Cloning Of Open Reading Frames Research Articles

A plasmid library of full-length zebrafish rab proteins for in vivo cell biology.

The zebrafish is an emerging model for highly sophisticated medium-throughput experiments such as genetic and chemical screens. However, studies of entire protein families within this context are often hampered by poor genetic resources such as clone libraries. Here we describe a complete collection of 76 full-length open reading frame clones for the zebrafish rab protein family. While the mouse genome contains 60 rab genes and the human genome 63, we find that 18 zebrafish rab genes have 2, and in the case of rab38, 3 paralogues. In contrast, we were unable to identify zebrafish orthologues of the mammalian Rab2b, Rab17 or Rab29. We make this resource available through the Addgene repository to facilitate cell biologic approaches using this model.

Evidence for a Dominant Negative Effect Conferred By the APOL1 G2 Sickle Cell Nephropathy Risk Allele in an in Vivo Model

BACKGROUND: Sickle cell disease (SCD) patients have a heterogeneous clinical course, and as patient survival has improved, end-organ damage has become an emergent clinical priority. End stage renal disease (ESRD), occurring in 5-18% of SCD patients, is a particular concern, because it is a major risk factor for early mortality (Platt et al. 1994). Detection of SCD nephropathy (SCDN) relies on relatively late-stage markers, namely proteinuria and reduced glomerular filtration rate (GFR). Consequently, at-risk SCD patients are not identified prior to end-organ damage. In non-SCD nephropathy, ESRD risk among African American individuals has been attributed to coding variants (termed G1 and G2) in apolipoprotein L1 (APOL1). The G1 allele consists of two nonsynonymous variants in perfect LD, rs73885319 and rs60910145 (encoding S384G and I384M), while the G2 variant consists of a six base pair deletion removing amino acids N388 and Y389 (~21% and ~13% allelic frequency in African Americans for G1 and G2, respectively). We demonstrated that these variants in APOL1 are strong predictors of risk for proteinuria in SCD (Ashley-Koch et al. 2011). Here, we use zebrafish as an in vivo model to both examine the role of apol1 in glomerular development and pronephric filtration and also to test the effects of APOL1 G1 and G2 expression in the developing kidney.METHODS: A morpholino (MO) was designed by Gene Tools, LLC (Philomath, OR) to target the translation initiation site of zebrafish apol1. APOL1 G1 and G2 allelic constructs were synthesized from a wild-type (WT) APOL1 human open reading frame clone (GenBank: BC112943) using site-directed mutagenesis (Stratagene, QuikChange II), subsequently transcribed (mMESSAGE mMACHINE®, Life Technologies) into capped mRNA and co-injected with apol1-MO into zebrafish embryos. To assay glomerular filtration, 70 kDa FITC-conjugated dextran was injected into the cardiac venous sinus of 48 hour post-fertilization embryos. The eye vasculature of individual fish was imaged at two, 12, and 36 hours after dextran injection. The average fluorescence intensity was measured across the eye, and changes in intensity relative to the 2-hour post-injection measurements were calculated for comparison. Electron microscopy sections of five days post-fertilization embryos were cut on a Leica-Reichert Ultracut E ultramicrotome, and semi-thin sections (1.0μm) were stained and examined on a Phillips CM12 electron microscope.RESULTS: As we showed previously (Anderson et al., ASH 2013), MO-induced suppression of apol1 in zebrafish embryos results in pericardial edema, glomerular filtration defects, and extensive podocyte loss. Importantly, complementation of apol1 morphants with WT human APOL1 mRNA rescues the observed kidney defects. However, we now show that neither APOL1 G1 nor G2 risk alleles ameliorate defects caused by apol1 suppression. Notably, injection of APOL1 G2 alone results in renal defects, as indicated by increased dextran clearance and the presence of microvillus protrusions in the urinary space. Injection of APOL1 G1 alone, however, does not induce noticeable kidney dysfunction. Furthermore, when APOL1 G2 injected embryos were titrated with increasing concentrations of human APOL1 WT mRNA, we observed a significant reduction of edema formation in developing embryos, suggesting a possible dominant-negative effect of the altered protein.CONCLUSIONS: Unlike the WT APOL1 mRNA, neither the APOL1 G1 or G2 risk alleles could rescue kidney dysfunction due to knockdown of apol1 in zebrafish embryos, suggesting that these SCDN risk alleles impact the normal function of APOL1 in the kidney. Furthermore, development of edema with concomitant defects in glomerular ultrastructure in zebrafish embryos injected with APOL1 G2 mRNA alone suggest that this allele may act in a dominant-negative manner to induce kidney defects. Interestingly, it has been shown that APOL1 may cause toxic renal effects through programmed cell death pathways leading to glomerulosclerosis (Wan et al. 2008). Thus, apol1 suppression could result in the dysregulation of autophagic pathways, causing podocyte malformation and thereby affecting the susceptibility of the pronephros to glomerular injury. In summary, these data provide essential insight into the biological mechanisms by which APOL1 variants confer disease risk in human SCDN and other nondiabetic nephropathies. DisclosuresNo relevant conflicts of interest to declare.

A Proteomic Strategy for Global Analysis of Plant Protein Complexes

Global analyses of protein complex assembly, composition, and location are needed to fully understand how cells coordinate diverse metabolic, mechanical, and developmental activities. The most common methods for proteome-wide analysis of protein complexes rely on affinity purification-mass spectrometry or yeast two-hybrid approaches. These methods are time consuming and are not suitable for many plant species that are refractory to transformation or genome-wide cloning of open reading frames. Here, we describe the proof of concept for a method allowing simultaneous global analysis of endogenous protein complexes that begins with intact leaves and combines chromatographic separation of extracts from subcellular fractions with quantitative label-free protein abundance profiling by liquid chromatography-coupled mass spectrometry. Applying this approach to the crude cytosolic fraction of Arabidopsis thaliana leaves using size exclusion chromatography, we identified hundreds of cytosolic proteins that appeared to exist as components of stable protein complexes. The reliability of the method was validated by protein immunoblot analysis and comparisons with published size exclusion chromatography data and the masses of known complexes. The method can be implemented with appropriate instrumentation, is applicable to any biological system, and has the potential to be further developed to characterize the composition of protein complexes and measure the dynamics of protein complex localization and assembly under different conditions.

A yeast one-hybrid and microfluidics-based pipeline to map mammalian gene regulatory networks.

The comprehensive mapping of gene promoters and enhancers has significantly improved our understanding of how the mammalian regulatory genome is organized. An important challenge is to elucidate how these regulatory elements contribute to gene expression by identifying their trans-regulatory inputs. Here, we present the generation of a mouse-specific transcription factor (TF) open-reading frame clone library and its implementation in yeast one-hybrid assays to enable large-scale protein-DNA interaction detection with mouse regulatory elements. Once specific interactions are identified, we then use a microfluidics-based method to validate and precisely map them within the respective DNA sequences. Using well-described regulatory elements as well as orphan enhancers, we show that this cross-platform pipeline characterizes known and uncovers many novel TF-DNA interactions. In addition, we provide evidence that several of these novel interactions are relevant in vivo and aid in elucidating the regulatory architecture of enhancers.

Efficient vectors for expression cloning of large numbers of PCR fragments in P. pastoris

The yeast vectors described, pYEV and pYEVB, were designed for parallel polymerase chain reaction (PCR) cloning of open reading frames (ORFs) and immediate protein expression in Pichia pastoris. The pYEV vector was used to clone PCR fragments obtained by using Taq or similar polymerase mixes, which leave an A-base overhang. The other vector pYEVB, with the same features for blunt-end ligation of PCR products, was developed to be complementary to pYEV. These two plasmids were linearized using the restriction enzymes BfuI and SchI, respectively. The purified PCR products, without any other treatments, were cloned into the linearized vectors, followed by selection on plates supplemented with X-gal. Only desired recombinants carrying the target gene in the correct orientation can give typical blue colonies. This screening technique is based on a lacO reconstruction strategy that can produce a full-length lacO to exhaust endogenous LacI and switch on the transcription of lacZ in the host. The recombinant plasmids extracted from the blue colonies can be linearized by SalI and transformed into P. pastoris for immediate expression. By using these two vectors, researchers could be saved from the tedious and time-consuming conventional cloning procedures.

High-Throughput Cloning of Open Reading Frames (ORFs): Assembling Large Sets of Expression Constructs

INTRODUCTIONNearly all methods for studying protein function begin with the expression of protein from cloned copies of the protein-coding sequences. Thus, obtaining a complete set of validated protein-coding clones is the first step toward establishing a functional proteomics platform for any organism of interest. Different protein functional studies demand different protein expression vectors; therefore, a flexible vector system should be used that enables the cloned sequences to be transferred rapidly to any vector. This can be achieved most efficiently by using vectors that employ recombinational cloning, a strategy that allows DNA fragments flanked by site-specific recombination sites to be moved from one vector to another in a single-step procedure, in frame and without mutation. There is now extensive experience with several such systems, including the Gateway system (Invitrogen) described here. These reactions are simple enough to allow high-throughput (HT) automation and are highly efficient. Once a "master" clone is created, the identical sequences can be transferred easily into all bacterial, mammalian, and viral vectors commonly used for protein analysis in vivo or in vitro. This protocol describes the process of transferring protein-coding sequences from a master vector into a protein expression (destination) vector for use in functional experiments.

A race through the maze of genomic evidence

A race through the maze of genomic evidence

Exploration of Human ORFeome: High-Throughput Preparation of ORF Clones and Efficient Characterization of Their Protein Products

In this study, we established new systematic protocols for the preparation of cDNA clones, conventionally termed open reading frame (ORF) clones, suitable for characterization of their gene products by adopting a restriction-enzyme-assisted cloning method using the Flexi® cloning system. The system has following advantages: (1) preparation of ORF clones and their transfer into other vectors can be achieved efficiently and at lower cost; (2) the system provides a seamless connection to the versatile HaloTag® labeling system, in which a single fusion tag can be used for various proteomic analyses; and (3) the resultant ORF clones show higher expression levels both in vitro and in vivo. With this system, we prepared ORF clones encoding 1929 human genes and characterized the HaloTag-fusion proteins of its subset that are expressed in vitro or in mammalian cells. Results thus obtained have demonstrated that our Flexi® ORF clones are efficient for the production of HaloTag-fusion proteins that can provide a new versatile set for a variety of functional analyses of human genes.

An efficient plasmid vector for expression cloning of large numbers of PCR fragments in Escherichia coli

The described plasmid pEamTA was designed for parallel polymerase chain reaction (PCR) cloning of open reading frames (ORFs) in Escherichia coli. It relies on the well-known TA-cloning principle, and the "T-vector" can be generated from a plasmid preparation by digestion with the restriction enzyme Eam1105I. The single 3'-T-overhangs of the vector fragment are positioned in a way that A-tailed PCR-products beginning with the start-ATG of an ORF end up in optimal position for expression from a strong tac-promoter when ligated in correct orientation. The orientation of the insert can be checked via a reconstituted NdeI site (catATG) present in correct clones. The protocol works regardless of internal restriction sites of the PCR fragment, a major advantage when cloning a number of fragments in parallel. It also does not require 5'-primer extensions and finally delivers an expression clone for the preparation of untagged protein in less than a week.

The Stardust Family Protein MPP7 Forms a Tripartite Complex with LIN7 and DLG1 That Regulates the Stability and Localization of DLG1 to Cell Junctions

MPP7, a previously uncharacterized member of the p55 Stardust family of membrane-associated guanylate kinase (MAGUK) proteins, was found in a tripartite complex with DLG1 and LIN7A or LIN7C. MPP7 dimerizes with all three LIN7 family members (LIN7A, -B, and -C) through interaction of the single L27 domain of LIN7 with the carboxyl-terminal L27 domain of MPP7, thereby stabilizing both proteins. The dimer of MPP7 with LIN7A or LIN7C associates with DLG1 through an interaction requiring the amino-terminal L27 domain of MPP7. The amino-terminal L27 domain of MPP7 is not sufficient for interaction with DLG1 but interacts efficiently only if MPP7 is in a complex with LIN7A or -C. Thus the specificity of interaction of DLG1 with the LIN7-MPP7 complex is determined by L27 interactions with both MPP7 and LIN7. The tripartite complex forms in a ratio of 1:1:1 and localizes to epithelial adherens junctions in a manner dependent upon MPP7. Expression of MPP7 stabilizes DLG1 in an insoluble compartment. Expression of MPP7 deleted of the PDZ or Src homology 3 domain redistributes MPP7, DLG1, and LIN7 out of adherens junctions and into the soluble cytoplasmic fraction without changing the localization of E-cadherin. Thus, the stability and localization of DLG1 to cell-cell junctions are complex functions determined by the expression and association of particular Stardust family members together with particular LIN7 family members.

Transcription factor families in Arabidopsis: major progress and outstanding issues for future research

Transcription factors (TFs) are a group of proteins that control cellular processes by regulating the expression of downstream target genes. Recent progress has been made in the cloning and characterization of Arabidopsis TFs on the genome scale, especially on the cloning of open reading frames (ORFs), sequence analysis and the expression profiling of different TF families. Huge difference in numbers of subfamily members were found for Arabidopsis MYB, C2H2 (Zn), C3H-type 1 (Zn), C3H-type 2 (Zn) TFs by independent research groups, mainly because of differences in bioinformatic search stringency. However, the Arabidopsis and rice genomes contain very different numbers of TFs in the WRKY, NAC, bZIP, MADS, ALFIN-like, GRAS and C2C2 (Zn)-dof families, indicating a possible divergence of biological functions from dicots to monocots. TFs have also been found to play key roles in the biosynthesis and signaling of plant hormones, in cell growth and differentiation, and in photomorphogenesis.

Simultaneous high‐throughput recombinational cloning of open reading frames in closed and open configurations

Comprehensive open reading frame (ORF) clone collections, ORFeomes, are key components of functional genomics projects. When recombinational cloning systems are used to capture ORFs in master clones, these DNA sequences can be easily transferred into a variety of expression plasmids, each designed for a specific assay. Depending on downstream applications, an ORF is cloned either with or without a stop codon at its original position, referred to as closed or open configuration, respectively. The former is preferred when the encoded protein is produced in its native form or with an amino-terminal tag; the latter is obligatory when the protein is produced as a fusion with a carboxyl-terminal tag. We developed a streamlined protocol for high-throughput, simultaneous cloning of both open and closed ORF entry clones with the Gateway recombinational cloning system. The protocol is straightforward to set up in large-scale ORF cloning projects, and is cost-effective, because the initial ORF amplification and the cloning in a pDONR vector are performed only once to obtain the two ORF configurations. We illustrated its implementation for the isolation and validation of 346 Arabidopsis ORF entry clones.

All systems go: launching cell simulation fueled by integrated experimental biology data

Biological simulation serves to unify the basic elements of systems biology, namely, model selection, experimentation and model refinement. To select biochemical models for simulation, metabolome analysis can be performed using capillary electrophoresis or liquid chromatography coupled with mass spectrometry. In this manner, selected models can be elaborated with temporal/spatial gene and protein expression data obtained from model organisms such as Escherichia coli. The E. coli single gene deletion mutant library (KO collection) and His-tag/GFP-fusion single open reading frame clone expression library (ASKA) are powerful resources for this task. The integration of parallel experimental datasets into dynamic simulation tools forms the remaining challenge for the systematic analysis and elucidation of biological networks and holds promise for biotechnological applications.

Preparation of a Set of Expression-Ready Clones of Mammalian Long cDNAs Encoding Large Proteins by the ORF Trap Cloning Method

Although we have so far identified and sequenced >2000 human long cDNAs, known as KIAA cDNAs, half of them have yet to be functionally annotated. Expression-ready cDNA clones derived from these genes, where the open reading frame (ORF) of the gene of interest is placed under the control of an appropriate promoter, are critical for functional characterization of these gene products. In this study, we attempted to systematically convert original cDNA clones to expression-ready forms for native and fusion proteins. For this purpose, we developed a new method for ORF cloning based on a homologous recombination in Escherichia coli to avoid laborious manipulations and artificial introduction of mutations in ORF. Using 1589 putative full-length ORFs (from 1002 KIAA genes, 119 human known genes and 468 mouse genes) with an average size of 2.8 kb, we successfully prepared expression plasmids for 1463 native proteins and for 1343 fusion proteins by this method. The resultant expression-ready clones were examined using an in vitro transcription/translation system followed by SDS-polyacrylamide gel electrophoresis and by transient expression of GFP-fusion proteins in human embryonic kidney (HEK) 293 cells. This set of expression-ready clones of long cDNAs encoding large proteins would open a new route to experimentally analyze their functions on a proteomic scale, since unavailability of expression-ready clones for mammalian large proteins has been a major obstacle to the functional analysis of these cDNAs.

High‐throughput protein localization in Arabidopsis using Agrobacterium‐mediated transient expression of GFP‐ORF fusions

We describe a streamlined and systematic method for cloning green fluorescent protein (GFP)-open reading frame (ORF) fusions and assessing their subcellular localization in Arabidopsis thaliana cells. The sequencing of the Arabidopsis genome has made it feasible to undertake genome-based approaches to determine the function of each protein and define its subcellular localization. This is an essential step towards full functional analysis. The approach described here allows the economical handling of hundreds of expressed plant proteins in a timely fashion. We have integrated recombinational cloning of full-length trimmed ORF clones (available from the SSP consortium) with high-efficiency transient transformation of Arabidopsis cell cultures by a hypervirulent strain of Agrobacterium. To demonstrate its utility, we have used a selection of trimmed ORFs, representing a variety of key cellular processes and have defined the localization patterns of 155 fusion proteins. These patterns have been classified into five main categories, including cytoplasmic, nuclear, nucleolar, organellar and endomembrane compartments. Several genes annotated in GenBank as unknown have been ascribed a protein localization pattern. We also demonstrate the application of flow cytometry to estimate the transformation efficiency and cell cycle phase of the GFP-positive cells. This approach can be extended to functional studies, including the precise cellular localization and the prediction of the role of unknown proteins, the confirmation of bioinformatic predictions and proteomic experiments, such as the determination of protein interactions in vivo, and therefore has numerous applications in the post-genomic analysis of protein function.

Many paths to many clones: a comparative look at high-throughput cloning methods.

The creation of genome-scale clone resources is a difficult and costly process, making it essential to maximize the efficiency of each step of clone creation. In this review, we compare the available commercial and open-source recombinational cloning methods with regard to their use in creating comprehensive open reading frame (ORF) clone collections with an emphasis on the properties requisite to use in a high-throughput setting. The most efficient strategy to the creation of ORF clone resources is to build a master clone collection that serves as a quality validated source for producing collections of expression clones. We examine the methods for recombinational cloning available for both the creation of master clones and their conversion into expression clones. Alternative approaches to creating clones involving mixing of cloning methods, including gap-repair cloning, are also explored.

Biochemical Investigations of Control of Replication Initiation of Plasmid R6K

The mechanistic basis of control of replication initiation of plasmid R6K was investigated by addressing the following questions. What are the biochemical attributes of mutations in the pi initiator protein that caused loss of negative control of initiation? Did the primary control involve only initiator protein-ori DNA interaction or did it also involve protein-protein interactions between pi and several host-encoded proteins? Mutations at two different regions of the pi-encoding sequence individually caused some loss of negative control as indicated by a relatively modest increase in copy number. However, combinations of the mutation P42L, which caused loss of DNA looping, with those located in the region between the residues 106 and 113 induced a robust enhancement of copy number. These mutant forms promoted higher levels of replication in vitro in a reconstituted system consisting of 22 purified proteins. The mutant forms of pi were susceptible to pronounced iteron-induced monomerization in comparison with the WT protein. As contrasted with the changes in DNA-protein interaction, we found no detectable differences in protein-protein interaction between wild type pi with DnaA, DnaB helicase, and DnaG primase on one hand and between the high copy mutant forms and the same host proteins on the other. The DnaG-pi interaction reported here is novel. Taken together, the results suggest that both loss of negative control due to iteron-induced monomerization of the initiator and enhanced iteron-initiator interaction appear to be the principal causes of enhanced copy number.

ORFDB: an information resource linking scientific content to a high-quality Open Reading Frame (ORF) collection.

The ORFDB (http://orf.invitrogen.com/) represents an ongoing effort at Invitrogen Corporation to integrate relevant scientific data with an evolving collection of human and mouse Open Reading Frame (ORF) clones (Ultimate ORF Clones). The ORFDB serves as a central data warehouse enabling researchers to search the ORF collection through its web portal ORFBrowser, allowing researchers to find the Ultimate ORF clones by blast, keyword, GenBank accession, gene symbol, clone ID, Unigene ID, LocusLink ID or through functional relationships by browsing the collection via the Gene Ontology (GO) Browser. As of October 2003, the ORFDB contains 6200 human and 2870 mouse Ultimate ORF clones. All Ultimate ORF clones have been fully sequenced with high quality, and are matched to public reference protein sequences. In addition, the cloned ORFs have been extensively annotated across six categories: Gene, ORF, Clone Format, Protein, SNP and Genomic links, with the information assembled in a format termed the ORFCard. The ORFCard represents an information repository that documents the sequence quality, alignment with respect to public protein sequences, and the latest publicly available information associated with each human and mouse gene represented in the collection.

Simultaneous cloning of open reading frames into several different expression vectors

Genomic sequencing has enabled the prediction of thousands of genes, most of which either cannot be assigned a function or can be only broadly categorized on the basis of sequence alone. High-throughput strategies for elucidating protein function are of high priority, and numerous approaches are being developed. Many of these approaches require the cloning of open reading frames (ORFs) into expression vectors that enable the encoded proteins to be tested for biological and biochemical activities. Typically, more than one type of vector must be employed, as different experiments require different conditions of protein production. Here we show that it is possible to simultaneously transfer a single ORF from a source vector to four target vectors using a commercially available in vitro recombination system. To test the approach, we constructed new vectors for expression of fusion proteins in yeast, including vectors for the LexA two-hybrid system. We show that individual ORFs can be efficiently transferred to four different vectors in a single in vitro reaction. The resulting expression plasmids can be separated using prototrophic markers specific to each vector. Using this system to produce multiple expression constructs simultaneously could greatly facilitate high-throughput subcloning and proteomic studies.

The Mycobacterium tuberculosis recA intein can be used in an ORFTRAP to select for open reading frames.

The DNA repair protein RecA of Mycobacterium tuberculosis contains an intein, a self-splicing protein element. We have employed this Mtu recA intein to create a selection system for successful intein splicing by inserting it into a kanamycin-resistance gene so that functional antibiotic resistance can only be restored upon protein splicing. We then proceeded to develop an ORFTRAP, i.e., a selection system for the cloning of open reading frames (ORFs). The ORFTRAP exploits the self-splicing properties of inteins (which depend on full-length in-frame translation of a precursor protein) by allowing protein splicing to occur when DNA fragments encoding ORFs are inserted into the Mtu recA intein, whereas DNA fragments containing non-ORFs are selected against. Regions of the Mtu recA intein that tolerate the insertion of additional amino acids were identified by Bgl II linker scanning mutagenesis, and a respective construct was chosen as the ORFTRAP. To test the maximum insert size that could be cloned into ORFTRAP, DNA fragments of increasing length from the Listeria monocytogenes hly gene as well as a genomic library of Haemophilus influenzae were inserted and it was found that the longest permissive inserts were 425 bp and 251 bp, respectively. The H. influenzae ORFTRAP library also demonstrated the strength (strong selection power) and weakness (insertion of very small fragments) of the system. Further modifications should make the ORFTRAP useful for protein expression, epitope mapping, and antigen screening.