Screening Of cDNA Expression Library Research Articles

Functional Screening of cDNA Expression Library for Novel Ctenophore Photoproteins.

The functional screening of cDNA libraries (or functional cloning) enables isolation of cDNA genes encoding novel proteins with unknown amino acid sequences. This approach is the only way to identify a protein sequence in the event of shortage of biological material for obtaining pure target protein in amounts sufficient to determine its primary structure, since sensitive functional test for a target protein is only required to successfully perform functional cloning. Commonly, bioluminescent proteins from representatives belonging to different taxa significantly differ in sequences due to independent origin of bioluminescent systems during evolution. Nonetheless, these proteins are frequently similar in functions and can use even the same substrate of bioluminescence reaction, allowing the use of the same functional test for screening. The cDNA genes encoding unknown light-emitting proteins can be identified during functional screening with high sensitivity, which is provided by modern light recording equipment making possible the detection of a very small amount of a target protein. Here, we present the protocols for isolation of full-size cDNA genes for the novel bioluminescent protein family of light-sensitive Ca2+-regulated photoproteins in the absence of any sequence information by functional screening of plasmid cDNA expression library. The protocols describe all the steps from gathering animals to isolation of individual E. coli colonies carrying full-size cDNA genes using photoprotein berovin from ctenophore Beroe abyssicola as an illustrative example.

CDNA expression library screening revealed novel functional genes involved in clear cell carcinogenesis of the ovary in vitro

In order to identify genes involved in the pathogenesis of clear cell carcinoma of the ovary (CCC), functional screening using a cDNA expression library was performed. We extracted mRNA from a CCC cell line (RMG-1), established a cDNA library using a retroviral vector, transfected that library into mouse NIH3T3 cells and sequenced the resultant foci. The tissue-type specific expression of isolated genes and their transforming activities were evaluated. Seven genes were isolated. Of these genes, the mRNA expression of SEC61B and DVL1 is significantly stronger in CCC than in other histological types (p < .05). Immunohistochemical staining reveals the stronger expression of SEC61B and C1ORF38 than normal ovarian tissues (p < .05). Focus formation is confirmed by the transfection of SEC61B, C1ORF38, and DVL1 into NIH3T3 cells. The present study identified novel genes including SEC61B, C1ORF38, and DVL1, involved in the pathogenesis of CCC. These genes may be additional therapeutic targets for CCC. Impact statement What is already known on this subject? Several important genetic abnormalities, including ARID1A and PIK3CA mutations, have been reported in ovarian clear cell carcinoma (CCC). What the results of this study add? SEC61B, C1ORF38, and DVL1 were newly detected as candidate genes involved in ovarian clear cell carcinogenesis. What the implications are of these findings for clinical practice and/or further research? Functional screening using a cDNA expression library may be a useful technique to identify functional genes for pathogenesis. The information obtained using this technique may provide new therapeutic targets of CCC.

Downstream components of the calmodulin signaling pathway in the rice salt stress response revealed by transcriptome profiling and target identification

BackgroundCalmodulin (CaM) is an important calcium sensor protein that transduces Ca2+ signals in plant stress signaling pathways. A previous study has revealed that transgenic rice over-expressing the calmodulin gene OsCam1–1 (LOC_Os03g20370) is more tolerant to salt stress than wild type. To elucidate the role of OsCam1–1 in the salt stress response mechanism, downstream components of the OsCam1–1-mediated response were identified and investigated by transcriptome profiling and target identification.ResultsTranscriptome profiling of transgenic ‘Khao Dawk Mali 105’ rice over-expressing OsCam1–1 and wild type rice showed that overexpression of OsCam1–1 widely affected the expression of genes involved in several cellular processes under salt stress, including signaling, hormone-mediated regulation, transcription, lipid metabolism, carbohydrate metabolism, secondary metabolism, photosynthesis, glycolysis, tricarboxylic acid (TCA) cycle and glyoxylate cycle. Under salt stress, the photosynthesis rate in the transgenic rice was slightly lower than in wild type, while sucrose and starch contents were higher, suggesting that energy and carbon metabolism were affected by OsCam1–1 overexpression. Additionally, four known and six novel CaM-interacting proteins were identified by cDNA expression library screening with the recombinant OsCaM1. GO terms enriched in their associated proteins that matched those of the differentially expressed genes affected by OsCam1–1 overexpression revealed various downstream cellular processes that could potentially be regulated by OsCaM1 through their actions.ConclusionsThe diverse cellular processes affected by OsCam1–1 overexpression and possessed by the identified CaM1-interacting proteins corroborate the notion that CaM signal transduction pathways compose a complex network of downstream components involved in several cellular processes. These findings suggest that under salt stress, CaM activity elevates metabolic enzymes involved in central energy pathways, which promote or at least maintain the production of energy under the limitation of photosynthesis.

Identification of immune protective genes of Eimeria maxima through cDNA expression library screening

BackgroundEimeria maxima is one of the most prevalent Eimeria species causing avian coccidiosis, and results in huge economic loss to the global poultry industry. Current control strategies, such as anti-coccidial medication and live vaccines have been limited because of their drawbacks. The third generation anticoccidial vaccines including the recombinant vaccines as well as DNA vaccines have been suggested as a promising alternative strategy. To date, only a few protective antigens of E. maxima have been reported. Hence, there is an urgent need to identify novel protective antigens of E. maxima for the development of neotype anticoccidial vaccines.MethodsWith the aim of identifying novel protective genes of E. maxima, a cDNA expression library of E. maxima sporozoites was constructed using Gateway technology. Subsequently, the cDNA expression library was divided into 15 sub-libraries for cDNA expression library immunization (cDELI) using parasite challenged model in chickens. Protective sub-libraries were selected for the next round of screening until individual protective clones were obtained, which were further sequenced and analyzed.ResultsAdopting the Gateway technology, a high-quality entry library was constructed, containing 9.2 × 106 clones with an average inserted fragments length of 1.63 kb. The expression library capacity was 2.32 × 107 colony-forming units (cfu) with an average inserted fragments length of 1.64 Kb. The expression library was screened using parasite challenged model in chickens. The screening yielded 6 immune protective genes including four novel protective genes of EmJS-1, EmRP, EmHP-1 and EmHP-2, and two known protective genes of EmSAG and EmCKRS. EmJS-1 is the selR domain-containing protein of E. maxima whose function is unknown. EmHP-1 and EmHP-2 are the hypothetical proteins of E. maxima. EmRP and EmSAG are rhomboid-like protein and surface antigen glycoproteins of E. maxima respectively, and involved in invasion of the parasite.ConclusionsOur results provide a cDNA expression library for further screening of T cell stimulating or inhibiting antigens of E. maxima. Moreover, our results provide six candidate protective antigens for developing new vaccines against E. maxima.

Functional Identification and Characterization of Genes Cloned from Halophyte Seashore Paspalum Conferring Salinity and Cadmium Tolerance.

Salinity-affected and heavy metal-contaminated soils limit the growth of glycophytic plants. Identifying genes responsible for superior tolerance to salinity and heavy metals in halophytes has great potential for use in developing salinity- and Cd-tolerant glycophytes. The objective of this study was to identify salinity- and Cd-tolerance related genes in seashore paspalum (Paspalum vaginatum), a halophytic perennial grass species, using yeast cDNA expression library screening method. Based on the Gateway-compatible vector system, a high-quality entry library was constructed, which contained 9.9 × 106 clones with an average inserted fragment length of 1.48 kb representing a 100% full-length rate. The yeast expression libraries were screened in a salinity-sensitive and a Cd-sensitive yeast mutant. The screening yielded 32 salinity-tolerant clones harboring 18 salinity-tolerance genes and 20 Cd-tolerant clones, including five Cd-tolerance genes. qPCR analysis confirmed that most of the 18 salinity-tolerance and five Cd-tolerance genes were up-regulated at the transcript level in response to salinity or Cd stress in seashore paspalum. Functional analysis indicated that salinity-tolerance genes from seashore paspalum could be involved mainly in photosynthetic metabolism, antioxidant systems, protein modification, iron transport, vesicle traffic, and phospholipid biosynthesis. Cd-tolerance genes could be associated with regulating pathways that are involved in phytochelatin synthesis, HSFA4-related stress protection, CYP450 complex, and sugar metabolism. The 18 salinity-tolerance genes and five Cd-tolerance genes could be potentially used as candidate genes for genetic modification of glycophytic grass species to improve salinity and Cd tolerance and for further analysis of molecular mechanisms regulating salinity and Cd tolerance.

Identification of RNA-binding Proteins by RNA Ligand-based cDNA Expression Library Screening

We previously reported when a portion of the Requiem (REQ/DPF2) messenger ribonucleic acid (mRNA) 3’ untranslated region (3’UTR), referred to as G8, was overexpressed in K562 cells, β-globin expression was induced, suggesting that the 3’UTR of REQ mRNA plays a physiological role (Kim et al., 2014). To identify trans-acting factors that bind to the REQ 3’UTR, we describe the RNA ligand based cDNA expression library screening method. This protocol could be adapted to detect specific RNA-protein interactions. Following this method, we identified six positive clones in the initial round of screening and four pure clones after sib-screening. This protocol was originally published in Kim et al. (2014).

A Bemisia tabaci midgut protein interacts with begomoviruses and plays a role in virus transmission.

Begomoviruses are a major group of plant viruses, transmitted exclusively by Bemisia tabaci (Gennadius) in a persistent circulative non-propagative manner. The information regarding molecular and cellular basis underlying Begomovirus - whitefly interaction is very scarce. Evidences have suggested that the insect gut possesses some crucial protein receptors that allow specific entry of virus into the insect haemolymph. We have performed yeast two hybrid gut cDNA expression library screening against coat protein of Tomato leaf curl New Delhi virus (ToLCV) and Cotton leaf curl Rajasthan virus (CLCuV) as bait. Midgut protein (MGP) was the common protein found interacting with both ToLCV and CLCuV. MGP was localized in whole mount B. tabaci as well as in dissected guts through confocal microscopy. Pull down and dot blot assays confirmed in vitro interaction between ToLCV/CLCuV coat protein and MGP. Immunolocalization analysis also showed colocalization of ToLCV/CLCuV particles and MGP within insect's gut. Finally, anti-MGP antibody fed B. tabaci, exhibited 70% reduction in ToLCV transmission, suggesting a supportive role for MGP in virus transmission.

RNPs and autoimmunity: 20 years later.

RNPs and autoimmunity: 20 years later.

Angiogenic cytokines are antibody targets during graft-versus-leukemia reactions.

The graft-versus-leukemia (GVL) reaction is an important example of immune-mediated tumor destruction. A coordinated humoral and cellular response accomplishes leukemia cell killing, but the specific targets remain largely uncharacterized. To learn more about the antigens that elicit antibodies during GVL reactions, we analyzed patients with advanced myelodysplasia (MDS) and acute myelogenous leukemia (AML) who received an autologous, granulocyte-macrophage colony-stimulating factor (GM-CSF)-secreting tumor cell vaccine early after allogeneic hematopoietic stem cell transplantation (HSCT). A combination of tumor-derived cDNA expression library screening, protein microarrays, and antigen-specific ELISAs were used to characterize sera obtained longitudinally from 15 patients with AML/MDS who were vaccinated early after allogeneic HSCT. A broad, therapy-induced antibody response was uncovered, which primarily targeted intracellular proteins that function in growth, transcription/translation, metabolism, and homeostasis. Unexpectedly, antibodies were also elicited against eight secreted angiogenic cytokines that play critical roles in leukemogenesis. Antibodies to the angiogenic cytokines were evident early after therapy, and in some patients manifested a diversification in reactivity over time. Patients that developed antibodies to multiple angiogenic cytokines showed prolonged remission and survival. These results reveal a potent humoral response during GVL reactions induced with vaccination early after allogeneic HSCT and raise the possibility that antibodies, in conjunction with natural killer cells and T lymphocytes, may contribute to immune-mediated control of myeloid leukemias.

Systematic mining of salt-tolerant genes in halophyte-Zoysia matrella through cDNA expression library screening

Though a large number of salt-tolerant genes were identified from Glycophyte in previous study, genes involved in salt-tolerance of halophyte were scarcely studied. In this report, an important halophyte turfgrass, Zoysia matrella, was used for systematic excavation of salt-tolerant genes using full-length cDNA expression library in yeast. Adopting the Gateway-compatible vector system, a high quality entry library was constructed, containing 3 × 106 clones with an average inserted fragments length of 1.64 kb representing a 100% full-length rate. The yeast expression library was screened in a salt-sensitive yeast mutant. The screening yielded dozens of salt-tolerant clones harboring 16 candidate salt-tolerant genes. Under salt-stress condition, these 16 genes exhibited different transcription levels. According to the results, we concluded that the salt-tolerance of Z. matrella might result from known genes involved in ion regulation, osmotic adjustment, as well as unknown pathway associated with protein folding and modification, RNA metabolism, and mitochondrial membrane translocase, etc. In addition, these results shall provide new insight for the future researches with respect to salt-tolerance.

'Sex' in the cancer cell.

'Sex' in the cancer cell.

Antibody Repertoire in Paraneoplastic Cerebellar Degeneration and Small Cell Lung Cancer

The goal of this study is to determine whether patients with paraneoplastic cerebellar degeneration (PCD) and small-cell lung cancer (SCLC) have a specific repertoire of antibodies, if SOX1 antibodies (SOX1-ab) can predict the presence of SCLC, and if antibodies to cell surface antigens occur in this syndrome. Antibody analysis was done using immunohistochemistry on rat brain, immunoblot with recombinant antigens, screening of cDNA expression libraries, and immunolabeling of live neurons in 39 patients with PCD and SCLC. VGCC-ab were measured by RIA, and SOX1-ab, Hu-ab, and ZIC4-ab by immunoblot. Lambert-Eaton myastenic syndrome (LEMS) was present in 10 of 23 patients with electrophysiological studies. At least one antibody was detected in 72% of patients. The individual frequencies were: 49% SOX1-ab, 44% VGCC-ab, 31% Hu-ab, and 13% ZIC4-ab. SOX1-ab occurred in 76% of patients with VGCC-ab and 27% of those without VGCC-ab (p = 0.0036). SOX1-ab were not found in 39 patients with sporadic late-onset cerebellar ataxia, 23 with cerebellar ataxia and glutamic acid decarboxylase antibodies, and 73 with PCD and cancer types other than SCLC (31 without onconeural antibodies, 25 with Yo-ab , 17 with Tr-ab). Five patients (13%) had antibodies against unknown neuronal cell surface antigens but none of them improved with immunotherapy. One serum immunoreacted against the axon initial segment of neurons and another serum against ELKS1, a protein highly expressed in the cerebellum that interacts with the beta4-subunit of the VGCC. In conclusion, 72% of patients with PCD and SCLC had one or more antibodies that indicate the presence of this tumor. In these patients, VGCC-ab and SOX1-ab occur tightly associated. SOX1-ab are predictors of SCLC in ataxia patients with a specificity of 100% and sensitivity of 49%. Unlike limbic encephalitis with SCLC, antibodies to cell surface antigens other than VGCC-ab, are infrequent and do not predict response to treatment.

Abstract 2742: Identification of tumor antigens in neu transgenic mice may provide biomarkers useful for the early detection and diagnosis of human breast cancer

Abstract Early detection of breast cancer is crucial for better prognosis and successful treatment of the patients. Development of novel methods for screening for breast cancer is needed for early detection and diagnosis. The human immune system responds to tumor-specific antigens in the pre-malignant stage of breast cancer and produces specific antibodies, which can be detected as potential breast cancer biomarkers. Due to the limited availability of well-annotated pre-cancer and post-cancer sera from human subjects, we performed serial serum collection in a neu transgenic mouse model from the age of 10 weeks to the terminal stage of disease. Using a technique called serological screening of cDNA expression library (SEREX), a high-throughput method to rapidly screen recombinant proteins expressed in a bacteriophage-based cDNA library, we have previously identified tumor antigens in the late stage of neu breast cancers. The mouse tumors have similar immunogenicity as their human counterparts due to the fact that some of these antigens are also immunogenic in human. Indeed, we have found that one mouse antigen called gelsolin, shares 93% homology with human gelsolin and has the highest response to human serum antibody among other antigens. In an ELISA analysis using the serum samples from 50 breast cancer cases and 50 normal donors, the area-under-curve (AUC) value of the receiver-operating-characteristic (ROC) curve of gelsolin ELISA detects more than 70% of cancer patients. Using the SEREX technique, we sought to further identify the early-stage serum antibody biomarkers by comparing the pre-cancer, early stage, and late stage sera from the same mouse. We have identified three early-stage biomarkers, A20/TNFaip3, Rpl5, and Pdhx, in neu mice. In summary, we conclude that identification of tumor antigens in the neu transgenic animal model is useful for the discovery of serum antibody biomarkers for early detection and diagnosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2742.

Inhibition of HIV-1 replication by eIF3f

Viruses often use host machinery in unusual ways to execute different steps during their replication. To identify host factors critical for virus replication, we screened cDNA expression libraries for genes or gene fragments that could interfere with HIV-1 vector transduction. The DNA clone that most potently inhibited HIV-1 expression encoded the N-terminal 91 aa of the eukaryotic initiation factor 3 subunit f (N91-eIF3f). Overexpression of N91-eIF3f or full-length eIF3f drastically restricted HIV-1 replication by reducing nuclear and cytoplasmic viral mRNA levels. N91-eIF3f and eIF3f specifically targeted the 3' long terminal repeat (3'LTR) region in the viral mRNA. We show that the 3' end cleavage of HIV-1 mRNA precursors is specifically reduced in N91-eIF3f expressing cells. Our results suggest a role of eIF3f in mRNA maturation and that it can specifically interfere with the 3' end processing of HIV-1 mRNAs.

Transmembrane Protein 18 Enhances the Tropism of Neural Stem Cells for Glioma Cells

The failure of current glioma therapies is mainly due to the ability of the tumor cells to invade extensively the surrounding healthy brain tissue, hence escaping localized treatments. Neural stem cells (NSC) are able to home in on tumor foci at sites distant from the main tumor mass, possibly enabling treatment of scattered glioma clusters. To make the strategy more effective, we performed a cDNA expression library screening to identify the candidate genes that once overexpressed would enhance the tropism of NSCs for gliomas. Here, we show that a previously unannotated gene, the one encoding transmembrane protein 18 (TMEM18), is one such gene. Overexpression of TMEM18 was seen in the current study to provide NSCs and neural precursors an increased migration capacity toward glioblastoma cells in vitro and in the rat brain. Functional inactivation of the TMEM18 gene resulted in almost complete loss of the migration activity of these cells. Thus, TMEM18 is a novel cell migration modulator. Overexpression of this protein could be favorably used in NSC-based glioma therapy.

CDNA expression library screening and identification of two novel antigens: Ubiquitin and receptor for activated C kinase (RACK) homologue, of the fish parasite Trypanosoma carassii

Trypanosoma carassii is a kinetoplastid parasite infecting cyprinid fish with a high prevalence in nature. Antibodies have been shown to play a protective role in the immune response against this parasite in common carp, Cyprinus carpio. To identify immunogenic and putative protective T. carassii antigens we constructed a λTriplEx2 expression library of the parasite and screened this with pooled carp immune serum collected 6 weeks post-infection. Screening of the library not only revealed ribosomal proteins but identified ubiquitin and a homologue of the receptor for activated C kinase (RACK) as immunogenic proteins. Equivalents of all these proteins have been identified as immunogenic in expression library screenings of other Trypanosomatida, suggesting an evolutionary conservation of their immunogenicity. The possibility that ubiquitin and/or the homologue of RACK could represent protective antigens and be targets for the design of novel therapies is discussed.

P83 Systematic validation of novel breast cancer progression-associated biomarkers via highthroughput antibody generation and application of tissue microarray technology: an initial report

A plethora of promising breast cancer-associated autoantigens have been cloned by immunoscreening cDNA expression libraries with breast cancer sera or identified using proteomics, yet no biomarkers, whether individual autoantigens or panels of antigens developed using antibody-based methods have been validated and incorporated to routine oncologic practice for the early diagnosis of breast cancer. Recently, the addition of genomics, proteomics and high throughput technology to traditional immunological techniques has revived the interest in this field, and some of the most promising breast cancer autoantigens are in the process of being validated prospectively in large cohorts of patients with breast cancer. In addition, some of the identified breast cancer-associated autoantigens are recognized by T-cells and may prove to have a role in the treatment of breast cancer in the future. Autoantibodies found in breast cancer patient sera provide important clues about their significance. The discovery of breast cancer-associated antigens has provocative implications beyond the quest for novel diagnostic biomarkers, because autoantibodies target molecules involved in signal transduction, cell cycle regulation, cell proliferation and apoptosis, all of them key processes in carcinogenesis. Molecular components of the DNA double-strand break repair machinery as well as several members of the rapamycin-sensitive pathway elicit an autoantibody response in breast cancer. Data obtained by screening cDNA expression libraries of breast cancer antigens with autoantibodies present in breast cancer sera suggest that autoantibodies in cancer sera may be linked to the process of apoptosis. The studies reviewed here, clearly demonstrate the participation of autoimmunity in breast cancer to an extent previously unsuspected, which may have broad implications for the discovery of molecular targets for drug therapy and cancer biomarkers in general.

Antitumor effects of vaccination with dendritic cells transfected with modified receptor for hyaluronan-mediated motility mRNA in a mouse glioma model

The receptor for hyaluronan-mediated motility (RHAMM) is frequently overexpressed in brain tumors and was recently identified as an immunogenic antigen by using serological screening of cDNA expression libraries. In this study, which was conducted using a mouse glioma model, the authors tested the hypothesis that vaccination with dendritic cells transfected with RHAMM mRNA induces strong immunological antitumor effects. The authors constructed a plasmid for transduction of the mRNAs transcribed in vitro into dendritic cells, which were then used to transport the intracellular protein RHAMM efficiently into major histocompatibility complex class II compartments by adding a late endosomal-lysosomal sorting signal to the RHAMM gene. The dendritic cells transfected with this RHAMM mRNA were injected intraperitoneally into the mouse glioma model 3 and 10 days after tumor cell implantation. The antitumor effects of the vaccine were estimated by the survival rate, histological analysis, and immunohistochemical findings for immune cells. Mice in the group treated by vaccination therapy with dendritic cells transfected with RHAMM mRNA survived significantly longer than those in the control groups. Immunohistochemical analysis revealed that greater numbers of T lymphocytes containing T cells activated by CD4+, CD8+, and CD25+ were found in the group vaccinated with dendritic cells transfected with RHAMM mRNA. These results demonstrate the therapeutic potential of vaccination with dendritic cells transfected with RHAMM mRNA for the treatment of malignant glioma.

Identification of Desmoglein as a Cadherin and Analysis of Desmoglein Domain Structure

In the 1980s and early 1990s, the ability to clone complementary DNA (cDNA) resulted in great advances in cell biology, including our understanding of cell adhesion. Cloning the cDNA encoding desmosomal molecules, and the determination of their deduced amino-acid sequences, provided novel insights into their structure and function. It had been shown by various biochemical and immunochemical techniques that desmosomal proteins consist of plaque proteins inside the cell and transmembrane glycoproteins. For desmosomal transmembrane glycoproteins, Koch et al. (1990) first isolated and characterized the cDNA encoding desmoglein in 1990. Using monoclonal antibodies against desmoglein, they screened cDNA expression libraries constructed from bovine muzzle epithelial mRNA. The major scientific advance from this work was that the deduced amino-acid sequences of their isolated clones showed high homology to cadherins, which had already been shown to be calciumdependent cell adhesion molecules. Thus, they identified desmoglein as a member of the cadherin supergene family of cell adhesion molecules. Similar to classic cadherins such as Eand N-cadherins, they, and subsequently others, showed that desmoglein is a type I transmembrane protein with an amino-terminal extracellular domain, a single transmembrane spanning region, and a carboxy-terminal cytoplasmic domain (Figure 1). The extracellular domain was shown to contain four subdomain repeating units that have homology to similar extracellular subdomains in cadherins and that, similar to those units, contained putative calcium-binding sites. The cytoplasmic domain of desmoglein, although it contained a subdomain (termed the intracellular cadherin segment or ICS) that was homologous to a cytoplasmic subdomain of cadherin, significantly differed in that it was longer than that of cadherins. In addition to the ICS subdomain, desmoglein contained a proline-rich linker (IPL) and a terminal repeating unit domain (RUD). Although the ICS domain has been shown to bind various intracellular molecules, such as plakoglobin, the function of the additional subdomains of desmoglein remains unclear. Progress, by cDNA cloning, was also made in characterizing the other major transmembrane molecule of the desmosome, desmocollin (Collins et al., 1991). cDNA cloning of desmocollin indicated that it also belonged to the cadherin family. The unique aspect of desmocollin was that the cytoplasmic domain contained a longer “a” form and a shorter “b” form, produced by alternative splicing of mRNA. To date, four isoforms of desmoglein (Dsg1–4) and three isoforms of desmocollin (Dsc1–3) have been identified, each arising from a different gene. The genes are clustered on the q arm of chromosome 18 for humans (Kljuic et al., 2003). Because of their homologies to classical cadherins, these glycoproteins are now termed “desmosomal cadherins”. The cDNA cloning of desmogleins and, subsequently, desmocollins,

Feedback loops in intracellular signal processing and their potential for identifying novel signalling proteins

Mapping of intracellular signalling networks has attracted a lot of attention during recent years. Approaches which have been used to characterise functional and physical relationships within signalling systems and to identify novel components have included bioinformatics, genomics, proteomics and genetics. However, the importance of feedback loops in the regulation of signal processing systems has often been ignored. Here we report a novel focus for our previous screening platform, which evaluates the potential of autocatalytic feedback for functional screening of cDNA expression libraries. We confirm that the use of such systems is feasible. We propose that the use of feedback loops in functional screens may introduce a bias towards the detection of signalling molecules, which are likely to be of key regulatory importance due to the sensitivity of the system towards their expression levels.