scholarly journals Evidence for two isoforms of the endoplasmic-reticulum Ca2+ pump in pig smooth muscle

1989 ◽  
Vol 260 (3) ◽  
pp. 757-761 ◽  
Author(s):  
J A Eggermont ◽  
F Wuytack ◽  
S De Jaegere ◽  
L Nelles ◽  
R Casteels
Keyword(s):  
Amino Acids ◽  
Endoplasmic Reticulum ◽  
Smooth Muscle ◽  
Cdna Clones ◽  
Gene Transcript ◽  
Muscle Tissues ◽  
Class 1 ◽  
Terminal Amino ◽  
Slow Twitch ◽  
Primary Gene

cDNA clones coding for the endoplasmic reticulum Ca2+-transport ATPase have been cloned from a pig smooth-muscle cDNA library. The transcripts can be divided into two classes which differ in their 3′ ends due to alternative splicing of the primary gene transcript. The class 1 cDNA encodes a protein of 997 amino acids (Mr 110,000). The class 2 protein (1042 amino acids; Mr 115,000) is completely identical to the class 1 protein except that the four C-terminal amino acids of the class 1 protein are replaced in the class 2 protein with a tail of 49 amino acids. Comparison of these sequences with other Ca2+ pump sequences reveals that the class 1 isoform corresponds to the sarcoplasmic reticulum Ca2+ pump of slow-twitch skeletal/cardiac muscle, whereas the class 2 protein corresponds to a Ca2+ pump recently detected in non-muscle tissues.

Sequence heterogeneity, multiplicity, and genomic organization of alpha- and beta-tubulin genes in sea urchins

1981 ◽  
Vol 1 (12) ◽  
pp. 1125-1137
Author(s):  
D Alexandraki ◽  
J V Ruderman
Keyword(s):  
Amino Acids ◽  
Ribonucleic Acid ◽  
In Vitro Translation ◽  
Cdna Clones ◽  
Beta Tubulin ◽  
Ribonucleic Acids ◽  
Alpha Tubulin ◽  
Messenger Ribonucleic Acid ◽  
Tubulin Genes ◽  
Terminal Amino

We analyzed the multiplicity, heterogeneity, and organization of the genes encoding the alpha and beta tubulins in the sea urchin Lytechinus pictus by using cloned complementary deoxyribonucleic acid (cDNA) and genomic tubulin sequences. cDNA clones were constructed by using immature spermatogenic testis polyadenylic acid-containing ribonucleic acid as a template. alpha- and beta-tubulin clones were identified by hybrid selection and in vitro translation of the corresponding messenger ribonucleic acids, followed by immunoprecipitation and two-dimensional gel electrophoresis of the translation products. The alpha cDNA clone contains a sequence that encodes the 48 C-terminal amino acids of alpha tubulin and 104 base pairs of the 3' nontranslated portion of the messenger ribonucleic acid. The beta cDNA insertion contains the coding sequence for the 100-C terminal amino acids of beta tubulin and 83 pairs of the 3' noncoding sequence. Hybrid selections performed at different criteria demonstrated the presence of several heterogeneous, closely related tubulin messenger ribonucleic acids, suggesting the existence of heterogeneous alpha- and beta-tubulin genes. Hybridization analyses indicated that there are at least 9 to 13 sequences for each of the two tubulin gene families per haploid genome. Hybridization of the cDNA probes to both total genomic DNA and cloned germline DNA fragments gave no evidence for close physical linkage of alpha-tubulin genes with beta-tubulin genes at the DNA level. In contrast, these experiments indicated that some genes within the same family are clustered.

ISOLATION AND CHARACTERIZATION OF cDNA CLONES FOR THE PLATELET MEMBRANE GLYCOPROTEINS IIb and IIIa

1987 ◽  
Author(s):  
A E Butler-Zimrin ◽  
J S Bennett ◽  
M Poncz ◽  
E Schwartz ◽  
S Surrey ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Transmembrane Domain ◽  
Platelet Membrane ◽  
Membrane Receptors ◽  
Cdna Clones ◽  
Single Chain ◽  
Platelet Protein ◽  
Isolation And Characterization ◽  
Terminal Amino

The platelet membrane GPIIb/GPIIIa complex on activated platelets contains receptors for fibrinogen, von Willebrand factor, and fibronectin. GPIIb and GPIlia also appear to be members of a family of membrane receptors involved in cell-cell and cell-matrix interactions. To study the structure of GPIIb and GPIIIa, we have constructed an expression library in the vector lambda gtll using mRNA from the HEL cell line and screened it with polyclonal antibody against each platelet protein. HEL cells constitutively express proteins similar to platelet GPIIb and GPIIIa. A 3.2kb GPIIb cDNA clone was identified that encodes for all 1008 amino acids of GPIIb including the known N-terminal amino acids of the α Cand βsubunits. This confirms that GPIIb is synthesized as a single chain polypeptide that is cleaved into two disulfide-linked subunits posttranslation. Analysis of the amino acid sequence revealed a major C-terminal transmembrane domain in the βsubunit, two potential transmembrane domains near the N-terminus of the αsubunit, and four possible N-linked glycosylation sites. Approximately 30% amino acid identity was found between GPIIb and the available amino acid sequences for the larger chains of the fibronectin and vitronectin receptors. Initial sequence analysis of a 3.8kb cDNA for GPIIIa included the known N-terminal amino acids of the platelet protein. Northern blot analysis was performed using HEL cell total RNA. The GPIIb cDNA hybridized to a 4.1kb mRNA while the GPIIIa cDNA hybridized to a 5.8kb mRNA. This indicates that the two cDNAs do not cross-hybridize and suggests that GPIIb and GPIIIa are encoded by separate genes. The availability of these cDNA for GPIIb and GPIIIa will facilitate study of the structure and function of the proteins and will aid in clarifying their relationship to other adhesive protein receptors.

scholarly journals Smooth muscle expresses a cardiac/slow muscle isoform of the Ca2+-transport ATPase in its endoplasmic reticulum

1989 ◽  
Vol 257 (1) ◽  
pp. 117-123 ◽  
Author(s):  
F Wuytack ◽  
Y Kanmura ◽  
J A Eggermont ◽  
L Raeymaekers ◽  
J Verbist ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Endoplasmic Reticulum ◽  
Smooth Muscle ◽  
Sarcoplasmic Reticulum ◽  
Cardiac Muscle ◽  
Muscle Enzymes ◽  
Transport Atpase ◽  
Slow Twitch ◽  
Transport Atpases ◽  
Fast Twitch

Smooth muscle expresses in its endoplasmic reticulum an isoform of the Ca2+-transport ATPase that is very similar to or identical with that of the cardiac-muscle/slow-twitch skeletal-muscle form. However, this enzyme differs from that found in fast-twitch skeletal muscle. This conclusion is based on two independent sets of observations, namely immunological observations and phosphorylation experiments. Immunoblot experiments show that two different antibody preparations against the Ca2+-transport ATPase of cardiac-muscle sarcoplasmic reticulum also recognize the endoplasmic-reticulum/sarcoplasmic-reticulum enzyme of the smooth muscle and the slow-twitch skeletal muscle whereas they bind very weakly or not at all to the sarcoplasmic-reticulum Ca2+-transport ATPase of the fast-twitch skeletal muscle. Conversely antibodies directed against the fast-twitch skeletal-muscle isoform of the sarcoplasmic-reticulum Ca2+-transport ATPase do not bind to the cardiac-muscle, smooth-muscle or slow-twitch skeletal-muscle enzymes. The phosphorylated tryptic fragments A and A1 of the sarcoplasmic-reticulum Ca2+-transport ATPases have the same apparent Mr values in cardiac muscle, slow-twitch skeletal muscle and smooth muscle, whereas the corresponding fragments in fast-twitch skeletal muscle have lower apparent Mr values. This analytical procedure is a new and easy technique for discrimination between the isoforms of endoplasmic-reticulum/sarcoplasmic-reticulum Ca2+-transport ATPases.

scholarly journals Alternative processing of the sarco/endoplasmic reticulum Ca2+-ATPase transcripts during muscle differentiation is a specifically regulated process

1996 ◽  
Vol 317 (3) ◽  
pp. 647-651 ◽  
Author(s):  
Ludo VAN DEN BOSCH ◽  
Luc MERTENS ◽  
Yvon CAVALOC ◽  
Martha PETERSON ◽  
Frank WUYTACK ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
B Cell ◽  
Alternative Splice ◽  
Polyadenylation Site ◽  
Cell Maturation ◽  
Gene Transcript ◽  
Alternative Processing ◽  
Splice Efficiency ◽  
B Cell Maturation ◽  
Primary Gene

Expression of the muscle-specific 2a isoform of the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2) requires activation of an otherwise inefficient splice process at the 3´-end of the primary gene transcript. We provide evidence that SERCA2 splicing is a specifically regulated process, rather than the result of an increase in general splice efficiency or a decrease in polyadenylation efficiency at the 5´-most polyadenylation site. This is indicated by the fact that changes in general splice and polyadenylation efficiency, as observed during B-cell maturation, did not affect SERCA2 splicing. Furthermore, expression and overexpression studies did not support the hypothesis that changes in the level of the alternative splice factor ASF/SF2 or other arginine and serine rich proteins are sufficient to obtain the regulation of muscle- and neuronal-specific splicing.

scholarly journals Sequence elements surrounding the acceptor site suppress alternative splicing of the sarco/endoplasmic reticulum Ca2+-ATPase 2 gene transcript

1997 ◽  
Vol 322 (3) ◽  
pp. 885-891 ◽  
Author(s):  
Ludo VAN DEN BOSCH ◽  
Luc MERTENS ◽  
Sofie GIJSBERS ◽  
Mark VER HEYEN ◽  
Frank WUYTACK ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Distal Part ◽  
Myogenic Differentiation ◽  
Acceptor Site ◽  
Gene Transcript ◽  
Muscle Type ◽  
Sequence Elements ◽  
Neuronal Type ◽  
Primary Gene ◽  
Broad Region

Expression of the muscle-specific 2a isoform of the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2) requires activation of an inefficient optional splice process at the 3´ end of the primary gene transcript. The sequence elements required for this regulated splice event were studied by modifying a minigene containing the 3´ end of the SERCA2 gene. An important requirement appears to be a strong muscle-specific acceptor site, as replacing it by a weak one prevented the induction of muscle-type splicing during myogenic differentiation. The induction of muscle-type splicing did not depend on positive cis-active sequences in the muscle-specific exon. On the other hand, replacement of a broad region around the acceptor site dramatically deregulated the expression pattern, as this modification strongly induced muscle-type splicing in undifferentiated muscle cells and in fibroblasts. This cis-active region is also involved in the suppression of the neuronal type of splicing. Furthermore selective replacement of the acceptor site as well as deletions or replacements in the muscle-specific exon induced muscle-type splicing to various extents in undifferentiated myogenic cells. Therefore sequence elements in the distal part of the optional intron and in the muscle-specific exon contribute to the suppression of muscle-specific SERCA2 splicing.

scholarly journals Expression of endoplasmic-reticulum Ca2+-pump isoforms and of phospholamban in pig smooth-muscle tissues

1990 ◽  
Vol 271 (3) ◽  
pp. 649-653 ◽  
Author(s):  
J A Eggermont ◽  
F Wuytack ◽  
J Verbist ◽  
R Casteels
Keyword(s):  
Endoplasmic Reticulum ◽  
Smooth Muscle ◽  
Pulmonary Artery ◽  
Western Blot ◽  
Mrna Levels ◽  
Protection Assay ◽  
Western Blots ◽  
Specific Antibodies ◽  
Muscle Tissues ◽  
Low Levels

The expression of the gene 2 sarcoplasmic/endoplasmic-reticulum Ca2(+)-pump isoforms (SERCA2a and SERCA2b) and of phospholamban was studied in pig smooth muscle of the stomach, longitudinal ileum, pulmonary artery and aorta. mRNA levels were determined using an RNAase protection assay. The SERCA2 isoforms and phospholamban were tested on Western blots with a panel of antibodies, some of which were isoform-specific. The pig smooth-muscle tissues all contained comparable SERCA2 mRNA levels, but these levels were 10-20-fold lower than SERCA2 mRNA levels in cardiac muscle. Of the SERCA2 mRNAs in smooth muscle, 72-81% encoded the non-muscle isoform (SERCA2b), and Western blot analysis with isoform-specific antibodies confirmed that the SERCA2b isoform is the predominant endoplasmic-reticulum Ca2(+)-pump in smooth muscle. In contrast with SERCA2 mRNA levels, phospholamban mRNA levels varied by 12-fold between the different pig smooth-muscle tissues, with low and very low levels in the pig pulmonary artery and the pig aorta respectively. The differential expression of phospholamban was also confirmed on Western blots. The finding that the phospholamban content varied between the different smooth-muscle tissues whereas the SERCA2 expression remained rather constant indicates that, in pig smooth muscle, the expression of phospholamban is not coupled with that of SERCA2.

scholarly journals Sequence heterogeneity, multiplicity, and genomic organization of alpha- and beta-tubulin genes in sea urchins.

1981 ◽  
Vol 1 (12) ◽  
pp. 1125-1137 ◽  
Author(s):  
D Alexandraki ◽  
J V Ruderman
Keyword(s):  
Amino Acids ◽  
Ribonucleic Acid ◽  
In Vitro Translation ◽  
Cdna Clones ◽  
Beta Tubulin ◽  
Ribonucleic Acids ◽  
Alpha Tubulin ◽  
Messenger Ribonucleic Acid ◽  
Tubulin Genes ◽  
Terminal Amino

We analyzed the multiplicity, heterogeneity, and organization of the genes encoding the alpha and beta tubulins in the sea urchin Lytechinus pictus by using cloned complementary deoxyribonucleic acid (cDNA) and genomic tubulin sequences. cDNA clones were constructed by using immature spermatogenic testis polyadenylic acid-containing ribonucleic acid as a template. alpha- and beta-tubulin clones were identified by hybrid selection and in vitro translation of the corresponding messenger ribonucleic acids, followed by immunoprecipitation and two-dimensional gel electrophoresis of the translation products. The alpha cDNA clone contains a sequence that encodes the 48 C-terminal amino acids of alpha tubulin and 104 base pairs of the 3' nontranslated portion of the messenger ribonucleic acid. The beta cDNA insertion contains the coding sequence for the 100-C terminal amino acids of beta tubulin and 83 pairs of the 3' noncoding sequence. Hybrid selections performed at different criteria demonstrated the presence of several heterogeneous, closely related tubulin messenger ribonucleic acids, suggesting the existence of heterogeneous alpha- and beta-tubulin genes. Hybridization analyses indicated that there are at least 9 to 13 sequences for each of the two tubulin gene families per haploid genome. Hybridization of the cDNA probes to both total genomic DNA and cloned germline DNA fragments gave no evidence for close physical linkage of alpha-tubulin genes with beta-tubulin genes at the DNA level. In contrast, these experiments indicated that some genes within the same family are clustered.

scholarly journals Antibodies against the non-muscle isoform of the endoplasmic reticulum Ca2+-transport ATPase

1989 ◽  
Vol 264 (3) ◽  
pp. 765-769 ◽  
Author(s):  
F Wuytack ◽  
J A Eggermont ◽  
L Raeymaekers ◽  
L Plessers ◽  
R Casteels
Keyword(s):  
Endoplasmic Reticulum ◽  
Smooth Muscle ◽  
Cdna Sequence ◽  
Polyclonal Antiserum ◽  
Transport Atpase ◽  
C Terminus ◽  
Muscle Tissues ◽  
Gastric Smooth Muscle ◽  
Liver And Kidney ◽  
Alternatively Spliced

We report here the production of a polyclonal antiserum which specifically recognizes an epitope confined to the ultimate 12-residue-long C-terminus of an alternatively spliced transcript of gene 2 encoding the sarcoplasmic reticulum Ca2+ pump in slow skeletal and cardiac muscle. This alternatively spliced transcript was shown to be mainly represented in non-muscle tissues. These antibodies have enabled us to show the presence of the unique C-terminus of this type of Ca2+ pump, as predicted from the cDNA sequence, in the endoplasmic reticulum of vascular and gastric smooth muscle, liver and kidney.

Prevalence of the pit and antipit in the genus Glycine

1987 ◽  
Vol 45 ◽  
pp. 986-987
Author(s):  
R. W. Yaklich ◽  
E. L. Vigil ◽  
W. P. Wergin
Keyword(s):  
Amino Acids ◽  
Endoplasmic Reticulum ◽  
Glycine Max ◽  
Seed Coat ◽  
Cell Types ◽  
Abaxial Surface ◽  
Legume Seed ◽  
Initial Report ◽  
Cone Cells ◽  
Glycine Max L

The legume seed coat is the site of sucrose unloading and the metabolism of imported ureides and synthesis of amino acids for the developing embryo. The cell types directly responsible for these functions in the seed coat are not known. We recently described a convex layer of tissue on the inside surface of the soybean (Glycine max L. Merr.) seed coat that was termed “antipit” because it was in direct opposition to the concave pit on the abaxial surface of the cotyledon. Cone cells of the antipit contained numerous hypertrophied Golgi apparatus and laminated rough endoplasmic reticulum common to actively secreting cells. The initial report by Dzikowski (1936) described the morphology of the pit and antipit in G. max and found these structures in only 68 of the 169 seed accessions examined.

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