A high-throughput screening of genes that encode proteins transported into the endoplasmic reticulum in mammalian cells

The compartments of eukaryotic cells maintain a distinct protein composition to perform a variety of specialized functions. We developed a new method for identifying the proteins that are transported to the endoplasmic reticulum (ER) in living mammalian cells. The principle is based on the reconstitution of two split fragments of enhanced green fluorescent protein (EGFP) by protein splicing with DnaE from Synechocystis PCC6803. Complementary DNA (cDNA) libraries fused to the N-terminal halves of DnaE and EGFP are introduced in mammalian cells with retroviruses. If an expressed protein is transported into the ER, the N-terminal half of EGFP meets its C-terminal half in the ER, and full-length EGFP is reconstituted by protein splicing. The fluorescent cells are isolated using fluorescence-activated cell sorting and the cDNAs are sequenced. The developed method was able to accurately identify cDNAs that encode proteins transported to the ER. We identified 27 novel proteins as the ER-targeting proteins. The present method overcomes the limitation of the previous GFP- or epitope-tagged methods, using which it was difficult to identify the ER-targeting proteins in a high-throughput manner.     

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.     

小鼠肝脏RNA编辑酶ADAR1 4种剪切体：克隆、表达及功能分析

RNA编辑是DNA转录为RNA后遗传信息发生改变的一种方式.A-to-IRNA编辑酶ADAR1(adenosinedeaminasethatactsonRNA1)具有将pre-mRNA中特定的腺嘌呤核苷转变为次黄嘌呤核苷的功能.通过RT-PCR技术从小鼠肝脏组织中克隆了小鼠A-to-IRNA编辑酶ADAR1的4种剪切体,采用荧光示踪技术研究其在细胞内定位,利用Bac-to-Bac杆状病毒表达系统构建了ADAR1重组杆状病毒并在sf9昆虫细胞内将其进行了表达,最后对表达产物进行了活性鉴定.结果发现,小鼠ADAR1在小鼠肝脏组织中主要以4种剪切方式存在,分别命名为ADAR1-La\Lb和ADAR1-Sa\Sb.这4种ADAR1剪切体在细胞内分布有着明显的区别,ADAR1-La\Lb主要分布于胞浆,而ADAR1-Sa\Sb主要分布于细胞核及核仁.Bac-to-Bac杆状病毒表达系统表达的4种ADAR1剪切体蛋白的双链RNA编辑活性明显不同,提示各个ADAR1剪切体的底物识别和特异性RNA编辑功能可能有所不同.ADAR1剪切体的克隆和表达以及它们在细胞内定位和编辑活性的差异的发现为进一步研究其结构和功能的关系及寻找它们的新底物奠定了基础.     

Screening of Rice Genes from the cDNA Catalog Using the Data Obtained by Protein Sequencing

The partial amino acid sequences of 121 rice proteins separated by two-dimensional gel electrophoresis (2D-PAGE), were determined for a protein sequence data file. In the Rice Genome Research Program (RGP), more than 20,000 cDNA clones randomly selected from rice cDNA libraries have been sequenced to construct a cDNA catalog. Complimentary DNAs encoding about 30% of proteins in the protein sequence data file could be identified in the catalog by computer search. It was deduced that 20,000–40,000 genes are present in the rice genome. Only half of about 20,000 cDNAs sequenced in the RGP, corresponding to 1/4–1/2 of genes present in the entire rice genome, should have unique sequences after considering gene redundancy. This is consistent with the fact that the cDNAs encoding about 30% of the sequenced proteins could be identified in the catalog. If the size of the cDNA catalog is enlarged further, cDNAs encoding all proteins separated by 2D-PAGE could be easily identified from the catalog by using the protein sequence data.     

Prediction of the coding sequences of mouse homologues of KIAA gene: IV. The complete nucleotide sequences of 500 mouse KIAA-homologous cDNAs identified by screening of terminal sequences of cDNA clones randomly sampled from size-fractionated libraries.

We have been conducting a mouse cDNA project to predict protein-coding sequences of mouse homologues of human KIAA and FLJ genes since 2001. As an extension of these projects, we herein present the entire sequences of 500 mKIAA cDNA clones and 4 novel cDNA clones that were incidentally identified during this project. We have isolated cDNA clones from the size-fractionated mouse cDNA libraries derived from 7 tissues and 3 types of cultured cells. The average size of the 504 cDNA sequences reached 4.3 kb and that of the deduced amino acid sequences from these cDNAs was 807 amino acid residues. We assigned the integrity of CDSs from the comparison with the corresponding human KIAA cDNA sequences. The comparison of mouse and human sequences revealed that two different human KIAA cDNAs are derived from single genes. Furthermore, 3 out of 4 proteins encoded in the novel cDNA clones showed moderate sequence similarity with human KIAA proteins, thus we could obtain new members of KIAA protein families through our mouse cDNA projects.     

Identification and characterization of three members of the human SR family of pre-mRNA splicing factors.

SR proteins have a characteristic C-terminal Ser/Arg-rich repeat (RS domain) of variable length and constitute a family of highly conserved nuclear phosphoproteins that can function as both essential and alternative pre-mRNA splicing factors. We have cloned a cDNA encoding a novel human SR protein designated SRp30c, which has an unusually short RS domain. We also cloned cDNAs encoding the human homologues of Drosophila SRp55/B52 and rat SRp40/HRS. Recombinant proteins expressed from these cDNAs are active in constitutive splicing, as shown by their ability to complement a HeLa cell S100 extract deficient in SR proteins. Additional cDNA clones reflect extensive alternative splicing of SRp40 and SRp55 pre-mRNAs. The predicted protein isoforms lack the C-terminal RS domain and might be involved in feedback regulatory loops. The ability of human SRp30c, SRp40 and SRp55 to modulate alternative splicing in vivo was compared with that of other SR proteins using a transient contransfection assay. The overexpression of individual SR proteins in HeLa cells affected the choice of alternative 5' splice sites of adenovirus E1A and/or human beta-thalassemia reporters. The resulting splicing patterns were characteristic for each SR protein. Consistent with the postulated importance of SR proteins in alternative splicing in vivo, we demonstrate complex changes in the levels of mRNAs encoding the above SR proteins upon T cell activation, concomitant with changes in the expression of alternatively spliced isoforms of CD44 and CD45.     

Alternative splicing and endoproteolytic processing generate tissue-specific forms of pituitary peptidylglycine alpha-amidating monooxygenase (PAM).

The pituitary is a rich source of peptidylglycine alpha-amidating monooxygenase (PAM). This bifunctional protein contains peptidylglycine alpha-hydroxylating monooxygenase (PHM) and peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL) catalytic domains necessary for the two-step formation of alpha-amidated peptides from their peptidylglycine precursors. In addition to the four forms of PAM mRNA identified previously, three novel forms of PAM mRNA were identified by examining anterior and neurointermediate pituitary cDNA libraries. None of the PAM cDNAs found in pituitary cDNA libraries contained exon A, the 315-nucleotide (nt) segment situated between the PHM and PAL domains and present in rPAM-1 but absent from rPAM-2. Although mRNAs of the rPAM-3a and -3b type encode bifunctional PAM precursors, the proteins differ significantly. rPAM-3b lacks a 54-nt segment encoding an 18-amino acid peptide predicted to occur in the cytoplasmic domain of this integral membrane protein; rPAM-3a lacks a 204-nt segment including the transmembrane domain and encodes a soluble protein. rPAM-5 is identical to rPAM-1 through nt 1217 in the PHM domain; alternative splicing generates a novel 3'-region encoding a COOH-terminal pentapeptide followed by 1.1 kb of 3'-untranslated region. The soluble rPAM-5 protein lacks PAL, transmembrane, and cytoplasmic domains. These three forms of PAM mRNA can be generated by alternative splicing. The major forms of PAM mRNA in both lobes of the pituitary are rPAM-3b and rPAM-2. Despite the fact that anterior and neurointermediate pituitary contain a similar distribution of forms of PAM mRNA, the distribution of PAM proteins in the two lobes of the pituitary is quite different. Although integral membrane proteins similar to rPAM-2 and rPAM-3b are major components of anterior pituitary granules, the PAM proteins in the neurointermediate lobe have undergone more extensive endoproteolytic processing, and a 75-kDa protein containing both PHM and PAL domains predominates. The bifunctional PAM precursor undergoes tissue-specific endoproteolytic cleavage reminiscent of the processing of prohormones.     

Two species of human CRK cDNA encode proteins with distinct biological activities.

Two distinct human CRK cDNAs, designated CRK-I and CRK-II, were isolated from human embryonic lung cells by polymerase chain reaction and by screening of a human placenta cDNA library, respectively. CRK-I differed from CRK-II in that it lacked a 170-nucleotide sequence, suggesting that CRK-I and CRK-II were the products of alternative splicing. The amino acid sequences deduced from these two cDNAs differed in the carboxyl termini and contained one SH2 and either one or two SH3 domains. RNAse protection analysis demonstrated both CRK-I and CRK-II mRNAs in various human cells. Three CRK proteins, of 42, 40, and 28 kDa, were identified in human embryonic lung cells by means of antibodies against the SH2 region and the SH3 region of the bacterially expressed CRK-I protein. Transient expression of CRK-I and CRK-II cDNAs in COS7 cells showed that the former encoded the 28-kDa protein and the latter encoded the 40- and 42-kDa proteins. All human cell lines so far examined expressed the 40-kDa protein; however, expression of the 28- and the 42-kDa proteins was variable. In a comparison of the biological activity of the two human CRK proteins, both proteins were stably expressed in rat 3Y1 cells. All cell lines expressing CRK-I protein showed altered morphology, proliferated in soft agar, and grew as massive tumors in nude mice. Although CRK-II-expressing cells showed a slight morphologic change, they did not make colonies in soft agar or grow in nude mice. These results demonstrate that the two species of human CRK cDNA encode proteins which differ in their biological activities.     

Construction and application of a yeast surface-displayed human cDNA library to identify post-translational modification-dependent protein-protein interactions

Although post-translational modifications such as phosphorylation mediate fundamental biological processes within the cell, relatively few methods exist that allow proteome-wide identification of proteins that interact with these modifications. We constructed a yeast surface-displayed human cDNA library and utilized it to identify protein fragments with affinity for phosphorylated peptides derived from the major tyrosine autophosphorylation sites of the epidermal growth factor receptor or focal adhesion kinase. We identified cDNAs encoding the Src homology 2 domains from adapter protein APS, phosphoinositide 3-kinase regulatory subunit 3, SH2B, and tensin, demonstrating the effectiveness of this approach. Our results suggest that large libraries of functional human protein fragments can be efficiently displayed on the yeast surface. In addition to the analysis of post-translational modifications, yeast surface-displayed human cDNA libraries have many potential applications, including identifying targets and defining potential cross-reactive proteins for small molecules or drugs.     

Transgenic silkworms produce recombinant human type III procollagen in cocoons

We describe the generation of transgenic silkworms that produce cocoons containing recombinant human collagen. A fusion cDNA was constructed encoding a protein that incorporated a human type III procollagen mini-chain with C-propeptide deleted, a fibroin light chain (L-chain), and an enhanced green fluorescent protein (EGFP). This cDNA was ligated downstream of the fibroin L-chain promoter and inserted into a piggyBac vector. Silkworm eggs were injected with the vectors, producing worms displaying EGFP fluorescence in their silk glands. The cocoons emitted EGFP fluorescence, indicating that the promoter and fibroin L-chain cDNAs directed the synthesized products to be secreted into cocoons. The presence of fusion proteins in cocoons was demonstrated by immunoblotting, collagenase-sensitivity tests, and amino acid sequencing. The fusion proteins from cocoons were purified to a single electrophoretic band. This study demonstrates the viability of transgenic silkworms as a tool for producing useful proteins in bulk.     

Automation of yeast two-hybrid screening

We have developed an automated format for screening yeast two-hybrid libraries for protein-protein interactions. The format consists of a liquid array in which pooled library subsets of yeast, expressing up to 1000 different cDNAs, are mated to a yeast strain of the opposite mating type, expressing a protein of interest. Interactors are detected by a liquid assay for beta-galacsidase following prototrophic selection. The method is demonstrated by the detection of interactions between two encoded yeast RNA polymerase subunits in simulated libraries of varied complexity. To demonstrate its utility for large scale screening of complex cDNA libraries, two nuclear receptor ligand-binding domains were screened through two cDNA libraries arrayed in pooled subsets. Screening these libraries yielded clones which had previously been identified in traditional yeast two hybrid screens, as well as several new putative interacting proteins. The formatting of the cDNA library into pooled subsets lends itself to functional subtraction of the promiscuous positive class of interactor from the library. Also, the liquid arrayed format enables electronic handling of the data derived from interaction screening, which, together with the automated handling of samples, should promote large-scale proteome analysis.     

The alpha 1 chain of type XIII collagen consists of three collagenous and four noncollagenous domains, and its primary transcript undergoes complex alternative splicing.

We have recently reported a characterization of cDNA clones that encode an apparently novel human collagen that undergoes alternative splicing. These cDNAs covered one-third of the corresponding 2.5-2.8-kilobase mRNAs. We have now determined the complete primary structure of the protein encoded by several overlapping cDNAs isolated from a human endothelial cell library. Since the deduced translation product of the cDNAs is different in structure from all other collagen types, we have given the collagen chain encoded by the cDNAs the designation alpha 1 (XIII). The deduced polypeptide consists of three collagenous domains and four noncollagenous domains, two of them separating the collagenous domains and two located at the N-terminal and C-terminal ends of the polypeptide. Cysteine residues are found in three of the noncollagenous domains and also in the extreme N-terminal collagenous domain. Surprisingly, comparison of the nucleotide sequences encoded by the overlapping cDNA clones demonstrates that there are several alpha 1 (XIII) collagen mRNAs in HT-1080 human fibrosarcoma cells and human endothelial cells which differ in coding potential. Nuclease S1 mapping experiments suggest that these different mRNAs arise through alternative splicing of the precursor RNA at five locations within the coding region. This property makes type XIII collagen unique among all the collagen types studied so far. Its polypeptide length, therefore, may vary between 614 and 526 amino acids, depending on what internal splicing has taken place.     

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.