scholarly journals Tissue-specific expression of a rat intestinal mucin-like peptide

1992 ◽  
Vol 286 (2) ◽  
pp. 335-338 ◽  
Author(s):  
G Xu ◽  
D Wang ◽  
L J Huan ◽  
E Cutz ◽  
G G Forstner ◽  
...  
Keyword(s):  
Splice Variants ◽  
Goblet Cells ◽  
Rat Tissues ◽  
Rat Intestine ◽  
Rich Region ◽  
Specific Expression ◽  
Intestinal Mucin ◽  
C Terminus ◽  
Hybridization In Situ

Expression of the gene for a rat intestinal mucin-like peptide (MLP) was studied by Northern-blot analyses of RNA prepared from a panel of rat tissues. Four probes (A-D) were constructed so as to span a 3.5 kb-long cDNA for rat MLP, and used for hybridization. Positive signals were obtained in intestine and colon, whereas lung, liver, stomach, submandibular gland and spleen were negative. The only transcript detected was approx. 9.5 kb in size. No mRNA splice variants were found. Hybridization in situ using probe B1, which corresponds to a cysteine-rich region near the C-terminus of MLP, confirmed that the gene for MLP is expressed by goblet cells of rat intestine and colon.

scholarly journals Molecular characterization and expression of the stratification-related cytokeratins 4 and 15.

1988 ◽  
Vol 106 (4) ◽  
pp. 1249-1261 ◽  
Author(s):  
R E Leube ◽  
B L Bader ◽  
F X Bosch ◽  
R Zimbelmann ◽  
T Achtstaetter ◽  
...  
Keyword(s):  
Basal Cell ◽  
Blot Hybridization ◽  
Northern Blot Hybridization ◽  
Type I ◽  
Specific Expression ◽  
Genes Encoding ◽  
Cell Layers ◽  
Hybridization In Situ ◽  
Stratified Epithelia

A number of human cytokeratins are expressed during the development of stratified epithelia from one-layered polar epithelia and continue to be expressed in several adult epithelial tissues. For studies of the regulation of the synthesis of stratification-related cytokeratins in internal tissues, we have prepared cDNA and genomic clones encoding cytokeratin 4, as a representative of the basic (type II) cytokeratin subfamily and cytokeratin 15, as representative of the acidic (type I) subfamily, and determined their nucleotide sequences. The specific expression of mRNAs encoding these two polypeptides in certain stratified tissues and cultured cell lines is demonstrated by Northern blot hybridization. Hybridization in situ with antisense riboprobes and/or synthetic oligonucleotides shows the presence of cytokeratin 15 mRNA in all layers of esophagus, whereas cytokeratin 4 mRNA tends to be suprabasally enriched, although to degrees varying in different regions. We conclude that the expression of the genes encoding these stratification-related cytokeratins starts already in the basal cell layer and does not depend on vertical differentiation and detachment from the basal lamina. Our results also show that simple epithelial and stratification-related cytokeratins can be coexpressed in basal cell layers of certain stratified epithelia such as esophagus. Implications of these findings for epithelial differentiation and the formation of squamous cell carcinomas are discussed.

Characteristics of rodent intestinal mucin Muc3 and alterations in a mouse model of human cystic fibrosis

2001 ◽  
Vol 280 (6) ◽  
pp. G1321-G1330 ◽  
Author(s):  
Ismat A. Khatri ◽  
Catherine Ho ◽  
Robert D. Specian ◽  
Janet F. Forstner
Keyword(s):  
Cystic Fibrosis ◽  
Mouse Model ◽  
Oligonucleotide Probe ◽  
Goblet Cells ◽  
Soluble Form ◽  
Density Fractions ◽  
Intestinal Mucin ◽  
Apical Membranes ◽  
Columnar Cells

Human mucin MUC3 and rodent Muc3 are widely assumed to represent secretory mucins expressed in columnar and goblet cells of the intestine. Using a 3′-oligonucleotide probe and in situ hybridization, we observed expression of rat Muc3 mostly in columnar cells. Two antibodies specific for COOH-terminal epitopes of Muc3 localized to apical membranes and cytoplasm of columnar cells. An antibody to the tandem repeat (TR) sequence (TTTPDV)3, however, localized to both columnar and goblet cells. On CsCl gradients, Muc3 appeared in both light- and heavy-density fractions. The lighter species was immunoreactive with all three antibodies, whereas the heavier species reacted only with anti-TR antibody. Thus Muc3 is expressed in two forms, a full-length membrane-associated form found in columnar cells (light density) and a carboxyl-truncated soluble form present in goblet cells (heavy density). In a mouse model of human cystic fibrosis, both soluble Muc3 and goblet cell Muc2 were increased in amount and hypersecreted. Thus Muc2 and Muc3 contribute to the excess intestinal luminal mucus of cystic fibrosis mice.

scholarly journals Human salivary cystatin S. Cloning, sequence analysis, hybridization in situ and immunocytochemistry

1991 ◽  
Vol 278 (3) ◽  
pp. 627-635 ◽  
Author(s):  
L A Bobek ◽  
A Aguirre ◽  
M J Levine
Keyword(s):  
Sequence Similarity ◽  
Leader Peptide ◽  
Cdna Clones ◽  
Specific Expression ◽  
Direct Dna Sequencing ◽  
Pcr Products ◽  
Cystatin Gene ◽  
Hybridization In Situ ◽  
Cystatin Sn

A human submandibular-gland (SMG) cDNA library was constructed in a lambda was constructed in a lambda gt11 Sfi-Not orientation-specific expression vector and then screened with antibody generated against human salivary cystatins. The clone C4-4 encoded an N-terminally truncated cystatin S, whereas the others encoded cystatin SN. The library was then rescreened with the C4-4, and the inserts of several positive clones were directly amplified from the eluted plaques by linear PCR and the PCR products analysed by Southern blotting and direct DNA sequencing. Two clones (C3 and C12) encoded a full-length secreted cystatin S and its leader peptide and included 5′- and 3′-untranslated regions. These clones showed a high degree of sequence similarity to cDNA clones encoding human salivary cystatin SN and genomic clones encoding cystatin SN and SA. Hybridization in situ of normal human SMG and parotid-gland (PG) tissue sections localized the cystatin-gene transcripts to the cytoplasm of serous acinar cells of both glands, with a much higher concentration of cystatin mRNA in the SMG. Immunocytochemistry localized the salivary cystatin gene products also to the serous cells, and the levels of cystatin protein correlated with the amount of cystatin mRNA, with a much stronger signal in the SMG than in the PG.

scholarly journals Tissue and cell-type specific expression of a splice variant in the II-III cytoplasmic loop ofCacna1b

2019 ◽  
Author(s):  
Bunda Alexandra ◽  
LaCarubba Brianna ◽  
Akiki Marie ◽  
Andrade Arturo
Keyword(s):  
Motor Neurons ◽  
Splice Variants ◽  
Gene Encoding ◽  
Specific Expression ◽  
Cell Type Specific Expression ◽  
Alternatively Spliced ◽  
Cell Type Specific ◽  
Alternatively Spliced Exons ◽  
Cerebellar Cells

ABSTRACTPresynaptic CaV2.2 (N-type) channels are fundamental for transmitter release across the nervous system. The gene encoding CaV2.2 channels,Cacna1b, contains alternatively spliced exons that originate functionally distinct splice variants (e18a, e24a, e31a and 37a/37b). Alternative splicing of the cassette exon 18a generates two mRNA transcripts (+e18a-Cacna1band Δe18a-Cacna1b). In this study, using novel mouse genetic models and in situ hybridization (BaseScope™), we confirmed that +e18a-Cacna1bsplice variants are expressed in monoaminergic regions of midbrain. We expanded these studies and identified +e18a-Cacna1bmRNA in deep cerebellar cells and spinal cord motor neurons. Furthermore, we determined that +e18a-Cacna1bis enriched in cholecystokinin expressing interneurons. Our results provide key information to understand cell-specific functions of CaV2.2 channels.

scholarly journals Developmental and cell-specific expression ofCacna1dsplice variants

2019 ◽  
Author(s):  
LaCarubba Brianna ◽  
Bunda Alexandra ◽  
Savage Kitty ◽  
Sargent Hannah ◽  
Akiki Marie ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Brain Development ◽  
Splice Variants ◽  
Cell Types ◽  
Adult Brain ◽  
Specific Expression ◽  
C Terminus ◽  
Dependent Inactivation ◽  
Alternatively Spliced ◽  
Domain I

ABSTRACTCaV1.3 is an L-type voltage-gated calcium channel implicated in several functions including gene expression, pacemaking activity, and neurotransmitter release. The gene that encodes the CaVα1-pore forming subunit of CaV1.3 (Cacna1d) is a multi-exon gene that undergoes extensive alternative splicing, which provides functional versatility to this gene across tissues and cell-types. The function and expression of severalCacna1dsplice variants within the C-terminus have been previously characterized. These splice variants differ in their voltage-dependence of activation, Ca2+-dependent inactivation, and their sensitivity to dihydropyridines. However, less is known about alternatively spliced exons inCacna1dlocated downstream of domain I and upstream of the C-terminus (e11, e22a/e22, e31a/e31b/e32). Here, we performed a systematic study to determine the developmental and cell-specific expression of severalCacna1dsplice variants. We found that the cassette e11 is upregulated during brain development, and in adult cortical tissue is more abundant in excitatory neurons relative to inhibitory interneurons. This exon is also upregulated upon nerve growth factor (NGF) induced differentiation of pheochromocytoma cells, PC12. At the functional level, the splice variants resulting from e11 alternative splicing (+e11-Cacna1dand Δe11-Cacna1d) form functional CaV1.3 channels with similar biophysical properties in expression mammalian systems. Of the pair of mutually exclusive exons, e22a and e22, the later dominates at all stages. However, we observed a slight upregulation of e22 from embryonic to adult human brain. A second pair of mutually exclusive exons, e31a and e31b, was also studied. We found that e31a increases during brain development. Finally, the cassette exon 32 is repressed in adult brain tissue.

scholarly journals Tenascin in bone morphogenesis: expression by osteoblasts and cell type-specific expression of splice variants

1992 ◽  
Vol 103 (3) ◽  
pp. 765-771 ◽  
Author(s):  
E.J. Mackie ◽  
R.P. Tucker
Keyword(s):  
In Situ Hybridization ◽  
Splice Variants ◽  
Cdna Probe ◽  
Cell Type ◽  
Specific Expression ◽  
Primary Osteoblast ◽  
Cell Type Specific ◽  
Enriched Cultures

The extracellular matrix glycoprotein, tenascin, is associated in vivo with mesenchyme undergoing osteogenesis and chondrogenesis, but is absent from mature bone and cartilage matrix. The expression of tenascin by osteoblastic cells in vitro has been investigated by immunoblotting and immunocytochemistry. Tenascin was secreted into the medium and deposited in the matrix by human and rat osteoblast-like cell lines, as well as by primary osteoblast-enriched cultures from chick embryo calvarial bones. In primary osteoblast-enriched cultures, extracellular tenascin was found only in cell aggregates expressing the osteoblast marker alkaline phosphatase. Chicken osteoblast cultures synthesized almost exclusively the largest tenascin subunit, whereas fibroblast cultures from periostea of chicken calvariae synthesized approximately equal amounts of all three subunits. In situ hybridization studies of developing chicken bones, using a cDNA probe that hybridizes to all chicken tenascin splice variants, showed specific labelling of both osteogenic and chondrogenic regions of developing endochondral bones. In contrast, a cDNA probe specific for the large tenascin splice variant showed specific hybridization in osteogenic but not chondrogenic regions. Within osteogenic regions, tenascin mRNA was expressed by osteoblasts. A comparison of in situ hybridization and immunohistochemical studies demonstrated that tenascin mRNA and protein were codistributed in osteogenic regions of endochondral and membrane bones, whereas protein was retained in regions of differentiating cartilage where mRNA was no longer detectable. The results presented here demonstrate that tenascin is synthesized by osteoblasts. Moreover, within developing bones, there are at least three different cell type-specific patterns of expression of tenascin splice variants.

Cell-specific expression of fibronectin and EIIIA and EIIIB splice variants after oxygen injury

1998 ◽  
Vol 274 (4) ◽  
pp. L599-L609
Author(s):  
William M. Maniscalco ◽  
Richard H. Watkins ◽  
Patricia R. Chess ◽  
Robert A. Sinkin ◽  
Stuart Horowitz ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Splice Variants ◽  
Large Vessel ◽  
Cell Phenotype ◽  
Type Ii Cells ◽  
Type Ii ◽  
Specific Expression ◽  
Type Ii Cell

Cellular fibronectin (cFN) expression is characteristic of injured tissues. Unlike plasma FN, cFN mRNA often contains the EIIIA or EIIIB domains. We examined the lung cell-specific expression of total cFN mRNA and the EIIIA and EIIIB splice variants in rabbits after acute oxygen injury. By in situ hybridization, control lung had low cFN mRNA. After exposure to >95% oxygen, mRNAs for total cFN and EIIIA were noted primarily in alveolar macrophages and large-vessel endothelial cells. By 3–5 days recovery, cFN and EIIIA mRNA abundance was increased in alveolar septal cells (i.e., alveolar epithelial, interstitial, or endothelial cells) and in some large-vessel endothelial cells but was low in bronchial epithelial cells. During recovery, EIIIB mRNA was low in alveolar septal cells but was noted mainly in chondrocytes. Immunostaining for EIIIA increased during recovery, paralleling the in situ hybridizations. Because FN may modulate alveolar type II cell phenotype, we investigated type II cell cFN mRNA expression in vivo. During recovery, neither isolated type II cells nor cells with surfactant protein C mRNA in vivo contained FN mRNA. In summary, these data suggest that cFN with the EIIIA domain has a role in alveolar cell recovery from oxygen injury and that type II cells do not express cFN during recovery.

scholarly journals A novel neuronal-specific splice variant of Type I phosphatidylinositol 4-phosphate 5-kinase isoform gamma

2004 ◽  
Vol 379 (2) ◽  
pp. 489-496 ◽  
Author(s):  
Maria-Luisa GIUDICI ◽  
Piers C. EMSON ◽  
Robin F. IRVINE
Keyword(s):  
Splice Variant ◽  
Granule Cells ◽  
Cerebellar Granule Cells ◽  
Splice Variants ◽  
Type I ◽  
Cellular Processes ◽  
C Terminus ◽  
Neuronal Processes ◽  
Phosphatidylinositol 4

Type I PIPkins (phosphatidylinositol 4-phosphate 5-kinases) are the enzymes that catalyse the major cellular route of synthesis of PtdIns(4,5)P2, and three isoforms (α, β and γ) with several splice variants have been found to date. In the present paper, we describe the discovery of a novel splice variant of the γ isoform, which we call PIPkin Iγc, and which is characterized by the inclusion of a 26-amino-acid insert near the C-terminus. Its transcript appears to be selectively expressed in brain, where it locates in the neurons of restricted regions, such as cerebellum, hippocampus, cortex and olfactory bulb, as indicated by in situ hybridization studies. Overexpression of two different catalytically inactive constructs of PIPkin Iγc in rat cerebellar granule cells causes a progressive loss of their neuronal processes, whereas equivalent kinase-dead versions of PIPkin Iγa did not induce any such effect, suggesting the possible existence of a specific PtdIns(4,5)P2 pool synthesized by PIPkin Iγc, which is involved in the maintenance of some neuronal cellular processes.

scholarly journals Molecular cloning and characterization of PLC-η2

2005 ◽  
Vol 391 (3) ◽  
pp. 667-676 ◽  
Author(s):  
Yixing Zhou ◽  
Michele R. Wing ◽  
John Sondek ◽  
T. Kendall Harden
Keyword(s):  
Splice Variants ◽  
Second Messenger ◽  
Binding Motif ◽  
Rt Pcr ◽  
Specific Expression ◽  
Catalytic Core ◽  
C Terminus ◽  
Rapid Amplification

PLC (phospholipase C) isoenzymes catalyse the conversion of PtdIns(4,5)P2 into the Ca2+-mobilizing second messenger, Ins(1,4,5)P3, and the protein kinase C-activating second messenger, diacylglycerol. With the goal of identifying additional mammalian PLC isoenzymes, we screened the NCBI non-redundant database using a BLAST algorithm for novel sequences with homology with the conserved PLC catalytic core. Two unique sequences corresponding to two unknown PLC isoenzymes were identified, and one of these, designated PLC-η2, was cloned and characterized. Most of the coding sequence of PLC-η2 was constructed from two ESTs (expressed sequence tags), which included an overlapping sequence that was confirmed by multiple ESTs and mRNAs. 5′-RACE (rapid amplification of cDNA ends) also identified an upstream exon not deduced from available EST or mRNA sequences. Sequence analysis of PLC-η2 revealed the canonical domains of a PLC isoenzyme with an additional long C-terminus that contains a class II PDZ-binding motif. Genomic analyses indicated that PLC-η2 is encoded by 23 exons. RT-PCR (reverse transcriptase-PCR) analyses illustrated expression of PLC-η2 in human retina and kidney, as well as in mouse brain, eye and lung. RT-PCR with exon-specific primers also revealed tissue-specific expression of four splice variants in mouse that represent alternative use of sequences in exons 21, 22 and 23. PLC-η2-specific antisera recognized one of these splice variants as an approx. 155 kDa species when expressed in COS-7 cells; PLC-η2 natively expressed in 1321N1 human astrocytoma cells also migrated as an approx. 155 kDa species. PLC activity was observed in vitro and in vivo for three different constructs of PLC-η2, each containing possible alternatively spliced first exons. Co-expression of PLC-η2 with Gβ1γ2 dimers of heterotrimeric G-proteins resulted in marked stimulation of inositol lipid hydrolysis. Thus PLC-η2 may in part function downstream of G-protein-coupled receptors.

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