为了进一步研究Elymus sibiricus L.、E．nutans Griseb．和E．burchan-buddae(Neuski)Tzelev [＝Roegneria nutans(Keng)Keng]的外部形态差异及其系统学关系,本文对这三种植物的6个穗部形 态性状进行了观测和比较,并对这三个Elymus种进行了种间杂交及杂种F1的减数分裂染色体配对行 为的分析研究。结果表明：这三个Elymus种的穗长及颖长等性状均变异很大,而内稃的长、宽则变异 不大并具有明显的种间差异。E. nutans×E．barchan-buddae及E.nutans×E．sibiricus的杂种F1均完 全不育,减数分裂不规则。E．nutans×E．burchan-buddae杂种F1的减数分裂构型为：7.70I+13.40 Ⅱ+0.06Ⅲ+O.08 Ⅳ,而E．sibiricus×E．nutans杂种F1的构型为11.98 Ⅰ+9.61Ⅱ+O.64Ⅲ+0.39 Ⅳ+0.01V。由本实验的形态学和细胞学的研究结果得出以下结论：1．利用内稃形态性状结合穗部 其它性状的差异能对这三个物种进行较准确的鉴定;2．E．nutans与E.burchan-buddae的亲缘关系较 近,而E．nutans与E．sibiricus的亲缘关系则较远;3. E,burchan-buddae×E．nutans的杂种Fl中存在着染色体配对控制因子。     

人脑髓鞘碱性蛋白cDNA体外扩增、克隆和鉴定

采用聚合酶链反应(PCR)从人脑cDNA文库中扩增出600bp的髓鞘碱性蛋白(MBP)cDNA片段,与载体pGEM-3Zf(+)平端连接.重组质粒DNA转化宿主菌JM109,在含X-gal和IPTG的平板上直接筛选阳性克隆.限制性内切酶分析和成套引物扩增鉴定证明,该克隆含有7个外显子的21.5kD人脑MBP全长编码序列.