scholarly journals Characterization of beta pat-3 heterodimers, a family of essential integrin receptors in C. elegans.

1995 ◽  
Vol 129 (4) ◽  
pp. 1127-1141 ◽  
Author(s):  
S N Gettner ◽  
C Kenyon ◽  
L F Reichardt
Keyword(s):  
Caenorhabditis Elegans ◽  
Cytoplasmic Domain ◽  
Beta Subunit ◽  
Beta Subunits ◽  
Integrin Receptors ◽  
Diverse Range ◽  
C Elegans ◽  
Alpha Subunits ◽  
Sds Page

Members of the integrin family of cell surface receptors have been shown to mediate a diverse range of cellular functions that require cell-cell or cell-extracellular matrix interactions. We have initiated the characterization of integrin receptors from the nematode Caenorhabditis elegans, an organism in which genetics can be used to study integrin function with single cell resolution. Here we report the cloning of an integrin beta subunit from C. elegans which is shown to rescue the embryonic lethal mutation pat-3(rh54) and is thus named beta pat-3. Analysis of the deduced amino acid sequence revealed that beta pat-3 is more similar to Drosophila integrin beta PS and to vertebrate integrin beta 1 than to other integrin beta subunits. Regions of highest homology are in the RGD-binding region and in the cytoplasmic domain. In addition, the 56 cysteines present in the majority of integrin beta subunits are conserved. A major transcript of approximately 3 kilo-base pairs was detected by RNA blot analysis. Immunoblot analysis using a polyclonal antiserum against the cytoplasmic domain showed that beta pat-3 migrates in SDS-PAGE with apparent M(r) of 109 k and 120 k under nonreducing and reducing conditions, respectively. At least nine protein bands with relative molecular weights in the range observed for known integrin alpha subunits coprecipitate with beta pat-3, and at least three of these bands migrate in SDS-PAGE with increased mobility when reduced. This behavior has been observed for a majority of integrin alpha subunits. Immunoprecipitations of beta pat-3 from developmentally staged populations of C. elegans showed that the expression of several of these bands changes during development. The monoclonal antibody MH25, which has been postulated to recognize the transmembrane component of the muscle dense body structure a (Francis, G. R., and R. H. Waterston. 1985. Muscle organization in Caenorhabditis elegans: localization of proteins implicated in thin filament attachment and I-band organization. J. Cell Biol. 101:1532-1549), was shown to recognize beta pat-3. Finally, immunocytochemical analysis revealed that beta pat-3 is expressed in the embryo and in many cell types postembryonically, including muscle, somatic gonad, and coelomocytes, suggesting multiple roles for integrin heterodimers containing this beta subunit in the developing animal.

scholarly journals Purification and characterization of protein kinase C from the nematode Caenorhabditis elegans

1992 ◽  
Vol 282 (1) ◽  
pp. 219-223 ◽  
Author(s):  
T Sassa ◽  
J Miwa
Keyword(s):  
Protein Kinase C ◽  
Caenorhabditis Elegans ◽  
Protein Kinase ◽  
Column Chromatography ◽  
Purification And Characterization ◽  
C Elegans ◽  
Kinase C ◽  
Sds Page ◽  
Cytosolic Extract

Protein kinase C (PKC) of Caenorhabditis elegans was identified by enzymatic activity and [3H]phorbol 12,13-dibutyrate binding after DEAE-Sephacel column chromatography of a crude cytosolic extract. Ca(2+)-dependent activation of nematode PKC was observed in the presence of phosphatidylserine. The enzyme was maximally activated by 1,2-dioleoylglycerol or phorbol 12-myristate 13-acetate in the presence of phosphatidylserine and Ca2+. Hydroxyapatite column chromatography showed only one peak of PKC activity with histone H1 and myelin basic protein as substrates. The enzyme was purified to near homogeneity by sequential chromatography on polylysine-agarose and phosphatidylserine affinity columns. The purified protein showed a molecular mass of 79 kDa on SDS/PAGE. The substrate specificity of the C. elegans enzyme was shown to be different from that of mammalian PKCs. Here we describe some of the properties of the nematode enzyme.

scholarly journals A human leukocyte differentiation antigen family with distinct alpha-subunits and a common beta-subunit: the lymphocyte function-associated antigen (LFA-1), the C3bi complement receptor (OKM1/Mac-1), and the p150,95 molecule.

1983 ◽  
Vol 158 (6) ◽  
pp. 1785-1803 ◽  
Author(s):  
F Sanchez-Madrid ◽  
J A Nagy ◽  
E Robbins ◽  
P Simon ◽  
T A Springer
Keyword(s):  
Human Leukocyte ◽  
Beta Subunit ◽  
Alpha Subunit ◽  
Beta Subunits ◽  
Complement Receptor ◽  
Lymphocyte Function ◽  
Subunit Dissociation ◽  
Molecular Weights ◽  
Alpha Subunits ◽  
Sds Page

The human lymphocyte function-associated antigen-1 (LFA-1), the complement receptor-associated OKM1 molecule, and a previously undescribed molecule termed p150,95, have been found to be structurally and antigenically related. Each antigen contains an alpha- and beta-subunit noncovalently associated in an alpha 1 beta 1-structure as shown by cross-linking experiments. LFA-1, OKM1, and p150,95 alpha-subunit designations and their molecular weights are alpha L = 177,000 Mr, alpha M = 165,000 Mr, and alpha X = 150,000 Mr, respectively. The beta-subunits are all = 95,000 Mr. Some MAb precipitated only LFA-1, others only OKM1, and another precipitates all three antigens. The specificity of these MAb for particular subunits was examined after subunit dissociation by high pH. MAb specific for LFA-1 or OKM1 bind to the alpha L- or alpha M-subunits, respectively, while the cross-reactive MAb binds to the beta-subunits. Coprecipitation experiments with intact alpha 1 beta 1-complexes showed anti-alpha and anti-beta MAb can precipitate the same molecules. In two-dimensional (2D) isoelectric focusing-SDS-PAGE, the alpha subunits of the three antigens are distinct, while the beta-subunits are identical. Biosynthesis experiments showed alpha L, alpha M, and alpha X are synthesized from distinct precursors, as is beta. The three antigens differ in expression on lymphocytes, granulocytes, and monocytes. During maturation of the monoblast-like U937 line, alpha M and alpha X are upregulated and alpha L is downregulated. Some MAb to the alpha subunit of OKM1 inhibited the complement receptor type three. LFA-1, OKM1, and p150,95 constitute a novel family of functionally important human leukocyte antigens that share a common beta-subunit.

scholarly journals Mapping of antigenic and functional epitopes on the alpha- and beta-subunits of two related mouse glycoproteins involved in cell interactions, LFA-1 and Mac-1.

1983 ◽  
Vol 158 (2) ◽  
pp. 586-602 ◽  
Author(s):  
F Sanchez-Madrid ◽  
P Simon ◽  
S Thompson ◽  
T A Springer
Keyword(s):  
Monoclonal Antibodies ◽  
Structural Features ◽  
Cell Interactions ◽  
Beta Subunit ◽  
Alpha Subunit ◽  
Beta Subunits ◽  
Complement Receptor ◽  
Receptor Function ◽  
Alpha Subunits ◽  
Sds Page

Mouse Mac-1, a complement receptor-associated surface structure on macrophages, and LFA-1, a function-associated structure on lymphocytes, comprise a novel family of leukocyte differentiation antigens participating in adhesive cell interactions. Mac-1 and LFA-1 contain alpha-subunits of 170,000 and 180,000 Mr, respectively, and beta-subunits of 95,000 Mr noncovalently associated in alpha 1 beta 1 complexes. The structural relation between the alpha- and between the beta-subunits, and the location of functionally important sites on the molecules, have been probed with antibodies. Both non-cross-reactive and cross-reactive monoclonal antibodies (MAb) and antisera prepared to the purified molecules or the LFA-1 alpha-subunits were used. Reactivity with individual subunits was studied by immunoprecipitation after dissociation induced by high pH treatment, or by immunoblotting after SDS-PAGE. Cross-reactive epitopes on Mac-1 and LFA-1 were found to be present on the beta-subunits, which were immunologically identical. Non-cross-reactive epitopes that are distinctive for Mac-1 or LFA-1 were localized to the alpha-subunits. MAb to LFA-1 alpha-subunit epitopes inhibited CTL-mediated killing. Two MAb to Mac-1 alpha-subunit epitopes but not a third MAb to a spatially distinct alpha-epitope inhibited complement receptor function. Neither function was inhibited by a MAb binding to a common beta-subunit epitope. Therefore, sites of Mac-1 and LFA-1 involved in their respective adhesion-related functions, as well as distinctive structural features, have been localized to the alpha-subunits.

The glycomes of Caenorhabditis elegans and other model organisms

2002 ◽  
Vol 69 ◽  
pp. 117-134 ◽  
Author(s):  
Stuart M. Haslam ◽  
David Gems ◽  
Howard R. Morris ◽  
Anne Dell
Keyword(s):  
Caenorhabditis Elegans ◽  
Current Knowledge ◽  
Structural Data ◽  
Model Organisms ◽  
Entire Genome ◽  
C Elegans ◽  
The Face ◽  
Area Of Concern ◽  
Nematode Worm

There is no doubt that the immense amount of information that is being generated by the initial sequencing and secondary interrogation of various genomes will change the face of glycobiological research. However, a major area of concern is that detailed structural knowledge of the ultimate products of genes that are identified as being involved in glycoconjugate biosynthesis is still limited. This is illustrated clearly by the nematode worm Caenorhabditis elegans, which was the first multicellular organism to have its entire genome sequenced. To date, only limited structural data on the glycosylated molecules of this organism have been reported. Our laboratory is addressing this problem by performing detailed MS structural characterization of the N-linked glycans of C. elegans; high-mannose structures dominate, with only minor amounts of complex-type structures. Novel, highly fucosylated truncated structures are also present which are difucosylated on the proximal N-acetylglucosamine of the chitobiose core as well as containing unusual Fucα1–2Gal1–2Man as peripheral structures. The implications of these results in terms of the identification of ligands for genomically predicted lectins and potential glycosyltransferases are discussed in this chapter. Current knowledge on the glycomes of other model organisms such as Dictyostelium discoideum, Saccharomyces cerevisiae and Drosophila melanogaster is also discussed briefly.

scholarly journals Characterization of Caenorhabditis elegans Homologs of the Down Syndrome Candidate Gene DYRK1A

Genetics ◽  
2003 ◽  
Vol 163 (2) ◽  
pp. 571-580 ◽  
Author(s):  
William B Raich ◽  
Celine Moorman ◽  
Clay O Lacefield ◽  
Jonah Lehrer ◽  
Dusan Bartsch ◽  
...  
Keyword(s):  
Down Syndrome ◽  
Caenorhabditis Elegans ◽  
Trisomy 21 ◽  
Gene Dosage ◽  
Inducible Promoters ◽  
C Elegans ◽  
Dual Specificity ◽  
Specificity Protein

Abstract The pathology of trisomy 21/Down syndrome includes cognitive and memory deficits. Increased expression of the dual-specificity protein kinase DYRK1A kinase (DYRK1A) appears to play a significant role in the neuropathology of Down syndrome. To shed light on the cellular role of DYRK1A and related genes we identified three DYRK/minibrain-like genes in the genome sequence of Caenorhabditis elegans, termed mbk-1, mbk-2, and hpk-1. We found these genes to be widely expressed and to localize to distinct subcellular compartments. We isolated deletion alleles in all three genes and show that loss of mbk-1, the gene most closely related to DYRK1A, causes no obvious defects, while another gene, mbk-2, is essential for viability. The overexpression of DYRK1A in Down syndrome led us to examine the effects of overexpression of its C. elegans ortholog mbk-1. We found that animals containing additional copies of the mbk-1 gene display behavioral defects in chemotaxis toward volatile chemoattractants and that the extent of these defects correlates with mbk-1 gene dosage. Using tissue-specific and inducible promoters, we show that additional copies of mbk-1 can impair olfaction cell-autonomously in mature, fully differentiated neurons and that this impairment is reversible. Our results suggest that increased gene dosage of human DYRK1A in trisomy 21 may disrupt the function of fully differentiated neurons and that this disruption is reversible.

The genetic and RFLP characterization of the left end of linkage group III in Caenorhabditis elegans

Genome ◽  
1993 ◽  
Vol 36 (4) ◽  
pp. 712-724 ◽  
Author(s):  
Dave Pilgrim
Keyword(s):  
Caenorhabditis Elegans ◽  
Linkage Group ◽  
Genetic Map ◽  
Physical Map ◽  
Group Iii ◽  
C Elegans ◽  
Genomic Regions ◽  
Caenorhabditis Elegans Genome ◽  
Selection Of

A genetic approach was taken to identify new transposable element Tc1 -dependent polymorphisms on the left end of linkage group III in the nematode Caenorhabditis elegans. The cloning of the genomic DNA surrounding the Tc1 allowed the selection of overlapping clones (from the collection being used to assemble the physical map of the C. elegans genome). A contig of approximately 600–800 kbp in the region has been identified, the genetic map of the region has been refined, and 10 new RFLPs as well as at least four previously characterized genetic loci have been positioned onto the physical map, to the resolution of a few cosmids. This analysis demonstrated the ability to combine physical and genetic mapping for the rapid analysis of large genomic regions (0.5–1 Mbp) in genetically amenable eukaryotes.Key words: Caenorhabditis elegans, genome analysis, RFLP, physical map, genetic map.

scholarly journals Baculovirus expression of two protein disulphide isomerase isoforms from Caenorhabditis elegans and characterization of prolyl 4-hydroxylases containing one of these polypeptides as their β subunit

1996 ◽  
Vol 317 (3) ◽  
pp. 721-729 ◽  
Author(s):  
Johanna VEIJOLA ◽  
Pia ANNUNEN ◽  
Peppi KOIVUNEN ◽  
Antony P. PAGE ◽  
Taina PIHLAJANIEMI ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Cloning And Expression ◽  
Β Subunit ◽  
Α Subunit ◽  
Protein Disulphide Isomerase ◽  
C Elegans ◽  
Baculovirus Expression ◽  
Expression Studies ◽  
Α Subunits

Protein disulphide isomerase (PDI; EC 5.3.4.1) is a multifunctional polypeptide that is identical to the β subunit of prolyl 4-hydroxylases. We report here on the cloning and expression of the Caenorhabditis elegans PDI/β polypeptide and its isoform. The overall amino acid sequence identity and similarity between the processed human and C. elegans PDI/β polypeptides are 61% and 85% respectively, and those between the C. elegans PDI/β polypeptide and the PDI isoform 46% and 73%. The isoform differs from the PDI/β and ERp60 polypeptides in that its N-terminal thioredoxin-like domain has an unusual catalytic site sequence -CVHC-. Expression studies in insect cells demonstrated that the C. elegans PDI/β polypeptide forms an active prolyl 4-hydroxylase α2β2 tetramer with the human α subunit and an αβ dimer with the C. elegans α subunit, whereas the C. elegans PDI isoform formed no prolyl 4-hydroxylase with either α subunit. Removal of the 32-residue C-terminal extension from the C. elegans α subunit totally eliminated αβ dimer formation. The C. elegans PDI/β polypeptide formed less prolyl 4-hydroxylase with both the human and C. elegans α subunits than did the human PDI/β polypeptide, being particularly ineffective with the C. elegans α subunit. Experiments with hybrid polypeptides in which the C-terminal regions had been exchanged between the human and C. elegans PDI/β polypeptides indicated that differences in the C-terminal region are one reason, but not the only one, for the differences in prolyl 4-hydroxylase formation between the human and C. elegans PDI/β polypeptides. The catalytic properties of the C. elegans prolyl 4-hydroxylase αβ dimer were very similar to those of the vertebrate type II prolyl 4-hydroxylase tetramer, including the Km for the hydroxylation of long polypeptide substrates.

Reporter gene constructs suggest that the Caenorhabditis elegans avermectin receptor beta-subunit is expressed solely in the pharynx.

1997 ◽  
Vol 200 (10) ◽  
pp. 1509-1514 ◽  
Author(s):  
D L Laughton ◽  
G G Lunt ◽  
A J Wolstenholme
Keyword(s):  
Caenorhabditis Elegans ◽  
Genomic Sequence ◽  
Gene Promoter ◽  
Pcr Amplification ◽  
Beta Subunit ◽  
Amino Acid Residues ◽  
C Elegans ◽  
Larval Stages ◽  
Major Mode ◽  
Flanking Sequences

Gene promoter/LacZ reporter constructs were made in order to analyse the expression of the beta-subunit of the Caenorhabditis elegans glutamate-gated Cl- channel (Glu-Cl) receptor. Southern blot analysis of the C. elegans cosmid C35E8 identified a 4kbp EcoRI fragment which contained the 5' portion of the Glu-Cl beta coding sequence together with 5' flanking sequences. This was subcloned and used as the template for polymerase chain reaction (PCR) amplification of a DNA fragment encoding the first 24 amino acid residues of Glu-Cl beta together with 1.4 kbp of 5' genomic sequence. The fragment was subcloned into the LacZ expression vector pPD22.11 to form a translational reporter fusion. After injection of the construct into worms, six stably transformed lines were established and assayed for beta-galactosidase activity. Stained nuclei were observed in the pharyngeal metacorpus in adults and in all larval stages, and stained nuclei were seen in many embryos undergoing morphogenesis. Additional stained nuclei towards the terminal bulb of the pharynx were observed in larval stages. These results provide further evidence that the Glu-Cl receptor mediates the glutamatergic inhibition of pharyngeal muscle via the M3 motor neurone and point to inhibition of pharyngeal pumping as a major mode of action for avermectins.

Open channel block of human heart hKv1.5 by the beta-subunit hKv beta 1.2

1997 ◽  
Vol 272 (6) ◽  
pp. H2932-H2941 ◽  
Author(s):  
M. De Biasi ◽  
Z. Wang ◽  
E. Accili ◽  
B. Wible ◽  
D. Fedida
Keyword(s):  
Human Heart ◽  
Open Channel ◽  
Beta Subunit ◽  
Beta Subunits ◽  
Channel Block ◽  
Open Channel Block ◽  
Pharmacological Characterization ◽  
Kinetics Of ◽  
K Currents

Voltage-gated K+ currents in human heart are likely to derive from multisubunit complexes of pore-forming alpha-subunits with one or more auxiliary beta-subunits. We recently cloned a novel beta-subunit from human atrium, hKv beta 1.2 (K. Majumder, M. De Biasi, Z. Wang, and B. A. Wible. FEBS Lett. 361: 13-16, 1995), and showed that it interacts with channels in the Kv1 family. Here we characterize the interaction of hKv beta 1.2 with hKv1.5 in terms of a two-closed-state and one-open-state open channel block model. After coexpression in Xenopus oocytes, hKv1.5 currents were reduced in the presence of hKv beta 1.2, and at positive potentials an inactivation process was introduced. Deactivation kinetics of hKv1.5 were slowed, and there was an increased steepness with a -14-mV hyperpolarizing shift in the midpoint of steady-state activation. The model was able to predict all the above features of the interaction of hKv1.5 and hKv beta 1.2 as a result of rapid open channel block of activated channels. Understanding the mechanism of hKv beta 1.2 action on heart K+ channels will further aid the development of the functional and pharmacological characterization of native cardiac K+ currents.

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