Expression of intermediate filament proteins during development of Xenopus laevis. III. Identification of mRNAs encoding cytokeratins typical of complex epithelia

Development ◽  
1988 ◽  
Vol 104 (4) ◽  
pp. 533-548 ◽  
Author(s):  
B. Fouquet ◽  
H. Herrmann ◽  
J.K. Franz ◽  
W.W. Franke
Keyword(s):  
Xenopus Laevis ◽  
Developmental Stages ◽  
Type I ◽  
Type Ii ◽  
S1 Nuclease ◽  
Intermediate Filament Proteins ◽  
Larval Stages ◽  
Adult Tissues ◽  
Adult Organism ◽  
Restricted Pattern

A Xenopus laevis mRNA encoding a cytokeratin of the basic (type II) subfamily that is expressed in postgastrulation embryos was cDNA-cloned and sequenced. Comparison of the deduced amino acid sequence of this polypeptide (513 residues, calculated mol. wt 55,454; Mr approximately 58,000 on SDS-PAGE) with those of other cytokeratins revealed its relationship to certain type II cytokeratins of the same and other species, but also remarkable differences. Using a subclone representing the 3′-untranslated portion of the 2.4 kb mRNA encoding this cytokeratin, designated XenCK55(5/6), in Northern blot experiments, we found that it differs from the only other Xenopus type II cytokeratin known, i.e. the simple epithelium-type component XenCK1(8), in that it is absent in unfertilized eggs and pregastrulation embryos. XenCK55(5/6) mRNA was first detected at gastrulation (stage 11) and found to rapidly increase during neurulation and further development. It was also identified in Xenopus laevis cultured kidney epithelial cells of the line A6 and in the adult animal where it is a major polypeptide in the oesophageal mucosa but absent in most other tissues examined. The pattern of XenCK55(5/6) expression during embryonic development was similar to that reported for the type I polypeptides of the ‘XK81 subfamily’ previously reported to be embryo-specific and absent in adult tissues. Therefore, we used a XK81 mRNA probe representing the 3′-untranslated region in Northern blots, S1 nuclease and hybrid-selection-translation assays and found the approximately 1.6 kb XK81 mRNA and the resulting protein of Mr approximately 48,000 not only in postgastrula embryos and tadpoles but also in the oesophagus of adult animals. Our results show that both these type II and type I cytokeratins are synthesized only on gastrulation and are very actively produced in early developmental stages but is continued in at least one epithelium of the adult organism. These observations raise doubts on the occurrence of Xenopus cytokeratins that are strictly specific for certain embryonic or larval stages and absent in the adult. They rather suggest that embryonically expressed cytokeratins are also produced in some adult tissues, although in a restricted pattern of tissue and cell type distribution.

scholarly journals Evaluation of Effective and Practical Euthanasia Methods for Larval African Clawed Frogs (Xenopus laevis)

2020 ◽  
Vol 59 (3) ◽  
pp. 269-274
Author(s):  
Ilana A Galex ◽  
Cameron M Gallant ◽  
Nicole D'Avignon ◽  
Lauren M Kuchenbrod ◽  
Craig A Fletcher ◽  
...  
Keyword(s):  
Xenopus Laevis ◽  
Developmental Stages ◽  
Age Group ◽  
Research Model ◽  
Larval Stages ◽  
Large Numbers ◽  
Guidance Documents ◽  
Stock Solutions ◽  
Tricaine Methanesulfonate ◽  
Tank Water

Larval, or tadpole-stage Xenopus laevis frogs are a popular research model for developmental biology and disease studies. Existing euthanasia guidance documents offer recommendations for both eggs and adult stages, yet do not specifically address the larval stage. Data evaluating effective euthanasia methods for groups of X. laevis tadpoles would therefore be useful. The goal of the current study was to evaluate the efficacy of various immersion euthanasia procedures on tadpoles: tricaine methanesulfonate (MS222) at 6 g/L, eugenol at 800 μL/L and rapid chilling (2 to 4 °C). We also evaluated tadpoles at various developmental stages (NF stages 46, 47 and 49). Tadpoles (n = 70) were exposed to euthanasia solution for 15 min, and controls (n = 40) were placed in housing tank water for 15 min. All animals were then placed in recovery tanks containing housing tank water for 4 h to confirm irreversibility of each agent. Cessation of the heartbeat was assessed at the end of euthanasia solution exposure and at each hour thereafter. We found that immersion in a 6 g/L solution of MS222 resulted in 100% euthanasia of all larval stages tested. Conversely, eugenol produced variable euthanasia rates that were affected by both age group and batches of stock solutions. Rapid chilling was completely ineffective as a euthanasia method in our study. Based on our findings, we recommend MS222 as an effective and practical means of euthanizing large numbers of X. laevis tadpoles.

Genes for intermediate filament proteins and the draft sequence of the human genome

2001 ◽  
Vol 114 (14) ◽  
pp. 2569-2575 ◽  
Author(s):  
Michael Hesse ◽  
Thomas M. Magin ◽  
Klaus Weber
Keyword(s):  
Human Genome ◽  
Dna Sequences ◽  
Intermediate Filament ◽  
Muscle Protein ◽  
Gene Families ◽  
Type I ◽  
Type Ii ◽  
Draft Sequence ◽  
Intermediate Filament Proteins ◽  
Processed Pseudogenes

We screened the draft sequence of the human genome for genes that encode intermediate filament (IF) proteins in general, and keratins in particular. The draft covers nearly all previously established IF genes including the recent cDNA and gene additions, such as pancreatic keratin 23, synemin and the novel muscle protein syncoilin. In the draft, seven novel type II keratins were identified, presumably expressed in the hair follicle/epidermal appendages. In summary, 65 IF genes were detected, placing IF among the 100 largest gene families in humans. All functional keratin genes map to the two known keratin clusters on chromosomes 12 (type II plus keratin 18) and 17 (type I), whereas other IF genes are not clustered. Of the 208 keratin-related DNA sequences, only 49 reflect true keratin genes, whereas the majority describe inactive gene fragments and processed pseudogenes. Surprisingly, nearly 90% of these inactive genes relate specifically to the genes of keratins 8 and 18. Other keratin genes, as well as those that encode non-keratin IF proteins, lack either gene fragments/pseudogenes or have only a few derivatives. As parasitic derivatives of mature mRNAs, the processed pseudogenes of keratins 8 and 18 have invaded most chromosomes, often at several positions. We describe the limits of our analysis and discuss the striking unevenness of pseudogene derivation in the IF multigene family. Finally, we propose to extend the nomenclature of Moll and colleagues to any novel keratin.

scholarly journals Developmentally regulated cytokeratin gene in Xenopus laevis.

1985 ◽  
Vol 5 (10) ◽  
pp. 2575-2581 ◽  
Author(s):  
J A Winkles ◽  
T D Sargent ◽  
D A Parry ◽  
E Jonas ◽  
I B Dawid
Keyword(s):  
Amino Acid ◽  
Xenopus Laevis ◽  
Intermediate Filament ◽  
Type I ◽  
Developmentally Regulated ◽  
Amino Acid Homology ◽  
Intermediate Filament Proteins ◽  
Functional Roles ◽  
Terminal Domain ◽  
Terminal Domains

We have determined the sequence of cloned cDNAs derived from a 1,665-nucleotide mRNA which transiently accumulates during Xenopus laevis embryogenesis. Computer analysis of the deduced amino acid sequence revealed that this mRNA encodes a 47-kilodalton type I intermediate filament subunit, i.e., a cytokeratin. As is common to all intermediate filament subunits so far examined, the predicted polypeptide, named XK70, contains N- and C-terminal domains flanking a central alpha-helical rod domain. The overall amino acid homology between XK70 and a human 50-kilodalton type I keratin is 47%; homology within the alpha-helical domain is 57%. The N-terminal domain, which is not completely contained in our cDNAs, is basic, contains 42% serine plus alanine, and includes five copies of a six-amino-acid repeating unit. The C-terminal domain has a high alpha-helical content and contains a region with sequence homology to the C-terminal domains of other type I and type III intermediate filament proteins. We suggest that different keratin filament subtypes may have different functional roles during amphibian oogenesis and embryogenesis.

scholarly journals The amino acid sequence of component 7c, a type II intermediate-filament protein from wool

1989 ◽  
Vol 261 (3) ◽  
pp. 1015-1022 ◽  
Author(s):  
L G Sparrow ◽  
C P Robinson ◽  
D T W McMahon ◽  
M R Rubira
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Intermediate Filament ◽  
Coiled Coil ◽  
British Library ◽  
Intermediate Filament Protein ◽  
Type I ◽  
Type Ii ◽  
Blocking Group ◽  
Intermediate Filament Proteins

Component 7c is one of the four homologous type II intermediate-filament proteins that, by association with the complementary type I proteins, form the microfibrils or intermediate filaments in wool. Component 7c was isolated as the S-carboxymethyl derivative from Merino wool and its amino acid sequence was determined by manual and automatic sequencing of peptides produced by chemical and enzymic cleavage reactions. It is an N-terminally blocked molecule of 491 residues and Mr (not including the blocking group) of 55,600; the nature of the blocking group has not been determined. The predicted secondary structure shows that component 7c conforms to the now accepted pattern for intermediate-filament proteins in having a central rod-like region of approximately 310 residues of coiled-coil alpha-helix flanked by non-helical N-and C-terminal regions. The central region is divided by three non-coiled-coil linking segments into four helical segments 1A, 1B, 2A and 2B. The N-and C-terminal non-helical segments are 109 and 71 residues respectively and are rich in cysteine. Details of procedures use in determining the sequence of component 7c have been deposited as a Supplementary Publication SUP 50152 (65 pages) at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1989) 257,5. The information comprises: (1) details of chemical and enzymic methods used for cleavage of component 7c, peptides CN1, CN2 and CN3, and various other peptides, (2) details of the procedures used for the fractionation and purification of peptides from (1), including Figures showing the elution profiles from the chromatographic steps used, (3) details of methods used to determine the C-terminal sequence of peptide CN3, and (4) detailed evidence to justify a number of corrections to the previously published sequence.

scholarly journals Nodal signalling and apoptosis

Reproduction ◽  
2007 ◽  
Vol 133 (5) ◽  
pp. 847-853 ◽  
Author(s):  
Hongmei Wang ◽  
Benjamin K Tsang
Keyword(s):  
Transforming Growth Factor ◽  
Biological Effects ◽  
Transforming Growth Factor Β ◽  
Cell Types ◽  
Type I ◽  
Type Ii ◽  
Human Trophoblast ◽  
Adult Tissues ◽  
Ovarian Epithelial Cancer ◽  
Smad Proteins

Nodal, a member of the transforming growth factor β family, was first cloned from a 7.5 day post-coitum mouse embryo cDNA library. Nodal exerts its biological effects by signalling through its types I and II serine/threonine kinase receptor complex and intracellular Smad proteins. The type II receptors for Nodal are Activin type II receptors ActRIIA and ActRIIB, whereas the putative type I receptors are Activin receptor like kinase (ALK) 4 and ALK7. The main Smad proteins involved in Nodal signalling are Smad2 and Smad3. Studies of Nodal in adult tissues indicate that it is pro-apoptotic in rat ovarian granulosa cells, human trophoblast cells and human ovarian epithelial cancer cells and is growth inhibitory in the latter two cell types. This review summarises the progress made on the functions of Nodal in the apoptosis of adult tissues, especially in the ovary and placenta.

The development of lampbrush chromosome-type transcription in the early diplotene oocytes of Xenopus laevis: an electron-microscope analysis

1980 ◽  
Vol 44 (1) ◽  
pp. 87-101
Author(s):  
R.S. Hill ◽  
H.C. Macgregor
Keyword(s):  
Linear Regression ◽  
Xenopus Laevis ◽  
Linear Regression Analysis ◽  
Transcription Unit ◽  
Type I ◽  
Type Ii ◽  
Type Iii ◽  
Significant Difference ◽  
Germinal Vesicles ◽  
The Mean

Oocytes of Xenopus laevis in pachytene and early diplotene of meiosis have been studied using the Miller spreading technique. Transcription first appears in germinal vesicles 25–40 micrometers in diameter, when the oocyte is in early diplotene. Transcription at this stage consists of arrays of short RNP transcripts, irregularly distributed along the DNP axis. Linear regression analysis has shown that many of these arrays are transcription units (Type I) with the transcripts having a common point of origin. The lengths of these early transcription units (mean = 7.06 +/− 5.06 micrometers), calculated from the linear regression data, are comparable to the lengths of transcription units from later stages, including Dumont stage 3. The polymerase granules of Type I transcription units are widely and irregularly spaced, having a mean spacing of 505 +/− 748 nm. More advanced transcription units (Type II, mean length = 8.72 +/− 3.77 micrometer) are usually found in the same chromosome set as the Type I units. Type II transcription units have closer and more regularly spaced polymerase granules than Type I transcription units (mean spacing = 92 +/− 49 nm). Both Type I and II transcription units have comparatively short RNP transcripts, the mean values for the slopes of their regression lines being 0.1336 and 0.1440 respectively. By the time the germinal vesicles are about 50–60 micrometers in diameter the transcription units have a quite different morphology (Type III). The lengths of the Type III transcription units are comparable to the Type I and II units, the mean length being 6.34 +/− 4.03 micrometers. The spacing of the polymerase granules in the Type III units is closer and more regular than the earlier stages (70 +/− 40 nm). Another significant difference between Type III and Types I and II transcription units is a decrease in the foreshortening of the Type III RNP transcripts. The mean slope of the regression lines for Type III transcription units is 0.2439. The morphological appearance of the Type III transcription unit is virtually identical to that of the transcription units from Dumont stage 3 oocytes, both with respect to the length and the spacing of the polymerase granules. However, the transcripts in Type III transcription units are still more foreshortened than those of Dumont stage 3 oocytes, having mean regression slopes of 0.4728. From the data obtained in the present study, it has been concluded that the pattern of lampbrush-type transcription is virtually fully established by the time most germinal vesicles are about 50 micrometers in diameter.

Vertebrate liver cytokeratins: a comparative study

1987 ◽  
Vol 65 (6) ◽  
pp. 547-557 ◽  
Author(s):  
R. G. Pankov ◽  
A.A. Uschewa ◽  
B. T. Tasheva ◽  
G. G. Markov
Keyword(s):  
Electron Microscopy ◽  
Molecular Weight ◽  
Intermediate Filaments ◽  
Methodological Approach ◽  
Type I ◽  
Type Ii ◽  
Electron Microscopic ◽  
Intermediate Filament Proteins ◽  
Lower Vertebrates ◽  
Microscopic Morphology

The structure and composition of intermediate filaments isolated from liver of representatives of different vertebrate classes have been studied by electron microscopy and biochemical and immunochemical methods. It has been shown that the methodological approach for isolation of rat liver intermediate filaments can be efficiently applied to all other classes of vertebrates. The intermediate filaments studied have the same electron microscopic morphology and are species undistinguishable. The molecular weight of intermediate filament proteins varies from 40 000 to 60 000 and their isoelectric point varies from 5.0 to 6.45. Immunological investigations show that in all animals studied the intermediate filaments are built up of cytokeratins belonging to both types of keratins: type I and type II. Only one protein of the type II cytokeratins is present in all vertebrate classes, whereas in lower vertebrates two or even three type I cytokeratins contribute to the structure of liver intermediate filaments. The biochemical and immunochemical results are discussed with regard to the evolution of liver cytokeratins.

scholarly journals Stage-specific keratins in Xenopus laevis embryos and tadpoles: the XK81 gene family.

1986 ◽  
Vol 103 (5) ◽  
pp. 1957-1965 ◽  
Author(s):  
S Miyatani ◽  
J A Winkles ◽  
T D Sargent ◽  
I B Dawid
Keyword(s):  
Xenopus Laevis ◽  
Cdna Clone ◽  
Cdna Clones ◽  
Type I ◽  
Larval Stages ◽  
Hybridization Data ◽  
Isolation And Characterization ◽  
Partial Cdna ◽  
Flanking Sequences ◽  
Partial Cdna Clone

This report describes the isolation and characterization of genomic and cDNA clones which define a subfamily of type I keratins in Xenopus laevis whose expression is restricted to embryonic and larval stages. The XK81 subfamily, named after the prototype cDNA clone DG81, contains four members arranged in two pairs of closely homologous loci; they were named 81A1, A2, B1, and B2. Genomic clones were obtained representing all of these regions. The A1 gene has been completely sequenced together with approximately 1 kb of flanking sequences at each end; this gene corresponds to the previously reported cDNA clone 8128 (Jonas, E., T. D. Sargent, and I. B. Dawid, 1985, Proc. Natl. Acad. Sci. USA, 82:5413-5417). The B2 gene is represented by a partial cDNA clone, DG118. Upstream sequences and about half of the coding regions have been sequenced for the B1 and B2 genes, whereas the A2 locus has been identified on the basis of hybridization data and could be a gene or pseudogene. Genomic Southern blotting indicates that all members of the subfamily have been isolated. The keratin proteins encoded by the B1 and B2 genes are 96% homologous in the central rod domain, whereas A/B gene homology in this region is 81%. During development mRNAs derived from A and B genes accumulate coordinately during gastrula and neurula stages; in the tadpole, 81A mRNA decays rapidly, whereas 81B mRNA shows a second abundance peak, persists for most of tadpole life, and decays by metamorphosis. RNAs derived from the XK81 keratin subfamily are undetectable in the adult, where different type I keratin genes are expressed.

scholarly journals Type II Keratins Are Phosphorylated on a Unique Motif during Stress and Mitosis in Tissues and Cultured Cells

2002 ◽  
Vol 13 (6) ◽  
pp. 1857-1870 ◽  
Author(s):  
Diana M. Toivola ◽  
Qin Zhou ◽  
Luc S. English ◽  
M. Bishr Omary
Keyword(s):  
Cultured Cells ◽  
Uv Light ◽  
Ex Vivo ◽  
Psoriatic Skin ◽  
Type I ◽  
Type Ii ◽  
Intermediate Filament Proteins ◽  
Phosphatase Inhibitors

Epithelial cell keratins make up the type I (K9–K20) and type II (K1–K8) intermediate filament proteins. In glandular epithelia, K8 becomes phosphorylated on S73 (71LLpSPL) in human cultured cells and tissues during stress, apoptosis, and mitosis. Of all known proteins, the context of the K8 S73 motif (LLS/TPL) is unique to type II keratins and is conserved in epidermal K5/K6, esophageal K4, and type II hair keratins, except that serine is replaced by threonine. Because knowledge regarding epidermal and esophageal keratin regulation is limited, we tested whether K4–K6 are phosphorylated on the LLTPL motif. K5 and K6 become phosphorylated in vitro on threonine by the stress-activated kinase p38. Site-specific anti-phosphokeratin antibodies to LLpTPL were generated, which demonstrated negligible basal K4–K6 phosphorylation. In contrast, treatment of primary keratinocytes and other cultured cells, and ex vivo skin and esophagus cultures, with serine/threonine phosphatase inhibitors causes a dramatic increase in K4–K6 LLpTPL phosphorylation. This phosphorylation is accompanied by keratin solubilization, filament reorganization, and collapse. K5/K6 LLTPL phosphorylation occurs in vivo during mitosis and apoptosis induced by UV light or anisomycin, and in human psoriatic skin and squamous cell carcinoma. In conclusion, type II keratins of proliferating epithelia undergo phosphorylation at a unique and conserved motif as part of physiological mitotic and stress-related signals.

Cloning of the human keratin 18 gene and its expression in nonepithelial mouse cells

1988 ◽  
Vol 8 (4) ◽  
pp. 1540-1550
Author(s):  
D A Kulesh ◽  
R G Oshima
Keyword(s):  
Cell Lines ◽  
Peptide Mapping ◽  
Cell Types ◽  
Type I ◽  
Type Ii ◽  
S1 Nuclease ◽  
Mouse Cells ◽  
Keratin 18 ◽  
Cloned Gene ◽  
Endodermal Cells

Human keratin 18 (K18) and the homologous mouse protein, Endo B, are intermediate filament subunits of the type I keratin class. Both are expressed in many simple epithelial cell types including trophoblasts, the first differentiated cell type to appear during mouse embryogenesis. The K18 gene was identified and cloned from among the 15 to 20 similar sequences identified within the human genome. The identity of the cloned gene was confirmed by comparing the sequence of the first two exons to the K18 cDNA sequence and transfecting the gene into various murine cell lines and verifying the encoded protein as K18 by immunoprecipitation and partial peptide mapping. The transfected K18 gene was expressed in mouse HR9 parietal endodermal cells and mouse fibroblasts even though the fibroblasts fail to express endogenous Endo B. S1 nuclease protection analysis indicated that mRNA synthesized from the transfected K18 gene is initiated at the same position as authentic K18 mRNA found in both BeWo trophoblastoma cells and HeLa cells. Pulse-chase experiments indicated that the human K18 protein is stable in murine parietal endodermal cells (HR9) which express EndoA, a complementary mouse type II keratin. Surprisingly, however, K18 was degraded when synthesized in cells which lack a type II keratin. This turnover of K18 may be an important mechanism by which epithelial cells maintain equal molar amounts of both type I and II keratins. In addition, the levels of the endogenous type I Endo B in parietal endodermal cells were compensatingly down regulated in the presence of the K18 protein, while the levels of the endogenous type II Endo A were not affected in any of the transfected cell lines.

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