Cloning and expression of two genes encoding auxin-binding proteins from tobacco

Two genes encoding the auxin-binding protein (ABP1) of tobacco (Nicotiana tabacum L.), both of which possess the characteristics of a luminal protein of the endoplasmic reticulum (ER), were isolated and sequenced. These genes were composed of at least five exons and four introns. The two coding exons showed 95% sequence homology and coded for two precursor proteins of 187 amino acid residues with molecular masses of 21 256 and 21 453 Da. The deduced amino acid sequences were 93% identical and both possessed an amino-terminal signal peptide, a hydrophilic mature protein region with two potential N-glycosylation sites and a carboxyl-terminal sorting signal, KDEL, for the ER. Restriction mapping of the cDNAs encoding tobacco ABP1, previously purified by amplification of tobacco cDNA libraries by polymerase chain reaction (PCR) using specific primers common to both genes, indicated that both genes were expressed, although one was expressed at a higher level than the other. Genomic Southern blot hybridization showed no other homologous genes except for these two in the tobacco genome. The apparent molecular mass of the mature form of tobacco ABP1 was revealed to be 25 kDa by SDS polyacrylamide gel electrophoresis using affinity-purified anti (tobacco ABP1) antibodies raised against a fusion protein with maltose-binding protein. Expression of the recombinant ABP1 gene in transgenic tobacco resulted in accumulation of the 25 kDa protein. A single point mutation of an amino acid residue at either of the two potential N-glycosylation sites resulted in a decrease in the apparent molecular mass and produced a 22 kDa protein. Mutations at both sites resulted in the formation of a 19.3 kDa protein, suggesting that tobacco ABP1 is glycosylated at two asparagine residues.     

碱蓬cDNA表达文库的构建

以盐生植物盐地碱蓬（Suaeda salsa)地上部分为材料,提取植物总RNA,纯化出mRNA,合成cDNA第一链,得到双链cDNA后,连接入植物表达载体,构建cDNA表达文库。该文库重组子约10^6。将该文库转化农杆菌GV3101,可直接用以转化拟南芥。     

Identification of a cDNA encoding 6-phosphogluconate dehydrogenase from a human heart cDNA library

A full-length cDNA clone for human 6-phosphogluconate dehydrogenase (PGD) was isolated from a human adult heart cDNA library. The clone encoded an open reading frame of 483 amino acids. When the amino acid sequences of human PGD and sheep PGD were aligned, 94.2% identity between these two proteins was found. Its calculated molecular weight is 53,149 daltons. The predicted isoelectric point is 6.85. When the secondary structure of human PGD was examined by the PROSIS software, 36% α-helix and 9% β-sheet were found.     

Generation of full-length cDNA libraries enriched for differentially expressed genes for functional genomics

Here, we describe the application of a RecA-based cloning technology to generate full-length cDNA libraries enriched for genes that are differentially expressed between tumor and normal tissue samples. First, we show that the RecA-based method can be used to enrich cDNA libraries for several target genes in a single reaction. Then, we demonstrate that this method can be extended to enrich a cDNA library for many full-length cDNA clones using fragments derived from a subtracted cDNA population. The results of these studies show that this RecA-mediated cloning technology can be used to convert subtracted cDNAs or a mixture of several cDNA fragments corresponding to differentially expressed genes into a full-length library in a single reaction. This procedure yields a population of expression-ready clones that can be used for further high-throughput functional screening.     

High-efficiency identification of genes by functional analysis from a retroviral cDNA expression library.

Retroviral gene transfer efficiently delivers genes of interest stably into target cells, and expression cDNA cloning has been shown to be highly successful. Considering these two advantages, we now report a method by which one can identify genes stimulating cell growth through functional analysis. The first step requires the construction of a retroviral cDNA expression library and the optimization of transfection of vector DNA into virus packaging cells. The second step involves the cocultivation of target cells with libraries of retrovirus-producing cells, resulting in the amplification of target cells transduced with a gene(s) stimulating cell growth. Under standardized conditions of transfection, we detected an average of 4,000 independent clones per dish, among which expression of a retroviral beta-galactosidase gene at an abundance of 0.2% could be detected. Next, we demonstrated the augmentation of the sensitivity of the assay by retroviral infection and functional analysis. We did this by cocultivating factor-dependent (FD) cells with dishes of GP/E cells transfected with plasmids containing various molar ratios of pN2-IL3 DNA and retroviral library cDNA and by determining the highest dilution of pN2-IL3 which still resulted in the conversion of FD cells to factor independence. The retroviral interleukin-3 gene at an abundance as low as 0.001% could be detected. Indeed, we were able to detect from FD cells the development of factor-independent colonies with different phenotypes after retroviral transfer of cDNAs from an immortalized hemopoietic stem cell line. Thus, the combination of a standardized high-efficiency DNA transfection and retrovirus-mediated gene transfer should facilitate the identification of genes capable of conferring to target FD cells a detectable new function or phenotype. By scaling up the size of the experiment realistically during screening, the assay can detect cDNA at an abundance of lower than 0.0001%.     

Differential expression of pathogen-responsive genes encoding two types of glycine-rich proteins in barley

Gene-specific probes (3' ends of cDNAs) were obtained from barley cDNAs encoding two types of glycine-rich proteins: HvGRP2, characterized by a cytokeratin-like and a cysteine-rich domain, and HvGRP3, whose main feature was an RNA-binding domain. Expression of genes Hvgrp2 and Hvgrp3, which are present at one (or two) copies per haploid genome, was ubiquitous and gene Hvgrp3 was under light/darkness modulation. Cold treatment increased Hvgrp2 and Hvgrp3 mRNA levels. Methyl jasmonate (10 M) switched off the two genes. Expression of Hvgrp2, but not that of Hvgrp3, was induced by ethylene treatment (100 ppm). Fungal pathogens Erysiphe graminis and Rhynchosporium secalis increased the mRNAs levels of the two genes, both in compatible and in incompatible interactions, while bacterial pathogens did not.     

Protein kinase activities in tonoplast and plasmalemma membranes from corn roots

Protein kinase and phosphatase activities were studied in plasmalemma and tonoplast membrane fractions from corn (Zea mays L.) roots in order to test the hypothesis that the tonoplast H⁺-ATPase is regulated by intrinsic protein phosphorylation (G Zocchi, SA Rogers, JB Hanson 1983 Plant Sci Lett 31: 215-221), and to facilitate future purification of kinase activities from these membranes. Kinase activity in the plasmalemma was about three-fold higher than in the tonoplast, and displayed Michaelis Menten-type behavior with a K_m value for MgATP²⁻ of about 50 micromolar. Both activities were optimal at 3 millimolar free Mg²⁺ and had pH optima at 6.6 and 7.0 for the plasmalemma and tonoplast, respectively. Kinase activities in both fractions were stimulated by 1 micromolar free Ca²⁺, but calmodulin had no stimulatory effect, and chlorpromazine was inhibitory only at high concentrations. The pattern of phosphopeptides on SDS polyacrylamide gel electrophoresis was similar in both fractions except for one band of 50 kilodaltons that was present only in the tonoplast. A partially purified H⁺-ATPase fraction was prepared from tonoplast membranes, incubated under conditions optimal for protein phosphorylation. The three polypeptides (of 67, 57, and 36 kilodaltons), enriched in this fraction, did not become phosphorylated, suggesting that this protein is not regulated by endogenous protein phosphorylation. Protein phosphatase activity was detected only in the plasmalemma fraction. These results indicate that a regulatory cycle of protein phosphorylation and dephosphorylation may operate in the plasmalemma. The activity in the tonoplast appears to originate from plasmalemma contamination.     

鸡胚成纤维细胞cDNA表达文库的构建

鸡胚成纤维细胞(CEF)是研究鸡传染性法氏囊病病毒(IBDV)的主要细胞材料,而构建CEF的cDNA表达文库是筛选IBDV在CEF中的细胞受体,研究细胞嗜性的基础平台。采用Gateway技术构建CEF的表达文库,避免使用限制性内切酶切割cDNA,能够解决常规方法构建cDNA文库的技术缺陷。该技术将CEF的mRNA分离纯化后,以5′端生物素标记的Oligo(dT)primer为引物反转录后连接Adapter,层析柱纯化,通过BP重组反应构建cDNA入门文库,其平均滴度为1.1×106cfu/mL,文库总容量为1.2×107cfu,平均插入片段为2243bp,重组率为100%。通过LR重组反应将入门文库转换为表达文库,经测定平均滴度为5×105cfu/mL,文库总容量为5.5×106cfu,平均插入片段为2411bp,重组率为100%。结果表明,所构建的文库具有较高的重组率和较大的库容量,可作为较高质量的文库来研究IBDV的相关基因,为研究病毒受体和病毒入侵途径,进一步了解IBDV的致病机理奠定了基础。     

Flow cytometric screening of cDNA expression libraries for fluorescent proteins

Fluorescence-activated cell sorting (FACS) was applied for quantitative screening of cDNA expression libraries in bacteria for rare fluorescent protein encoding cDNAs. Rare fluorescent cells, observed at a frequency of 1 in 200,000 bacteria in a cDNA expression library constructed from Astrangia lajollaensis, were detected, enriched, and purified by sorting, yielding three distinct green fluorescent proteins. Two of the isolated fluorescent proteins were found to be 2.5-fold brighter in whole cell fluorescence than the widely used and already optimized EGFP variant and possessed a novel cysteine-containing chromophore. FACS can possess significant advantages in the screening of cDNA libraries in bacteria, since desired genes may occur at low frequencies and possess unexpected properties. This strategy provides a high-throughput, quantitative approach for isolating fluorescent proteins from a more diverse range of organisms and should be extendable to proteins that are not intrinsically fluorescent with the use of available fluorescent indicators.     

Construction of a quinoa (Chenopodium quinoa Willd.) BAC library and its use in identifying genes encoding seed storage proteins

Quinoa (Chenopodium quinoa Willd.) is adapted to the harsh environments of the Andean Altiplano region. Its seeds have a well-balanced amino acid composition and exceptionally high protein content with respect to human nutrition. Quinoa grain is a staple in the diet of some of the most impoverished people in the world. The plant is an allotetraploid displaying disomic inheritance (2n=4x=36) with a di-haploid genome of 967 Mbp (megabase pair), or 2C=2.01 pg. We constructed two quinoa BAC libraries using BamHI (26,880 clones) and EcoRI (48,000 clones) restriction endonucleases. Cloned inserts in the BamHI library average 113 kb (kilobase) with approximately 2% of the clones lacking inserts, whereas cloned inserts in the EcoRI library average 130 kb and approximately 1% lack inserts. Three plastid genes used as probes of high-density arrayed blots of 73,728 BACs identified approximately 2.8% of the clones as containing plastid DNA inserts. We estimate that the combined quinoa libraries represent at least 9.0 di-haploid nuclear genome equivalents. An average of 12.2 positive clones per probe were identified with 13 quinoa single-copy ESTs as probes of the high-density arrayed blots, suggesting that the estimate of 9.0× coverage of the genome is conservative. Utility of the BAC libraries for gene identification was demonstrated by probing the library with a partial sequence of the 11S globulin seed storage protein gene and identifying multiple positive clones. The presence of the 11S globulin gene in four of the clones was verified by direct comparison with quinoa genomic DNA on a Southern blot. Besides serving as a useful tool for gene identification, the quinoa BAC libraries will be an important resource for physical mapping of the quinoa genome.     

Identification of novel proteins and phosphorylation sites in a tonoplast enriched membrane fraction of Arabidopsis thaliana

Plant vacuoles play essential roles in many physiological processes, particularly in mineral nutrition, turgor provision and cellular signalling. The vacuolar membrane, the tonoplast, contains many membrane transporters that are critical in the execution of these processes. However, although increasing knowledge is available about the identity of proteins involved in these processes very little is known about the regulation of tonoplast transporters. By studying the phosphoproteome of tonoplast-enriched membranes, we identified 66 phosphorylation sites on 58 membrane proteins. Amongst these, 31 sites were identified in 28 membrane transporters of various families including tonoplast anion transporters of the CLC family, potassium transporters of the KUP family, tonoplast sugar transporters and ABC transporters. In a number of cases, the detected sites were well conserved across isoforms of one family pointing to common mechanisms of regulation. In other cases, isoform-unique sites were present, suggesting regulatory mechanisms tailored to the function of individual proteins. These results provide the basis for future studies to elucidate the mechanistic regulation of tonoplast membrane transporters.     

Organization and expression of two Arabidopsis DREB2 genes encoding DRE-binding proteins involved in dehydration- and high-salinity-responsive gene expression

In plants, a cis-acting element, DRE/CRT, is involved in ABA-independent gene expression in response to dehydration and low-temperature stress. To understand signal transduction pathways from perception of the dehydration stress signal to gene expression, we characterized a gene family for DRE/CRT-binding proteins DREB2A and DREB2B in Arabidopsis thaliana. Northern analysis showed that both genes are induced by dehydration and high-salt stress. Organ-specific northern analysis with gene-specific probes showed that these genes are strongly induced in roots by high-salt stress and in stems and roots by dehydration stress. The DREB2A gene is located on chromosome 5, and DREB2B on chromosome 3. We screened an Arabidopsis genomic DNA library with cDNA fragments of DREB2A and DREB2B as probes, and isolated DNA fragments that contained 5-flanking regions of these genes. Sequence analysis showed that both genes are interrupted by a single intron at identical positions in their leader sequence. Several conserved sequences were found in the promoter regions of both genes. The -glucuronidase (GUS) reporter gene driven by the DREB2 promoters was induced by dehydration and high-salt stress in transgenic Arabidopsis plants.     

Preparation of a differentially expressed, full-length cDNA expression library by RecA-mediated triple-strand formation with subtractively enriched cDNA fragments.

We have developed a fast and general method to obtain an enriched, full-length cDNA expression library with subtractively enriched cDNA fragments. The procedure relies on RecA-mediated triple-helix formation of single-stranded cDNA fragments with a double-stranded cDNA plasmid library. The complexes were then captured from the solutions using the digoxigenin-antidigoxigenin paramagnetic beads followed by recovery of the enriched double-stranded cDNA expression library. We have observed a linear relation between the capture of full-length cDNAs in the library and the fold enrichment in the subtracted cDNA population.