Mapping of deletions and substitutions in heteroduplex DNA molecules of bacteriophage lambda by electron microscopy

Electron microscopy of heteroduplex DNA molecules, composed of one strand of Escherichia coli phage lambda(+) DNA annealed to the complementary DNA strand of a lambda deletion or substitution mutant, permits visualization, as well as precise measurements and mapping, of the unpaired single-stranded regions of nonhomology in the otherwise double-stranded molecules. In the lambdab2 mutant, the central segment (13 percent) of the lambda(+) DNA molecule is shown to be deleted. In the hybrid phages lambda(i434) and lambda(i21) a segment of the right arm of the lambda(+) genome (5.5 or 7.6 to 9 percent) is replaced by the corresponding immunity regions of phage 434 (3.3 percent or phage 21 (4 percent) DNA. The b5 region in the lambdab5 mutant appears to be identical to the i(21) segment. From these data it is possible to estimate the size and posiion of those lambda genes which are replaced by the i(434) and i(21) segments. The method permits preparing complete physical maps of viral genomes with a precision heretofore unattainable.     

J genes for heavy chain immunoglobulins of mouse

A 15,8-kilobase pair fragment of BALB/c mouse liver DNA, cloned in the Charon 4A lambda phage vector system, was shown to contain the mu heavy chain constant region (CHmu) gene for the mouse immunoglobulin M. In addition, this fragment of DNA contains at least two J genes, used to code for the carboxyl terminal portion of heavy chain variable regions. These genes are located in genomic DNA about eight kilobase pairs to the 5' side of the CHmu gene. The complete nucleotide sequence of a 1120-base pair stretch of DNA that includes the two J genes has been determined.     

番鸭cDNA-AFLP体系的建立

以番鸭脑垂体为材料,用Trizol法提取总RNA,用Superscipt Ⅱ-RT合成第一链cDNA,RNase H和DNA Polymerase合成第二链cDNA,最后用EcoR Ⅰ/Mse Ⅰ分步酶切cDNA各2h,经T4连接酶合成双链cDNA,预扩增反应后产物稀释40倍进行选择性扩增反应.经1％琼脂糖电泳及6％变...     

Plus and minus single-stranded DNA separately encapsidated in adeno-associated satellite virions

Based on physical and chemical determinations, the mnolecular weight of the type 4 adeno-satellite virus is 5.4 X 10(6) daltons, and the virion contains 1.4 X 10(6) daltons of DNA. Denaturation and renaturation studies indicate that the viral genome is a single-stranded DNA molecule and that each virion contains either a minus or a plus strand. Upon extraction, the minus and plus strands unite to form double-stranded DNA molecules with no obvious excess of unpaired strands.     

条斑紫菜TPS基因TPP结构域的缺失突变

条斑紫菜6-磷酸海藻糖合成酶(TPS)基因(PyTPS)具有TPS及TPP双结构域,以该基因的原核表达载体pET22b-PyTPS为材料,利用双酶切片段替换法对该基因TPP结构域磷酸酶基序的编码框Box1与Box2进行了缺失突变.第一,用715bp缺失Box1的KpnI-EcoRI人工合成片段替换pET22b-PyTPS中相应的片段,获得了缺失Box1的突变基因PyTPS-Box1.第二,用661bp缺失Box2的EcoRI-HindII片段进行替换,获得了缺失Box2的突变基因PyTPS-Box2.第三,在缺失Box1后,再通过替换缺失Box2,获得了同时缺失Box1与Box2的突变基因PyTPS-Box1-Box2.本研究既获得了三种突变基因,又获得了其原核表达载体,为研究PyTPS基因的编码活性提供了材料,奠定了基础.     

Duchenne muscular dystrophy involving translocation of the dmd gene next to ribosomal RNA genes

Duchenne muscular dystrophy (DMD) is a severe X-linked disorder leading to early death of affected males. Females with the disease are rare, but seven are known to be affected because of a chromosomal rearrangement involving a site at or near the dmd gene on the X chromosome. One of the seven has a translocation between the X and chromosome 21. The translocation-derived chromosomes from this patient have been isolated, and the translocation is shown to have split the block of genes encoding ribosomal RNA on the short arm of chromosome 21. Thus ribosomal RNA gene probes may be used to identify a junction fragment from the translocation site, allowing access to cloned segments of the X at or near the dmd gene and presenting a new approach to the study of this disease.     

Visualization of nucleolar genes

The presence of extrachromosomal nucleoli in amphibian oocytes has permitted isolation and electron microscopic observation of the genes coding for ribosomal RNA precursor molecules. Visualization of these genes is possible because many precursor molecules are simultaneously synthesized on each gene. Individual genes are separated by stretches of DNA that apparently are not transcribed at the time of synthesis of precursor rRNA in the extrachromosomal nucleoli.     

The DNA binding arm of lambda repressor: critical contacts from a flexible region.

Segments of protein that do not adopt a well-ordered conformation in the absence of DNA can still contribute to site-specific recognition of DNA. The first six residues (NH2-Ser1-Thr2-Lys3-Lys4-Lys5-Pro6-) of phage lambda repressor are flexible but are important for site-specific binding. Low-temperature x-ray crystallography and codondirected saturation mutagenesis were used to study the role of this segment. All of the functional sequences have the form [X]1-[X]2-[Lys or Arg]3-[Lys]4-[Lys or Arg]5-[X]6. A high-resolution (1.8 angstrom) crystal structure shows that Lys3 and Lys4 each make multiple hydrogen bonds with guanines and that Lys5 interacts with the phosphate backbone. The symmetry of the complex breaks down near the center of the site, and these results suggest a revision in the traditional alignment of the six lambda operator sites.     

水稻白叶枯病菌hrp调节基因hrpXoo的克隆与序列分析

 用化学方法诱变水稻白叶枯病菌PXO99A 菌株 ,获得 6株hrp-突变体 ,此突变体除丧失在非寄主烟草上激发过敏反应和在感病寄主水稻上的致病能力外 ,有些缺乏激发烟草产生HR的信号物质 ,有些在胞内存在此信号物质 ,而不能泌至胞外。来自Xanthomonasoryzaepv .oryzaeJXOIII粘粒基因文库的hrp基因克隆pUHRX2 4 5 ,所携hrp基因片段大小为 36 .8kb。系列亚克隆 36 .8kbhrp基因片段及各亚克隆对hrp-突变体功能互补作用的结果显示 ,3.3kbSacI片段为最小酶切功能片段。序列测定和分析结果表明 ,3.3kbhrp片段含hrpX oo基因和含与热激蛋白 90家族有关的 2个开放阅读框hspORF1和hspORF2。HspORF1在蛋白质数据库中未发现序列蛋白。HspORF2与Hsp90 Xo的同源性达 99%。hrpXoo与黄单胞菌中已报道的hrpX基因有很高的同源性(90 %以上 )。在黄单胞菌中高度保守的编码α 螺旋 转 α 螺旋结构的 6 0 bp核苷酸序列 ,在交叉功能互补时是必需的。不含此结构的hrpXoo (1.1kb)片段 ,可使JXOIII的hrp-突变体在水稻上具致病性和在非寄主烟草上激发产生HR ,但不能使来自PXO99A 和RS10 5的hrp-突变体恢复在烟草上激发产生HR的功能。黄单胞菌中已知HrpX的同列比较显示 ,X .oryzae和X .campestris种间在 88、196和 2 4 7位点的氨基酸上有     

Increasing the multiplicity of ribosomal RNA genes in Drosophila melanogaster

In wild-type Drosophila melanogaster females there are about 250 ribosomal RNA genes in each nucleolus organizer region of the two X chromosomes. When this same nucleolus organizer region is present in flies in only a single dose, the number of ribosomal RNA genes increases to approximately 400. This increase is most easily explained by disproportionate replication of these genes.     

北方梭囊孔菌漆酶基因DNA片段的克隆与序列分析

根据报道的10几种真菌漆酶氨基酸序列的保守区域设计了一对简并引物,以北方梭囊孔菌总DNA为模板,通过PCR扩增到一个1 201 bp的基因片段.将片段序列在NCBI的BLAST软件上进行同源性搜寻,显示99个序列,全部为漆酶基因及其类似基因,因而认为克隆到的片段就是北方梭囊孔菌漆酶基因片段.经过与同源性最高的粗毛革孔菌（laccase lcc1）同一区段编码区序列进行ClustalW比较,其中有6个间隔区,确定为内含子区.去除内含子后,推导的氨基酸序列为286个残基.经与其他几种真菌漆酶的氨基酸序列进行ClustalW比对,同源性均在51%以上.