芜菁花叶病毒（TUMV）核酸cDNA的合成与克隆

从接种芜菁花叶病毒后发病芥菜(Brassica Juneaen)中提取病毒,然后提取其核酸(TuMV-RNA)。以纯化的TuMV-RNA为模板,以Oligo(dT)_(12-18)及小牛胸腺DNA水解物为引物,合成双链cDNA,双链cDNA长度约500—4300bp。将双链cDNA补齐后,钝端连接到pUC19质粒的Smal位点,转化E.coli DH,获得500多个白色克隆。菌落原位杂交及酶切分析表明,重组质粒中的插入片段大多数为芜菁花叶病毒RNA的互补DNA,长度为500—4000bp。     

cDNA approaches to isolation of the mouse mutant weaver gene

The mouse autosomal recessive mutant gene weaver (wv) results in abnormalities in cerebellum, substantia nigra and testis. Although a subtracted cDNA library prepared by removing P31 (wv/wv) sequences from a P1 (wv/+) library should contain mainly nonrepetitive neonatal sequences, unfortunately, repetitive sequences still appear during screening. Two clones, one repetitive, the other not, are used to illustrate the problems encountered in attempting to isolate the weaver gene from a subtracted cDNA library.Special issue dedicated to Dr. Sidney Ochs.     

Isolation,characterization, and chromosomal mapping of a novel cDNA clone encoding human selenium binding protein

We have isolated the full-length human 56 kDa selenium binding protein (hSP56) cDNA clone, which is the human homolog of mouse 56 kDa selenium binding protein. The cDNA is 1,668 bp long and has an open reading frame encoding 472 amino acids. The calculated molecular weight is 52.25 kDa and the estimated isoelectric point is 6.13. Using Northern blot hybridization, we found that this 56 kDa selenium binding protein is expressed in mouse heart with an intermediate level between those found in liver/lung/kidney and intestine. We have also successfully expressed hSP56 in Escherichia coli using the expression vector-pAED4. The hSP56 gene is located at human chromosome 1q21–22. J. Cell. Biochem. 64:217–224. © 1997 Wiley-Liss, Inc.     

cDNA cloning and overexpression of acidic ribosomal phosphoprotein P1 gene (RPLP1) from the giant panda

RPLP1 is one of acidic ribosomal phosphoproteins encoded by RPLP1 gene, which plays an important role in the elongation step of protein synthesis. The cDNA of RPLP1 was cloned successfully for the first time from the Giant Panda (Ailuropoda melanoleuca) using RT-PCR technology, which was also sequenced, analyzed preliminarily and expressed in E.coli. The cDNA fragment cloned is 449bp in size, containing an open reading frame of 344bp encoding 114 amino acids. Alignment analysis indicated that the nucleotide sequence and the deduced amino acid sequence are highly conserved to other five species studied, including Homo sapiens, Mus musculus, Rattus norvegicus, Bos Taurus and Sus scrofa. The homologies for nucleotide sequences of Giant Panda PPLP1 to that of these species are 92.4%, 89.8%, 89.0%, 91.3% and 87.5%, while the homologies for amino acid sequences are 96.5%, 94.7%, 95.6%, 96.5% and 88.6%. Topology prediction showed there are three Casein kinase II phosphorylation sites and two N-myristoylation sites in the RPLP1 protein of the Giant Panda (Ailuropoda melanoleuca). The RPLP1 gene was overexpressed in E. coli and the result indicated that RPLP1 fusion with the N-terminally His-tagged form gave rise to the accumulation of an expected 18kDa polypeptide, which was in accordance with the predicted protein and could also be used to purify the protein and study its function.     

Characterization of size-fractionated cDNA libraries generated by the in vitro recombination-assisted method.

We here modified a previously reported method for the construction of cDNA libraries by employing an in vitro recombination reaction to make it more suitable for comprehensive cDNA analysis. For the evaluation of the modified method, sets of size-selected cDNA libraries of four different mouse tissues and human brain were constructed and characterized. Clustering analysis of the 3' end sequence data of the mouse cDNA libraries indicated that each of the size-fractionated libraries was complex enough for comprehensive cDNA analysis and that the occurrence rates of unidentified cDNAs varied considerably depending on their size and on the tissue source. In addition, the end sequence data of human brain cDNAs thus generated showed that this method decreased the occurrence rates of chimeric clones by more than fivefold compared to conventional ligation-assisted methods when the cDNAs were larger than 5 kb. To further evaluate this method, we entirely sequenced 13 human unidentified cDNAs, named KIAA1990-KIAA2002, and characterized them in terms of the predicted protein sequences and their expression profiles. Taking all these results together, we here conclude that this new method for the construction of size-fractionated cDNA libraries makes it possible to analyze cDNAs efficiently and comprehensively.     

长臂光敏生物素标记葡萄扇叶病毒外壳蛋白基因cDNA探针的制备及其在检测上的应用

由葡萄扇叶病毒（Ｇｒａｐｅｖｉｎｅｆａｎｌｅａｆｖｉｒｕｓ,ＧＦＬＶ）导致的葡萄扇叶病在世界各地葡萄园内均有发生,是最为普遍和严重的病毒病害之一,感病葡萄可减产２０％～８０％。近年来,许多国家开展了葡萄脱毒苗木的培育和推广工作,同时进行了葡萄扇叶病毒...     

Cloning and Characterization of the cDNA Encoding a Novel Brain-Specific 14-kDa Protein

A new acidic protein with a molecular weight of 14,000 was purified from rat brain, in which it was specifically expressed, and partially sequenced by protein sequencing. On the basis of results obtained from the amino acid sequences, mixed oligonucleotides were synthesized and used as probes to clone a cDNA from a rat brain cDNA library. The cloned cDNA provided the full-length sequence of the 14-kDa protein. Northern blot hybridization using total RNA from several tissues of the rat provided evidence that the 14-kDa protein was expressed specifically in rat brain. Transfection of this cDNA into mammalian cells resulted in expression of the 14-kDa protein. The amino acid sequence predicted from the cDNA of the rat brain 14-kDa protein contained 137 amino acid residues. A hydropathy profile revealed a hydrophobic domain (amino acids 60-80) flanked by highly hydrophilic stretches on both sides. Whereas the N-terminal region of the 14-kDa protein contained four repeating motifs, EKTKEGV, the C-terminal domain was rich in glutamic acid and proline. A computer search of the amino acid sequence of the 14-kDa protein indicated no homology to any other protein reported so far.     

大鼠FMR1同源基因cDNA的克隆、测序与选择剪接的初步分析

应用ＲＴ－ＰＣＲ方法,从新生大鼠脑组织总ＲＮＡ扩增大鼠ＦＭＲ１同源基因的ｃＤＮＡ片段,克降至ｐＵＣ１８质粒中进行序列分析．获得从终止密码子起共１６８１ｂｐ的编码序列,尚缺少约２００ｂｐ的５′序列．所克隆的这部分大鼠ＦＭＲ１ｃＤＮＡ,不含有对应于人ＦＭＲ１基因的外显子１２及外显子１７第一和第三剪接受点之间的序列,提示大鼠ＦＭＲ１基因也有选择剪接表达．同源性分析显示,大鼠ＦＭＲ１与小鼠ＦＭＲ１基因的同源性为９７．７％,与人ＦＭＲ１基因的同源性为９４．９％;与小鼠ＦＭＲＰ（ＦＭＲ１蛋白）的氨基酸序列同源性为９８．４％,与人ＦＭＲＰ的氨基酸序列同源性为９７．９％．以大鼠ＦＭＲ１ｃＤＮＡ片段为探针检测到大鼠不同组织中ＦＭＲ１基因的选择剪接表达．上述结果为以大鼠为动物模型深入研究ＦＭＲ１基因功能奠定了基础．     

Study on fibroin heavy chain of the silkwormBombyx mori by fluorescencein situ hybridization (FISH)

The sericulture industry plays a very important role in our national economy. Silkworm (Bombyx mori) is always regarded as a model animal and biological reactor. There have been detailed studies on the structure, expression and control and molecular evolution of silk genes. However, few, if any, reports are available on the localization of structural genes in silkworm by molecular cytogenetics. The present experiment has tentatively localized theFib-H gene at the distal end of the 25th linkage group, namely at the 25-0.0 position, and verified thatFib-H has only one locus, thus providing a temporary solution to the problem about its localization.     

Human cardiomyocytes express high level of Na+/glucose cotransporter 1 (SGLT1)

We have quantitatively measured gene expression for the sodium-dependent glucose cotransporters 1 and 2 (SGLT1 and SGLT2) in 23 human tissues using the method of real time PCR. As predicted, our results revealed that the expression of SGLT1 was very high in the small intestine (1.2E + 6 molecules/microg total RNA) relative to that in the kidney (3E + 4 molecules/microg total RNA). Surprisingly, we observed that the expression of SGLT1 in human heart was unexpectedly high (3.4E + 5 molecules/microg total RNA), approximately 10-fold higher than that observed in kidney tissue. DNA sequencing confirmed that the PCR amplified fragment was indeed the human SGLT1 gene. Moreover, in situ hybridization studies using a digoxigenin (DIG)-labeled antisense cRNA probe corresponding to human SGLT1 cDNA confirm that human cardiomyocytes express SGLT1 mRNA. In contrast, the expression of SGLT2 in human tissues appears to be ubiquitous, with levels ranging from 6.7E + 4 molecules/microg total RNA (in skeletal muscle) to 3.2E + 6 molecules/microg total RNA (in kidney), levels 10-100-fold higher than the expression of SGLT1 in the same tissues. Our finding that human cardiomyocytes express high levels of SGLT1 RNA suggests that SGLT1 may have a functional role in cardiac glucose transport. Since several SGLT inhibitors are currently in development as potential anti-diabetic agents, it may be important to assess the functional consequences of inhibition of SGLT1 in the heart.     

Construction of BAC-based physical map and analysis of chromosome rearrangement in Chinese hamster ovary cell lines

Chinese hamster ovary (CHO) cells have frequently been used in biotechnology for many years as a mammalian host cell platform for cloning and expressing genes of interest. A detailed physical chromosomal map of the CHO DG44 cell line was constructed by fluorescence in situ hybridization (FISH) imaging using randomly selected 303 BAC clones as hybridization probes (BAC-FISH). The two longest chromosomes were completely paired chromosomes; other chromosomes were partly deleted or rearranged. The end sequences of 624 BAC clones, including 287 mapped BAC clones, were analyzed and 1,119 informative BAC end sequences were obtained. Among 303 mapped BAC clones, 185 clones were used for BAC-FISH analysis of CHO K1 chromosomes and 94 clones for primary Chinese hamster lung cells. Based on this constructed physical map and end sequences, the chromosome rearrangements between CHO DG44, CHO K1, and primary Chinese hamster cells were investigated. Among 20 CHO chromosomes, eight were conserved without large rearrangement in CHO DG44, CHO K1, and primary Chinese hamster cells. This result suggested that these chromosomes were stable and essential in CHO cells and supposedly conserved in other CHO cell lines.     

Localization of the calcium release channel gene in cattle and horse by in situ hybridization: evidence of a conserved synteny with glucose phosphate isomerase

In situ hybridization techniques were used to localize regionally the calcium release channel (CRC) gene on cattle and horse chromosomes, using a porcine CRC cDNA probe. In cattle, the hybridization signal peaked on the 18q23-q26 bands and in horse on the 10pter region. Previous studies have shown that the glucose phosphate isomerase (GPI) gene localizes at the same site in both species, indicating that the two loci are syntenic. As CRC and GPI are syntenic in human, pig and mouse, the present results in cattle and horse represent another example of synteny conservation in the evolution of mammalian chromosomes.     

阿片受体样受体ORL1基因在大鼠脑内的表达

我们采用地主辛标记的寡聚核苷酸探针和原位杂产技术研究了新发现的阿片受体样受体ＯＲＬ１基因在正常大鼠脑仙的表达和分布。发现ＯＲＬ１基因在大鼠脑内广泛表达。尤其在大脑皮层、丘脑、下丘脑、杏仁核、海马、隔核、缰核、导水管周围灰质、中缝核群及蓝斑等区域。提示ＯＲＬ１除参与痛觉调制外,还可能参与脑内多项生理功能的调控。