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 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.

scholarly journals A cDNA clone for human glucosamine-6-sulphatase reveals differences between arylsulphatases and non-arylsulphatases

1992 ◽  
Vol 288 (2) ◽  
pp. 539-544 ◽  
Author(s):  
D A Robertson ◽  
C Freeman ◽  
C P Morris ◽  
J J Hopwood
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Sequence Similarity ◽  
Genetic Disorder ◽  
Heparan Sulphate ◽  
Leader Peptide ◽  
Cdna Clones ◽  
Amino Acid Residues ◽  
Lysosomal Storage ◽  
Glycosylation Sites

Glucosamine-6-sulphatase is an exo-hydrolase required for the lysosomal degradation of heparan sulphate and keratan sulphate. Deficiency of glucosamine-6-sulphatase activity leads to the lysosomal storage of the glycosaminoglycan, heparan sulphate and the monosaccharide sulphate N-acetylglucosamine 6-sulphate and the autosomal recessive genetic disorder mucopolysaccharidosis type IIID. Glucosamine-6-sulphatase can be classified as a non-arylsulphatase since, relative to arylsulphatase B, it shows negligible activity toward 4-methylumbelliferyl sulphate. We have isolated human cDNA clones and derived amino acid sequence coding for the entire glucosamine-6-sulphatase protein. The predicted sequence has 552 amino acids with a leader peptide of 36 amino acids and contains 13 potential N-glycosylation sites, of which it is likely that 10 are used. Glucosamine-6-sulphatase shows strong sequence similarity to other sulphatases such as the family of arylsulphatases, although the degree of similarity is not as high as that between members of the arylsulphatase family. This pattern of inter- and intra-family similarity delineates regions and amino acid residues that may be critical for sulphatase function and substrate specificity.

scholarly journals Identification of two cDNA clones encoding small proline-rich proteins expressed in sheep ruminal epithelium

1996 ◽  
Vol 317 (1) ◽  
pp. 225-233 ◽  
Author(s):  
Luquan WANG ◽  
Ransom L. BALDWIN VI ◽  
Barry W. JESSE
Keyword(s):  
Epidermal Keratinocytes ◽  
Cdna Clones ◽  
Type Ii ◽  
Stratum Granulosum ◽  
Specific Expression ◽  
Cornified Envelope ◽  
Human Type ◽  
Epithelial Development ◽  
Ruminal Epithelium

Small proline-rich (SPRR) proteins are markers frequently associated with squamous cell differentiation. They have been proposed to be a novel group of precursor polypeptides for the cornified envelope in epidermal keratinocytes. A plus/minus screening procedure was used to identify cDNA clones expressed in mature but not in neonatal sheep ruminal epithelium. Two clones encoding SPRR proteins were identified and are reported here. Clone 27 encodes an ovine SPRR protein corresponding to the human type-II SPRR protein. Clone 26 encodes an ovine SPRR protein similar to human type-II SPRR protein, but which also contains an N-terminal His-Pro repeat similar to the paired repeats found in the Drosophila paired proteins. The unique combination of a paired domain and an SPRR protein has not been reported prior to this study. The tissue distribution indicates that specific expression of the genes corresponding to these two clones occurs in the epithelium of the ruminant forestomach, and to a lesser extent in skin epithelium. In situ hybridization demonstrated that the SPRR mRNA for both clones were localized in the stratum granulosum, in support of their putative physiological function, i.e. formation of the cornified envelope. Based on Northern blot analysis, mRNA complementary to the two clones appears in the ruminal epithelium by 1 week of age, corresponding to the formation of the stratum granulosum during ruminal epithelial development. The different patterns of changes in amount of mRNA corresponding to these clones during rumen epithelial development indicate that they play different roles in rumen epithelial development.

scholarly journals cDNA cloning and tissue-specific expression of a novel basic helix-loop-helix/PAS factor (Arnt2) with close sequence similarity to the aryl hydrocarbon receptor nuclear translocator (Arnt).

1996 ◽  
Vol 16 (4) ◽  
pp. 1706-1713 ◽  
Author(s):  
K Hirose ◽  
M Morita ◽  
M Ema ◽  
J Mimura ◽  
H Hamada ◽  
...  
Keyword(s):  
Aryl Hydrocarbon Receptor ◽  
Sequence Similarity ◽  
Cdna Clones ◽  
Mobility Shift ◽  
Phylogenetic Groups ◽  
Specific Expression ◽  
Basic Helix Loop Helix ◽  
Helix Loop Helix ◽  
Aryl Hydrocarbon ◽  
Adult Mice

We isolated mouse cDNA clones (Arnt2) that are highly similar to but distinct from the aryl hydrocarbon receptor (AhR) nuclear translocator (Arnt). The composite cDNA covered a 2,443-bp sequence consisting of a putative 2,136-bp open reading frame encoding a polypeptide of 712 amino acids. The predicted Arnt2 polypeptide carries a characteristic basic helix-loop-helix (bHLH)/PAS motif in its N-terminal region with close similarity (81% identity) to that of mouse Arnt and has an overall sequence identity of 57% with Arnt. Biochemical properties and interaction of Arnt2 with other bHLH/PAS proteins were investigated by coimmunoprecipitation assays, gel mobility shift assays, and the yeast two-hybrid system. Arnt2 interacted with AhR and mouse Sim as efficiently as Arnt, and the Arnt2-AhR complex recognized and bound specifically the xenobiotic responsive element (XRE) sequence. Expression of Arnt2 successfully rescued XRE-driven reporter gene activity in the Arnt-defective c4 mutant of Hepa-1 cells. RNA blot analysis revealed that expression of Arnt2 mRNA was restricted to the brains and kidneys of adult mice, while Arnt mRNA was expressed ubiquitously. In addition, whole-mount in situ hybridization of 9.5-day mouse embryos showed that Arnt2 mRNA was expressed in the dorsal neural tube and branchial arch 1, while Arnt transcripts were detected broadly in various tissues of mesodermal and endodermal origins. These results suggest that Arnt2 may play different roles from Arnt both in adult mice and in developing embryos. Finally, sequence comparison of the currently known bHLH/PAS proteins indicates a division into two phylogenetic groups: the Arnt group, containing Arnt, Arnt2, and Per, and the AhR group, consisting of AhR, Sim, and Hif-1alpha.

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 Structure, alternative splicing and chromosomal localization of the cystatin-related epididymal spermatogenic gene

1999 ◽  
Vol 340 (1) ◽  
pp. 85-93 ◽  
Author(s):  
Gail A. CORNWALL ◽  
Nelson HSIA ◽  
H. Gayle SUTTON
Keyword(s):  
Cystatin C ◽  
Sequence Similarity ◽  
Protein Structures ◽  
Chromosomal Mapping ◽  
Cdna Clones ◽  
Chromosome 2 ◽  
Specific Expression ◽  
Intron Splice ◽  
The Family ◽  
Cystatin Family

The cystatin superfamily of cysteine protease inhibitors consists of three major families, including the stefins, cystatins and kininogens. However, the recent identification of several genes that possess sequence similarity with the cystatins but have different gene or protein structures indicates that several new cystatin families or subgroups of families might exist. We previously identified the cystatin-related epididymal spermatogenic (Cres) gene, which is related to the family 2 cystatins but exhibits highly tissue-specific expression in the reproductive tract. In the studies presented here, an analysis of gene structure as well as chromosomal mapping studies suggest that the Cres gene might represent a new subgroup within the family 2 cystatins. Although the Cres gene possesses an additional exon encoding 5ʹ untranslated sequences, its coding exons are similar in size to the three coding exons of the cystatin family 2 genes, and the Cres exon/intron splice junctions occur in identical locations as in the cystatin C gene. Furthermore, chromosomal mapping studies show that the Cres gene co-segregates with the cystatin C gene on mouse chromosome 2. Similar to the cystatin family 2 proteins, the Cres protein possesses the type A and B disulphide loops that are necessary for cystatin folding. Interestingly, Cres protein also possesses half of a type C disulphide loop. Although probably related to the cystatin genes, the Cres gene is distinct in that its promoter contains consensus motifs typical of regulated genes. Finally, reverse transcriptase-mediated PCR studies and the identification of new Cres cDNA clones indicate that the Cres mRNA is alternatively spliced, resulting in two Cres

MPTP delta, a putative murine homolog of HPTP delta, is expressed in specialized regions of the brain and in the B-cell lineage

1993 ◽  
Vol 13 (9) ◽  
pp. 5513-5523
Author(s):  
K Mizuno ◽  
K Hasegawa ◽  
T Katagiri ◽  
M Ogimoto ◽  
T Ichikawa ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Type A ◽  
Extracellular Domain ◽  
Fine Tuning ◽  
Leader Peptide ◽  
Reticular Nucleus ◽  
Cdna Clones ◽  
Protein Tyrosine ◽  
Type C

Protein tyrosine phosphatases (PTPs), together with protein tyrosine kinases (PTKs), are involved in the regulation of cell activation, growth, and differentiation. To further elucidate the fine tuning of cell growth and differentiation through tyrosine phosphorylation, we tried to isolate mouse receptor-type PTP (RPTP) cDNA clones by screening mouse brain cDNA libraries with mouse CD45 PTP domain probes under reduced-stringency conditions. Characterization of isolated cDNA clones for RPTP showed that the cytoplasmic region contains two tandem repeats of PTP domain of about 230 amino acids with intrinsic phosphatase activity. The extracellular region was composed of immunoglobulin (Ig)-like domains and fibronectin type III (FN-III)-like domains. The gene was highly homologous to human PTP delta (HPTP delta) and thus was named MPTP delta (murine counterpart of HPTP delta). The MPTP delta gene appeared to generate at least three species of mRNA, which differ in the composition of the extracellular domain: type A, one Ig-like and four FN-III-like domains; type B, one Ig-like and eight FN-III-like domains; and type C, three Ig-like and eight FN-III-like domains. Interestingly, the 5' untranslated region and the leader peptide of types A and B were completely different from those of type C. Northern (RNA) blot analysis demonstrated that brain, kidney, and heart cells express three mRNA species of about 7 kb. Antibody directed against part of the extracellular domain of type A MPTP delta recognized a 210-kDa protein in brain and kidney lysates. In situ hybridization of brain samples revealed that MPTP delta mRNA is present in the hippocampus, thalamic reticular nucleus, and piriform cortex, where some Src family PTKs have been also demonstrated to exist. Although MPTP delta mRNA was not detected in lymphoid tissues, all of the pre-B-cell lines tested and one of three B-cell lines tested expressed MPTP delta mRNA, whereas antibody-producing B-cell hybridomas and T-cell and macrophage lines did not. Finally, the MPTP delta locus was tightly linked to the brown (b) locus on mouse chromosome 4.

Spinalin, a new glycine- and histidine-rich protein in spines of Hydra nematocysts

1998 ◽  
Vol 111 (11) ◽  
pp. 1545-1554 ◽  
Author(s):  
A.W. Koch ◽  
T.W. Holstein ◽  
C. Mala ◽  
E. Kurz ◽  
J. Engel ◽  
...  
Keyword(s):  
External Surface ◽  
Insoluble Fraction ◽  
Cdna Clones ◽  
Mature Protein ◽  
Capsule Wall ◽  
The Matrix ◽  
Acidic Region ◽  
Basic Region ◽  
Full Length Clone

Here we present the cloning, expression and immunocytochemical localization of a novel 24 kDa protein, designated spinalin, which is present in the spines and operculum of Hydra nematocysts. Spinalin cDNA clones were identified by in situ hybridization to differentiating nematocytes. Sequencing of a full-length clone revealed the presence of an N-terminal signal peptide, suggesting that the mature protein is sorted via the endoplasmic reticulum to the post-Golgi vacuole in which the nematocyst is formed. The N-terminal region of spinalin (154 residues) is very rich in glycines (48 residues) and histidines (33 residues). A central region of 35 residues contains 19 glycines, occurring mainly as pairs. For both regions a polyglycine-like structure is likely and this may be stabilized by hydrogen bond-mediated chain association. Similar sequences found in loricrins, cytokeratins and avian keratins are postulated to participate in formation of supramolecular structures. Spinalin is terminated by a basic region (6 lysines out of 15 residues) and an acidic region (9 glutamates and 9 aspartates out of 32 residues). Western blot analysis with a polyclonal antibody generated against a recombinant 19 kDa fragment of spinalin showed that spinalin is localized in nematocysts. Following dissociation of the nematocyst's capsule wall with DTT, spinalin was found in the insoluble fraction containing spines and the operculum. Immunocytochemical analysis of developing nematocysts revealed that spinalin first appears in the matrix but then is transferred through the capsule wall at the end of morphogenesis to form spines on the external surface of the inverted tubule and the operculum.

A gene with sequence similarity to Drosophila engrailed is expressed during the development of the neural tube and vertebrae in the mouse

Development ◽  
1988 ◽  
Vol 104 (2) ◽  
pp. 305-316 ◽  
Author(s):  
D. Davidson ◽  
E. Graham ◽  
C. Sime ◽  
R. Hill
Keyword(s):  
In Situ Hybridization ◽  
Neural Tube ◽  
Mouse Embryo ◽  
Sequence Similarity ◽  
Anterior Part ◽  
Limb Bud ◽  
Tissue Type ◽  
Restricted Domains ◽  
Restricted Region

The mouse genes En-1 and En-2 display sequence similarity, in and around the homeobox region, to the engrailed family in Drosophila. This paper describes their pattern of expression in the 12.5-day mouse embryo as determined by in situ hybridization. En-2 is expressed in a subset of cells expressing En-1. Both genes are expressed in the developing midbrain and its junction with the hindbrain. In addition, En-1 is expressed in the floor of the hindbrain, a restricted ventrolateral segment of the neural tube throughout the trunk and anterior part of the tail, the dermatome of tail somites, the centrum and costal processes in developing vertebrae, a restricted region of facial mesenchyme and the limb-bud ectoderm. Supplementary studies of 9.5-day and 10.5-day embryos showed that the same pattern of expression pertained in the neural tube, but that expression in the somites is at first confined to the dermatome and later found at a low level in restricted sclerotomal regions. Both genes are expressed in restricted domains which do not cross tissue-type boundaries. In several instances, however, boundaries of expression lie within morphologically undifferentiated tissue. These results suggest that En-1 and En-2 may be involved in the establishment or maintenance of the spatial integrity of specific domains within developing tissues.

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