HLA-DR和DQ cDNA亚探针减少由限制片段长度多态性作DR分型的复杂性

在DNA水平上鉴定HLA-DR等位基因是一种适用于任何有核细胞的分型技术。DNA分型的主要问题是解释Southern印迹分析中杂交片段的复杂格局,这在杂合个体尤为困难。为此,我们从DRβ、DQβ和DQα全长cDNA探针建立了亚探针,以便减少杂交片段的数目,从而降低限制片段长度多态性(RFLP)的复杂性。我们发现内切酶PvuⅡ和DRβ3'端不翻译区亚探针,而对一些DR等位基因作出鉴定。这些简化了的杂交片段格局有利于对一些杂合个体作DNA分型。     

慢粒白血病中c-abl 癌基因的重排

作者采用α-³²P-dCTP标记的v-abl癌基因探针与7例慢粒白血病细胞DNA的PvuⅡ、Hind Ⅲ和Bg 1 Ⅱ酶切片段杂交。结果发现其中3例慢粒病例的Pvu Ⅱ和Hind Ⅲ酶切杂交图谱与正常对照相比有明显变化,且Pvu Ⅱ酶切位点有多处改变。表明在慢粒白血病中,Ph染色体重排亦可累及 c-abl癌基因内部,并可能有多次多点重排发生。     

Multicopy expression vector based on temperature-regulated lac repressor: expression of human immunodeficiency virus env gene in Escherichia coli

A new expression vector (pBB1) has been constructed for the regulated expression of genes in Escherichia coli. Based on the pUC plasmids, the pBB 1 carries lacIts allele of the lac repressor gene. This makes it possible to control expression of cloned genes by shifting the temperature from 30°C to 42°C. Thus the vector combines advantages of the pUC plasmids with convenient regulation by temperature. Expression of a fragment of HIV-1 env gene was achieved with the help of this vector and shown by enzyme-linked immunosorbent assay and Western-blot analysis.     

单眼视网膜摘除后鸽左右前脑基因的差异表达

利用消减抑制杂交（SSH）技术分析左眼视网膜摘除后成鸽左右前脑基因差异表达情况,对14个重线子经反Northern杂交去除假阳性,最终获得4个阳性重组子,对阳性重组子进行克隆和测序,其中PFB／SSH－8片段长226bp,它的138bp与人CaM I基因外显子有85％的同泊性,PFB／SSH－15片段长252bp,与nexin I alpha非表达序列有低的同源性,另外2个片段未发现有同源物。     

Detection of dehydrin-like proteins in embryos and endosperm of mature Euterpe edulis seeds

Summary. Euterpe edulis Martius, a tropical palm species characterized as highly recalcitrant, accumulated dehydrin proteins in both the endosperm and the embryo of the mature seed, as detected by Western blot analysis and immunogold electron microscopy. Three major bands at molecular masses of approximately 16, 18, and 24 kDa were identified in both samples analysed. Immunogold electron microscopy studies detected the presence of dehydrins in the embryo and endosperm. In both cases, dehydrins were immunolocalized in cytoplasm and chromatin. No labelling associated with either membranes or organelles was detected. It is known that dehydrins are produced as part of the developmental program of orthodox seeds and are also present in some recalcitrant seeds of temperate regions. The constitutive presence of dehydrins in embryos of extremely recalcitrant species of tropical origin has not been previously reported. Correspondence and reprints: Departamento de Biodiversidad y Biología Experimental, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Pabellón 2, Ciudad Universitaria, C1428EHA, Ciudad Autónoma de Buenos Aires, Argentina.     

RNA-蛋白质印迹筛选HBsAg转录后调节因子表达克隆

RNA结合蛋白是基因表达的重要调节因子.RNA-蛋白质印迹（Northwestern blot）是近年来国外建立的筛选这类因子的重要方法之一.应用这一方法从肝细胞株HepG2 cDNA文库中成功筛选到乙型肝炎病毒表面抗原转录后调节片段互相作用蛋白表达克隆.结果显示：该蛋白与探针结合特异性强,经三轮筛选后,100%克隆为阳性克隆;PCR和EcoRⅠ酶切初步鉴定, 编码该蛋白的cDNA长约1 kb.     

Characterization and site-directed mutagenesis of the putative novel acyl carrier protein Rv0033 and Rv1344 from Mycobacterium tuberculosis

Mycolic acids are generated in Mycobacterium tuberculosis as a result of the interaction of two fatty acid biosynthetic systems: type I fatty acid synthase (FAS) and type II fatty acid synthase. Acyl carrier protein (ACP) is a small, acidic protein in type II FAS systems. It plays a central role in mycolic acid biosynthesis by transferring the acyl groups from one enzyme to another for the completion of the fatty acid synthesis cycle. The nature of the proper recognition between ACPs and its many interactive proteins is not understood. Here, we report the over-expression, purification, and characterization of two putative ACPs: Rv0033 and Rv1344 in M. tuberculosis. In order to study the role of the conserved residues and the conformation of whole protein, some site-directed mutations of recombinant Acp1344 were made and the 3D structure of Acp1344 was modeled.     

Bacterial diversity in maize rhizospheres: conclusions on the use of genetic profiles based on PCR-amplified partial small subunit rRNA genes in ecological studies

A cultivation-independent approach based on polymerase chain reaction (PCR)-amplified partial small subunit rRNA genes and genetic profiling by single-strand conformation polymorphism (SSCP) was used to characterize the bacterial diversity inhabiting the rhizosphere of maize plants grown on an agricultural field. The community structures of two cultivars, a genetically engineered and a nonengineered variety, different herbicide regimes and soil tillage were compared with each other at two sampling dates. SSCP-profiles were generated with DNA from bacterial cell consortia with primers hybridizing to evolutionarily highly conserved rRNA gene regions. On silver-stained gels, each profile consisted of approx. 50 distinguishable bands. Similarity analyses of patterns recorded by digital image analyses could not detect any difference between cultivars or treatments that was greater than the variability between replicates. A total of 54 sequences recovered from different bands were identified and grouped into operational taxonomical units (OTUs). Surprisingly, only five of 40 OTUs contained sequences of both samplings. Three different bands from a profile were selected to test whether this small overlap was due to an incomplete recovery of sequences. From a faint band, two different OTUs were found when 12 clones were analysed, and from two strong bands 24 and 22 OTUs were detected from a total of 26 and 36 clones, respectively. The OTUs belonged to phylogenetically different groups of bacteria. Gene probes that were developed to target different bands of the profiles, however, indicated in Southern blot analyses that patterns between treatments, replicates and samplings, and even from two different growing seasons were highly conserved. Our study demonstrates that community profiles can consist of more sequences than detectable by staining and that gene probes in Southern blot can be a useful control to investigate the composition of microbial communities by genetic profiles.