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We have adapted the protein fragment complementation assay (PCA) to the screening and selection of antibodies in the single-chain Fv (scFv) format. In this assay, two interacting proteins (target and antibody) are genetically fused to the two halves of the dissected enzyme dihydrofolate reductase. Binding of the two partners reassembles this enzyme and reconstitutes its activity, thus allowing growth on minimal medium. We have optimized this system with regard to linker length and orientation, and can reach an efficiency for antigen/antibody interactions similar to that with fused leucine zippers. Using several model antibodies specific for peptides and proteins, we show that cognate interactions give rise to about seven orders of magnitude more colonies than non-specific interactions. When transforming mixtures of plasmids encoding different antigens and/or antibodies, all colonies tested contained plasmids encoding cognate pairs. We believe that this system will be very powerful as a routine system for generating antibodies, especially in functional genomics, since it does not require purification and immobilization of the antigen. The identification of an antibody specific for a cDNA or EST-encoded protein will require only cloning, transformation and plating of bacteria.
J Mol Biol 2001 Apr 27
PMID:Fast selection of antibodies without antigen purification: adaptation of the protein fragment complementation assay to select antigen-antibody pairs. 1132 56

The spliceosomal protein, UIA, is a component of the U1snRNP essential to pre-mRNA splicing. From the ubiquitous nature of the splicing machinery, expression of U1A genes is expected to be constitutive. However, many plant genes are organised in multigene families that exhibit variation in expression profiles. Without detailed knowledge of the size of the U1A gene family or their degree of sequence variation, we examined the expression of the U1A genes using a novel approach. The approach was based on 5' RACE with [32P]-labelled primers and separation of products on high-resolution DNA sequencing gels to give a 'snapshot' of the expression of U1A genes. This was followed by sequencing of cloned 5' RACE products and of products re-amplified from excised bands. In combination with RT-PCR/SSCP, these analyses allowed the rapid identification of different gene transcripts and assessment of their relative expression profiles. Transcripts from four U1A genes (U1A-1 to U1A-4) were identified, of which U1A-1 and U1A-2 were expressed much more highly than U1A-3 and U1A-4. Differential expression of the two most highly expressed genes, U1A-1 and UIA-2, was observed in that only U1A-2 was expressed in flowers. Upstream sequences of U1A-1 and U1A-2 were cloned and the gene-specific promoters identified on the basis of the sequence variation defined from the 5' RACE products. The differential expression of these genes may be due to a 1.3 kb insertion less than 200 bp upstream of the U1A-1 coding sequence. This approach can be used more generally to examine expression profiles of multigene families, and in particular to refine information from EST or microarray analyses, and to isolate rapidly gene-specific promoters.
Plant Mol Biol 2001 Mar
PMID:Differential expression of potato U1A spliceosomal protein genes: a rapid method for expression profiling of multigene families. 1135 63

The human HFE gene was identified in 1996 as the gene whose mutations are responsible for hereditary hemochromatosis in most patients. Expression analysis by Northern blot indicated that the gene was approximately 4.1 kb in length. However, the cDNA reported was only 2716 bp. These results implied that at least 1.4 kb of the mRNA remained to be identified. In the present study, we detected several 3' EST clones while screening the genomic region of the gene in search of potential additional HFE mRNA sequences. Subsequent sequencing of these EST clones and RT-PCR experiments revealed that exon 7 of the HFE gene has, in fact, a length of 1944 bp and it presents two polyadenylation signals. The new human HFE exon 7 region has been screened in non-C282Y HH patients in search for new putative mutations. Mouse 3' RACE experiments also further extend the previously reported mouse HFE exon 6 sequence. Additionally, we report two novel end forms of the human HFE gene detected by 3' RACE experiments and several novel splicing forms identified in the HepG2 cell line.
Blood Cells Mol Dis
PMID:Complete characterization of the 3' region of the human and mouse hereditary hemochromatosis HFE gene and detection of novel splicing forms. 1135 57

Parasitic nematodes, like all aerobic organisms, require antioxidant enzymes to cope with reactive oxygen species (ROS) generated during cellular metabolism. Additionally, they have to protect themselves against ROS produced by the host. Parasitic nematode enzymes that deal with the superoxide anion radical, the superoxide dismutases (SODs), have been described in every species examined, whereas enzymes that deal with hydrogen peroxide have been difficult to identify. A major family of enzymes in mammals, the selenium-containing glutathione peroxidases (GPXs), appears to be absent, although a selenium-independent GPX family exists. These enzymes demonstrate little or no activity with hydrogen peroxide. Catalase (CAT) activity has been detected, but sequences encoding a typical CAT polypeptide have only been identified in a few species, despite the active EST sequencing projects. However, a new family of enzymes has recently been described, the peroxiredoxins (PRXs), which are abundant in parasitic nematodes and have been shown to react with hydrogen peroxide. This review summarizes the major characteristics of each of these enzyme families in general and in parasitic nematodes, emphasizing and comparing the newer data on the family of PRXs.
Mol Biochem Parasitol 2001 May
PMID:Antioxidant enzyme families in parasitic nematodes. 1137 93

The peritrophic matrix lines the midgut of most insects and has important roles in digestion, protection of the midgut from mechanical damage and invasion by micro-organisms. Although a few intrinsic peritrophic matrix proteins have been characterised, no direct homologues of any of these proteins have been found in other insect species, even closely related species, suggesting that the peritrophic matrix proteins show considerable sequence divergence. We now report the identification of the cDNA and genomic DNA sequences of a Chrysomya bezziana homologue of the Lucilia cuprina intrinsic peritrophic matrix protein, peritrophin-48. The gene for C. bezziana peritrophin-48 spans 1315 bp and consists of three exons (65, 560 and 690 bp, respectively) separated by introns of 566 and 72 bp. The transcriptional start site, identified by a consensus of cDNA clones and primer extension analysis, is probably located 58 bp upstream from the start codon. However, there may be multiple start sites for transcription. Two potential TATA boxes and a consensus arthropod transcription initiator are located within 134 bp of sequence upstream of the putative transcriptional start site suggesting that this region contains the gene promoter. Immuno-fluorescence localization demonstrated that C. bezziana peritrophin-48 was localised to the larval peritrophic matrix. Protein fold recognition analysis indicated structural similarities between peritrophin-48 and wheatgerm lectin. As wheatgerm lectin binds chitin, this result suggested that C. bezziana peritrophin-48 may also bind chitin, a constituent of the peritrophic matrix. Chitin binding studies with a recombinant peritrophin-48 protein confirmed that it binds chitin. A Drosophila melanogaster homologue of peritrophin-48 encoded in an EST and a genomic sequence was also identified. The pairwise percentage identities of the deduced amino acid sequences for the peritrophin-48 homologues from the three higher Dipteran species were relatively low, ranging between 32 and 42%. Despite this sequence variability, the predicted structure of these proteins, dictated by five domains, each containing a characteristic distribution of six cysteines, was strictly conserved. It is concluded that considerable sequence variation can be tolerated in this protein because of the constraints imposed on the structure of the protein by an extensive disulphide bonded framework.
Insect Biochem Mol Biol 2001 Jul 26
PMID:Identification and molecular characterisation of a peritrophin gene, peritrophin-48, from the myiasis fly Chrysomya bezziana. 1143 51

COPI-coated vesicles are involved in retrograde-directed selective transport of proteins from the Golgi complex to the endoplasmic reticulum (ER) as well as mediate anterograde transport of cargo proteins within the Golgi or in endosomal trafficking. The COPI protein complex contains an ADP-ribosylation factor (ARF1) and seven coatamer subunits (alpha, beta, beta', gamma, delta, epsilon, zeta-COP). The localization and function of human beta subunit of coatamer (COPB) suggests it is likely a candidate gene of ruby-eye-2 (ru2), which is a mouse model of human Hermansky-Pudlak syndrome characterized by the dysfunction of several subcellular organelles. In this study, we determined the entire coding sequence of mouse (Copb) cDNA by combining an overlapping mouse EST contig with EST walking. beta-COP was found highly conserved in mouse, rat, and human, and it is ubiquitously expressed in mouse. The Copb gene was mapped to mouse Chr 7 at a position of 53.3 cM by radiation hybrid mapping. Our RH mapping data, sequencing of RT-PCR products, and Western blotting exclude the Copb gene as a candidate for ru2.
Somat Cell Mol Genet 1999 May
PMID:cDNA sequence and mapping of the mouse Copb gene encoding the beta subunit of the COPI coatomer complex. 1144 37

The human hydroxysteroid sulfotransferase, dehydroepiandrosterone sulfotransferase (DHEA-ST), is highly expressed in liver and adrenal cortex and displays reactivity towards a broad range of hydroxysteroids including 3 beta-hydroxysteroids, 3 alpha-hydroxysteroids, estrogens with a 3-phenolic moiety, and 17-hydroxyl group of androgens. In contrast, characterization of the newly described human hydroxysteroid sulfotransferase SULT2B1 isoforms shows that these enzymes are selective for the sulfation of 3 beta-hydroxysteroids, such as pregnenolone, epiandrosterone, DHEA, and androstenediol. There was no activity detected towards testosterone, dexamethasone, beta-estradiol, androsterone, or p-nitrophenol. The SULT2B1 gene encodes two isoforms, SULT2B1a and SULT2B1b, which are generated by alternate splicing of the first exon; therefore the SULT2B1 isoforms differ at their N-terminals. Northern Blot analysis detected a SULT2B1 message in RNA isolated from the human prostate and placenta. No SULT2B1 message was observed in RNA isolated from human liver, colon, lung, kidney, brain, or testis tissue. Purified SULT2B1a was used to generate a specific rabbit polyclonal anti-SULT2B1 antibody. The anti-SULT2B1 antibody did not react with expressed human EST, P-PST-1, M-PST, DHEA-ST, or ST1B2, during immunoblot analysis. The substrate specificity of the expressed SULT2B1 isoforms suggests that these enzymes are capable of regulating the activity of adrenal androgens in human tissues via their inactivation by sulfation.
J Steroid Biochem Mol Biol 2001 Jun
PMID:Expression and characterization of the human 3 beta-hydroxysteroid sulfotransferases (SULT2B1a and SULT2B1b). 1145 64

DNA fragments containing genetic information for five secretion-related small GTPases of Aspergillus niger (srgA-E) were isolated and identified as members of different Rab/Ypt subfamilies. This isolation and the search for similar sequences in fungal genomic and EST databases showed that, in contrast to Saccharomyces cerevisiae, filamentous fungi also possess homologues of mammalian Rab2 GTPases. Multiple transcripts with unusually long 5' and 3' untranslated regions were found for all srg genes. Their level of expression was independent of the type of carbon source used for growth. Although the transcripts of srgA and srgB were abundant to the same extent throughout the cultivation, that of the other genes peaked during the early growth phase and then declined. Two genes, srgA and srgB, were characterized further. The protein encoded by srgA exhibited relatively low identity (58%) to its closest S. cerevisiae homologue SEC4, whereas the protein encoded by srgB showed 73% identity with S. cerevisiae YPT1. In contrast to other SEC4 homologues, srgA was unable to complement an S. cerevisiae sec4 mutant, and its disruption was not lethal in A. niger. SrgA mutants displayed a twofold increase in their hyphal diameter, unusual apical branching and strongly reduced protein secretion during growth on glucose.
Mol Microbiol 2001 Jul
PMID:Identification and characterization of a family of secretion-related small GTPase-encoding genes from the filamentous fungus Aspergillus niger: a putative SEC4 homologue is not essential for growth. 1148 35

The number of genes encoded by the human genome has long sought to be determined. With the recent publications of the complete sequence of the human genome, the number of genes encoded by the human genome has now been estimated to be approximately 32,000-38,000. Now the next step will be to determine which of these genes are expressed in a given cell, tissue or organ. Using three unique approaches taking advantage of our current cardiovascular EST database and the complete nucleotide sequence of human chromosomes 21 and 22 as well as cDNA microarray hybridization, we estimate that between 20,930-27,160 genes are expressed in the cardiovascular system.
J Mol Cell Cardiol 2001 Oct
PMID:Cardiovascular genomics: estimating the total number of genes expressed in the human cardiovascular system. 1160 29

Clara cell secretory protein (CCSP) is one of the most abundant proteins present in airway lining fluid of mammals. In an effort to elucidate the function of CCSP, we established CCSP-null [CCSP(-/-)] mice and demonstrated altered sensitivity to various environmental agents including oxidant pollutants and microorganisms. Although CCSP deficiency itself may be central to the observed changes in environmental susceptibility, altered lung gene expression associated with CCSP deficiency may contribute to the observed phenotype. To determine whether CCSP deficiency results in altered lung gene expression, high-density cDNA microarrays were used to profile gene expression in the total lung RNA of wild-type and CCSP(-/-) mice. Genes that were differentially expressed between wild-type and CCSP(-/-) mice included a previously non-annotated expressed sequence tag (EST W82219) and immunoglobulin A (IgA), both of which were elevated with CCSP deficiency. mRNA expression of EST W82219 and IgA was localized in the lungs of wild-type and CCSP(-/-) mice to airway Clara cells and peribronchial lymphoid tissues, respectively. We conclude that CCSP deficiency is associated with 1) altered gene expression in Clara cells of the conducting airway epithelium and 2) alterations to peribronchial B lymphocytes. These findings identify new roles for Clara cells and their secretions in airway homeostasis.
Am J Physiol Lung Cell Mol Physiol 2001 Dec
PMID:Altered lung gene expression in CCSP-null mice suggests immunoregulatory roles for Clara cells. 1170 49


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